PLANT SMALL RNA IN FOOD CROPS This page intentionally left blank PLANT SMALL RNA IN FOOD CROPS Edited by PRAVEEN GULERIA Plant Biotechnology and Genetic Engineering Lab, Department of Biotechnology, DAV University, Jalandhar, Punjab, India VINEET KUMAR Department of Biotechnology, School of Bioengineering and Biosciences, Lovely Professional University, Phagwara, Punjab, India BEIXIN MO Guangdong Provincial Key Laboratory for Plant Epigenetics, College of Life Sciences and Oceanography, Shenzhen University, Shenzhen, Guangdong, China Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright © 2023 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. ISBN: 978-0-323-91722-3 For information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals Publisher: Nikki Levy Acquisitions Editor: Nancy Maragioglio Editorial Project Manager: Kathrine Esten Production Project Manager: Sruthi Satheesh Cover Designer: Victoria Pearson Esser Typeset by TNQ Technologies Contents List of contributors Editors biography Preface xi xv xvii SECTION 1 Basics 1. Food crops, growth and productivity as an important focus for sustainable agriculture 3 Amina A. Aly and Zeyad M. Borik 1. Most popular food crops 2. Future challenges 3. Increasing growth and productivity of crops foods 4. Future perspectives References Further reading 2. Challenges and opportunities to sustainable crop production 3 5 8 16 18 22 25 Simerjeet Kaur and Bhagirath S. Chauhan 1. Present scenario of food production 2. Challenges and opportunities for sustainable food production 3. Conclusions References 3. Molecular mechanisms and strategies in response to abiotic stresses for the sustainability of crop production under changing climate 25 28 41 42 45 K.L. Bhutia, Biswajit Pramanick, Sagar Maitra, Saipayan Ghosh and Akbar Hossain 1. 2. 3. 4. 5. Introduction Abiotic stresses and signal transduction: perceiving the stress signal Molecular mechanism of abiotic stress tolerance Role of small RNAs in abiotic stress tolerance Strategies to develop abiotic stress tolerance for sustainability of crop production under changing climate 45 48 50 51 56 v vi Contents 6. Conclusion References 4. Small RNAs as emerging regulators of agricultural traits of food crops 58 60 69 Jinyuan Tao and Yu Yu 1. Introduction 2. Biogenesis, classification, and modes of action of small RNAs 3. Small RNAs contribute to the agricultural traits of food crops 4. Conclusion and future perspectives References 69 70 72 86 95 SECTION 2 Small RNA from food crops: mechanism and regulation 5. Exploring small RNA in food crops: techniques and approaches 109 Saurabh Chaudhary 1. Introduction 2. High throughput next generation small RNA sequencing 3. Future perspectives Acknowledgment References 6. Plant small RNAs: biogenesis, mechanistic functions and applications 109 110 123 124 124 129 S.V. Ramesh, S. Rajesh and T. Radhamani 1. Introduction 2. Small RNA biogenesis and mode of action 3. Plant small RNAs in response to stress 4. Small RNAs of field crops 5. Small RNAs of horticultural crops 6. Cross-kingdom transfer of small RNAs 7. Small RNAs in the food and nutritional security 8. Concluding remarks References 129 130 132 133 155 156 164 165 165 Contents 7. RNAi based approaches for abiotic and biotic stresses tolerance of crops vii 183 Neha Patwa, Om Prakash Gupta, Vanita Pandey and Anita Yadav 1. Introduction 2. Mechanism and biogenesis of sRNA 3. Role of small RNA in abiotic stress tolerance 4. Role of small RNA during biotic stress 5. Applications of RNAi technology in crop development 6. Conclusion and future prospective References 8. Regulation of morphogenesis and development in food crops: role of small RNA 183 186 187 193 199 204 206 215 Jayanti Jodder 1. Introduction 2. Modes of sRNA transport to cell 3. Role of small RNAs in development, morphogenesis, and yield of the plant 4. Small RNA regulating growth and development related hormone signaling 5. Small RNA in disease resistance and stress tolerance in plants 6. Conclusion and future prospects References 9. Small RNA e regulator of biotic stress and pathogenesis in food crops 215 217 218 221 222 223 224 233 Ilamathi Raja and Jebasingh Tennyson Introduction Plant immunity and small RNAs in plant defense MicroRNA of plants Small interfering RNA Importance of small RNAs in biotic stress regulation Circular RNA and its biogenesis Small RNA movement between organisms to regulate plant-pathogen interactions 8. RNA based technologies for plant disease control 9. Conclusion References 1. 2. 3. 4. 5. 6. 7. 233 234 235 246 249 252 256 257 260 261 viii Contents 10. Small RNA networking: host-microbe interaction in food crops 271 Uzma Afreen, Manish Kumar and Kunal Mukhopadhyay 1. Introduction 2. Differentially expressed stress responsive small RNA 3. Pathogenic interaction-triggers plant immunity 4. MiRNAs 5. MicroRNA like RNA 6. Si-RNA 7. Plant-microbe beneficial interactions 8. Small RNA in crop improvement 9. Conclusion References 11. Small RNAs involved in salt stress tolerance of food crops 271 272 272 274 278 281 283 285 287 287 295 Zahra-Sadat Shobbar, Nazanin Amirbakhtiar, Raheleh Mirdar Mansuri, Fatemeh Loni, Alireza Akbari and Mahboube Sasaninezhad 1. Salt stress is a main abiotic stress restricting growth and production of crops 2. Salt-responsive small RNAs are identified in different food crops 3. Common small RNAs involved in salt stress response of different food crops and their target genes 4. Salt tolerance improvement of food crops through manipulation of small RNAs References 12. miRNAs perspective in mitigating waterlogging stress in plants 295 297 323 326 336 347 Garima Singroha and Pradeep Sharma 1. Introduction 2. Waterlogging effect on plant growth and development 3. miRNA biogenesis in plants 4. Expression and modulation of miRNAs and morphological adaptations 5. Conclusion References 347 349 349 350 355 359 Contents 13. Molecular mechanisms alleviating drought stress tolerance in crop plants ix 365 Kolluru Viswanatha Chaitanya, Akbar Ali Khan Pathan and Reddymalla Nikhila Reddy Introduction Drought stress tolerance in plants Transcription factors engaged in abiotic stress resistance Non-coding RNAs and drought stress RNAi technology for the improvement of drought stress tolerance in crop plants 6. Conclusions Acknowledgments References 1. 2. 3. 4. 5. 14. Grain development and crop productivity: role of small RNA 365 366 370 372 378 379 380 380 385 Md Fakhrul Azad, Heshani de Silva Weligodage, Anuradha Dhingra, Pranav Dawar and Christopher D. Rock 1. Overview 2. Fruit development; case study in Darwin’s “abominable mystery” 3. miRNAs in citrus 4. miRNAs in Brassica 5. miRNAs in asterids (Solanaceae) 6. miRNAs in other asterids Oleaceae, Sesame, and Actinidiaceae 7. miRNAs in rosids 8. miRNAs in monocots 9. Conclusions and future prospects References 385 390 392 397 403 414 415 424 434 450 SECTION 3 Applications and future scope 15. Food crops improvement: comparative biotechnological approaches 471 Ting Shi 1. Introduction 2. miRNA-based biotechnology for food crop improvement 3. miRNAs roles in food crops improvement 4. Future perspectives References 471 472 483 492 494 x Contents 16. Small RNA transgenesis for abiotic stress tolerant food crops 507 Jie Cui 1. Introduction 2. Introduction of sRNA transgenic technologies 3. Challenges: safety and specificity 4. Perspectives: better strategies and efficiency 5. Conclusion Acknowledgments References 507 509 524 526 530 531 531 17. Scope of small RNA technology to develop biotic stress tolerant food crops 545 Urvashi Mittal, Vijay Kumar, Sarvjeet Kukreja, Baljeet Singh and Umesh Goutam 1. Introduction 2. Effect of biotic stress on food crops 3. Role of small RNA in eliminating various biotic stress 4. Viral stress 5. Fungal stress 6. Conclusion References 18. Future prospective of small RNA molecules: food crop improvement and agricultural sustainability 545 548 553 558 562 564 564 571 Jafar K. Lone, Muntazir Mushtaq, Om Prakash Gupta and Gayacharan 1. Introduction 2. Classification of sRNA 3. Micro RNA (miRNA) 4. Short-interfering RNAs (siRNA) 5. Biogenesis and mode of action of sRNA in plants 6. Comparison between miRNA and siRNA 7. Role of miRNA in food crop improvement 8. Role of siRNA in crop improvement 9. siRNA for nutritional enhancement in food crops 10. sRNA in developing climate-resilient food crop plants 11. sRNA-based approaches to develop biotic stress tolerance in crop plants 12. Pest and nematode resistance 13. Conclusion References Index 571 572 574 575 575 577 577 579 580 582 582 591 592 592 601 List of contributors Uzma Afreen Department of Bioengineering and Biotechnology, Birla Institute of Technology, Ranchi, Jharkhand, India Alireza Akbari Department of Systems Biology, Agricultural Biotechnology Research Institute of Iran (ABRII), Agricultural Research Education and Extension Organization (AREEO), Karaj, Iran Amina A. Aly Egyptian Atomic Energy Authority, National Center for Radiation and Technology, Natural Products Department, Cairo, Egypt Nazanin Amirbakhtiar Genetic Research Department, Seed and Plant Improvement Institute, Agricultural Research, Education and Extension Organization, Karaj, Iran Md Fakhrul Azad Department of Biological Sciences, Texas Tech University, Lubbock TX, United States K.L. Bhutia Department of Agricultural Biotechnology and Molecular Biology, Dr. Rajendra Prasad Central Agricultural University, Samastipur, Bihar, India Zeyad M. Borik October University for Modern Science and Arts (MSA), Faculty of Biotechnology, Cairo, Egypt Kolluru Viswanatha Chaitanya Department of Microbiology and Food Science Technology, GITAM Institute of Science, GITAM University, Visakhapatnam, India Saurabh Chaudhary School of Biosciences, Cardiff University, Cardiff, United Kingdom Bhagirath S. Chauhan The University of Queensland, Queensland Alliance for Agriculture and Food Innovation (QAAFI) and School of Agriculture and Food Sciences (SAFS), Gatton, QLD, Australia Jie Cui Guangdong Provincial Key Laboratory for Plant Epigenetics, Shenzhen University, College of Life Sciences and Oceanography, Shenzhen, Guangdong, China Pranav Dawar Department of Biological Sciences, Texas Tech University, Lubbock TX, United States Anuradha Dhingra Department of Biological Sciences, Texas Tech University, Lubbock TX, United States xi xii List of contributors Gayacharan ICAR-National Bureau of Plant Genetic Resources, Division of Germplasm Evaluation, Hyderabad, Telangana, India Saipayan Ghosh Department of Agricultural Biotechnology and Molecular Biology, Dr. Rajendra Prasad Central Agricultural University, Samastipur, Bihar, India Umesh Goutam School of Bioengineering and Biosciences, Lovely Professional University, Phagwara, Punjab, India Om Prakash Gupta ICAR-Indian Institute of Wheat and Barley Research, Karnal, Haryana, India Akbar Hossain Department of Agronomy, Bangladesh Wheat and Maize Research Institute, Dinajpur, Bangladesh Jayanti Jodder Presidency University, Institute of Health Sciences, Kolkata, India Simerjeet Kaur Punjab Agricultural University, Ludhiana, Punjab, India Akbar Ali Khan Pathan Department of Biotechnology, GITAM Institute of Science, GITAM University, Visakhapatnam, India Sarvjeet Kukreja School of Agriculture, Lovely Professional University, Phagwara, Punjab, India Manish Kumar Department of Bioengineering and Biotechnology, Birla Institute of Technology, Ranchi, Jharkhand, India Vijay Kumar School of Bioengineering and Biosciences, Lovely Professional University, Phagwara, Punjab, India Jafar K. Lone ICAR-National Bureau of Plant Genetic Resources, Division of Germplasm Evaluation, Hyderabad, Telangana, India Fatemeh Loni Department of Systems Biology, Agricultural Biotechnology Research Institute of Iran (ABRII), Agricultural Research Education and Extension Organization (AREEO), Karaj, Iran Sagar Maitra Department of Agronomy, CUTM, Bhubaneswar, Odisha, India List of contributors xiii Raheleh Mirdar Mansuri Department of Systems Biology, Agricultural Biotechnology Research Institute of Iran (ABRII), Agricultural Research Education and Extension Organization (AREEO), Karaj, Iran Urvashi Mittal School of Bioengineering and Biosciences, Lovely Professional University, Phagwara, Punjab, India Kunal Mukhopadhyay Department of Bioengineering and Biotechnology, Birla Institute of Technology, Ranchi, Jharkhand, India Muntazir Mushtaq ICAR-National Bureau of Plant Genetic Resources, Division of Germplasm Evaluation, Hyderabad, Telangana, India Vanita Pandey ICAR-Indian Institute of Wheat and Barley Research, Karnal, Haryana, India Neha Patwa ICAR-Indian Institute of Wheat and Barley Research, Karnal, Haryana, India; Department of Biotechnology, Kurukshetra University, Kurukshetra, Haryana, India Biswajit Pramanick Department of Agronomy, Dr. Rajendra Prasad Central Agricultural University, Samastipur, Bihar, India T. Radhamani Centre for Plant Molecular Biology and Biotechnology, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India Ilamathi Raja Department of Plant Sciences, School of Biological Sciences, Madurai Kamaraj University, Madurai, Tamil Nadu, India S. Rajesh Centre for Plant Molecular Biology and Biotechnology, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India S.V. Ramesh ICAR-Central Plantation Crops Research Institute, Kasaragod, Kerala, India Reddymalla Nikhila Reddy Department of Biotechnology, GITAM Institute of Science, GITAM University, Visakhapatnam, India Christopher D. Rock Department of Biological Sciences, Texas Tech University, Lubbock TX, United States Mahboube Sasaninezhad Department of Systems Biology, Agricultural Biotechnology Research Institute of Iran (ABRII), Agricultural Research Education and Extension Organization (AREEO), Karaj, Iran xiv List of contributors Pradeep Sharma ICAR-Indian Institute of Wheat and Barley Research, Karnal, Haryana, India Ting Shi College of Horticulture, Nanjing Agricultural University, Nanjing, China Zahra-Sadat Shobbar Department of Systems Biology, Agricultural Biotechnology Research Institute of Iran (ABRII), Agricultural Research Education and Extension Organization (AREEO), Karaj, Iran Baljeet Singh School of Bioengineering and Biosciences, Lovely Professional University, Phagwara, Punjab, India; ICAR-Central Potato Research Institute, Shimla, Himachal Pradesh, India Garima Singroha ICAR-Indian Institute of Wheat and Barley Research, Karnal, Haryana, India Jinyuan Tao Shenzhen University, Guangdong Provincial Key Laboratory for Plant Epigenetics, College of Life Sciences and Oceanography, Shenzhen, Guangdong, China Jebasingh Tennyson Department of Plant Sciences, School of Biological Sciences, Madurai Kamaraj University, Madurai, Tamil Nadu, India Heshani de Silva Weligodage Department of Biological Sciences, Texas Tech University, Lubbock TX, United States Anita Yadav Department of Biotechnology, Kurukshetra University, Kurukshetra, Haryana, India Yu Yu Shenzhen University, Guangdong Provincial Key Laboratory for Plant Epigenetics, College of Life Sciences and Oceanography, Shenzhen, Guangdong, China Editors biography Praveen Guleria is presently working as an Assistant Professor in the Department of Biotechnology at DAV University, Jalandhar, Punjab, India. She has worked in the areas of plant biotechnology, plant metabolic engineering, and plant stress biology at CSIR-Institute of Himalayan Bioresource Technology, Palampur, H.P., India. Her research interests include plant stress biology, plant small RNA biology, plant epigenomics, and nanotoxicity. She has published several research articles in various peer-reviewed journals. She is also serving as the editorial board member and reviewer for certain international peer-reviewed journals. She has been awarded the SERBStart Up Grant by DST, GOI. She has also been awarded the prestigious “Bharat Gaurav Award” by the India International Friendship Society, New Delhi. She has also received various awards like CSIR/ICMR-Junior Research Fellowship, CSIR-Senior Research Fellowship, and state level merit scholarship awards. She has published five books with Springer, two with CRC Taylor & Francis, and one with Elsevier. Vineet Kumar is currently working as an Associate Professor in the Department of Biotechnology, Lovely Professional University, Jalandhar, Punjab, India. He has worked in different areas of biotechnology and nanotechnology in various institutes and universities in India namely, Panjab University Chandigarh, CSIR-Institute of Microbial Technology, Chandigarh, India, CSIR-Institute of Himalayan Bioresource Technology, and Himachal Pradesh University. He has published many articles in xv xvi Editors biography these areas featuring in peer-reviewed journals. He is also serving as editorial board member and reviewer for international peer-reviewed journals. He has received various awards like Dr. DSK-Postdoctoral Fellowship, senior research fellowship, and best poster awards. He has published four books for CRC, Taylor & Francis, five books for Springer, and two books for Elsevier. Beixin Mo is a “Distinguished Professor of Guangdong Province Talent Special Support Plan.” She received her bachelor’s degree in Plant Developmental and Molecular Biology from Peking University, China, and received her PhD degree from Guelph University, Canada. Her research area includes elucidation of molecular mechanisms involved in the biogenesis, degradation, and movement of plant small RNAs. She has received extensive research funding and published over 50 papers in distinguished journals, such as Nature Communications, Nature Plants, The Plant Cell, and Genome Biology, four books, and three book chapters. Preface The RNA world referred to a hypothetical stage in the origin of life on Earth e Sidney Altman Food crops are the prime producers and essential staple food for the majority of population around the globe. Plant growth and productivity is a prime concern in the present times. Alarming increase in the human population and commercialization has induced significant and rapid change in environmental parameters. The adversities in air, water, and soil have collectively led to the most harmful effect, the global climate change. This has ultimately affected the growth, development, and survival of plants including food crops. Significant adversities in food crops lead to considerable yield losses that risk the sustainability of growing population. These changing environmental parameters are known to affect the molecular structure of plant genome, transcriptome, proteome, and metabolome. Plant small RNAs are the key regulatory molecules affecting the molecular functioning of plants and their responses for biotic and abiotic environmental factors. Small RNAs are significantly involved in the plant growth, development, and environmental interactions. Hence, understanding the role of small RNA in food crops during varying environmental conditions can help in developing molecular framework to support agricultural sustainability and growing human population. RNAi as a silencing tool in plant defense against viral pathogens (DNA/RNA). Here both the miRNAs and vsiRNAs (virus-derived siRNAs) are involved. (Adapted from Lone et al. from Chapter 18). xvii xviii Preface This book, entitled Plant Small RNA in Food Crops, is written by 18 international contributors from 8 countries. The first two chapters by Aly and Borik and Kaur and Chauhan summarize the need and importance of food crops in the present scenario of increasing world population. These chapters give a detailed description of future challenges that might be met in cultivating food crops necessary for human survival and development. Chapter 3 by Bhutia et al. describes the molecular mechanisms of abiotic stress tolerance in food crops. Tao and Yu in Chapter 4 summarize the distribution and significance of diverse types of small RNAs that modulate important agricultural traits in major food crops. Chapters 5 and 6 describe the biogenesis, mode of action, and techniques of small RNA exploration in food crops. Patwa et al. and Jodder in Chapters 7 and 8 discuss the mechanism of small RNA to regulate morphogenesis and development of various food crops. Raja and Tennyson and Afreen et al. in Chapters 9 and 10 demonstrate the small RNA networking to regulate biotic pathogenesis for the growth and sustainability of food crops. These chapters cover the mechanisms and regulations of small RNAs in both pathogens and plants extensively to provide a deep insight into exploring an efficient way of disease management against biotic stress and pathogenesis in food crops. The detailed description of small RNAemediated regulation of abiotic stresses, namely salt, waterlogging, and drought in food crops is described in Chapters 11e13. The integrated and orchestrated activities of the factors enabling the crop plants to adapt and survive the adverse environmental conditions are explained in these chapters. Azad et al. in chapter 14 draw the potential and limitations of small RNA research. Given that small RNAs are integral to plant growth, and development, the idea of understanding the undesirable pleiotropic side effects of small RNAs manipulation while translating basic knowledge to crop improvement is significantly put forward. Further, the roles of small RNAs in fruit development are described. Chapter 15 presents a comparative discussion on the various biotechnological approaches for food crop improvement with an emphasis on small RNA transgenic technology for abiotic and biotic stress tolerance in Chapters 16 and 17. Lone et al. in Chapter 18 present the understanding of research gaps and utilization of small RNAs for crop improvement. Early biotechnology was focused to improve agronomic or input traits resulting in first-generation genetically modified crops. Currently, the focus is on improvement of output traits, which have more direct impact on consumers just like enhancement in the nutritional content of plant-derived foods. Here we have outlined the evidence for the involvement of small Preface xix RNAs in plant development, growth, and response to environmental stress through transgenic techniques and other functional analyses. Evolutionary perspective and evidence show that plant small RNA functions and mechanisms of action for cell fate and cellular homeostasis are largely conserved; however, the question arises: how accurate or justified are predictions based on orthology of small RNAs across model organisms to translate the similar processes in crops versus speciation and neofunctionalization of small RNAs? Small RNAs have the potential to improve nutrition by modifying food allergies and toxic chemicals, as well as increasing tolerance to biotic and abiotic challenges. Small RNAemediated genome manipulation is considered as a ground-breaking technique to decipher plant function and to create plants with better and unique features. However, small RNAe mediated genetic engineering has not been explored effectively till date, and still additional research is needed. This book, thus, presents the new trends in small RNA research on biogenesis, classification, role, and target genes, as well as the mechanistic relationship between them, in order to apprise researchers with adequate information on small RNA biology advancements. We, therefore, present the scope and challenges associated with small RNA research to regulate the growth, development, and sustainability of food crops. This page intentionally left blank SECTION 1 Basics 1 This page intentionally left blank CHAPTER 1 Food crops, growth and productivity as an important focus for sustainable agriculture Amina A. Alya and Zeyad M. Borikb a Egyptian Atomic Energy Authority, National Center for Radiation and Technology, Natural Products Department, Cairo, Egypt; bOctober University for Modern Science and Arts (MSA), Faculty of Biotechnology, Cairo, Egypt 1. Most popular food crops Food crops are any plants that are grown for the purpose of feeding humans or animals and it mostly consists of fruits, vegetables, seeds, spices, herbs, legumes, grains, nuts, etc. Other examples of food crops are those that are used for beverages such as coffee, tea, or any other plant extract-based beverages. Crops, in general, are divided based on their application to: oil crops, from which we extract oils used in cooking or for industrial application, food crops that are used for sustenance to the human population such as rice and wheat, ornamental crops which only serves esthetic purposes, fiber crops which are grown for their fibers that are used in textile in clothes and in other application such as fillings of pillows or for creating cordages, feed crops which are mostly used to feed livestock like cows and camels, and industrial crops. “Industrial crops” are sometimes synonymously used with “non-food crops” (Balasubramanian, 2014). Examples of the most popular food crops that are used for human consumption are grains (wheat, rice, and corn), fruits (apples, bananas, and dates), and vegetables (carrots, spinach, and broccoli). The primary source of food to the human populations for thousands of years and a major source of income to the agricultural sector to many countries are grains. The most popularly consumed grains are wheat, rice, oats, maize, barley, millet, and rye. Grains are grown more efficiently in different climate regions. For example, barely, wheat, rye, and oats are favored in regions with temperate climates, while rice and maize are favored in tropical and sub-tropical regions. Grains are used for different purposes other than feeding the human population e.g., for feeding cattle, oil production, etc. (Awika, 2011). Plant Small RNA in Food Crops ISBN 978-0-323-91722-3 https://doi.org/10.1016/B978-0-323-91722-3.00002-6 © 2023 Elsevier Inc. All rights reserved. 3 4 Plant Small RNA in Food Crops Moreover, Fruits are also major product in the agricultural industry. Examples of fruits are mangoes, bananas, and citrus fruits like oranges. Fruits are major source of important minerals and vitamins. Fruits are also a major source of phytochemicals with potent antioxidant activity, such as phenolic compounds, carotenoids, and terpenoids. For such reasons, fruits are sometimes recommended for the purpose of aiding avoidance of chronic diseases like cancer and heart illness. Fruit produce plays imperative task in the agricultural sector of many states as they can sometimes be more favorably grown in a country where the climate makes cultivating strategic crops (like wheat, rice as well as sugar) are hard. Fruits in this case are sold by said country to purchase the strategic food crops that’s in demand. The total global market for fruits has grown by an average 40% over the past decade or so. According to the Fresh Plaza, the fruit markets have increase from 45 million tons to 63 million tons over the past decade. The biggest importer of fruits is the United States, as the total increase in the US’ import of fruits has increased by 3.2 million tons over the past decade, estimated to be a 77% growth-Mexico being the largest exporter of fruits to the US (USDA FAS, 2022). Furthermore, vegetables are one of the essential food crops that’s needed for a stable diet and a cornerstone for the agricultural sector of most countries. Vegetables are rich in fibers, minerals, antioxidants, and vitamins (e.g. Vitamin A, B6, E, and B9). Vegetable’s antioxidant activities help protect against chronic diseases and damage from free radicals. They also help in prevention of caner and may have debilitating influence over tumor cells, thus help prevention of tumor progression. A variety of vegetables are grown worldwide, as 402 vegetable crops are cultivated around the globe, representing 69 families and 230 genera (Das, 2012). Food crops also include seeds and nuts, which are very rich in proteins and fats. Different types of nuts have slightly variable amounts of minerals and vitamins, but most of them have very similar content in carbohydrates, proteins, and fats). Seeds and nuts are good source of cis-fats, which based on population studies, may aid in weight loss. Therefore, seeds and nuts are sometimes included in weight-loss diet in reasonable amounts (Zec & Glibetic, 2018). Further, herbs and spices are also very important form of food crops. They are mostly seeds, berries, buds, vegetables, or barks that had been dried and used in flavoring, coloring of food, or used in medicine formulation. Mostly, spice can be any part of a plant (other than the leaves), while herbs are mostly leaves of plants, both are mostly dried. Spices are mostly used for cooking and flavoring, while herbs are used for medicinal purposes, although not strictly. Spices have many physiological and Food crops, growth and productivity as an important focus for sustainable agriculture 5 pharmaceutical benefits for the human body, and many chronic and neurodegenerative diseases can be prevented by having spices in regular diet in reasonable amounts (Carlsen et al., 2010). India and Pakistan are one of the largest spice producers around the world. Herbs are very important in the pharmaceutical industry, even in ancient times they were considered the cornerstone of medicine. Herbs are widely praized for having less side effects than synthetic drugs when dealing with an ailment, and are publicly more accepted for being more ”natural” (Balammal et al., 2012). Finally, food crops include legumes, which are plants that are part of the family Fabaceae that forms beans. Their beans are mostly dried to serve longer shelf life that’s cheap and more available to the general public in different seasons. Legumes are in fact a type of vegetable, and examples of it are beans, peas and lentils. They are high in protein, fibers, carbohydrates, and are low in fats. They are containing necessary vitamins for the human growth and development. Legumes have plenty health benefits, as they are shown to decrease the blood cholesterol levels. They are also able to decrease inflammatory markers in the blood and blood pressure (Figueira et al., 2019). 2. Future challenges With the rapidly increasing populations and urbanization, agriculture of food crops and food security worldwide may be facing multiple challenges in the near future due to many reasons, among them are rapid changes in the global climate, mismanagement of fertilizers, and the declining resources required for mass production of food crops. It is evaluated that by the year 2050, the people growth on earth will grow to become 9.7 billion, which will require an ample amount of food products, estimated to be 70% increase in production of food crops, and in order to achieve that goal, multiple challenges needs to be overcome (Ngoune et al., 2020). For starters, climate change is the most popular factor affecting food crops production worldwide, and for good reasons. As the global temperature increases, water levels and extreme rainfall events increase, which contribute to flooding in coastal areas. It had been estimated that within 100 km of the world’s coastline about 23% of the human population lives which is expected to double to 50% by the next decade or so (Adger et al., 2005). Not only does climate change affects coastal cities, it will also affect crop production of many countries, such as the tropical Asian countries who’s food security is extremely important on the cropping patterns of 6 Plant Small RNA in Food Crops coastal deltas. Although these coastal cropping patterns aren’t very productive due to it being exposed to stresses from salinity to drought, it provides millions of jobs to low-income families. Crop lands of the coastal regions are highly affected by the tidal river’s influence, from changes in flow and salinity in different seasons to the changes in river heights seen during the diurnal cycle (Johnson & Humphreys, 2021). Rising sea levels causes floods and increases the risks of saline intrusion into groundwater and rivers, which will create unfavorable conditions for farming in coastal regions (Milliman, 1992). Moreover, Egypt and Nigeria are one of the African countries who’s crop security will very likely be affected from the rising sea level. For Egypt, a 1-m increase in sea level will very likely cause 28.93% of the whole area of the Nile Delta to be submerged underwater, with diminishing surrounding crop areas due to saline intrusion in the soil. The fisheries located in the lagoons (which account for around one-third of Egypt’s fish production) will also be seriously impacted, affecting millions of people whose livelihoods depends heavily on such areas of agriculture’s. Nigeria will also experience similar challenges from the variable climate change, as increased rainfall will cause floods and the increased water levels will likely submerge a large portion of the Niger delta underwater (El-Nahry & Doluschitz, 2009; Eregha et al., 2014). Other countries in Europe, such as the Netherlands, will also be heavily affected from the rising sea level (specifically in the agricultural sector), as Netherlands uses excessive polder systems for agriculture. A polder is a patch of land that’s reclaimed from a body of water (such as a lake or the sea), surrounded by an embankment called dikes. The embankment is usually gated by a sluice gate, which regulates water level and flow in the embanked land. Polders are mostly below the surrounding water level, which makes it very susceptible to flooding, especially if the embankment was broken from extreme flooding pressure. It was calculated that 60% of the Netherland is below or at sea level, due to excessive reclamations of land from the sea and river (De Mulder et al., 1994). Asian countries like Vietnam, Bangladesh, and China also use excessive polder systems for agriculture, where the coastal regions of Bangladesh, for example, has more than 129 polders with areas of few thousands to 50,000 ha (Tuong et al., 2014). Climate change impact doesn’t only stop there, it will also affect the ecosystems and the diversity of animals and crops, resulting in an increase in the rates of biodiversity loss, which will be discussed later (FAO, 2017a). Rising sea level and generally climate change will likely cause tremendous impact on food security of such countries and the major delta regions of Asian Food crops, growth and productivity as an important focus for sustainable agriculture 7 countries, and considering that Asian countries produce large portions of the world’s supply of rice and other major food crops, this impact on food security will ripple across the world, likely to cause famines in already vulnerable communities. Another key issue that required to be addressed is the declining resources needed to produce fertilizers, particularly phosphate (FAO, 2017b). 2.1 Establishing priorities Although there has been great improvement in the productivity and yield of food crops over the past 50 years or so, it’s still a far cry from what was envisioned at the united nation’s food and agriculture organization (FAO); “a world free from hunger, malnutrition. A world in which food and agriculture contribute to the standards of living of all, in a socially, economically, and environmentally sustainable manner” (FAO, 2017b). Sustainable agricultural development requires 3 main key challenges that need to be overcome: maximizing economic profitability, maintaining environment integrity, and reducing social inequality; all working in unison. It’s important that any agricultural system is able to provide a comprehensive approach that addresses these challenges, and research in the respective field should also give more focus on such challenges to meet sustainable outcomes. Setting priorities for research is the systematic approach that is followed in order to find general agreement among several stakeholders regarding challenges important enough to receive focus and resources. Priority is then made by the stakeholders towards the most promising research that maximize the most efficient use of minute resources. Furthermore, many local food systems, particularly in low- and middle-income countries, might benefit from a rational and transparent definition of research priority issues (Nguyen et al., 2021). Because Food systems encompasses many sectors and disciplines, from socio-economic to environmental sustainability and health, and thus requires immense amount of interdisciplinary data in order to approach such interconnected challenges and constraints (Battersby, 2020). As a result, setting a priority for food system research is more difficult than prioritizing a particular study subject. Examining the priority setting process for research may help future research in avoiding wasting time and resources by neglecting what is already known or already being investigated (Chalmers et al., 2014). Determining research priorities doesn’t follow a general method owing to different contexts, and can adopt many approaches used in health research priority setting, but the general consensus is 8 Plant Small RNA in Food Crops that the methods should be transparent, based on evidences, fair, genuine, and inclusive of the wide range of stakeholders (Tong et al., 2019). Stakeholders, as well as other factors such as budgets and local cultures/ traditions, can greatly impact the priority setting process for research (Bukachi et al., 2014). Perhaps the most troubling, climate change is a priority concern for many countries, especially coastal countries, as the impact of climate change on agriculture is truly immense, as it can lead to creation of inhabitable environment for many crop species, as well as endangering other species that aid in agriculture and impact many economies in the process. Furthermore, attempts are currently being made to embrace sustainable approaches to boost yields, variety, and seed systems, as well as to assure long-term plant health. Other sub-sectors’ actions, particularly those connected to land use and water supplies, should be taken into account as well, to ensure that adaptation initiatives do not clash with one another (FAO UN, 2018). Supporting agriculture in developing countries is one of the most effective methods in improving the lives of millions living in poverty, and in most of those countries, agriculture is most of the times the main economic driver, meaning that supporting those countries will not only help such countries develop, but also increase their export of food crops. Developing the agriculture sector in general creates job opportunities, improve economies, and reduce malnutrition. In reality, practically every industrialized nation began its economic development with a shift in agriculture. Brazil, China, and Vietnam are three recent examples of countries that have quadrupled the value of their agriculture sectors in the last 20 years. Various other countries in African, Asian, and Latin American have also begun the change process. 3. Increasing growth and productivity of crops foods After climate change, feeding the constantly rising population is likely the second most serious challenge confronting humanity. Many scholars recommend increasing the output of current agricultural lands and investing in minimizing food waste and improving food storage as a means of feeding the next generation responsibly. To feed a rising and increasingly demanding human population, agricultural intensification is essential. Intensification is related with a simultaneous rise in both resource consumption and resource use efficiency, i.e. with an increase in both resource use and resource use efficiency. Resource usage efficiency is influenced by agronomic, environmental, economic, social, transgenerational, and global Food crops, growth and productivity as an important focus for sustainable agriculture 9 issues. The notion of sustainable intensification and current agronomy are in dispute. Sustainable intensification requires clarity on priority-setting principles and procedures, an all-inclusive and clear cost-benefit analysis, and subsequent weighing of trade-offs based on scientifically accepted, agreed standards, in order to re-green agriculture (Struik & Thomas, 2017). Future food security is thus dependent on the development of technologies that improve resource efficiency while preventing cost externalization order to maximize input efficiency, the current trend is toward intensification, namely higher output per production unit. As a result, future food security is contingent on the development of technologies that enhance resource efficiency while avoiding cost externalization. The present trend is toward intensification, or increased output per production unit, in order to maximize input efficiency. Whether or not this tendency can be sustained is a hot topic among scientists and policymakers alike. The key concern is how to make more food with a lot less resources (Struik et al., 2014). People engaged in sustainable agricultural and food systems employ a variety of techniques. On the farm, growers can utilize measures to improve soil health, reduce water consumption, and reduce pollution. Consumers and retailers interested about sustainability should seek out “values-based” foods farmed in ways that promote farm worker well-being, are environmentally friendly, or help the local economy. Researchers in sustainable agriculture frequently combine biology, economics, engineering, chemistry, community development, and other disciplines in their work. Sustainable agriculture, on the other hand, is more than a set of procedures. It’s also a negotiating process: a tug of war between an individual farmer’s or a community’s frequently conflicting interests as they strive to address complicated concerning how we grow our food (Velten et al., 2015). 3.1 Strategies for increasing crop yield 3.1.1 Cover crops They are any non-cash crop that is farmed in addition to the major cash crop. These crops have the ability to increase soil organic matter and fertility, as well as minimize erosion, improve soil structure, boost water infiltration, and prevent insect and disease outbreaks. Cover cropping has various advantages, however, like with any management strategy, there are tradeoffs and constraints to consider. Cover cropping may reduce dependency on fossil fuels and increase agricultural production in many cases. In the early part of the twentieth century, cover crops were widely 10 Plant Small RNA in Food Crops employed across the country. In the 1950s, many farmers abandoned cover crops due to the introduction of selective pre- and post-emergent herbicides. With a renewed focus on soil quality and decreasing chemical inputs in recent years, there has been renewed interest in the possible benefits of using cover crops in agricultural production systems (Concerned Scientists, 2019). Slowing erosion, improving soil health, increasing water penetration, smothering weeds, controlling pests and diseases, and increasing biodiversity are all reasons to use cover crops in your crop management plan. Growing cover crops to provide nitrogen to the soil is a typical cause for organic farmers and farms that do not use chemical fertilizers. Leguminous cover crops may deliver a significant quantity of nitrogen. Crops sown after a cover crop can make better use of this plant-available nitrogen. Cover crops are used in traditional farming systems to increase soil organic matter and improve the physical structure of the soil. Farmers can use fastgrowing grasses or species with broad root systems as a cover crop in these conditions. Determining the aims and function of a chosen cover crop is one of the most important aspects of conducting a successful cover crop management programmed. The site specifications, timing, and cropping history all play a role in determining which cover crop species to use in a system. While there is evidence to support the multiple benefits of incorporating cover crops into a system, producers may experience obstacles in installation or management. Cover crops’ water requirements, especially in desert or drought-prone areas, may reduce the quantity of water available to the primary crop or necessitate the use of supplemental irrigation. In these cases, it’s critical to examine if the anticipated advantages of cover cropping justify the expenditure. There are additional economic considerations to consider in addition to potential increases in irrigation. With the addition of a cover crop, seed and soil preparation costs, as well as labor needs, will vary. There is no direct benefit to the farmer for harvested agricultural goods since cover crops are left in the field. While certain cover crops can help to lessen the effect of specific pests and pathogens, they can also function as a reservoir for other insects, rodents, weeds, and illnesses. When the field is transitioned and readied for later plantings, some cover crops might persist as weeds if badly selected or maintained (MacLaren et al., 2020). 3.1.2 Microorganisms for soil amelioration, and sustainable crop production The latest face of increasing food production throughout agricultural amplification in the challenge of shrinking per capita arable lands, decline Food crops, growth and productivity as an important focus for sustainable agriculture 11 the global economy, and irregular climate alteration has resulted into an over-reliance on agrochemical efforts, which are frequently expensive and dangerous to human and animal health as well as the environment. To enhance healthy crop production, an alternative eco-friendly. Alternative eco-friendly solutions that best match smallholder methods have been presented to promote healthy crop production practices using of biological factors, especially plant growth-promoting microorganisms (PGPMs), which offer significant for agricultural ecosystems in the overall vision of improving agriculture production and the environment protecting, is the most familiar and broadly prevalent solution. The PGPMs are important components of agroecological cycle that include soil nutrients improvement, improving crops nutrient, plant tolerance to biotic and abiotic stresses, biological control of insects and diseases and water absorption. Plant-related microbiomes offer great promise for improving for increasing plant resilience and productivity in agricultural settings. Biological approaches that employ bacteria or their metabolites are increasingly being shown to improve nutrients absorption and production, insect management, and ameliorate stress response of plants. However, in order to fully fulfill of microbial technology’s promise, its efficacy and consistency under a wide range of real-world settings must be enhanced. While microbial biofertilizers and biological control are fast being improved for usage in a variety of soils, crop types, and settings, but for plant breeding, with beneficial plants for breeding “microbially optimized plants and does not yet contain a selection of microbial interactions. Recent research into microbiome engineering might result in microbial consortia that are more adapted for supporting plants. To address food security, all three techniques might be used to produce greatest advantages and greatly higher crop yields (Singh & Trivedi, 2017). Breeding plants that select for helpful microorganisms is another strategy for increasing agricultural output. Plant-microbe interactions and microbial community formation may be explained using a combination of top-down and bottom-up investigations using multiomics and visualization technologies, as well as a synthetic community’s method. These essential ideas and expertize may be applied to the creation of next-generation agriculture in order to address food security issues (Sun et al., 2021). 3.1.3 Nuclear technology Crop breeding using nuclear knowledge can provide enhanced varieties that are more resilient to climate change and assist vulnerable nations secure 12 Plant Small RNA in Food Crops food and nutritional security. Electromagnetic-wave irradiation has been utilized in agriculture to generate crop types, control insect pests, evaluate fertilizer efficacy, and protect agricultural production. Electromagnetic waves are split into eight spectral groups based on their frequencies and wavelengths, containing audio waves, radio waves, microwaves, infrared, visible light, ultraviolet, X-rays, and gamma rays (Aly et al., 2022; Zhong et al., 2021; Aly et al., 2021). Agriculture, horticulture, ecology, and space science are all interested in the impact of ionizing radiation on higher plants. Ionizing radiation, such as gamma rays, cooperates with atoms or molecules to form free radicals in cells. These radicals may harm or change essential components of plant cells, and they have been shown to have varied effects on plant morphology, anatomy, biochemistry, and physiology depending on the intensity of irradiation. Changes in plant cellular structures and metabolism, such as thylakoid membrane dilatation, photosynthetic modification, antioxidative system regulation, and phenolic compound accumulation, are among these consequences (Khalil et al., 2015; El-Beltagi et al., 2013; Aly et al., 2010). Gamma irradiation has been more essential in plant breeding and genetic research aimed at improving yield and other desirable characteristics in a variety of crops (El-Beltagi et al., 2022; Aly et al., 2019a and 2019b; El-Beltagi et al., 2013). Following modest dosages of g-rays, primary branches and yield quality such as pods number/plant, flowers number/plant, seed index, and others were stimulated, whereas the same attributes were inhibited at greater rates (Jan et al., 2010). Furthermore, Aly et al. (2018) found that various gamma ray dosages had distinct impacts on plant morphological and biochemical properties, such as improving germination and seedling development as well as boosting proline concentration. Gamma rays may clearly be utilized to create mutations that are resistant to environmental stresses, such as salt. As investigated by Aly et al. (2019c), gamma ray can be applied for stimulating the crops production as well as increasing the secondary metabolites that have a profitable significance and a high assessment. Gamma irradiation at a dose level of 50 Gy improved the growth traits such as germination proportion, plant height, root length, fruit length and fruit diameter in eggplant. Likewise, this dose increased the total phenolic, flavonoid and tannin contents for pulp, peel and whole fruits in comparison to the 100 Gy dose level. It is worth mentioning that the activity of phenylalanine ammonialyase (PAL) enzyme and polyphenol oxidase enzyme increased by applying gamma irradiation dose level of 50 Gy. While increasing irradiation dose level to 100 Gy reduces both enzyme activities. Every year, the Food crops, growth and productivity as an important focus for sustainable agriculture 13 FAO/IAEA Mutant Variety Database registers a large number of novel cultivars, containing those that were created by ionizing radiation. This type of mutagenesis is also significant in the production of new horticultural types. Plants can swiftly repair a considerable percentage of the harm generated by acute IR exposure, according to mutagenesis tests. For instance, Taheri et al. (2014) found that Curcuma alismatifolia picked up considerably from a 10 Gy dosage within 24 h and advised that 20 Gy or more may be used for meaningful net rates of mutagenesis in horticultural breeding. The high potential of plants for DNA repair is a common constraint to the use of comet assays in plant investigations of the effects of IR on DNA (Lanier et al., 2015). This also serves as a prompt that an acute exterior dosage of IR is one of the only mutagenic circumstances in which an acute dosage is genuinely conceivable, as opposed to chemical mutagens, which frequently remain in biological systems after exposure has ended. Also, instead of synthetic growth hormones and pesticides, gammaradiation-processed chitosan and sodium alginate might be employed as natural plant growth promoters and antifungal mediators, with no negative effects on human health or the environment. There have been several researches on the effects of chitosan and sodium alginate on plant development (Acemi et al., 2018; Hossain et al., 2013; Parvin et al., 2013). 3.1.4 Genetically modified crops Increasing the use of genetically engineered crops (Van Hesse et al., 2020) or organic insecticides (Kalkura et al., 2021) might be realistic options for reducing crop output losses caused by pests and illnesses. However, using genetically engineered plants raises issues about human health and biodiversity loss (Raman, 2017; Tsatsakis et al., 2017). Furthermore, there are some facts that herbicide-tolerance crops do not give higher yields or require less herbicide treatment. Since the beginning of agriculture, farmers and food dealers have been worried about losses. However, as global food consumption rises, the issue of how much food is wasted after harvest due to handing out, spoilage, insects and rodents, or other reasons becomes more pressing. Lowering postharvest losses might theoretically contribute a significant amount to the world food supply, reducing the need to increase output in the future. 3.1.5 Prevention of postharvest loss The decrease in both quantity and quality of a food crop from harvest to consumption is known as postharvest loss. Losses in feature include those 14 Plant Small RNA in Food Crops that influence a product’s nutrient/calorie content, suitability, and edibility. In industrialized countries, these losses are more prevalent (Kader, 2002). Amount losses are those that result in the loss of a product’s quantity. In underdeveloped nations, quantity loss is more prevalent (Kitinoja & AlHassan, 2012). According to FAO research, global quantities of lost and discarded food are larger in downstream phases of the food chain in highincome countries, while the converse is true in low income counties, where more food is gone and wasted in upstream phases (FAO, 2012). Food and Agricultural Organization (FAO) reported that 25e35% of fruits and vegetables are lost through natural causes by pests, microbes, and insects (Ihsanullah & Rashid, 2017). Therefore, preservation of produce is a prerequisite for enhancing both food safety and quality. Different postharvest management techniques, like low temperature storage, controlled environment packing, and surface treatment with synthetic chemicals, have been widely used to prevent post-harvest losses and increase the shelf life of fresh fruit (Kaushik et al., 2010; Maraei & Elsawy, 2017). In numerous reports, it has been recognized that controlled atmosphere packaging and low temperature storage techniques are effective and popular strategies for extending the shelf life of fresh commodities. However, in the prevailing storage conditions, these methods may not be able to control pathogenic fungi and bacteria as Fusarium spp., Aspergillus spp., Bacillus spp., and other bacteria from the Enterobacteriaceae family (Hussain et al., 2021; Linda et al., 2020). Damage, which is a visible symptom of degradation, should not be mistaken with loss. Damage limits the use of a thing, whereas loss prevents it from being used. Quantity (weight or volume) and value (physical condition or attributes) losses can happen at any time. Gamma radiation has proven to be an effective alternative therapy for microbial disinfection and extending the shelf life of fresh products (Kaushik and Chauhan, 2008; Prakash et al., 2000). Furthermore, g-irradiation is a promising and successful alternative technique of food processing and preservation (Bidawid et al., 2000). Irradiation has been documented as a choice to chemicals for treating fresh and dried agricultural products to overcome quarantine barriers in international trade, as a mode of decontamination, disinfestations, delaying ripening, and senescence of fruits and vegetables to improve nutritional attributes and shelf-life (Hong et al., 2008). The joint study group on high dose irradiation “FAO/IAEA/WHO (1997)” found that irradiation of any commodity up to an overall average dosage of 10 kGy has no toxicological risk, and so there is no demand for toxicological testing of foods treated at dose levels of 10 kGy or less. Similarly, Hussain et al. (2021) Food crops, growth and productivity as an important focus for sustainable agriculture 15 found that irradiation up to 10 kGy had no significant nutritional or microbiological consequences. Ionizing radiation is a cost-effective method for decreasing post-harvest losses, prolonging the shelf life of fresh goods, and ensuring the sanitary quality of fresh products (Gonzalez-Aguilar et al., 2004). Irradiation has been demonstrated to extend the shelf life of a variety of tropical and subtropical fruits like papayas, mangoes, bananas, tomatoes, and kiwis in previous studies (Linda et al., 2020; Hussain et al., 2021). Gamma irradiation has been shown to be an effective approach to boost storability and extend the shelf life of a variety of fruits and vegetables, including blueberries, wild edible mushrooms, as well as ash gourd, both alone and in conjunction with refrigeration as a hurdle technology (Tripathi et al., 2013; Sharma et al., 2020). Gamma radiation will soon become a useful postharvest tool, as the method gains widespread acceptability (Mahto & Das, 2013). Since the beginning of agriculture, farmers and food dealers have been worried about losses. However, as global food consumption rises, the issue of how much food is wasted after harvest due to processing, spoilage, insects and rodents, or other reasons becomes more pressing. Lowering postharvest losses might theoretically contribute a significant amount to the world food provide, decreasing the need to increase output in the future. 3.2 Sustainable agriculture innovation Agriculture faces the daunting challenge of producing more food to fulfill the nutrition demands of increasing global population (Valin et al., 2021), whereas dealing with climate alteration and ever-tightening natural resource constraints. This challenge is made even more complex by the fact that unless safe and nutritious food is affordable, and reliably accessible, food insecurity and malnutrition will persist (FAO, 2020). In addition, unless farmers and farm workers make decent incomes, poverty will grow, and farming will fail (World Bank Group, 2015). Accelerate agricultural productivity growth must be considered to meet the complex, multi-objective challenge of transformation to more sustainable food systems. The need for agricultural productivity growth to meet food and conservation needs is not a minority view of a subsector of academia or interest groups. Diverse groups have come to the same conclusion. Food systems transformation, according to the EAT-Lancet Commission (Willet et al., 2019), requires “at least a 75% decrease of yield gaps on present farmland.” Climate change is increasing the urgency of accelerating sustainable productivity growth. Through its impact on drought, floods, pests, weather variability, and even 16 Plant Small RNA in Food Crops human health, climate change will, and in many cases already is, challenging farmers to produce more with reduced and less reliable natural resource inputs. Innovative approaches to agricultural productivity growth will be critical to adaptation and to limiting the food security impacts of climate change (USGCRP, 2018). Although the significant labors made in latest decades to establish strategies and rules aimed at achieving global food security, nearly one out of every ten people globally presently faces acute food insecurity (FAO et al., 2020). Malnutrition is anticipated to rise as a result of population expansion (Hall et al., 2017), whereas extensive resource extraction may result in land degradation and lower soil production (Tóth et al., 2018). Climate change can cause crop failure or break by increasing severe occurrences (e.g., droughts and floods) and increasing the frequency of pests and diseases (Richardson et al., 2018; Spence et al., 2012). Lastly, changing feeding patterns and increased require for additional items are raising pressure for land and water resources, depleting them and increasing food security uncertainty. As a result, the food and nutritional security agenda need immediate international cooperation and the implementation of efficient global food security insurance (FAO et al., 2020; Ruben et al., 2018). In South Asia and Sub-Saharan Africa, an increase of 1% in food production is predicted to reduces poverty by 0.48% and 0.72%, respectively. Furthermore, increasing agricultural effectiveness of the major crops might significantly enhance farmers’ income, encourage domestic commerce, and promote excellent health and nutrition (World Bank Group, 2015). To get more effective in agricultural management and lessen the influence on ecosystem services, it is required to combine several aspects (e.g., environmental, institutional, organizational, and socioeconomic) (Millennium Ecosystem Assessment, 2005). Pests and diseases, for example, are thought to be responsible for around 35% of agricultural output reduction (Popp et al., 2013). Agriculture covers around 38% of the land area. Nevertheless, food security is failing in some places (for example, urban and peri-urban areas) (Foley et al., 2011; Grundy et al., 2016; Radwan et al., 2019). As a result, estimate the alterations in agricultural areas, as well as the forces liable for such changes, in the agricultural land field of research. 4. Future perspectives By 2050, the world’s population is predicted to reach approximately 9 billion people, resulting in a 100% increase in food demand. Presently one Food crops, growth and productivity as an important focus for sustainable agriculture 17 in every eight people, or 842 million people, faces daily hunger. Even more so, around one billion people worldwide are food insecure, meaning they do not have enough affordable, nutritional food. Farmers would need to boost food output by 70e100% to fulfill global nutritious requires, despite the fact that agriculture has developed to handle these rapidly increasing food demands. According to forecasts from the United Nations Food and Agriculture Organization (FAO), 80% of the further food needed to fulfill demand in 2050 would have coming up from land already under agriculture. Farmers and food producers have to achieve such greater yields with the equal (or less) area and less natural resources as they do now. Consumers all over the world are forced to spend 50% or more of their income on food, which is disastrous for them. We must build a more stable society by giving people with the skills they need to raise themselves out of poverty, which is accomplished by first combating hunger. Humans have an intrinsic drive to learn more about a wide range of topics, and biotechnology is no exception. Farmers that use these contemporary farming practices contribute to a healthy, nutritious, and long-term food supply. Farmers have to know more about biotechnology’s security and real-world uses, the more likely the general public will embrace it as a key part of agricultural output. Farmers may generate more end products per acre when they grow genetically modified crops. This is due to the fact that the modified plants were created to produce more while also being tolerance to pests, weeds, and illnesses. They’ve also been developed to be drought resistant, using less water. Farmers can apply less pesticides and herbicides because of the resistance programmed in the genetics of the crops, resulting in a lower environmental effect. It also has a higher nutritional content than similar products, making it healthier to consume. The GMOs’ shelf life can also be increased to reduce waste. All of this improves food producers’ efficiency and output ability, making it simpler to feed the globe. Water and nutrients can now be measured, tested, and distributed more efficiently through the advances in science and research. Moisture, pH, phosphorus, potassium, calcium, and magnesium levels may all be measured to determine exactly what is required to produce crops. Moreover, farmers understand which crops resolve thrive where, how to minimize environmental damage, and how to best maintain soil nutrients ultimately. Using sophisticated technologies, today’s farmer can control every aspect of their property. 18 Plant Small RNA in Food Crops References Acemi, A., Bayrak, B., Çakır, M., et al. (2018). Comparative analysis of the effects of chitosan and common plant growth regulators on in vitro propagation of Ipomoea purpurea (L.) Roth from nodal explants. In Vitro Cellular & Developmental Biology Plant, 54, 537e544. https://doi.org/10.1007/s11627-018-9915-0 Adger, W., Hughes, T. P., Folke, C., Carpenter, S. R., & Rockström, J. (2005). Socialecological resilience to coastal disasters. Science (New York, N.Y.), 309(5737), 1036e1039. https://doi.org/10.1126/science.1112122 Aly, A. A., El-Desouky, W., & Abou El-Leel, O. F. (2022). Micropropagation, phytochemical content and antioxidant activity of gamma irradiated blackberry (Rubus fruticosus L.) plantlets. In Vitro Cellular & Developmental Biology-Plant, 58, 457e469. Aly, A. A., Eliwa, N. E., Borik, Z. M., & Safwat, G. (2021). Physiological variation of irradiated red radish plants and their phylogenic relationship using SCoT and CDDP markers. Notulae Botanicae Horti Agrobotanici Cluj-Napoca, 49, 12396. Aly, A. A., Eliwa, N. E., & AbdEl-Megid, M. H. (2019a). Stimulating effect of gamma radiation on some active compounds in eggplant fruits. Egyptian Journal of Radiation Sciences and Applications, 32(1), 61e73. https://doi.org/10.21608/ejrsa.2019.10024.1066 Aly, A. A., Maraei, R. W., & Aldrussi, I. (2019b). Changes in peroxidase and polyphenol oxidase activity and transcript levels of related genes in two Egyptian bread wheat cultivars (Triticum aestivum L.) affected by gamma irradiation and salinity stress. Bangl J Botany, 48, 177e186. https://doi.org/10.3329/bjb.v48i1.47482 Aly, A. A., Maraei, R. W., & Ayadi, S. (2018). Some biochemical changes in two Egyptian bread wheat cultivars in response to gamma irradiation and salt stress. Bulgarian Journal of Agriculture Science, 24, 50e59. Aly, A. A., Noha, E. E., & Rabab, W. M. (2019c). Genetic variation based on ISSR in two wheat cultivars after exposing to gamma radiation. ScienceAsia, 45(5), 436e445. https:// doi.org/10.2306/scienceasia1513-1874.2019.45.43 Aly, A. (2010). Biosynthesis of phenolic compounds and water soluble vitamins in culantro (Eryngium foetidum L.) plantlets as affected by low doses of gamma irradiation. Nalele Universit
atii Din Oradea-Fascicula Biologie Tom. XVII, 2, 356e361. Awika, J. M. (2011). Major cereal grains production and use around the world. American Chemical Society, 1089, 1e13. Balammal, G., Sekar, B. M., & Reddy, J. P. (2012). Analysis of herbal medicines by modern chromatographic techniques. International Journal of Preclinical and Pharmaceutical Research, 3(1), 50e63. Balasubramanian, A. (2014). Classification of agricultural crops. Technical Report. Battersby, J. (2020). In A. Blay-Palmer, D. Conare, K. Meter, & A. Di Battista (Eds.), “Data gaps and the politics of data” in sustainable food system Assessment: Lessons from global practice. London: C. Johnston Newgen Publishing UK. Bidawid, S., Farber, S., & Sttar, A. (2000). Inactivation of hepatitis A virus (HAV) in fruits and vegetables by gamma irradiation. International Journal of Food Microbiology, 57, 91e97. Bukachi, S. A., Onyango-Ouma, W., Siso, J. M., Nyamongo, I. K., Mutai, J. K., Hurtig, A. K., et al. (2014). Healthcare priority setting in Kenya: A gap analysis applying the accountability for reasonableness framework. The International Journal of Health Planning and Management, 29, 342e361. https://doi.org/10.1002/hpm.2197 Carlsen, M. H., Halvorsen, B. L., Holte, K., Bohn, S. K., Dragland, S., Sampson, L., Willey, C., Senoo, H., Umezono, Y., Sanada, C., Barikmo, I., Berhe, N., Willett, W. C., Phillips, K. M., Jacobs, D. R., & Blomhoff, R. (2010). The total antioxidant content of more than 3100 foods, beverages, spices, herbs and supplements used worldwide. Nutrition Journal, 9(3), 1e11. Food crops, growth and productivity as an important focus for sustainable agriculture 19 Chalmers, I., Bracken, M. B., Djulbegovic, B., Garattini, S., Grant, J., Gülmezoglu, A. M., et al. (2014). How to increase value and reduce waste when research priorities are set. Lancet, 383, 156e165. https://doi.org/10.1016/S0140-6736(13)62229-1 Concerned Scientists. (2019). What is sustainable agriculture?. Available from: https://www. ucsusa.org/food-agriculture/advancesustainableagriculture/what-issustainableagriculture. (Accessed 5 June 2019). Das, A., Raychaudhuri, U., Chakraborty, R., et al. (2012). Cereal based functional food of Indian subcontinent: A review. Journal of Food Science and Technology, 46(9), 665e672. https://doi.org/10.1007/s13197-011-0474-1 de Mulder, E. F. J., van Bruchem, A. J., Claessen, F. A. M., Hannink, G., Hulsbergen, J. G., & Satijn, H. M. C. (1994). Environmental impact assessment on land reclamation projects in the Netherlands: A case history. 37(1), 15e23. https://doi.org/10.1016/ 0013-7952(94)90078-7 El-Beltagi, H. S., Maraei, R. W., Shalaby, T. A., & Aly, A. A. (2022). Metabolites, nutritional quality and antioxidant activity of red radish roots affected by gamma rays. Agronomy, 12, 1916. https://doi.org/10.3390/agronomy12081916 El-Beltagi, H. S., Mohamed, H. I., Mohammed, A. M. A., Zaki, L. M., & Mogazy, A. M. (2013). Physiological and biochemical effects of g-irradiation on cowpea plants (Vigna sinensis) under salt stress. Notulae Botanicae Horti Agrobotanici Cluj-Napoca, 41(1), 104e114. El-Nahry, A. H., & Doluschitz, R. (2009). Climate change and its impacts on the coastal zone of the Nile Delta, Egypt. Environmental Earth Sciences, 59(7), 1497e1506. https:// doi.org/10.1007/s12665-009-0135-0 Eregha, P. B., Babatolu, J. S., & Akinnubi, R. T. (2014). Climate change and crop production in Nigeria: An error correction modelling approach. International Journal of Energy Economics and Policy, 4(2), 297e311. FAO. (2012). World agriculture towards 2030/2050: The 2012 revision, Nikos Alexandratos and Jelle Bruinsma. ESA Working Paper No. 12-03, June 2012. Food and Agriculture Organization of the United Nations. Retrieved on 10 February from: www.fao.org/ economic/esa. FAO. (2017a). The future of food and agriculture: Trends and challenges, 2522-722. FAO. (2017b). The future of food and agriculture: Trends and challenges, ISBN 978-92-5109551-5. Rome. FAO. (2020). The state of agricultural commodity markets 2020, the state of agricultural commodity markets 2020. Rome, Italy: FAO. https://doi.org/10.4060/cb0665en FAO, IFAD, UNICEF, WFP, & WHO. (2020). The state of food security and nutrition in the world 2020. Transforming food systems for affordable healthy diets. Rome: FAO. https:// doi.org/10.4060/ca9692en Figueira, N., Curtain, F., Beck, E., & Grafenauer, S. (2019). Consumer understanding and culinary use of legumes in Australia. Nutrients, 11(7), 1575. https://doi.org/10.3390/ nu11071575. Published online 2019 Jul 12. Foley, J. A., Ramankutty, N., Brauman, K. A., Cassidy, E. S., Gerber, J. S., Johnston, M., Mueller, N. D., O’Connell, C., Ray, D. K., West, P. C., Balzer, C., Bennett, E. M., Carpenter, S. R., Hill, J., Monfreda, C., Polasky, S., Rockström, J., Sheehan, J., Siebert, S., Tilman, D., & Zaks, D. P. M. (2011). Solutions for a cultivated planet. Nature, 478, 337e342. https://doi.org/10.1038/nature10452 Food and Agriculture Organization of the United Nations. (2018). www.fao.org/in-action/ naps. Gonzalez-Aguilar, G., Wang, C. Y., & Buta, G. J. (2004). UV-C irradiation reduces breakdown and chilling injury of peaches during cold storage. Journal of the Science of Food and Agriculture, 84, 415e422. 20 Plant Small RNA in Food Crops Grundy, M. J., Bryan, B. A., Nolan, M., Battaglia, M., Hatfield-Dodds, S., Connor, J. D., & Keating, B. A. (2016). Scenarios for Australian agricultural production and land use to 2050. Agricultural Systems, 142, 70e83. https://doi.org/10.1016/j.agsy.2015.11.008 Hall, J., Jeggo, P. A., West, C., Gomolka, M., Quintens, R., Badie, C., Laurent, O., Aerts, A., et al. (2017). Ionizing radiation biomarkers in epidemiological studies e an update. Mutation Research, 771, 59e84. Hong, Y., Park, J., Park, J., Chung, M., Kwon, K., Chung, K., Won, M., & Song, K. (2008). Inactivation of Enterobacter sakazakii, Bacillus cereus, and Salmonella typhimurium in powdered weaning food by electron-beam irradiation. Radiation Physics and Chemistry, 77(9), 1097e1100. https://doi.org/10.1016/j.radphyschem.2008.05.004 Hossain, M. A., Hoque, M. M., Khan, M. A., et al. (2013). Foliar application of radiation processed chitosan as plant growth promoter and anti-fungal agent on tea plants. International Journal of Scientific Engineering and Research, 4, 1693. Hussain, P. R., Rather, S. A., Suradkar, P. P., & Omeera, A. (2021). Gamma irradiation treatment of minimally processed kiwi fruit to maintain physicochemical quality and prevent microbial proliferation during refrigerated storage. Journal of Food Processing and Preservation, 45, 15309. Ihsanullah, I., & Rashid, A. (2017). Current activities in food irradiation as a sanitary and phytosanitary treatment in the Asia and the Pacific region and a comparison with advanced countries. Food Control, 72, 345e359. Jan, S., Parween, T., Siddiqi, T., O, & Mahmooduzzafar, X. (2010). Gamma radiation effects on growth and yield attributes of Psoralea corylifolia L. with reference to enhanced production of psoralen. Plant Growth Regulation, 64(2), 163e171. Johnson, D. E., & Humphreys, E. (2021). Enhancing the productivity and sustainability of cropping systems in the coastal zones of tropical deltas of Asia. Field Crops Research, 263, 108059. https://doi.org/10.1016/j.fcr.2021.108059 Kader, A. A. (2002). Postharvest Technology of Horticultural Crops (3rd, p. 535). Oakland, CA: UC Publication 3311. University of California, Division of Agriculture and Natural Resources. Kalkura, P., Raj, B. P., Kashyap, N. S., & Surya, R. (2021). Pest control management system using organic pesticides. Global Transitions Proceedings. https://doi.org/10.1016/ j.gltp.2021.08.058 Kaushik, P., & Chauhan, A. (2008). Antibacterial potential of aqueous and organic extracts of N. Commune: A Cyanobacterium. VEGETOS, 21(1), 77e80. Kaushik, P., Goyal, P., Chauhan, A., & Chauhan, G. (2010). In vitro evaluation of antibacterial potential of dry fruit extracts of Elettaria cardamomum Maton (ChhotiElaichi). Iranian Journal of Pharmaceutical Research, 9(3), 287. Khalil, S. A., Ahmad, N., & Zamir, R. (2015). Gamma radiation induced variation in growth characteristics and production of bioactive compounds during callogenesis in Stevia rebaudiana (Bert.). New Negat. Plant Science, 1-2, 1e5. Kitinoja, L., & AlHassan, H. A. (2012). Identification of appropriate postharvest technologies for improving market access and incomes for small horticultural farmers in SubSaharan Africa and South Asia. Part 1: Postharvest losses and quality assessments. Acta Horticulturae (IHC 2010), 934, 31e40. Lanier, C., Manier, N., Cuny, D., & Deram, A. (2015). The comet assay in higher terrestrial plant model: Review and evolutionary trends. Environment and Pollution, 207, 6e20. https://doi.org/10.1016/j.envpol.2015.08.020 Linda, J. H., Feng, Y., Vanessa, M. L., & Jung, J. (2020). Growth and survival of foodborne pathogens during soaking and drying of almond (Prunus dulcis) kernels. Journal of Food Protection, 83(12), 2122e2133. MacLaren, C., Storkey, J., Menegat, A., et al. (2020). An ecological future for weed science to sustain crop production and the environment. A review. Agronomy for Sustainable Development, 40, 24. https://doi.org/10.1007/s13593-020-00631-6 Mahto, R., & Das, M. (2013). Effect of gamma irradiation on the physico-chemical and visual properties of mango (Mangifera indica L.), cv. ‘Dushehri’ and ‘Fazli’ stored at 20  C. Postharvest Biology and Technology, 447e455. Food crops, growth and productivity as an important focus for sustainable agriculture 21 Maraei, R. W., & Elsawy, K. M. (2017). Chemicalquality and nutrient composition of strawberry fruits trea-ted by g-irradiation. Journal of Radiation Research and Applied Sciences, 10, 80e87. Millennium Ecosystem Assessment, (2005). Island Press, Washington, DC. Milliman, J. D. (1992). Sea-level response to climate change and tectonics in the Mediterranean Sea. In L. Jeftic, J. D. Milliman, & G. Sestini (Eds.), Climatic change and the mediterranean (pp. 45e57). Edward Arnold. Ngoune Liliane, T., & Shelton Charles, M. (2020). Factors affecting yield of crops. Agronomyclimate change and food security. https://doi.org/10.5772/intechopen.90672 Nguyen, T., van den Berg, M., Raneri, J. E., & Huynh, T. (2021). Improving food systems: A participatory consultation exercise to determine priority research and action areas in Viet Nam. Frontiers in Sustainable Food Systems, 5, 717786. https://doi.org/10.3389/ fsufs.2021.717786 Parvin, F., Yeasmin, F., Islam, J. M. M., Molla, E., & Khan, M. A. (2013). Effect of gamma irradiated sodium alginate on Malabar spinach (Basella alba) and spinach (Spinacia oleracea) as plant growth promoter. American Academic & Scholarly Research Journal, 5(5), 63. Popp, J., PetT, K., & Nagy, J. (2013). Pesticide productivity and food security. A review. Agronomy for Sustainable Development, 33, 243e255. https://doi.org/10.1007/s13593012-0105-x Prakash, A., Guner, A. R., Caporaso, F., & Foley, D. M. (2000). Effects of low-dose gamma irradiation on the shelf life and quality characteristics of cut romaine lettuce packaged under modified atmosphere. Journal of Food Science, 65(3), 549e553. Radwan, T. M., Blackburn, G. A., Whyatt, J. D., & Atkinson, P. M. (2019). Dramatic loss of agricultural land due to urban expansion threatens food security in the Nile Delta, Egypt. Remote Sensing, 11, 332. https://doi.org/10.3390/rs11030332 Raman, R. (2017). The impact of genetically modified (GM) crops in modern agriculture: A review. GM Crops & Food, 8, 195e208. Richardson, K. J., Lewis, K. H., Krishnamurthy, P. K., Kent, C., Wiltshire, A. J., & Hanlon, H. M. (2018). Food security outcomes under a changing climate: Impacts of mitigation and adaptation on vulnerability to food insecurity. Climate change, 147, 327e341. https://doi.org/10.1007/s10584-018-2137-y Ruben, R., Verhagen, J., & Plaisier, C. (2018). The challenge of food systems research: What difference does it make? Sustainability, 11, 171. https://doi.org/10.3390/ su11010171 Sharma, P., Sharma, S. R., & Mittal, T. C. (2020). Effect and application of ionizing radiation on fruits and vegetables: A review. Journal of Agriculture Engineering, 57(2), 97e126. Singh, B. K., & Trivedi, P. (2017). Microbiome and the future for food and nutrient security. Microbial Biotechnology, 10, 50e53. https://doi.org/10.1111/1751-7915.12592 Spence, A., Poortinga, W., & Pidgeon, N. (2012). The psychological distance of climate change. Risk Analysis, 32, 957e972. https://doi.org/10.1111/j.15396924.2011.01695.x Struik, P, & Thomas, K. (2017). Sustainable intensification in agriculture: The richer shade of green. A review. Agronomy for Sustainable Development, 37, 39. Struik, P. C., Kuyper, T. W., Brussaard, L., & Leeuwis, C. (2014). Deconstructing and unpacking scientific controversies in intensification and sustainability: Why the tensions in concepts and values? Current Opinion in Environmental Sustainability, 8, 80e88. Sun, H., Jiang, S., Jiang, C., Wu, C., Gao, M., & Wang, Q. (2021). A review of root exudates and rhizosphere microbiome for crop production. Environmental Science and Pollution Research. https://doi.org/10.1007/s11356-021-15838-7 Taheri, S., Abdullah, T. L., Ahmad, Z., & Abdullah, N. A. P. (2014). Effect of acute gamma irradiation on Curcuma alismatifolia varieties and detection of DNA polymorphism through SSR Marker. BioMed Research International, 2014, 631813. https://doi.org/ 10.1155/2014/631813 22 Plant Small RNA in Food Crops Tong, A., Synnot, A., Crowe, S., Hill, S., Matus, A., Scholes-Robertson, N., et al. (2019). Reporting guideline for priority setting of health research (REPRISE). BMC Medical Research Methodology, 19, 1e11. https://doi.org/10.1186/s12874-019-0889-3 Tóth, G., Hermann, T., Ravina da Silva, M., & Montanarella, L. (2018). Monitoring soil for sustainable development and land degradation neutrality. Environmental Monitoring and Assessment, 190, 57. https://doi.org/10.1007/s10661-017-6415-3 Tripathi, J., Chatterjee, S., Vaishnav, J., Variyar, P. S., & Sharma, A. (2013). Gamma irradiation increases storability and shelf life of minimally processed readyto-cook (RTC) ash gourd (Benincasa hispida) cubes. Postharvest Biology and Technology, 76, 17e25. Tsatsakis, A. M., Amjad Nawaz, M., Kouretas, D., Balias, G., Savolainen, K. M., Tutelyan, V. A., Golokhvast, K. S., Dong Lee, J., Hwan Yang, S., & Chung, G. (2017). Environmental impacts of genetically modified plants: A review. Environmental Research, 156, 818e833. Tuong, T. P., Humphreys, E., Khan, Z. H., Nelson, A., Mondal, M., Buisson, M. C., & George, P. (2014). Unlocking the production potential of the polders of the coastal zone of Bangladesh through water management investment and reform. Messages from the Ganges Basin Development Challenges. CGIAR challenge program on water and food (CPWF) (p. 24). Dhaka, Bangladesh: WorldFish. USDA. Foreign Agricultural Service (USDA FAS). (2022). Global agricultural trade system online. https://apps.fas.usda.gov/GATS/default.aspx (Accessed 12 May 2022). USGCRP. (2018). In D. R. Reidmiller, C. W. Avery, D. R. Easterling, K. E. Kunkel, K. L. M. Lewis, T. K. Maycock, & B. C. Stewart (Eds.), Impacts, risks, and adaptation in the United States: Fourth national climate assessment, volume II (p. 1515). Washington, DC, USA: U.S. Global Change Research Program. https://doi.org/10.7930/NCA4.2018 Valin, T. H., Benjamin, L. B, Tomoko, H., & Elke, S. (2021). Achieving zero hunger by 2030, A review of quantitative assessments of synergies and tradeoffs amongst the UN sustainable development goals. A paper from the Scientific Group of the UN Food Systems Summit, 26 May 2021. Van Hesse, H. P., Reuber, T. L., & Van Der Does, D. (2020). Genetic modification to improve disease resistance in crops. New Phytologist, 225, 70e86. Velten, S., Leventon, J., Jager, N., & Newig, J. (2015). What is sustainable agriculture? A systematic review. Sustainability, 7, 7833e7865. Willet, et al. (2019). Food in the anthropocene: The EATelancet commission on healthy diets from sustainable food systems. Lancet, 393, 447e492. https://doi.org/10.1016/ WorldBank.2007 World Bank Group. (2015). Ending poverty and hunger by 2030 an agenda for the global food system. Zec, M., & Glibetic, M. (2018). Health benefits of nut consumption. In Reference module in food science (pp. 1e13). Elsevier. https://doi.org/10.1016/B978-0-08-100596-5.22511-0 Zhong, Z., Wang, X., Yin, X., Tian, J., & Komatsu, S. (2021). Morphophysiological and proteomic responses on plants of irradiation with electromagnetic waves. International Journal of Molecular Sciences, 22(22), 12239. https://doi.org/10.3390/ijms222212239 Further reading El-Beltagi, H. S., Ahmed, O. K., & El-Desouky, W. (2011). Effect of low doses g-irradiation on oxidative stress and secondary metabolites production of rosemary (Rosmarinus offcinalis L.) callus culture. Radiation Physics and Chemistry, 80, 968e976. Guner, A. R., Caporaso, F., & Foley, D. M. (2000). Effects of low-dose gamma irradiation on the shelf life and quality characteristics of cut romaine lettuce packaged under modified atmosphere. JFS: Sensory and Nutritive Qualities of Food, 65(3), 549e553. Food crops, growth and productivity as an important focus for sustainable agriculture 23 Kenner, B. (2020). U.S. Fruit Imports Grew by $8.9 Billion Over the Last Decade to Meet Rising Demand. Retrieved from 10 February 2022 https://www.ers.usda.gov/amber-waves/ 2020/september/us-fruit-imports-grew-by-89-billion-over-the-last-decade-to-meetrising-demand/. Kumar, A. K., Kumar, S., Kumari, K., & Singh, D. (2018). Medicinal uses of spices used in our traditional culture: Worldwide. Journal of Medicinal Plants Studies, 6, 116e122. Moher, D. (2009). Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement. Annals of Internal Medicine, 151, 264. https://doi.org/10.7326/ 0003-4819- 151-4-200908180-00135 Natalie, F., Felicity, C., Eleanor, B., & Sara, G. (July 2019). Consumer understanding and culinary use of legumes in Australia. Nutrients, 11(7), 1575. https://doi.org/10.3390/ nu11071575 This page intentionally left blank CHAPTER 2 Challenges and opportunities to sustainable crop production b Simerjeet Kaura and Bhagirath S. Chauhan a b Punjab Agricultural University, Ludhiana, Punjab, India; The University of Queensland, Queensland Alliance for Agriculture and Food Innovation (QAAFI) and School of Agriculture and Food Sciences (SAFS), Gatton, QLD, Australia 1. Present scenario of food production Most of the food consumed in many countries is produced locally. Since 1960, the significant expansion of area being used for agriculture, coupled with improved varieties, seeds, fertilizers, and agro-technologies, resulted in the tripling of agricultural production (Table 2.1). During the green revolution period, huge investments were observed in crop breeding, research, infrastructure for the market, and suitable policy support (Khush, 2001). This resulted in the tripling of cereal crop production (mainly rice and wheat) with only a 30% increase in area under cultivation. Worldwide, about 78% of the average per capita energy needs are fulfilled from cropTable 2.1 Global crop production (in million tonnes) trends. Crop production (in million tonnes) Low-income fooddeficit countries World Crop 1961 2019 1961 2019 Rice Wheat Barley Millet Maize Cottonseed (2018) Cotton lint (2018) Potato Flax Fresh fruits Fresh vegetables 215.65 222.36 72.41 25.71 205.03 17.44 9.46 270.55 0.69 6.57 62.30 755.47 765.77 158.98 28.37 1148.49 42.35 24.65 368.25 1.09 39.51 311.82 84.22 15.19 4.68 11.85 14.60 2.78 1.40 4.79 e 3.17 18.89 309.12 128.94 8.25 22.29 90.79 14.31 7.08 80.30 e 19.28 80.89 Data from Food and Agriculture Organization of the United Nations (FAO) (2020). Plant Small RNA in Food Crops ISBN 978-0-323-91722-3 https://doi.org/10.1016/B978-0-323-91722-3.00006-3 © 2023 Elsevier Inc. All rights reserved. 25 26 Plant Small RNA in Food Crops based food products and 20% is fulfilled from other food sources, such as milk, eggs, and meat (Brevik, 2013). However, eradication of hunger and malnutrition by 2050 is not possible, even at the current rate of production. Projections indicate that a 70% increase in overall food production would be needed to feed that population by 2050. Previous increases in food production since the 1960s have come at the expense of natural resources and the environment. The share of agriculture in GDP is declining at different rates across different regions. The intensive agricultural practices, large-scale deforestation, and fossil fuel burning have resulted in the deterioration of natural resources such as fresh/underground water depletion and biodiversity erosion. Various anthropogenic activities have resulted in pollution of the air and water. Agriculture or crop production is reeling under pressure due to degradation of natural resources (soil, water, etc.), climate change, change in land use, multiple pest complexes, and the evolution of resistance against pesticides. There is a need to sustain the agricultural growth by producing ‘more crop per drop’ with energy-efficient and climate-smart crop production practices. The 2030 agenda on sustainable development acknowledges that there are many challenges (that are inter-dependent) so that the second sustainable development goal (SDG) of the Food and Agriculture Organization of the United Nations (FAO) can be fulfilled, which aims at eradicating hunger and ensuring food and nutritional security by promoting sustainable agriculture (FAO, 2017). Significant transformations in agricultural practices are required to address these challenges and to ensure sustainable food production for all inhabitants of this planet earth. 1.1 Scenario of food 1.1.1 Food supply Food supply in terms of daily per capita caloric, protein, and fat supply has increased both globally and in low-income food-deficit countries since the 1960s (Table 2.2). However, these increases have not been uniform across different regions of the world, with significant improvement witnessed in lowincome Asian and African countries while a slower rate, or almost no increase, is being experienced in high-income countries. Over the past few decades, the food supply has reached a plateau in Europe, Oceania, and North America, and major increases have come from improvements in the food supply in Africa, Asia, and South America. In terms of caloric supply, there is more equality across different regions, but there is a significant regional difference in Challenges and opportunities to sustainable crop production 27 Table 2.2 The daily average supply of calories across the population. Low-income food-deficit countries World Particulars 1961 2017 Relative change (%) Daily caloric supply (kilocalories per person per day) Protein supply quantity (g/capita/ day) Fat supply quantity (g/capita/day) 2196 2884 þ31 1997 2444 þ22 61.46 81.23 þ32 51.73 60.82 þ18 47.52 82.76 þ74 31.81 49.78 þ56 1961 2017 Relative change (%) Data from United Nations Food and Agricultural Organization, FAO, 2017; http://www.fao.org/ faostat/en/#data. protein and fat supplies. There is a remarkable improvement in per capita fat supply from 48 g in 1961 to 83 g in 2017. Although this increase has come from all regions of the world over this period. Still, there is a great regional disparity in fat supply. United Nations Children’s Fund examined malnutrition in children and observed the ‘triple burden’ of malnutrition which refers to the co-existence of underweight, micronutrient deficiency (hidden hunger), and obesity in children (Unicef, 2020). 1.1.2 Food patterns Due to the growth of income in low- and middle-income countries, there is a change in dietary habits from cereal-based food to fruits, vegetables, and meat. Due to the globalization of food and agriculture, the consumption of processed and packaged food has increased manifold; resulting in more scope for food processing and value addition. Moreover, this expansion has resulted in more resource- and/or energy use by agriculture. The dietary transition requires an equivalent shift in agricultural production which will put pressure on natural resources. The big question is whether to sustain the present agriculture production levels, given the observed negative effects of climate change and scarce land and water resources. 1.1.3 Food wastage Food is wasted in numerous ways and it is estimated that one-third of food produced for human consumption is lost or wasted every year on a global 28 Plant Small RNA in Food Crops scale. This food loss and waste, occurring at various levels of supply chains, range from primary food production at the field level to the last level of household consumption (FAO, OECD, 2018). About 40% of food is wasted and lost at the later stages in the supply chain (retail and consumer levels) in middle-, upper-middle- and high-income countries. While a major portion of agricultural produce is wasted due to post-harvest losses in low- and middle-income countries where there is a lack of proper storage and processing facilities. To contain these food losses and wastage at different levels, solutions should focus on strengthening the supply chain through awareness and investments in infrastructure. 2. Challenges and opportunities for sustainable food production Sustainable food production is a universal challenge and there are multiple challenges for the agriculture sector in different income-group countries. However, it is the collective responsibility of all countries to bring about fundamental changes in the ways of crop production for feeding the everincreasing world population. There is a Kenyan proverb, “If there are to be problems, may they come during my lifetime so that I can resolve them and give my children the chance of a good life” (Hickey et al., 2012). In 2017, the FAO outlined in a report the challenges for sustainable crop production which must be addressed to meet the growing food demand of the world population in a cost-effective manner (The future of food and agriculture: trends and challenges). Over the last century, the world has seen a remarkable achievement in terms of crop production. However, much more effort is required to achieve the vision of the FAO. There is a huge regional disparity in terms of the challenges and opportunities as outlined in Table 2.3. Despite significant economic growth since 2000, African citizens earn only 5% of the average income of citizens living in the United States. This reveals deep income imbalances and huge regional inequalities and its implications are hard to predict. 2.1 Challenges and opportunities related to food demand 2.1.1 Population growth The ever-increasing population growth is putting pressure on food security and the demand for high-quality nutritional food. FAO has considered population dynamics, not the absolute population, as a key driver and a challenge for sustainable food production. Population dynamics included Challenges and opportunities to sustainable crop production 29 Table 2.3 Challenges and opportunities for the agriculture sector in different countries. Positive (þ) and negative ( ) symbols indicate the presence and absence, respectively of a challenge or opportunity. Challenge Limited capital and access to affordable credit Less human resources in agriculture Cashing in on high demographic dividends Large displacements and migration flows High cost of key inputs Low and declining soil fertility Pre- and post-harvest crop losses Inadequate storage and processing facilities Spread of transboundary pests and diseases Invasive species Migration and employment outside agriculture Evolution of multiple resistance against pesticides Inadequate markets and marketing infrastructure Inappropriate legal and regulatory framework Inadequate budgetary allocation Low absorption of modern technology Loss of biodiversity Conflicts, crises and natural disaster More capital-intensive food systems Degradation of natural resources Low- and middleincome countries High-income countries þ e e þ þ e þ e þ þ þ þ þ þ e e þ þ e þ þ e e þ þ e þ e þ þ e e e þ þ þ e þ þ þ þ þ e þ Opportunity Abundant human resources New and expanding markets Continued 30 Plant Small RNA in Food Crops Table 2.3 Challenges and opportunities for the agriculture sector in different countries. Positive (þ) and negative ( ) symbols indicate the presence and absence, respectively of a challenge or opportunity.dcont'd Challenge Potential for increasing production Vast irrigation potential Value addition and export Immigrants for agriculture Low- and middleincome countries High-income countries þ þ þ þ þ e þ e regional disparity, the population of working age (aging), and urbanization. The world population increased from 1.0 billion in 1800 to 7.7 billion today. In general, the world population growth is slowing down; however, the global population is expected to reach 9.7 billion by 2050. It is a relief that the world population growth rate has declined from 2.2% per year 50 years ago to 1.05% per year in present times. Still, the population growth rate is higher in low-income countries (Table 2.4). It is interesting to note that differences within the region are larger as compared to differences across different regions. Globally, it is estimated that around 800 million people are chronically hungry while about 2 billion people have micronutrient deficiencies. About 700 million people, of which mostly are rural populations are extremely poor and cannot afford meals. The results of the 26th round of the UN’s global population estimates and projections indicated that the population is growing the fastest in lowincome countries. The report about population projections indicates that nine countries (India, Nigeria, Pakistan, the Democratic Republic of the Congo, Ethiopia, the United Republic of Tanzania, Indonesia, Egypt, and the United States of America) will make up more than half the projected growth of the global population between now and 2050. Out of the projected 11 billion people by the year 2100, two continents namely Asia and Africa are expected to be home to a 9 billion population. The countries in these two continents are dependent upon agriculture mainly for their GDP. Therefore, population growth will put extra pressure on agriculture and more efforts are necessary to increase crop production using the limited land and water resources available in low- and middle-income countries. To tackle the challenges of equality, poverty, hunger, and malnutrition, population growth needs to be taken care of and maximum investments are required to ensure food security and to achieve development goals. Challenges and opportunities to sustainable crop production 31 Table 2.4 Annual global population increase from 1950 to 2019 and the projections until the end of this century. Population growth Country 1950 2019 Low-income countries Lower-middleincome countries Middle-income countries Upper-middleincome countries High income countries World 2.06 million 13.49 million 37.06 million 23.59 million 7.97 million 47.16 million 19.56 million 40.61 million 56.99 million 16.37 million 5.27 million 81.86 million Relative change (%) Projection (%) for 2100 þ849 36 þ201 100 þ54 117 31 161 34 97 þ74 96 Data from United Nations, Department of Economic and Social Affairs, Population Division (2017). World Population Prospects: The 2017 Revision, DVD Edition; https://population.un.org/wpp2019/ Download/Standard/Interpolated/. Otherwise, based on the current population growth trend, these countries could be threatened with a neo-Malthusian future. Follett (2020) cautioned that concerns about over-population may result in coercive actions as the world has witnessed neo-Malthusianism and coercive population control measures in the form of China’s one-child policy (1979e2015) and India’s forced sterilizations during its “Emergency” (1975e77). One of the main challenges faced by agriculture is land fragmentation. Population growth especially in middle- and low-income countries results in more fragmentation of agricultural farms. On a global scale, 72% farms of 570 million farms are less than 1-ha and control only 8% of total agricultural land (World Bank, IFAD, 2017). Identifying this challenge, rural transformation, and labor productivity to increase agricultural productivity of small-scale farms is a specific goal of the 2030 Agenda. 2.1.2 Aging Globally, people will not only increase in number but will also be older in the future. The faster growth of the working-age group (15e64 years old population as defined by the UN) as compared to other ages from 1950 to 2020 in low- and middle-income countries created avenues for better 32 Plant Small RNA in Food Crops education, health, and accelerated economic growth (Table 2.5). However, the world’s population is growing older and aging is more accelerated in low-income countries. It is estimated that the number of older people (aged 65 years or over) will double between 2019 and 2050 in Northern Africa and Western Asia, Central and Southern Asia, Eastern and South-Eastern Asia, Latin America, and the Caribbean (FAO, 2017). This falling proportion of the working-age group will put extra pressure on health care and social protection. This aging, especially of rural peoples, will affect crop production through its implications on the composition of the labor force, patterns of crop production, adaptability to new agro-techniques, and socio-economic development. 2.1.3 Migration 2.1.3.1 Migration from low-income countries to high-income countries Another major component in the population change pattern is due to migration (Table 2.6). The population trend shows that migration is increasing as people escape the insufficient employment opportunities in low- and middle-income countries. Other drivers are the demand for workers in high-income countries, as well as violence, insecurity, and armed conflict in low-income countries. Fourteen countries have a net inflow of nearly one million migrants, while a net outflow of a similar magnitude was witnessed in 10 countries between 2010 and 2020. Various natural disasters, violent conflicts, and cross-boundary terrorism are forcing many affected people back into poverty and fueling distress migration. The violent conflicts and crises in low-income countries reduce food availability, restrict access to health care, and sabotage social protection systems. The growth of poor people along with social protection is required to address the root cause of migration by eradicating poverty, hunger, malnutrition, income imbalance, and inequality both within and between countries. 2.1.3.2 Migration from the rural area to the urban area In the early 1950s, most of the global population (approximately 70%) lived in rural areas (Table 2.7). Nearly 90% and 60% of the population lived in rural areas in low-income and high-income countries, respectively. Since then, there has been a marked increase in urbanization. Presently, 56% of the global population lives in urban areas and it is projected to increase to 68% (FAO, 2017). It is interesting to note that earlier in the 1950s, Table 2.5 Total population aged 15e64 years and 65þ years old based on historic estimates from 1950 to 2015, and UN medium scenario projections to 2100. Population Country Elderly group (>65 years) Relative change (%) 1950 2020 1950 2020 Working age (15e64 years) 75.32 million 446.46 million 1.02 billion 569.31 million 446.58 million 1.54 billion 429.53 million 2.01 billion 3.83 billion 1.83 billion 821.12 million 5.08 billion 3.97 million 29.55 million 71.65 million 42.10 million 53.15 million 128.81 million 24.79 million 185.47 million 478.42 million 292.95 million 225.83 million 729.38 million Projection (%) for 2100 þ470 Elderly group (>65 years) þ524 þ298 Elderly group (>65 years) þ1412 þ349 þ528 þ49 þ458 þ277 þ568 þ13 þ265 þ221 þ596 28 þ143 þ84 þ325 10 þ74 þ231 þ466 þ32 Working age (15e64 years) þ245 Data from United Nations, Department of Economic and Social Affairs, Population Division (2017). World Population Prospects: The 2017 Revision, DVD Edition; https://esa.un.org/unpd/wpp/Download/Standard/Population/. Challenges and opportunities to sustainable crop production Low-income countries Lower-middleincome countries Middle-income countries Upper-middleincome countries High income countries World Working age (15 e64 years) 33 34 Plant Small RNA in Food Crops Table 2.6 World migration report 2020. Year Migrants (millions) Percent of total population 1990 1995 2000 2005 2010 2020 153 161 174 192 221 281 2.87 2.81 2.83 2.93 3.17 3.60 As per data from FAO. Table 2.7 Share of the population living in urban areas. Urbanization (%) Country 1950 2020 Relative change Projection for 2050 Low-income countries Lower-middle-income countries Middle-income countries Upper-middle-income countries High income countries World 9.32 17.21 33.17 41.60 þ256 þ142 þ51 þ42 19.89 22.08 53.74 68.20 þ170 þ209 þ27 þ21 58.51 29.61 81.85 56.17 þ40 þ90 þ8 þ22 Data source: UN World Urbanization Prospects 2018 and others. urbanization was a phenomenon in high-income countries. However, this urbanization is now prominent in low-income countries and is projected to increase in the 2050s. This increased urbanization will have a marked influence on agriculture in two ways, such as the decreasing agricultural labor force and changing food consumption patterns. Urbanization has resulted in a transition in dietary patterns with more intake of protein and fat. Further, there is an increased intake of fruits, vegetables, animal-source protein, and processed foods by the urban population which has high purchasing power as compared to the rural population. These changed dietary patterns will lead to a shift in employment from primary activities (food/crop production) in agriculture to secondary or tertiary activities, such as transportation, value addition, processing, and marketing. Challenges and opportunities to sustainable crop production 35 2.2 Challenges and opportunities related to food production 2.2.1 Natural resources The increased agricultural production and urbanization have been achieved at the expense of limited natural resources. The judicious use and conservation of natural resources such as soil and water are vital for sustainable crop production The availability and management of plant nutrients and water play a major role in crop production, along with weather factors (Kaur et al., 2018). Conservation or climate-resilient agriculture is being practiced as an adaptation and mitigation strategy for sustainable crop production under climate change conditions. 2.2.1.1 Soil Soils are just one of the natural resources (1.53 billion hectares) that create the foundation of food production; however, there is little scope to increase the agricultural area (Alexandratos & Bruinsma, 2012). Moreover, about 33% of the world’s soil resources are degraded. In the 2030 Agenda, one of the sustainable development goals is focused on the restoration of degraded soils and improving soil health. Different soil factors (clay content, organic matter, cation exchange capacity, and pH) affect water and nutrient availability. Various soil-related problems such as soil erosion, soil organic carbon change, and nutrient imbalances are observed at the global level which has a marked influence on crop productivity. There is an annual 0.3% yield loss due to soil erosion (Biggelaar et al., 2003) and it is projected that in 2050, a total yield reduction of 10.25% is possible if no conservation agriculture practices are adopted by the farmers. This loss is enormous and is equivalent to the removal of 150 million hectares from crop production globally (FAO-FAO ITPS, 2015). Soils have the largest terrestrial carbon pool. But, the decline of this organic carbon stock is negatively affecting soil fertility. Nutrient imbalances negatively affect food production and may result in water and air pollution. An investment of US$4.6 trillion is needed over 6 years to completely restore land degraded due to changes in land use globally (Nkonya et al., 2016). Carbon sequestration (storing atmospheric carbon dioxide in soils) in soils is one of the important mitigation strategies for climate change. Numerous and diverse farming approaches such as agroforestry, conservation agriculture, and organic farming may be used for sustainable soil management. Sustainable soil management practices may promote carbon sequestration and enhance soil biodiversity which will help in the improvement of soil health. Improved soil fertility can lead to 36 Plant Small RNA in Food Crops sustainable food production, food security, and nutrition. Further, soils, because of stored carbon reserves, are both influenced by and reason for climate change. 2.2.1.2 Water Water, the second natural resource, is the essence of life on this mother earth. Freshwater is under pressure and is depleting at an alarming rate. Agriculture accounts for 70% of total withdrawals of renewable freshwaters. About 20% of fresh and groundwater is being extracted and depleted for various agriculture and aquaculture activities in Asia. However, water for the service sector may increase due to the rise in the urban population. Countries may be considered water-stressed if the withdrawal of freshwater is more than 25%. UN-Water Integrated Monitoring Initiative for their SDG 6 in their meeting in March 2021 compiled country-level data on water and impressed upon doubling the current rate of progress to meet the goals of SDG (FAO, UN Water, 2021). Water use efficiency (expressed as value/volume) in agriculture, industrial, and services sectors were 0.60, 32, and 112 USD/m3, respectively, in 2018. Water is physically and economically scarce in many Asian and African countries. To meet SDG 6, and to promote water use efficiency in such areas, multi-disciplinary and partnership efforts from different stakeholders are needed. 2.2.2 Change in land use The anthropogenic activities are leading to changes in land cover/use occurring on a spatial and temporal scale. The high-input and capitalintensive food systems currently in use have caused deforestation, depleted soil resources, physical and economic water scarcities, and high greenhouse gas emissions. Agricultural activities are conducted on approximately 40% of available global land and are one of the major uses for land (Ramankutty et al., 2008). The global expansion in agricultural land has occurred at the expense of the loss of approximately 100 million hectares of forest land. More food was needed to feed expanding population, which was fulfilled by both horizontal and vertical expansion of agriculture. Intensive farming and area expansion through deforestation (80% due to agriculture) resulted in a many-fold increase in crop production (FAO, 2017). Deforestation has severe repercussions in the form of increased soil erosion, degraded environment, loss of biodiversity, and large emissions of greenhouse gases due to changes in land use (from forest land Challenges and opportunities to sustainable crop production 37 to agricultural fields). In present times, it is possible to ensure food security along with forest conservation. Earlier, the global expansion in the agricultural area was the reason for deforestation (forests to croplands). However, lately, the swelling urban population is the main driver for the change in land use (Lambin & Geist, 2006). Urbanization has also resulted in a change in land use from forest cover or agricultural fields to industrial areas or cities. Various natural (forest fire) and socio-economic (demography, poverty, demand for wood & construction material, market and credit facilities) factors manipulate the land-use change over an area and these factors vary between and within the regions and countries. The change in land use is occurring on a global scale leading to negative ecological consequences such as land degradation, loss of plant and animal biodiversity, habitat destruction, extinction of native flora and fauna, and climate change. Recently, agricultural land is being used for other human activities such as industries and housing (due to urban expansion) which is resulting in a decrease in area under crop production. The information on the change in land cover is required on a local level for framing sound environmental policies. To enhance global crop production through agricultural intensification, policy-makers must analyze the potential trade-offs between food production and SDGs of zero hunger, climate mitigation, and biodiversity. 2.2.3 Climate change One of the largest effects of population growth and other anthropogenic activities is global warming. Climate change is a change in the climatic variables over long periods and global warming is one sign of climate change. Along with fossil fuel burning, various agricultural production activities, such as rice cultivation, dairy production, and land-use change are contributing to climate change through the emission of greenhouse gases (25% of all man-made emissions) such as carbon dioxide, nitrogen oxides, and methane. It is important to understand that sustainable crop production is affected by climate change. The increasing levels of atmospheric carbon dioxide affect crop production through its effect on elevated surface temperature and photosynthesis. It is estimated that surface temperatures will rise by 3e12 C on both geographical and temporal scales (IPCC, 2007). Along with the rise in the earth’s temperature, there will be more extreme weather phenomena, such as excessive precipitation, drought, and strong windstorms. It is important to mention here that these climate change 38 Plant Small RNA in Food Crops patterns related to temperatures and rainfall are highly variable and their impacts on crop production are highly site-specific. Climate change will have negative impacts on crop growth and yield. Temperature and water stress will affect crop duration and the overall cop production over that area. These impacts of abiotic stresses are location-, plant-, and crop stage-specific. Carbon dioxide is important for the photosynthetic activities of plants and its increased concentration will have a positive impact on crop production. The increased carbon dioxide concentrations are coupled with elevated surface temperatures due to the greenhouse effects. By the end of the 21st century, crop yield is predicted to decrease by 30%e46% and 63%e82% under the slowest- and the most rapid-warming scenario, respectively (Schlenker & Roberts, 2009). In general, these negative impacts will be lower at higher latitudes, or even neutral yield gains can be observed while more yield losses will be observed in lower latitude regions. Precisely speaking, these impacts on crop production will be location-, crop-, and region-specific. Furthermore, it is indicated that the nutritional quality (protein content) of food crops could decrease due to changing climate conditions. It is estimated that marine phytoplankton concentrations have decreased by 40% due to climate change. Climate change will have a negative impact on crop production due to its effect on the intensity and range of infestation of pests (insects, weeds, diseases, viruses, and arthropods), in addition to the increasing problems of pest resistance, thus putting more pressure on plants. Extreme weather events, like strong wind currents, rainfall, and snowfall variability, may help in the migration and long-distance dispersal of invasive pest species to new areas through wind and water. It is a well-known fact that crop-pest interactions at any given place are mainly governed by climate and management practices. The growth of crop plants will be badly affected by climate change and the resultant weak plants will be more vulnerable to different pests. Therefore, an improved understanding of the effects of climate change on pests and their control strategies is a prerequisite for sustainable crop production. 2.2.4 Pest pressure on crops and evolution of resistance against pesticides It is pertinent to recognize that pests are dynamic, persistent, and hardy, and therefore will adapt to climate change more easily than crop plants. It will be essential to breed new germplasms with desired evolutionary traits, such Challenges and opportunities to sustainable crop production 39 as tolerance to biotic (pests) and abiotic (temperature and water) stresses or improved water and/or nutrient use efficiency. To supplement this, advanced agricultural practices need to be developed, which will help in improved soil and water management and thus mitigate these pressures. Climate-resilient agriculture practices, such as conservation agriculture, zero tillage, and cover cropping are currently being practiced in many countries as a mitigation strategy to climate change. Pesticides are important, effective, and efficient tools for preventing and controlling various types of pests, such as insects, weeds, and disease-causing organisms (fungi, viruses, bacteria, etc.). The reports of the over-use of pesticides in agricultural production and their negative impacts on the environment are widespread throughout the world. Pesticide resistance is another major problem that is increasing at a faster pace and refers to “the inherited, reduced susceptibility of a pest population to an agricultural applied rate of pesticide that was previously effective at controlling the pest”. Among various types of pests, insects are well-known to evolve resistance at alarming rates. Insecticide resistance has been confirmed in more than 600 insect-pest and related arthropod species (IRAC, 2021). Herbicide resistance has been confirmed in 266 weed species (153 dicots and 113 monocots) with 508 unique cases at the global level (Heap, 2021). Pesticide resistance is a population evolutionary process, which depends upon genetics and selection pressure (repeated application of the same pesticides). It is pertinent to note that pest populations may adapt to nonchemical methods of control through a change in their biology and phenology. The loss of pesticides during application (because of resistance) is a major problem that will have a great impact on the lives of all. If resistance to a pesticide or complete family/mode of action (due to cross or multiple resistance) evolves, these pesticides can no longer be used for pest management. Pesticide resistance management strategies involve a balanced and integrated use of different chemical and non-chemical control measures. Integrated pest management approaches are highly desirable, economical, and eco-friendly methods to avoid, delay and manage pesticide resistance. In this approach, various preventive measures (crop sanitation and quarantine measures) and multiple control measures (cultural, biological, and chemical) are adopted to keep pest populations below economic threshold levels. During earlier crop breeding programs, improved yield and quality were the only criteria that were selected, and less focus was put on crop-defense 40 Plant Small RNA in Food Crops capabilities. To obtain sustainable crop production from such varieties, a huge amount of inputs such as fertilizer, water, and pesticides are required. Presently, the development of pest-resistant crop varieties and improved quality are two important criteria for crop breeding programs. The use of insect- or disease-resistant or herbicide-resistant crop varieties is an economical and eco-friendly practice. However, the evolution of resistance to pests may reduce the beneficial nature of such varieties. Further, pesticide resistance can evolve at a faster rate due to the indiscriminate use of pesticides in these pest-resistant crop varieties. For example, the cultivation of herbicide-resistant crops requires the application of the same molecule year after year, which can select for resistant weed individuals. Therefore, to prevent the evolution of pest resistance, stewardship of pest-resistant crop varieties is necessary. The evolution of resistance in pests may reduce the usefulness of insecticidal proteins observed in transgenic Bacillus thuringiensis (Bt) crops. Sustainable crop production depends on effective pest management strategies that use various control methods that allow for reduced chemical inputs. Pests are reported to have the ability to aggressively flourish under higher carbon dioxide levels, elevated temperatures, and increased water stress conditions (Patterson et al., 1999). Recently, the focus of crop breeders has shifted toward the identification of plant defensive traits for keeping pests under check. To reduce the chemical loads in the agroecosystems, biological pest control methods that employ bioagents or biopesticides derived from bacteria and fungi should be exploited. Biopesticides should make an important component of integrated pest management in both conventional and organic crop production. 2.2.5 Interaction of climate change and pest pressure The crop yield is a function of the local climate, the variety grown, and management practices followed to raise the crop. It is pertinent to note that crop production has increased markedly since the green revolution through the use of high-yielding varieties coupled with improved water, fertilizer, and pesticide technologies. To sustain this level of crop productivity, more precise climate-smart agricultural techniques and integrated nutrient and pest management practices will need to be developed and adopted. Climate change can alter the durability and effectiveness of pest-resistant traits of such crop varieties (Kaur et al., 2020). Elevated carbon dioxide levels and temperature are likely to increase the pest pressure on crops. Changing climate conditions may affect the fecundity, growth, and fitness Challenges and opportunities to sustainable crop production 41 of weeds and crops and will thus change the nature of crop-weed competitive interactions (Patterson, 1995). Extreme weather events such as snowfall variability, strong wind currents, and elevated temperature may help in the migration and dissemination of various pests to far new areas. Invasive pest species may invade new areas under the impacts of climate change (Song et al., 2009), thus increasing the burden on pest control (Korres et al., 2016). Weather events such as temperature, rainfall, and relative humidity may alter the pesticide efficacy. For example, unfavorable weather conditions may result in either reduced herbicide efficacy or increased risk of crop phytotoxicity (Patterson et al., 1999). 2.3 Challenges and opportunities related to policy-making The world is facing challenges in food and agriculture that are interconnected. To address these challenges on the path to sustainable development, integrated policy approaches are required at both national and international levels. The interdependent challenges were fully established while framing the 2030 Agenda for Sustainable Development and many other global agreements. Given the vast regional disparity, it is not easy to frame policies that could be applicable to all environments and crops. FAO is supporting the 2030 agenda which stressed the importance of “leaving no one behind”. It is very important to protect the rights of the poor while framing policies and institutions that are dedicated to attaining sustainable development goals. Various challenges in agriculture are transboundary in nature and they need to be managed with international cooperation. 3. Conclusions Sustainable food production is a multidimensional global challenge. Today, agriculture faces a triple challenge of food security, employment generation, and sustainability of natural resources. In low-income and lower-middleincome countries, a major part of the population, around 60%, depends upon agriculture. The agriculture sector must generate jobs and contribute to poverty eradication and rural economic growth. Furthermore, farming practices need to ensure the sustainability of natural resources under climate change scenarios. The present-day intensive, energy-rich agricultural systems cannot deliver sustainable food and agricultural production. A holistic approach is needed to restore ecological balance and sustainable agricultural production. There is a shift of energy sources from fossil fuels to biofuels, renewable energy sources which will further intensify the competition on 42 Plant Small RNA in Food Crops natural resources for their production. The food systems should be more efficient and resilient for the improvement of smallholder livelihoods along with reducing the environmental footprint and impacts on biodiversity through holistic approaches including agroecology and integrated nutrient and pest management. Therefore, multi-disciplinary and international collaborations are needed for inclusive and equitable global development. References Alexandratos, N., & Bruinsma, J. (2012). World agriculture towards 2030/2050: The 2012 revision. Rome: FAO. ESA Working paper No. 12-03. Biggelaar, D. C., Lal, R., Eswaran, H., Breneman, V. E., & Reich, P. F. (2003). Crop losses to soil erosion at regional and global scales: Evidence from plot-level and GIS data. In K. Wiebe (Ed.), Land quality, agricultural productivity, and food security (pp. 223e261). Cheltenham, UK: Edward Elgar. Brevik, E. C. (2013). Soils and human health-an overview. In E. C. Brevik, L. C. Burgess, & B. Raton (Eds.), Soils and human health (pp. 29e56). Boca Raton: CRC. FAO. (2017). The future of food and agriculture - trends and challenges. Rome. FAO, ITPS. (2015). Status of the world’s soil resources (SWSR)- main report. Rome, Italy Rome: Food and Agriculture Organization of the United Nations and Intergovernmental Technical Panel on Soils. https://fao.org/3/i5199e.pdf. FAO, OECD. (2018). Food security and nutrition: Challenges for agriculture and the hidden potential of soil a report to the G20 agriculture deputies. http://www.g20.utoronto.ca/2018/ ois_report. FAO, UN Water. (2021). Progress on change in water-use efficiency. Rome: Global Status and Acceleration needs for SDG Indicator 6.4.1, 2021. https://doi.org/10.4060/cb6413en Follett, C. (2020). Neo-malthusianism and coercive population control in China and India (july 21, 2020). Cato Institute, Policy Analysis, No. 897. Heap, I. (2021). The international survey of herbicide resistant weeds. http://www.weedscience. com. (Accessed 31 October 2021). Hickey, G. M., Pelletier, B., Brownhill, L., Kamau, G. M., & Maina, I. N. (2012). Preface: Challenges and opportunities for enhancing food security in Kenya. Food Security, 4, 333e340. IPCC. (2007). Climate change 2007: Impacts, adaptation and vulnerability. In M. L. Parry, O. F. Canziani, J. P. Palutikof, P. J. van der Linden, & C. E. Hanson (Eds.), Contribution of working group II to the fourth assessment report of the intergovernmental panel on climate change (p. 976). Cambridge: Cambridge University Press. IRAC. (2021). The insecticide resistance action committee. https://irac-online.org. (Accessed 31 October 2021). Kaur, S., Jabran, K., Florentine, S., & Chauhan, B. S. (2020). Assuring crop protection in the face of climate change through an understanding of herbicide metabolisms and enhanced weed control strategies. In K. Jabran, et al. (Eds.), Crop protection under changing climate (pp. 17e56). Springer Nature Switzerland AG. Kaur, S., Kaur, R., & Chauhan, B. S. (2018). Understanding crop-weed-fertilizer-water interactions and their implications for weed management in agricultural systems. Crop Protection, 103, 65e72. Khush, G. S. (2001). Green revolution: The way forward. Nature Reviews Genetics, 2, 815e822. Challenges and opportunities to sustainable crop production 43 Korres, N. E., Norsworthy, J. K., Tehranchian, P., Gitsopoulos, T. K., Loka, D. A., Oosterhuis, D. M., Gealy, D. R., Moss, S. R., Burgos, N. R., Miller, M. R., & Palhano, M. (2016). Cultivars to face climate change effects on crops and weeds: A review. Agronomy for Sustainable Development, 36, 12. Lambin, E. F., & Geist, H. J. (Eds.). (2006). Land-use and Land-cover change. Local processes and global impacts. Berlin: Springer. Nkonya, E., Mirzabaev, A., & von Braun, J. (Eds.). (2016). Economics of land degradation and improvement- a global assessment for sustainable development. International Food Policy Research Institute (IFPRI) and Center for Development Research (ZEF). University of Bonn. Springer Open. Patterson, D. T. (1995). Effects of environmental stress on weed/crop interactions. Weed Science, 43, 483e490. Patterson, D. T., Westbrook, J. K., Joyce, R. J. V., Lingren, P. D., & Rogasik, J. (1999). Weeds, insects and diseases. Climate Change, 43, 711e727. Ramankutty, N., Evan, A. T., Monfreda, C., & Foley, J. A. (2008). Farming the planet: 1. Geographic distribution of global agricultural lands in the year 2000. Global Biogeochemical Cycles, 22, GB1003. https://doi.org/10.1029/2007gb002952 Schlenker, W., & Roberts, M. J. (2009). Nonlinear temperature effects indicate severe damages to US crop yields under climate change. Proceedings of the National Academy of Sciences of the United States of America, 106, 15594e15598. Song, L., Wu, J., Changhan, L., Furong, L., Peng, S., & Chen, B. (2009). Different responses of invasive and native species to elevated CO2 concentration. Acta Oecologica, 35, 128e135. Unicef. (2020). New insights: 21st century malnutrition. New York: Unpacking the triple burden for children nutritional wellbeing. World Bank, IFAD. (2017). Rural youth employment: A synthesis study. Report to the G20 Development Working Group. This page intentionally left blank CHAPTER 3 Molecular mechanisms and strategies in response to abiotic stresses for the sustainability of crop production under changing climate K.L. Bhutiaa, Biswajit Pramanickb, Sagar Maitrac, Saipayan Ghosha and Akbar Hossaind a Department of Agricultural Biotechnology and Molecular Biology, Dr. Rajendra Prasad Central Agricultural University, Samastipur, Bihar, India; bDepartment of Agronomy, Dr. Rajendra Prasad Central Agricultural University, Samastipur, Bihar, India; cDepartment of Agronomy, CUTM, Bhubaneswar, Odisha, India; dDepartment of Agronomy, Bangladesh Wheat and Maize Research Institute, Dinajpur, Bangladesh 1. Introduction There has been immense impact of climate change in Agriculture over the years. As per the reports of Intergovernmental Panel on Climate Change (IPCC), various types of stresses have been incurred by the crops leading toward reduction in crop production due to the drastic alterations in climate (Bhutia et al., 2018). Climate change has bought the increased incidence of abiotic stresses to crop plants. They impose tremendous effect on different plant species affecting the crop cycle. The different abiotic stressors which has been increased by change in climate includes extreme high and low temperature, elevated CO2, drought, waterlogging, chemical factors (heavy metals and pH), offseason rainfall and sunshine intensity (Bhutia et al., 2018). The uptake of water, which is a crucial element required by the plants for nutrients transportation and for other vital activities (Ashkavand et al., 2018) is hampered under stress conditions. Higher salt concentrations (soil salinity stress) reduces soil water potential making the uptake of water by roots problematic resulting into drought like stress to plant. Drought stress can also be elevated due to high temperature. Thus, in general, drought stress takes place because of various causes which results in the discharge of the water content of the cell, which causes plasmolysis and death of the cell. Plant Small RNA in Food Crops ISBN 978-0-323-91722-3 https://doi.org/10.1016/B978-0-323-91722-3.00004-X © 2023 Elsevier Inc. All rights reserved. 45 46 Plant Small RNA in Food Crops Drought stress is highly deleterious as it damages the thylakoid membranes thereby inhibits the process of photosynthesis. Similarly, under drought stress, the stress cell gets damaged due to the increased concentration of toxic ions. Generally, the plants in arid and semi-arid climatic regions with higher rates of evapotranspiration as compared to volume of precipitation throughout the year experiences adverse affects of soil salinity. When the increase in concentration of salts in the subsoil is caused in natural manner it is known as primary soil salinity and when it occurs anthropogenically through environmental pollution such as alteration of soil content, an increased use of fertilizer or due to the application of saline water for irrigation it is referred to as secondary soil salinity (Carillo et al., 2011). Soil salinity is a world-wide problem as it is responsible for reduction in crop yield around the world. There are several ways through which crop growth and development is hindered by salinity stress. Two relevant effects in crops result from increased salt concentration are osmotic stress and ionic toxicity. Under salt stress, osmotic pressure of the soil solution surpasses the osmotic pressure of the plant cells. The osmotic pressure is increased due to the osmotic stress leading to poor absorption of essential minerals including Kþ (Potassium Ion) and Ca2þ (Calcium ion) along with water from the soil into the root cells while Naþ (Sodium ion) and Cl (Chlorine ion) enters into the cells leading to adverse effects on the plasma membrane and different metabolic processes in cytoplasm. Salinity stress is responsible for different adverse effects including decreased cell growth, membrane dysfunction as well as decreased cytoplasmic metabolism and ROS production. Reports also infer that the higher levels of salt stress adversely impacts quality as well as quantity of plant production by inhibition of seed germination, retarding growth and development phases due to the combined effects of increased osmotic potential as well as particular ion toxicity (Akbari et al., 2007). Extremes of temperatures (High and low temperatures) are among the causes of various abiotic stresses especially drought. It has been observed that abnormal temperature variations drastically affect overall development of the plants leading to poor yield. When plants are exposed to chilling temperatures, plant experiences cold stress. It is a severe abiotic stress which hampers the growth, development and productivity of crop plants along with affecting the quality and post-harvest life. Cold stress affects all characteristics of crops affecting cell functions (Bhutia et al., 2018). Based on how plant reacts to cold temperature, plants are classified into three types. These include firstly cold susceptible or chilling delicate plants which are extremely sensitive to temperatures between 0 and below 15 C. Secondly, Response to abiotic stresses 47 the chilling or cold resistant plants which has the capability to resist chilling temperatures, however gets wounded due to the development of ice in the tissues. Thirdly, the plants which are resistant to frost and have the capability to resist chilling temperatures stress. Cold stress results in cellular injury due to the alterations in the structure of membrane and reduction in the electrolyte leakage, protoplasmic flow and plasmolysis (Bhutia et al., 2018). The metabolism at cellular level is inhibited by the alteration in the rate of respiration and based on the severity of the abnormal rate of respiration, unusual metabolites are synthesized. Simultaneously, due to the destruction of cell and alteration of metabolic activities, plant growth is stunted; ripening of fruits is abnormal, browning of vascular takes place including enhanced susceptibility to deterioration leading to the permanent damage of the plant (Devasirvatham et al., 2018). Similarly, when the crops are exposed to high temperatures, plants experiences heat stress. It is reported that due to increased rate of water loss by evapotranspiration under elevated temperatures, plants also experiences drought along with heat stress. Increased soil temperatures can deteriorate the germination or field emergence of the plants as well as damages the plants (Takahashi et al., 2013). Heavy metal stress (HMs) are metallic chemicals having relatively high density and poses immense adverse effects on plant cells and important cellular components like genes, resulting in mutagenic effects on plants by affecting and polluting soil, potable water and the entire ecosystem (Flora et al., 2008). In the soil, two categories of metals are found which are referred to as vital micronutrients for normal and appropriate growth of the plant (Mo Fe, Zn, Mg, Cu, Mn and Ni) and non-vital or non-essential elements with unknown biological function (Cd, Ag, As, Sb, Cr, Hg, Co and Pb, Se) (Schutzendubel et al., 2002). Both under and above ground surface of plants can uptake HM. In the structural components of protein and enzyme, the vital microelements have a significant role. Plants require vital elements in very less amount for their metabolic pathways and for normal growth and development. The significant factor is the concentration of vital metals in enhancing crop cycle and increased concentration can result in declination and retardation of plant growth. HM at deleterious doses inhibits general cellular processes in plants. They act as hindrance to different metabolisms in various ways, including the disruption of construction blocks of protein structure which arise by the formation of bonds among HM and sulfhydryl groups, thereby affecting the functional groups of relevant molecules (Yadav et al., 2019). 48 Plant Small RNA in Food Crops 2. Abiotic stresses and signal transduction: perceiving the stress signal The plant experiencing environmental stresses tries to cope up by adjusting several cellular metabolic processes. The process involved to cope up with the environmental stresses is complex involving metabolites, genes and proteins. The initial step is the establishment of signaling cascade after the perception of stress making molecular or structural changes in the cell by alteration of DNA and RNA sequences, adaptive changes in proteins and change in membrane fluidity responses (Lohani et al., 2020). The signal transduction starts with the sensing of stress mostly at the plasma membranes and subsequent triggering of signaling pathways of reactive oxygen species (ROSs) (Mittler et al., 2011), hydraulic waves (Christmann et al., 2013), electric signal (Nguyen et al., 2018), oscillations of cytoplasmic Caþ2 level by activated plasma membrane Caþ2 channels (Toyota et al., 2018) (Fig. 3.1). The increased level of Caþ2 in the cytoplasm acts as secondary Figure 3.1 A general view of signal transduction and molecular mechanism of abiotic stress tolerance in plants. ABA, abscisic acid; Ca2þ, calcium ion; CDPKs/CPKs, calciumdependent protein kinases; CBLs, calcineurin B-like proteins; CAM, calmodulin; CMLs, calmodulin-like proteins; MAPKs, mitogen-activated protein kinase; NADPH, nicotinamide adenine dinucleotide phosphate; ROS, reactive oxygen species; RLCKs, receptorlike cytoplasmic kinase. Response to abiotic stresses 49 messenger and activates the downstream pathways via several Caþ2 sensors of the cell such as such as CMLs (calmodulin-like proteins), CaMs (calmodulin), CDPKs/CPKs (calcium-dependent protein kinases), CBLs (calcineurin B-like proteins), MAPK (mitogen-activated protein kinase), GPCRs (G protein coupled receptors), MMPs (matrix metalloproteinases), PYR/PYL (pyrabactin resistance 1-like protein) and so on (Ahmad et al., 2021). CaM and CML are members of Ca2þ sensors family in plants and it contains helix-loop-helix EF-hand domains to control downstream pathways (Lohani et al., 2020). Its role under abiotic stress have been established in several crop plants such as under abiotic stress conditions the members of CAMTAs showed diversified expression in Brassica napus (Rahman et al., 2016) including heat stress (He et al., 2020). The CaM and CML proteins were found to be playing role in several cellular and metabolic processes, including cellular defense and rescue (Wang et al., 2012). CBLs, another Ca2þ sensor in plants have been found to be playing role in signal transduction under abiotic stress conditions in B. napus by interacting with CIPKs (calcineurin B-like protein kinases) (Chen et al., 2012; Yuan et al., 2014). Calcium dependent protein kinases (CDPKs) with its dual ability to detect Ca2þ and responding to signal through phosphorylation have been reported playing an essential role in plant cell under stress environment through modulation of the signaling pathways involving ABA and ROS homeostasis in brassica napus (Wang et al., 2018), in soybean (Wang et al., 2017). While responding to stress, the increase concentration of Ca2þ and CDPK in stress cell activates the RBOHD (plasma membrane-located protein called oxidase homolog D) which is involved in production of ROS i.e. hydrogen peroxide (H2O2) through NADPH (nicotinamide adenine dinucleotide phosphate) oxidase phosphorylation and activation of downstream signal like MAPKs (mitogen-activated protein kinases) in response to heat stress (Ahmad et al., 2021). The MAPKs is involved in linking different mechanism of abiotic stress tolerance and its signaling cascade integrates the signals and establishes the signal transduction. The established signal transduction provides a specific response to the stress by activating the expression of genes involved in stress response basically via phosphorylation (Chinnusamy et al., 2004). The role of MAPKs and other components of signaling pathways under abiotic stress tolerance in plants have been established by several such as MAPKs (Krysan & Colcombet, 2018), GPCR (Gao, Bai, et al., 2010; Gao, Zhao, et al., 2010; Nongpiur et al., 2019), PYR/PYL Protein (Di et al., 2018), HSPs (heat shock 50 Plant Small RNA in Food Crops proteins) and HSTFs (heat shock transcription factors) (Saidi et al., 2011), Matrix Metalloproteinases (Liu et al., 2018), Phytochrome A and B (Junior et al., 2021). 3. Molecular mechanism of abiotic stress tolerance At molecular level, an environmental stress modifies the activities of certain enzymes and cellular processes resulting in the generation of ROSs (reactive oxygen species). In response to it, the plant with tolerance capacity expresses an antioxidant enzymes gene which regulates the level of ROS to protect the plant cells from oxidative damages (Menezes-Benavente et al., 2004). Many genes encoding transporter proteins are also gets induced expression to maintain optimum ion homeostasis such as Naþ/Kþ balance (Jiang et al., 2018). Upon receiving the stress signal, many defense related genes gets over-expressed and defends the plants cell during stress period (Fig. 3.1), such as ABA responsive element (ABRE) binding protein 2 (ABP2) (Na et al., 2018) which is involved in regulating the expression of stress responsive genes like RD20, RD21, ERD, NHX3, LEA2, Rab 18 and GEA6 etc. (Ma et al., 2018), WRKY transcription factor proteins (Bo et al., 2020), RAV1 proteins (Min et al., 2014), MYB proteins (Wu et al., 2019), miRNAs (Fu et al., 2017) and several proteins mostly involved in lignin synthesis and anti-oxidation (Chen et al., 2014). Up or downregulation of stress responsive genes leads to metabolic adjustment including the osmotic adjustment at cellular level makes the plants able to tolerate the environmental stresses (Serraj & Sinclair, 2002). Similarly, several cellular compounds called as osmolytes such as soluble sugars, glycine betaine, trehalose, proline, sugar alcohols, organic acids have been found to be engaged in osmotic adjustment when plants are experiencing environmental stresses (Rohman et al., 2019). Generally, the abiotic stresses are encountered first from the soil i.e. at the root level that leads to altered root architecture and change in biomass such as primary root of Arabidopsis seedlings ceases to grow when the seedlings were grown under phosphorus deficient media. The ceases in growth of primary roots were reported to be a result of a signal transduction pathway involving LPR2, ALMT1 and STOP1 (Balzergue et al., 2017). Under stress conditions like nutrient deficiency, the plants with tolerant mechanism combines the energy/nutrient status and regulates the responding mechanism and growth of the plants mediated by proteins Snf1RELATED PROTEIN KINASE1 (SnRK1) and TARGET OF Response to abiotic stresses 51 RAPAMYCIN (TOR) (Hanson et al., 2008). SnRK1 upon activation affects the gene expression through phosphorylation of transcription factor like BZIP and targets downstream cytoplasmic regulatory and metabolic enzymes (Nukarinen et al., 2016) and TOR helps in growth and proliferation of the cell by phosphorylation of the target proteins upon receiving the signals (Schepetilnikov & Ryabova, 2018). Likewise, guanosine tetra- and penta-phosphate or (p)ppGpp, a signaling nucleotides plays an essential role in responding to stress signals. The accumulation of (p)ppGpp in the cells under abiotic stress have been found in several plants and its accumulation was found to be inhibiting chloroplast transcription and affecting the function of chloroplast in response to different abiotic stresses (Field, 2018). Under temperature stress, the photoreceptor phytochrome B (PHYB) showed responses and found to be imparting in proper growth and development of crop plants under environmental stresses. Similarly, expression of HSP/HSTF increases the tolerance capacity in plants to cope up against the abiotic stresses (Bechtold et al., 2018) with its default capacity to control the expression of complex network of stress related genes (Albihlal et al., 2018). 4. Role of small RNAs in abiotic stress tolerance Under stress conditions, plants cell turns on several mechanisms and triggers the several threads of genetic regulations such as up or down-regulation of various genes as a measure of protection (Ku et al., 2015). Eukaryotes including plants have numerous sophisticated way of controlling gene expression (Phillips, 2008). These ways of controlling gene expression needs to be precisely regulated in the complex cellular environment. In the complex group of mechanisms that are regulating the pattern of gene expression, there is special class of small RNA molecules such as siRNA (small interfering RNA) and miRNA (micro-RNA) which are known for playing roles in regulations of pre and posttranscriptional gene expression (Morris & Mattick, 2014). Both classes of small RNAs are associated with the argonautes family of proteins to form a functional complex, but both are different in terms of their origin, processing pathways, target and mechanism of action. Gene silencing by small RNA depends on the ability of the small RNAs to inhibit transcription of the gene coding for a particular mRNA or inducing mRNA degradation or inhibition of mRNA translation. Genes that encode proteins and other forms of RNAs are transcribed by RNA polymerase. The post transcriptional process such as 52 Plant Small RNA in Food Crops splicing and other processing mechanisms processed the primary transcript and forms a mature mRNA to form polypeptide chain that folds into proteins (Alberts et al., 2002). This is where some small RNA molecules can have their silencing effects and this RNA mediated inhibition of gene expression is termed as RNA interference or in short RNAi (Filipowicz et al., 2005). These post-transcriptional regulations plays important role in restoring plants cellular homeostasis and thereby helping the plant cell to recover from stress phases (Sunkar et al., 2012). 4.1 Biogenesis of small RNA in plants Although, miRNAs show differential expression in plants under environmental stress, miRNAs are not involved in plant response to environmental stress directly. Instead, it regulates the key components of gene network complex through RISC (RNA-induced silencing complex), a miRNA loaded to an argonaute protein which binds to the target mRNA for silencing (Iwakawa & Tomari, 2013). The miRNAs are transcribed from actual endogenous genes present in chromosomes of all multicellular plants and animals by RNA polymerase-II. The resulting immediate RNA molecules are called as pri-miRNAs. The pri-miRNAs folds into a structure called stem loop with single stranded extension at both 3ʹ and 5ʹ ends. This pri-miRNA becomes a substrate for nuclear RNAse-3 enzyme (DROSHA). Nuclear RNAse-3 enzyme forms a microprocessor complex by interacting with the specialized RNA binding protein and removes the 5ʹ and 3ʹ extension of pri-miRNAs and liberates a 60e70 nucleotides long transcript called pre-miRNA. The nuclear export factor recognizes the premiRNA and transports it frok nucleus to the cytoplasm for subsequent post transcriptional modifications/processing. In the cytoplasm, another RNAse-3 enzyme known as DICER performs the second endonucleatic cleavage reaction generally refers to as dicing resulting in miRNA:miRNA duplex carrying 5ʹ monophosphate and 3ʹ overhangs of two nucleotides. In the duplex strands, one strand is called as guide and the other one is called as passenger strands. The guide and passenger strand duplex then interact with the argonaute protein. Once the duplex is loaded into argonaute in an appropriate orientation, guide strand remains with the complex while the passenger strand is selectively excised out by the process called sorting. The sequence of the guide strand will determine which RNA will be silenced (Liu, Fortin, & Mourelatos, 2008). Argonaute protein charged with the guide miRNA are referred as miRISC or micro RNA induced silencing complex (Xu et al., 2019). Response to abiotic stresses 53 In contrast to miRNAs, small interfering RNAs (siRNAs) generally do not transcribed from the genes encoded in the genome; instead, it is produced from dsRNA (double stranded RNA) of numerous sources like virus, artificially injected dsRNA and some other endogenous sources like aberrant RNA (Liu & Chen, 2016). Irrespective of their origins, the duplex of siRNAs become substrate for the cytoplasmic RNA processing enzymes similar to miRNAs except the activity of DROSHA. Dicer, the cytoplasmic RNAse-3 enzyme sequentially cleaves the siRNA precursor long duplex into 20-25bp RNA similar to miRNA:miRNA duplex but no 5ʹ and 3ʹ overhangs. Like in miRNAs, siR:siR duplex is also loaded into the argonoute protein and guide strand is retained and the passenger strand is selectively removed. In plants, worms and some other eukaryotic organisms, the aberrant RNAs which are the products of RISC mediated degradation of targeted mRNA may become template for RDRP (RNA dependent RNA polymerase) that leads to unprimed RNA synthesis resulting into double stranded RNA, a precursor for siRNA (Fig. 3.2A). In another amplification process, the single stranded siRNA after the cleavage by cytoplasmic RNAse-3 (DICER), which do not associates with the RISC binds to the target mRNA as a primer for RDRP to polymerize the antisense RNA strand resulting into double stranded RNA, a precursor for siRNA (Forrest et al., 2004). The double stranded RNA molecules serves as a substrate for cytoplasmic RNAse-3 (DICER) and generates more siRNA and in-turn serves as primer for RDRP after unwinding or together with RISC mediates the silencing of target mRNA (Fig. 3.2B). Upon binding small RNAs induces degradation of mRNA and inhibition of mRNA translation. Majority of the targeted mRNA are cleaved in plants, as almost all miRNAs are perfectly binds to the target mRNA (Zhang, 2015). However, there are reports of miRNAs inhibiting protein translation from the targeted mRNA (Bartel, 2004; Zhang, 2015; Zhang et al., 2007). 4.2 Micro-RNAs in plants under abiotic stress Among the small RNAs involved in post transcriptional regulations, miRNAs belongs to a class of small endogenous RNA molecules widely distributed throughout the plant kingdom (Zhang, 2015) many of which are species specific (Xie et al., 2015). Abiotic stresses such as salinity, high temperature, low temperature, deficiency of nutrient and mineral toxicity, drought etc. Induces stress and stress dose specific aberrant expression of 54 Plant Small RNA in Food Crops A RDRP Double stranded RNA RISC mediated cleavage of mRNA Aberrant RNA DICER Degradation of mRNA siRISC ARGONOUTE B siRNA RDRP Primer siRNA not associated with RISC Binding with target mRNA Degradation of target mRNA siRISC Double stranded RNA DICER ARGONOUTE siRNA Figure 3.2 Biogenesis of small RNA particularly siRNA. (A) Aberrant RNA serves as template for synthesis of siRNA by RNA dependent RNA polymerase (RDRP). (B) siRNA serves as primer for synthesis of more siRNA by RDRP. miRNA, although, several miRNAs are up or down-regulated under all abiotic stresses (Zhang, 2015). Stress specific responses of miRNAs were reported in several crop plants. The expression of miR169 was induced by salinity but drought stress suppressed its expression in Arabidopsis. It was found that inhibition of miR169 under drought stress was through pathway involving abscisic acid (ABA) (Li et al., 2008; Xu et al., 2014). In salinity induced miR169, the expression of nfya5 (a subunit NFYA5 of nuclear factor NF-Y) which is the target of miR 166 was found to be inhibited in Arabidopsis (Xu et al., 2014). The expression of nfya5 was increased under drought stress where the expression of miR169 was impeded. The transgenic plants over-expressing miR169 and plants with nfya5 knockout both showed increased susceptibility to drought stress (Li et al., 2008). Similarly, in Arabidopsis miR398 expression was impeded by oxidative stress, salinity Response to abiotic stresses 55 and cold stress but UVB light induced the expression of miR398 and targets CSD1 and CSD2, the Cu/Zn superoxide dismutase coding genes (Jia et al., 2009; Sunkar et al., 2006; Sunkar and Zhu et al., 2004). The miR 395 which target the AST68 (sulfate transporter) and miR399 which target the PHO1 (phosphate transporter) were identified in Arabidopsis and found to be reactive to abiotic stresses (Sunkar & Zhu, 2004). Over the years, many miRNAs responsive to drought and salinity stress have been identified in several plants like soybean, rice, cowpea, Phaseolus vulgaris, Saccharum spp., barley etc. (Banerjee, Banerjee, et al., 2017; Banerjee, Sirohi, et al., 2017; Gentile et al., 2015; Zhou et al., 2010) (Fig. 3.3). Micro RNAs in plant are also involved in tolerance to extreme temperature (Chilling/Heat), another abiotic stress that hampers the crop productivity. Several miRNAs responsive to high and low temperature have been identified in crop plants like rice (Lv et al., 2010), wheat (Xin et al., 2011), Tea (Zhang et al., 2014), Wild tomato (Cao et al., 2014). Some of the identified miRNAs showing response to environmental stresses are miR156a miR156g, miR157d, miR158a, miR159a miR167a/c, miR169, miR 172a,b, miR 390, miR391, miR394a/b/c, miR396, miR397, miR397b, miR398, miR408, miR 528, miR775, miR812q, miR827, miR1425, miR1508, miR1515, miR 1510/ 2110, miR1532, and so on. In Rice miR812q showed significant response to cold stress miR391, miR159a, miR158a, miR775, miR172a,b, miR157d and miR156g showed significant up regulation under low oxygen stress in Arabidopsis (Moldovan et al., 2010); miR1425, miR827, miR397 and miR169 showed response to hydrogen peroxide treatment (H2O2) in Figure 3.3 Versatile roles of small RNAs particularly micro RNAs in regulating gene expression and enabling plants to cope up with adverse environmental stresses. 56 Plant Small RNA in Food Crops Arabidopsis (Li et al., 2011); miR156a and miR167a/c showed response to cadmium (Cd)toxicity stress in Brassica napus (Huang et al. 2019), Under manganese (Mn) toxicity stress, miR1508, miR1515, miR1510/2110, and miR1532 showed up-regulation in Phaselous vulgaris (Valdés-López et al., 2010), under Arsenic (As) toxicity stress miR408, miR528, and miR397b showed up regulation in rice (Liu & Zhang, 2012), under Alum inum (Al) toxicity stress miR396 and miR390 showed up-regulation in soybean (Zhao et al., 2019). Similarly, the role of miRNAs in tolerance against heavy metals is also established by some of the reports (Ding et al., 2020). In maize, the expression of several miRNAs were influenced by salt stress such as miR 205, miR17, miR 250, miR 330 showed down-regulation resulting into the upregulation of their targets mRNA thereby activating salt stress tolerant pathways (Fu et al., 2017). These altered expressions of many miRNAs seems to be different in susceptible and tolerant genotypes and paving a way toward understanding the genotype specific expression of miRNAs (Ding et al., 2009). 5. Strategies to develop abiotic stress tolerance for sustainability of crop production under changing climate 5.1 Use of PGRs in developing abiotic stress tolerance in crop plants One of the most severe hazards of global agro-system is the abiotic stresses. PGRs are well-known for the developing the stress-tolerance in the plants itself by improving the antioxidative defense system, osmoprotectants production and regulating stress tolerance genes (Dey et al., 2021; Pramanick et al., 2018). Conventional breeding techniques are efficient enough to develop a plant with abiotic-stress-tolerance. However, this process is time consuming and costly. In this situation, application of the PGRs is very beneficial to mitigate the stress (Sabagh Ayman et al., 2021). Plant-hormones (PGRs) like IAA (auxins), ABA (abscisic acids), BRs (brassinosteroids), Gas (gibberellic acid), ethylene, CKs (cytokinins), JA (jasmonic acid) etc. are very important in performing many important physiological processes involved in the abiotic stress tolerances (Pramanick et al., 2015; Singh et al., 2017). Even in small amount, these PGRs can regulate almost all the physiological activities of the plants from their entire life-cycle. Salt plus Cu-stress can many detrimental impacts on plants viz. Response to abiotic stresses 57 Reduction in photosynthesis which ultimately resulted in lower biomass production. Such stress can be mitigated by epibrassinolide (EBL) which can enhance the plant growth, photosynthetic-rate and overall biomass production under such stress (Fariduddin et al., 2013). Application of CKs can also mitigate the salinity stress of many plants by increasing the proline content in the plant (Wu et al., 2013). Cd induced stress of congress grass can be mitigated through spraying GA3 to the plants (Mohammad, 2018). Cold stress in the plants causes huge yield loss and this stress can be mitigated applying ABA on the crop canopy during the stress condition (Mohammad & Lakshmisri, 2018). Fig. 3.4 represents the role of PGRs on plant activities. 5.2 Molecular and biotechnological approaches for developing stress tolerance crop plants for sustainability of crop production under changing climate Change in the pattern of climatic factors such as off season heavy precipitation, extreme low or high temperatures, increase in soil salinity, soil acidity, soil nutrient deficiency and mineral toxicity and as well as other Figure 3.4 Role of different PGRs on plant activities. (Adapted from Sabagh Ayman, E. L., Mbarki, S., Hossain, A., Iqbal Muhammad, A., et al. (2021). Potential role of plant growth regulators in administering crucial processes against abiotic stresses. Frontiers in Agronomy, 3. https://www.frontiersin.org/article/10.3389/fagro.2021.648694.) 58 Plant Small RNA in Food Crops climatic factors like wind storms, drought and floods all these severely affects the yield of the crop plants (Bhutia et al., 2018). Among several approaches to overcome or to develop abiotic stress tolerance crop plants, biotechnological techniques is gaining greater acceleration due to the recent advancement in sequencing and molecular data analysis technologies. The development in DNA markers has helped breeders to do marker assisted selection to bring useful changes in the plants including abiotic stress tolerance (Kumawat et al., 2020). Simultaneously, the use of novel genetic designs based on bi and multi-parent has helped in association mapping and linkage analysis (Varshney et al., 2021) along with high-throughput phenotyping (HTP) platforms (Leng et al., 2017). Parallel with genome sequencing, transcriptome sequencing has also helped in identification of genes through differential gene expression analysis by setting the experiments accordingly. The genes or QTL (quantitative trait loci) which are identified using sequencing, mapping or other biotechnological tools are being frequently used in developing crop plants to withstand abiotic stresses (Leng et al., 2017). Focusing on small RNA, plant responds to abiotic stresses using numerous biosynthetic pathways including gene expression regulation by the miRNA. Transgenic approach showed potential application of miRNA in imparting tolerance against several stress in plants including abiotic stress tolerance. Several reports highlighted that the over-expression or suppression of miRNAs have enhances the tolerance to abiotic stresses in plants. Transgenic rice with over-expression of miR398 and suppressing the expression of miR396c showed improved tolerance against salinity (Gao, Bai, et al., 2010; Gao, Zhao, et al., 2010; Lu et al., 2010). The overexpression of miR169 and miR319 showed enhanced tolerance to drought by inhibiting stomatal opening (Zhang et al., 2011) and improved cold stress tolerance in tomato (Yang et al., 2013), respectively. Some of the reports on transgenic plants over-expressing miRNAs which established the role of miRNAs against abiotic stress tolerance are listed in Table 3.1. 6. Conclusion From the above discussion in the current chapter, it may be revealed that abiotic stresses are the major problem to reduce crop productivity globally. However, there are two ways to manage the abiotic stresses, such as development of crop cultivars and through management approaches. Tolerant plants can survive against abiotic stresses through genetic and Response to abiotic stresses 59 Table 3.1 Transgenic plants over-expressing miRNAs showed enhanced tolerance against abiotic stresses. Source plant Transgenic plant Response References miR169 Solanum lycopersicum Solanum lycopersicum Increased tolerance to drought miR394a Glycine max Arabidopsis thaliana Arabidopsis thaliana Arabidopsis thaliana miR319 Oryza sativa Oryza sativa Agrostis stolonifera Increased tolerance to drought Increased tolerance to extreme temperature Increased tolerance to cold Tolerance to drought and salt Zhang et al. (2011) Ni et al. (2012) Guan et al. (2013) miR397 Arabidopsis thaliana Arabidopsis thaliana Increased tolerance to cold miR399 Arabidopsis thaliana Solanum lycopersicum miR408 Arabidopsis thaliana Cicer arietinum Increased tolerance to phosphorus deficiency and cold stress Increased tolerance to drought miR 393 Arabidopsis thaliana Glycine max Arabidopsis thaliana Arabidopsis thaliana Arabidopsis thaliana Oryza sativa Oryza sativa Oryza sativa Arabidopsis thaliana Oryza sativa Oryza sativa Agrostis stolonifera Musa species Musa species miRNA miR398 miR 172 miR394a miR529a miR 268 miR393a miR397 Increased tolerance to salt Increased tolerance to Water deficit and salinity Increased tolerance to cold Increased resistance to oxidative stress Increased tolerance to cadmium Increased tolerance to multiple abiotic stress Increased biomass accumulation and mineral deficiency tolerance Yang et al. (2013) Zhou et al. (2013) Dong et al. (2014) Gao et al. (2015) Hajyzadeh et al. (2015) Chen et al. (2015) Li et al. (2016) Song et al. (2016) Yue et al. (2017) Ding et al. (2017) Zhao et al. (2019) Patel et al. (2019) Continued 60 Plant Small RNA in Food Crops Table 3.1 Transgenic plants over-expressing miRNAs showed enhanced tolerance against abiotic stresses.dcont'd miRNA miR319d miR408 miR160a.5p miR1861h Source plant Transgenic plant Response References Solanum habrochaites Oryza sativa Arabidopsis thaliana Oryza rufipogon Solanum lycopersicum Lolium perenne Solanum tuberosum Oryza sativa Chilling and heat stress tolerance Enhanced drought tolerance Enhanced heat and drought tolerance Enhanced salt tolerance Shi et al. (2019) Hang et al. (2021) Sanlı et al. (2021) Ai et al. (2021) molecular mechanisms; although these mechanisms are associated with advanced crop breeding programs. The current chapter highlighted that the traditional breeding approaches are not sufficient to survive against abiotic stresses and therefore in recent decade, several advanced genomics approaches have been improved, especially those traits relevant to abiotic stress management. The chapter aim to provide an update and all-inclusive information about all possible combinations of advanced genomics tools and gene regulatory network of ROS homeostasis for the suitable strategy that will promote to improve the productivity of crops under the hostile environments. References Ahmad, M., Waraich, E. A., Skalicky, M., Hussain, S., Zulfiqar, U., Anjum, M. Z., & EL Sabagh, A. (2021). Adaptation strategies to improve the resistance of oilseed crops to heat stress under a changing climate: An overview. Frontiers of Plant Science, 12, 767150. Ai, B., Chen, Y., Zhao, M., Ding, G., Xie, J., & Zhang, F. (2021). Over-expression of miR1861h increases tolerance to salt stress in rice (Oryza sativa L.). Genetic Resources and Crop Evolution, 68(1), 87e92. Akbari, G., Sanavy, S. A., & Yousefzadeh, S. (2007). Effect of auxin and salt stress (NaCl) on seed germination of wheat cultivars (Triticum aestivum L.). Pakistan Journal of Biological Sciences: PJBS, 10(15), 2557e2561. Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., & Walter, P. (2002). From DNA to RNA. In Molecular biology of the cell (4th ed.). Garland Science. Albihlal, W. S., Obomighie, I., Blein, T., Persad, R., Chernukhin, I., Crespi, M., Bechtold, U., & Mullineaux, P. M. (2018). Arabidopsis HEAT SHOCK TRANSCRIPTION FACTORA1b regulates multiple developmental genes under benign and stress conditions. Journal of Experimental Botany, 69(11), 2847e2862. Ashkavand, P., Zarafshar, M., Tabari, M., Mirzaie, J., Nikpour, A., Bordbar, S. K., Struve, D., & Striker, G. G. (2018). Application of SiO2 nanoparticles as pretreatment Response to abiotic stresses 61 alleviates the impact of drought on the physiological performance of Prunus mahaleb (Rosaceae). Boletín de la Sociedad Argentina de Botánica, 53(2), 207e219. Balzergue, C., Dartevelle, T., Godon, C., Laugier, E., Meisrimler, C., Teulon, J. M., Creff, A., Bissler, M., Brouchoud, C., Hagège, A., & Müller, J. (2017). Low phosphate activates STOP1-ALMT1 to rapidly inhibit root cell elongation. Nature Communications, 8(1), 1e16. Banerjee, S., Banerjee, A., Gill, S. S., Gupta, O. P., Dahuja, A., Jain, P. K., & Sirohi, A. (2017). RNA interference: A novel source of resistance to combat plant parasitic nematodes. Frontiers of Plant Science, 8, 834. Banerjee, S., Sirohi, A., Ansari, A. A., & Gill, S. S. (2017). Role of small RNAs in abiotic stress responses in plants. Plant Gene, 11, 180e189. Bartel, D. P. (2004). MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell, 116(2), 281e297. Bechtold, U., Ferguson, J. N., & Mullineaux, P. M. (2018). To defend or to grow: Lessons from arabidopsis C24. Journal of Experimental Botany, 69(11), 2809e2821. Bhutia, K. L., Khanna, V. K., Meetei, T. N. G., & Bhutia, N. D. (2018). Effects of climate change on growth and development of chilli. Agrotechnology, 7(2), 1e4. Bo, C., Chen, H., Luo, G., Li, W., Zhang, X., Ma, Q., Cheng, B., & Cai, R. (2020). Maize WRKY114 gene negatively regulates salt-stress tolerance in transgenic rice. Plant Cell Reports, 39(1), 135e148. Cao, X., Wu, Z., Jiang, F., Zhou, R., & Yang, Z. (2014). Identification of chilling stressresponsive tomato microRNAs and their target genes by high-throughput sequencing and degradome analysis. BMC Genomics, 15(1), 1e16. Carillo, P., Annunziata, M. G., Pontecorvo, G., Fuggi, A., & Woodrow, P. (2011). Salinity stress and salt tolerance. Abiotic Stress in Plants-Mechanisms and Adaptations, 1, 21e38. Chen, Z., Hu, L., Han, N., Hu, J., Yang, Y., Xiang, T., & Wang, L. (2015). Overexpression of a miR 393-resistant form of transport inhibitor response protein 1 (mTIR1) enhances salt tolerance by increased osmoregulation and Naþ exclusion in Arabidopsis thaliana. Plant and Cell Physiology, 56(1), 73e83. Chen, L., Ren, F., Zhou, L., Wang, Q. Q., Zhong, H., & Li, X. B. (2012). The Brassica napus calcineurin B-Like 1/CBL-interacting protein kinase 6 (CBL1/CIPK6) component is involved in the plant response to abiotic stress and ABA signalling. Journal of Experimental Botany, 63, 6211e6222. Chen, Z., Tonnis, B., Morris, B., Wang, R. R. B., Zhang, A. L., & Pinnow, D. (2014). Variation in seed fatty acid composition and sequence divergence in the FAD2 gene coding region between wild and cultivated sesame. Journal of Agricultural and Food Chemistry, 62, 11706e11710. Chinnusamy, V., Schumaker, K., & Zhu, J. K. (2004). Molecular genetic perspectives on cross-talk and specificity in abiotic stress signalling in plants. Journal of Experimental Botany, 55, 225e236. Christmann, A., Grill, E., & Huang, J. (2013). Hydraulic signals in long-distance signaling. Current Opinion in Plant Biology, 16(3), 293e300. Devasirvatham, V., & Tan, D. K. (2018). Impact of high temperature and drought stresses on chickpea production. Agronomy, 8(8), 145. Dey, P., Mahapatra, B. S., Pramanick, B., Kumar, A., Negi, M. S., Paul, J., Shukla, D. K., & Singh, S. P. (2021). Quality optimization of flax fibre through durational management of water retting technology under sub-tropical climate. Industrial Crops and Products, 162, 113277. https://doi.org/10.1016/j.indcrop.2021.113277 Di, F., Jian, H., Wang, T., Chen, X., Ding, Y., Du, H., et al. (2018). Genome wide analysis of the PYL gene family and identification of PYL genes that respond to abiotic stress in Brassica napus. Genes, 9, 156. 62 Plant Small RNA in Food Crops Ding, Y., Wang, Y., Jiang, Z., Wang, F., Jiang, Q., Sun, J., & Zhu, C. (2017). MicroRNA268 over-expression affects rice seedling growth under cadmium stress. Journal of Agricultural and Food Chemistry, 65(29), 5860e5867. Ding, D., Zhang, L., Wang, H., Liu, Z., Zhang, Z., & Zheng, Y. (2009). Differential expression of miRNAs in response to salt stress in maize roots. Annals of Botany, 103(1), 29e38. Dong, C. H., & Pei, H. (2014). Over-expression of miR397 improves plant tolerance to cold stress in Arabidopsis thaliana. Journal of Plant Biology, 57(4), 209e217. Fariduddin, Q., Khalil, R. R., Mir, B. A., Yusuf, M., & Ahmad, A. (2013). 24Epibrassinolide regulates photosynthesis, antioxidant enzyme activities and proline content of Cucumis sativus under salt and/or copper stress. Environmental Monitoring and Assessment, 185, 7845e7856. Field, B. (2018). Green magic: Regulation of the chloroplast stress response by (p) ppGpp in plants and algae. Journal of Experimental Botany, 69(11), 2797e2807. Filipowicz, W., Jaskiewicz, L., Kolb, F. A., & Pillai, R. S. (2005). Post-transcriptional gene silencing by siRNAs and miRNAs. Current Opinion in Structural Biology, 15(3), 331e341. Flora, S. J. S., Mittal, M., & Mehta, A. (2008). Heavy metal induced oxidative stress & its possible reversal by chelation therapy. Indian Journal of Medical Research, 128(4), 501. Forrest, E. C., Cogoni, C., & Macino, G. (2004). The RNA-dependent RNA polymerase, QDE-1, is a rate-limiting factor in post-transcriptional gene silencing in Neurospora crassa. Nucleic Acids Research, 32(7), 2123e2128. Fu, R., Zhang, M., Zhao, Y., He, X., Ding, C., Wang, S., Feng, Y., Song, X., Li, P., & Wang, B. (2017). Identification of salt tolerance-related microRNAs and their targets in maize (Zea mays L.) using high-throughput sequencing and degradome analysis. Frontiers of Plant Science, 8, 864. Gao, P., Bai, X., Yang, L., Lv, D., Li, Y., Cai, H., & Zhu, Y. (2010). Over-expression of osa-MIR396c decreases salt and alkali stress tolerance. Planta, 231(5), 991e1001. Gao, N., Qiang, X. M., Zhai, B. N., Min, J., & Shi, W. M. (2015). Transgenic tomato overexpressing ath-miR399d improves growth under abiotic stress conditions. Russian Journal of Plant Physiology, 62(3), 360e366. Gao, Y., Zhao, Y., Li, T., Ren, C., Liu, Y., & Wang, M. (2010). Cloning and characterization of a G protein b subunit gene responsive to plant hormones and abiotic stresses in Brassica napus. Plant Molecular Biology Reporter, 28, 450e459. Gentile, A., Dias, L. I., Mattos, R. S., Ferreira, T. H., & Menossi, M. (2015). MicroRNAs and drought responses in sugarcane. Frontiers of Plant Science, 6, 58. Guan, Q., Lu, X., Zeng, H., Zhang, Y., & Zhu, J. (2013). Heat stress induction of miR 398 triggers a regulatory loop that is critical for thermo tolerance in Arabidopsis. The Plant Journal, 74(5), 840e851. Hajyzadeh, M., Turktas, M., Khawar, K. M., & Unver, T. (2015). miR408 over-expression causes increased drought tolerance in chickpea. Gene, 555(2), 186e193. Hang, N., Shi, T., Liu, Y., Ye, W., Taier, G., Sun, Y., & Zhang, W. (2021). Overexpression of Os-microRNA408 enhances drought tolerance in perennial ryegrass. Physiologia Plantarum, 172(2), 733e747. Hanson, J., Hanssen, M., Wiese, A., Hendriks, M. M., & Smeekens, S. (2008). The sucrose regulated transcription factor bZIP11 affects amino acid metabolism by regulating the expression of ASPARAGINE SYNTHETASE1 and PROLINE DEHYDROGENASE2. Plant Journal, 53, 935e949. He, X., Liu, W., Li, W., Liu, Y., Wang, W., Xie, P., et al. (2020). Genome-wide identification and expression analysis of CaM/CML genes in Brassica napus under abiotic stress. Journal of Plant Physiology, 255, 153251. Response to abiotic stresses 63 Huang, C. Y., Wang, H., Hu, P., Hamby, R., & Jin, H. (2019). Small RNAs: Big players in plant-microbe interactions. Cell Host & Microbe, 26(2), 173e182. Iwakawa, H. O., & Tomari, Y. (2013). Molecular insights into microRNA-mediated translational repression in plants. Molecular Cell, 52(4), 591e601. Jiang, Z., Song, G., Shan, X., Wei, Z., Liu, Y., Jiang, C., Jiang, Y., Jin, F., & Li, Y. (2018). Association analysis and identification of ZmHKT1; 5 variation with salt-stress tolerance. Frontiers of Plant Science, 9, 1485. Jia, X., Wang, W. X., Ren, L., Chen, Q. J., Mendu, V., Willcut, B., & Tang, G. (2009). Differential and dynamic regulation of miR398 in response to ABA and salt stress in Populus tremula and Arabidopsis thaliana. Plant Molecular Biology, 71(1), 51e59. Junior, C. A. S., D’Amico-Damião, V., & Carvalho, R. F. (2021). Phytochrome type B family: The abiotic stress responses signaller in plants. Annals of Applied Biology, 178, 135e148. Krysan, P. J., & Colcombet, J. (2018). Cellular complexity in MAPK signaling in plants: Questions and emerging tools to answer them. Frontiers of Plant Science, 9, 1674. Kumawat, G., Kumawat, C. K., Chandra, K., Pandey, S., Chand, S., Mishra, U. N., & Sharma, R. (2020). Insights into marker assisted selection and its applications. In I. Y. Abdurakhmonov (Ed.), Plant breeding-current and future views (Edited). IntechOpen. https://doi.org/10.5772/intechopen.95004 Ku, Y. S., Wong, J. W. H., Mui, Z., Liu, X., Hui, J. H. L., Chan, T. F., & Lam, H. M. (2015). Small RNAs in plant responses to abiotic stresses: Regulatory roles and study methods. International Journal of Molecular Sciences, 16(10), 24532e24554. Leng, P. F., Lübberstedt, T., & Xu, M. L. (2017). Genomics-assisted breedingea revolutionary strategy for crop improvement. Journal of Integrative Agriculture, 16(12), 2674e2685. Li, T., Li, H., Zhang, Y. X., & Liu, J. Y. (2011). Identification and analysis of seven H₂O₂responsive miRNAs and 32 new miRNAs in the seedlings of rice (Oryza sativa L. ssp. indica). Nucleic Acids Research, 39(7), 2821e33. https://doi.org/10.1093/nar/gkq1047 Li, W. X., Oono, Y., Zhu, J., He, X. J., Wu, J. M., Iida, K., … Zhu, J. K. (2008). The Arabidopsis NFYA5 transcription factor is regulated transcriptionally and posttranscriptionally to promote drought resistance. The Plant Cell Online, 20(8), 2238e2251. Liu, L., & Chen, X. (2016). RNA quality control as a key to suppressing RNA silencing of endogenous genes in plants. Molecular Plant, 9(6), 826e836. Liu, X., Fortin, K., & Mourelatos, Z. (2008). MicroRNAs: Biogenesis and molecular functions. Brain Pathology, 18(1), 113e121. Liu, S., Jia, Y., Zhu, Y., Zhou, Y., Shen, Y., Wei, J., et al. (2018). Soybean matrix metalloproteinase Gm2-MMP relates to growth and development and confers enhanced tolerance to high temperature and humidity stress in transgenic Arabidopsis. Plant Molecular Biology Reporter, 36, 94e106. Li, W., Wang, T., Zhang, Y., & Li, Y. (2016). Over-expression of soybean miR 172c confers tolerance to water deficit and salt stress, but increases ABA sensitivity in transgenic Arabidopsis thaliana. Journal of Experimental Botany, 67(1), 175e194. Liu, Q., & Zhang, H. (2012). Molecular identification and analysis of arsenite stressresponsive miRNAs in rice. Journal of Agricultural and Food Chemistry, 60(26), 6524e6536. https://doi.org/10.1021/jf300724t Lohani, N., Jain, D., Singh, M. B., & Bhalla, P. L. (2020). Engineering multiple abiotic stress tolerance in canola, Brassica napus. Frontiers in Plant Science, 11, 3. Lu, Y., Feng, Z., Bian, L., Xie, H., & Liang, J. (2010). miR398 regulation in rice of the responses to abiotic and biotic stresses depends on CSD1 and CSD2 expression. Functional Plant Biology, 38(1), 44e53. 64 Plant Small RNA in Food Crops Lv, D. K., Bai, X., Li, Y., Ding, X. D., Ge, Y., Cai, H., … Zhu, Y. M. (2010). Profiling of cold-stress-responsive miRNAs in rice by microarrays. Gene, 459(1e2), 39e47. Ma, H., Liu, C., Li, Z., Ran, Q., Xie, G., Wang, B., Fang, S., Chu, J., & Zhang, J. (2018). ZmbZIP4 contributes to stress resistance in maize by regulating ABA synthesis and root development. Plant Physiology, 178(2), 753e770. Menezes-Benavente, L., Kernodle, S. P., Margis-Pinheiro, M., & Scandalios, J. G. (2004). Salt-induced antioxidant metabolism defenses in maize (Zea mays L.) seedlings. Redox Report, 9(1), 29e36. Min, H., Zheng, J., & Wang, J. (2014). Maize ZmRAV1 contributes to salt and osmotic stress tolerance in transgenic arabidopsis. Journal of Plant Biology, 57(1), 28e42. Mittler, R., Vanderauwera, S., Suzuki, N., Miller, G. A. D., Tognetti, V. B., Vandepoele, K., Gollery, M., Shulaev, V., & Van Breusegem, F. (2011). ROS signaling: The new wave? Trends in Plant Science, 16(6), 300e309. Mohammad, S. M. (2018). Streamlining devops automation for cloud applications. International Journal of Creative Research Thoughts, 6(4), 955e959. https://www.ijcrt.org/ papers/IJCRT1133443.pdf. Mohammad, S. M., & Lakshmisri, S. (2018). Security automation in information technology. International Journal of Creative Research Thoughts, 6(2), 901e905. https://www.ijcrt. org/papers/IJCRT1133434.pdf. Moldovan, D., Spriggs, A., Yang, J., Pogson, B. J., Dennis, E. S., & Wilson, I. W. (2010). Hypoxia-responsive microRNAs and trans-acting small interfering RNAs in Arabidopsis. Journal of Experimental Botany, 61(1). https://doi.org/10.1093/jxb/erp296, 165-77. Morris, K. V., & Mattick, J. S. (2014). The rise of regulatory RNA. Nature Reviews Genetics, 15(6), 423e437. Na, Z. O. N. G., LI, X. J., Lei, W. A. N. G., Ying, W. A. N. G., WEN, H. T., Ling, L. I., ZHANG, X., FAN, Y. L., & Jun, Z. H. A. O. (2018). Maize ABP2 enhances tolerance to drought and salt stress in transgenic Arabidopsis. Journal of Integrative Agriculture, 17(11), 2379e2393. Nguyen, C. T., Kurenda, A., Stolz, S., Chételat, A., & Farmer, E. E. (2018). Identification of cell populations necessary for leaf-to-leaf electrical signaling in a wounded plant. Proceedings of the National Academy of Sciences, 115(40), 10178e10183. Ni, Z., Hu, Z., Jiang, Q., & Zhang, H. (2012). Over-expression of gma-MIR394a confers tolerance to drought in transgenic Arabidopsis thaliana. Biochemical and Biophysical Research Communications, 427(2), 330e335. Nongpiur, R. C., Singla-Pareek, S. L., & Pareek, A. (2019). The quest for osmosensors in plants. Journal of Experimental Botany, 71, 595e607. Nukarinen, E., Nägele, T., Pedrotti, L., Wurzinger, B., Mair, A., Landgraf, R., Börnke, F., Hanson, J., Teige, M., Baena-Gonzalez, E., & Dröge-Laser, W. (2016). Quantitative phosphoproteomics reveals the role of the AMPK plant ortholog SnRK1 as a metabolic master regulator under energy deprivation. Scientific Reports, 6(1), 1e19. Patel, P., Yadav, K., Srivastava, A. K., Suprasanna, P., & Ganapathi, T. R. (2019). Overexpression of native Musa-miR397 enhances plant biomass without compromising abiotic stress tolerance in banana. Scientific Reports, 9(1), 1e15. Phillips, T. (2008). Regulation of transcription and gene expression in eukaryotes. Nature Education, 1(1), 199. Pramanick, B., Bera, P. S., Kundu, C. K., Badopadhyay, P., & Brahmachari, K. (2015). Effect of different herbicides used in transplanted rice on weed management in ricee lathyrus cropping system. Journal of Crop and Weed, 10(2), 433e436. Pramanick, B., Brahmachari, K., Ghosh, D., & Bera, P. S. (2018). Influence of foliar application seaweed (Kappaphycus and Gracilaria) saps in rice (Oryza sativa)epotato Response to abiotic stresses 65 (Solanum tuberosum)eblackgram (Vigna mungo) sequence. Indian Journal of Agronomy, 63(1), 7e12. Rahman, H., Xu, Y. P., Zhang, X. R., & Cai, X. Z. (2016). Brassica napus genome possesses extraordinary high number of CAMTA genes and CAMTA3 contributes to PAMP triggered immunity and resistance to Sclerotinia sclerotiorum. Frontiers of Plant Science, 7, 581. Rohman, M., Islam, M., Monsur, M. B., Amiruzzaman, M., Fujita, M., & Hasanuzzaman, M. (2019). Trehalose protects maize plants from salt stress and phosphorus deficiency. Plants, 8(12), 568. Sabagh Ayman, E. L., Mbarki, S., Hossain, A., Iqbal Muhammad, A., et al. (2021). Potential role of plant growth regulators in administering crucial processes against abiotic stresses. Frontiers in Agronomy, 3. https://www.frontiersin.org/article/10.3389/fagro.2021. 648694. Saidi, Y., Finka, A., & Goloubinoff, P. (2011). Heat perception and signalling in plants: A tortuous path to thermos-tolerance. New Phytologist, 190, 556e565. Sanlı, B. A., & Öztürk Gökçe, Z. N. (2021). Investigating effect of miR 160 through overexpression in potato cultivars under single or combination of heat and drought stresses. Plant Biotechnology Reports, 15(3), 335e348. Schepetilnikov, M., & Ryabova, L. A. (2018). Recent discoveries on the role of TOR (target of rapamycin) signaling in translation in plants. Plant Physiology, 176(2), 1095e1105. Schutzendubel, A., & Polle, A. (2002). Plant responses to abiotic stresses: Heavy metalinduced oxidative stress and protection by mycorrhization. Journal of Experimental Botany, 53(372), 1351e1365. Serraj, R., & Sinclair, T. R. (2002). Osmolyte accumulation: Can it really help increase crop yield under drought conditions? Plant, Cell and Environment, 25(2), 333e341. Shi, X., Jiang, F., Wen, J., & Wu, Z. (2019). Over-expression of Solanum habrochaites microRNA319d (sha-miR319d) confers chilling and heat stress tolerance in tomato (S. lycopersicum). BMC Plant Biology, 19(1), 1e17. Singh, R., Pramanick, B., Singh, A. P., Neelam, Kumar, S., Kumar, A., & Singh, G. (2017). Bio-efficacy of fenoxaprop-P-ethyl for grassy weed control in onion and its residual effect on succeeding maize crop. Indian Journal of Weed Science, 49(1), 63e66. Song, Y. H., Shim, J. S., Kinmonth-Schultz, H. A., & Imaizumi, T. (2016). Photoperiodic flowering: time measurement mechanisms in leaves. Annual Review of Plant Biology, 66, 441e464. https://doi.org/10.1146/annurev-arplant-043014-115555 Sunkar, R., Kapoor, A., & Zhu, J. K. (2006). Post-transcriptional induction of two Cu/Zn superoxide dismutase genes in Arabidopsis is mediated by down-regulation of miR398 and important for oxidative stress tolerance. The Plant Cell Online, 18(8), 2051e2065. Sunkar, R., Li, Y. F., & Jagadeeswaran, G. (2012). Functions of microRNAs in plant stress responses. Trends in Plant Science, 17(4), 196e203. Sunkar, R., & Zhu, J. K. (2004). Novel and stress-regulated microRNAs and other small RNAs from Arabidopsis. The Plant Cell Online, 16(8), 2001e2019. Takahashi, D., Li, B., Nakayama, T., Kawamura, Y., & Uemura, M. (2013). Plant plasma membrane proteomics for improving cold tolerance. Frontiers of Plant Science, 4, 90. Toyota, M., Spencer, D., Sawai-Toyota, S., Jiaqi, W., Zhang, T., Koo, A. J., Howe, G. A., & Gilroy, S. (2018). Glutamate triggers long-distance, calcium-based plant defense signaling. Science, 361(6407), 1112e1115. Valdés-López, O., Yang, S. S., Aparicio-Fabre, R., Graham, P. H., Reyes, J. L., Vance, C. P., & Hernández, G. (2010). MicroRNA expression profile in common bean (Phaseolus vulgaris) under nutrient deficiency stresses and manganese toxicity. New Phytologist, 187(3), 805e18. https://doi.org/10.1111/j.1469-8137.2010.03320.x 66 Plant Small RNA in Food Crops Varshney, R. K., Bohra, A., Yu, J., Graner, A., Zhang, Q., & Sorrells, M. E. (2021). Designing future crops: Genomics-assisted breeding comes of age. Trends in Plant Science, 26(6), 631e649. Wang, Q. L., Chen, J. H., He, N. Y., & Guo, F. Q. (2018). Metabolic reprogramming in chloroplasts under heat stress in plants. International Journal of Molecular Sciences, 19, 849. Wang, L., Ma, H., Song, L., Shu, Y., & Gu, W. (2012). Comparative proteomics analysis reveals the mechanism of pre-harvest seed deterioration of soybean under high temperature and humidity stress. Journal of Proteomics, 75, 2109e2127. Wang, S., Tao, Y., Zhou, Y., Niu, J., Shu, Y., Yu, X., et al. (2017). Translationally controlled tumor protein GmTCTP interacts with GmCDPKSK5 in response to high temperature and humidity stress during soybean seed development. Plant Growth Regulation, 82, 187. Wu, X., He, J., Chen, J., Yang, S., & Zha, D. (2013). Alleviation of exogenous 6benzyladenine on two genotypes of egg-plant (Solanum melongena Mill.) growth under salt stress. Protoplasma, 251, 169e176. Wu, J., Jiang, Y., Liang, Y., Chen, L., Chen, W., & Cheng, B. (2019). Expression of the maize MYB transcription factor ZmMYB3R enhances drought and salt stress tolerance in transgenic plants. Plant Physiology and Biochemistry, 137, 179e188. Xie, F., Jones, D. C., Wang, Q., Sun, R., & Zhang, B. (2015). Small RNA sequencing identifies miRNA roles in ovule and fibre development. Plant Biotechnology Journal, 13(3), 355e369. Xin, M., Wang, Y., Yao, Y., Song, N., Hu, Z., Qin, D., & Sun, Q. (2011). Identification and characterization of wheat long non-protein coding RNAs responsive to powdery mildew infection and heat stress by using microarray analysis and SBS sequencing. BMC Plant Biology, 11(1), 1e13. Xu, W., Jiang, X., & Huang, L. (2019). RNA interference technology. Comprehensive Biotechnology, 560. Xu, M. Y., Zhang, L., Li, W. W., Hu, X. L., Wang, M. B., Fan, Y. L., … Wang, L. (2014). Stress-induced early flowering is mediated by miR169 in Arabidopsis thaliana. Journal of Experimental Botany, 65(1), 89e101. Yadav, S., Vijapura, A., Dave, A., Shah, S., & Memon, Z. (2019). Genetic diversity analysis of different wheat [Triticum aestivum (L.)] varieties using SSR markers. International Journal of Current Microbiology and Applied Sciences, 8(02), 839e846. Yang, C., Li, D., Mao, D., Liu, X. U. E., Ji, C., Li, X., & Zhu, L. (2013). Over-expression of micro RNA 319 impacts leaf morphogenesis and leads to enhanced cold tolerance in rice (Oryza sativa L.). Plant, Cell and Environment, 36(12), 2207e2218. Yuan, F., Yang, H., Xue, Y., Kong, D., Ye, R., Li, C., et al. (2014). OSCA1 mediates osmotic-stress-evoked Ca2þ increases vital for osmosensing in Arabidopsis. Nature, 514, 367e371. Yue, E., Liu, Z., Li, C., Li, Y., Liu, Q., & Xu, J. H. (2017). Over-expression of miR529a confers enhanced resistance to oxidative stress in rice (Oryza sativa L.). Plant Cell Reports, 36(7), 1171e1182. Zhang, B. (2015). MicroRNA: A new target for improving plant tolerance to abiotic stress. Journal of Experimental Botany, 66(7), 1749e1761. Zhang, B., Wang, Q., & Pan, X. (2007). MicroRNAs and their regulatory roles in animals and plants. Journal of Cellular Physiology, 210(2), 279e289. Zhang, Y., Zhu, X., Chen, X., Song, C., Zou, Z., Wang, Y., & Li, X. (2014). Identification and characterization of cold-responsive microRNAs in tea plant (Camellia sinensis) and their targets using high-throughput sequencing and degradome analysis. BMC Plant Biology, 14(1), 1e18. Response to abiotic stresses 67 Zhang, X., Zou, Z., Gong, P., Zhang, J., Ziaf, K., Li, H., & Ye, Z. (2011). Over-expression of microRNA169 confers enhanced drought tolerance to tomato. Biotechnology Letters, 33(2), 403e409. Zhao, J., Yuan, S., Zhou, M., Yuan, N., Li, Z., Hu, Q., & Luo, H. (2019). Transgenic creeping bentgrass over-expressing Osa-miR393a exhibits altered plant development and improved multiple stress tolerance. Plant biotechnology journal, 17(1), 233e251. Zhou, M., Li, D., Li, Z., Hu, Q., Yang, C., Zhu, L., & Luo, H. (2013). Constitutive expression of a miR319 gene alters plant development and enhances salt and drought tolerance in transgenic creeping bentgrass. Plant Physiology, 161(3), 1375e1391. Zhou, L., Liu, Y., Liu, Z., Kong, D., Duan, M., & Luo, L. (2010). Genome-wide identification and analysis of drought-responsive microRNAs in Oryza sativa. Journal of Experimental Botany, 61(15), 4157e4168. Ding, Y., Ding, L., Xia, Y., Wang, F., Zhu, C. 2020. Emerging roles of microRNAs in plant heavy metal tolerance and homeostasis. Journal of Agricultural and Food Chemistry 68(7), 1958-1965. https://doi.org/10.1021/acs.jafc.9b074688 This page intentionally left blank CHAPTER 4 Small RNAs as emerging regulators of agricultural traits of food crops Jinyuan Tao and Yu Yu Shenzhen University, Guangdong Provincial Key Laboratory for Plant Epigenetics, College of Life Sciences and Oceanography, Shenzhen, Guangdong, China 1. Introduction Food crops meet the majority of the caloric and nutritional needs of the global population. However, the current trend of increasing food crop yields will be insufficient to meet the global demand by 2050 due to the rising human population, global climate change, the deterioration of cultivated soils, and other environmental concerns (Ray et al., 2013; Takeda & Matsuoka, 2008). Consequently, the stability of food crop production, together with sustainable agricultural systems, has been receiving increased attention. To ensure the successful delivery of the required volume and stable supply of food, it is necessary to incorporate elite genetic materials into crop lines of interest, particularly those for agronomic traits that favor crop growth, which has always been used as selection criteria and are usually controlled by single or multiple genes, or many quantitative trait loci (QTLs). The development and establishment of superior crops require the integration of advances in plant physiology, genetics, biotechnology, and genomic research. In recent decades, thousands of genes modulating various agronomic traits have been isolated and characterized, some of which have been introduced into the crop lines via genetic engineering to improve the agronomic traits (Varshney et al., 2006). However, the precise gene network involved in the regulation of agronomic traits of food crops remains poorly understood, and the number of genes suitable for use in conventional or molecular breeding of crops is limited due to the fact that many other genes exhibit adverse side effects despite correcting the primary defect (Gratten & Visscher, 2016). Therefore, it is urgent to identify new key regulatory genes and gain insights into the complicated processes of plant growth and development to accelerate the breeding procedure with greater accuracy and efficiency. Plant Small RNA in Food Crops ISBN 978-0-323-91722-3 https://doi.org/10.1016/B978-0-323-91722-3.00005-1 © 2023 Elsevier Inc. All rights reserved. 69 70 Plant Small RNA in Food Crops Current advances in high-throughput deep sequencing technologies have allowed for the study of genome-wide transcripts in eukaryotes, of which only 2% encode proteins (Rai et al., 2019; Wilusz et al., 2009). The remaining transcripts include a myriad of non-coding RNAs (ncRNAs) derived from intergenic regions, repetitive sequences, transposons, and pseudogenes, which are likely to have potential regulatory functions (Lloyd et al., 2018). ncRNAs can be categorized into three groups based on their lengths: small RNAs (sRNAs) (18e30 nt), medium-sized RNAs (31e200 nt), and long ncRNAs (lncRNAs) (>200 nt) (Yu et al., 2019). In this review, we highlight recent studies that reinforce our understanding of sRNA-mediated regulation of important agronomic traits in major food crops, with an emphasis on the molecular mechanisms of sRNAs orchestrating diverse agronomic traits, as well as their biogenesis, modes of action, and conserved and diverse functions in different food crops, in the hope of using them as candidates for improving future food crop production. 2. Biogenesis, classification, and modes of action of small RNAs sRNAs are ubiquitous components of endogenous plant transcriptomes and play crucial roles in various intracellular processes by modulating gene silencing at the transcriptional or post-transcriptional level (Chen, 2009; Komiya et al., 2014; Tang & Chu, 2017; Yu et al., 2019). Endogenous sRNAs can be classified into four types, based on their differences in biogenesis, function, and modes of action in plants: microRNAs (miRNAs), heterochromatic small interfering siRNA (hc-siRNA), phased small interfering RNAs (phasiRNAs), and natural antisense transcript small interfering RNAs (NAT-siRNAs) (Axtell, 2013). miRNAs have been proved to play versatile roles in regulating the agronomic traits of various food crops via either target transcript cleavage or translation repression in the sequence complementarity manner. Primary miRNA transcripts (pri–miRNAs) are transcribed from MIR genes to form the 5ʹ-capped and 3ʹ-polyadenylated, single-stranded hairpin structure, which are further processed into precursor miRNAs (pre–miRNAs) by an RNase III enzyme DCL (DICER-LIKE), usually DCL1 in Arabidopsis with the assist of HYL1 (HYPONASTIC LEAVES 1) and SE (SERRATE), and finally give rise to the miRNA:miRNA* duplexes. The duplexes undergo HEN1 (HUA ENHANCER 1)-catalyzed 2-O-methylation, which protects the mature sRNAs from uridylation and degradation (Chen, 2009; Small RNAs as emerging regulators of agricultural traits of food crops 71 Li et al., 2005; Voinnet, 2009; Yu et al., 2017a, 2017b). The functional mature miRNA strand is subsequently integrated into the AGO (ARGONAUTE) protein, mostly AGO1, to form an RNA-induced silencing complex (RISC) in the nucleus, followed by the export of the complex to the cytoplasm where it exhibits the post-transcriptional target gene silencing function (Bologna et al., 2018; Xie et al., 2021). Notably, over half of the miRNA targets are transcription factors (TFs) that play a vital role in crop development (Tang & Chu, 2017). Hc-siRNAs are mainly derived from transposable elements and repetitive genomic regions and serve as the major effectors in the RNA-directed DNA methylation (RdDM) pathway. The plant-specific RNA POLYMERASE IV (Pol IV), aided by the chromatin remodeling factors CLASSY and SHH1 (SAWADEE HOMEODOMAIN HOMOLOG 1), generates short single-stranded RNAs (ssRNAs) (Wendte & Pikaard, 2017), followed by the conversion of the ssRNAs to double-stranded RNAs (dsRNAs) by RDR2 (RNA-DEPENDENT RNA POLYMERASE 2). DCL3 processes the dsRNAs into 24 nt mature siRNA:siRNA* duplexes; subsequently, the 24 nt siRNAs generated from Pol IV transcripts are preferentially loaded into AGO4 protein. Another plant-specific RNA polymerase Pol V produces the scaffold transcripts at the nearby loci to recruit siRNA-AGO4 complexes, which further recruit DRM2 (DNA REARRANGED METHYLASE 2) to direct DNA methylation and trigger transcriptional gene silencing (Erdmann & Picard, 2020; Li et al., 2015; Wierzbicki et al., 2012; Zhou et al., 2018). PhasiRNAs are a class of secondary siRNAs relied on miRNAmediated cleavage of transcripts from long non-coding or protein-coding genes transcribed by Pol II. The 5ʹ or 3ʹ cleavage fragments are protected against degradation by SGS3 (SUPPRESSOR OF GENE SILENCING 3) and converted to dsRNAs via RDR6. DCL proteins then process the dsRNAs into 21 nt or 24 nt siRNAs following the phase mode of head-totail arrangement. Trans-acting siRNAs (tasiRNAs) belong to a subclass of phasiRNAs that are generated from TAS genes loci and act in trans (Axtell, 2013; Chen, 2009; Fei et al., 2013; Yu et al., 2019). High-throughput sequencing analyses reveal that the phasiRNAs are abundant and widespread in numerous plant species, suggesting that phasiRNAs are conserved and may play essential roles in plants (Komiya, 2017). NAT-siRNAs, including cis- and trans-NATs-siRNAs, are another subset of siRNAs, and their biogenesis is RDR-independent but requires transcripts from both protein-coding and noncoding loci to form dsRNAs. 72 Plant Small RNA in Food Crops For instance, dsRNA precursors of cis-NAT-siRNAs are transcribed from opposite strands at the same genomic regions, while trans-NATs-siRNAs originate from transcripts at different genomic regions but can form the imperfectly complemented dsRNAs. Although the generation of primary NAT-siRNAs does not rely on RDRs, the accumulation of NAT-siRNAs still requires RDRs, DCLs, and other factors to trigger the secondary siRNA amplification process and action (Axtell, 2013; Thody et al., 2020). 3. Small RNAs contribute to the agricultural traits of food crops Optimal agricultural traits enhance food crop growth and yields. In recent years, increasing attention has been paid to determining the function of sRNAs in the regulation of food crop traits. Herein, we will exemplify six major food crops: rice (Oryza sativa), maize (Zea mays), wheat (Triticum aestivum), barley (Hordeum vulgare), soybean (Glycine max), and potato (Solanum tuberosum), which are among the most important food crops worldwide, to emphasize the importance of various types of sRNAs in regulating food crop traits (Fig. 4.1). 3.1 Seed dormancy and germination Higher plants germinate from seeds, which comprise the embryo, the endosperm that stores the necessary amount of nutrients to support seedling Figure 4.1 Functions of miRNAs in plant development and stress responses. An overview of the current understanding of miRNA-mediated regulation during development and responses to biotic and abiotic stresses in rice. Small RNAs as emerging regulators of agricultural traits of food crops 73 establishment, and the seed coat that protects the embryo and endosperm. In food crops, both seed dormancy and germination are regarded as important agricultural traits for securing yields and are regulated by a complex internal network of biochemical and molecular mechanisms, as well as impacted by external stimuli (Penfield, 2017; Shu et al. 2015, 2016; Yan & Chen, 2020). Although scientists have elucidated the regulatory pathways of seed dormancy and germination, most previous studies have focused on phytohormone network modulation, such as gibberellic acid (GA) and abscisic acid (ABA). sRNAs are emerging effectors that modulate seed dormancy and germination via post-transcriptional regulation, gradually giving rise to a new field of study. Seed dormancy is an intrinsic feature of the late embryogenesis stage and an indicator of seed maturation, preventing seeds from pre-harvest sprouting and ensuring the yield and quality of agricultural production. In contrast, seed germination occurs when the seed dormancy is at low level or removed by favorable environmental conditions, such as temperature, light, and humidity. Numerous studies have uncovered the function of miRNAs in regulating seed dormancy and germination in the model plant Arabidopsis (Tognacca & Botto, 2021) as well as food crops. For instance, mutation in rice OsMIR156 strengthens seed dormancy through its target gene IPA1 (IDEAL PLANT ARCHITECTURE 1), which represses the gibberellin (GA) pathway (Miao et al., 2019). OsmiR168a and OsmiR164c have been shown to positively and negatively regulate seed germination rates in rice, respectively (Zhou et al., 2020). OsmiR393a, which is involved in the auxin signaling pathway, inhibits coleoptile elongation during rice seed germination and seedling establishment under submergence (Guo et al., 2016). In maize and soybean, although the modulatory pathway of sRNA in the regulation of seed dormancy and germination is poorly understood, advanced sequencing approaches have enabled the identification of numerous, highly abundant sRNAs that appear in the early stage of seed germination, suggesting their potential significance (Sun et al., 2016; Wang et al., 2011). Liu et al. sequenced sRNAs in maize embryos treated with exogenous phytohormones (ABA and GA) during seed germination to investigate the dynamic changes in miRNA, and found that ZmmiR159 and ZmmiR160 may regulate seed dormancy by influencing ABA signaling through their target genes ZmMYB33/ZmMYB101 (R2R3 MYB domain proteins) and ZmARF10 (AUXIN RESPONSE FACTOR 10), respectively, in addition, six members of the ZmmiR156 family were found to be differentially expressed after ABA/GA treatment, indicating 74 Plant Small RNA in Food Crops that they may potentially affect seed germination through SPLs (SQUAMOSA PROMOTER-BINDING PROTEIN-LIKEs) (Liu et al., 2020). However, experimental evidences are required for future research. In wheat, wheat-specific TamiR9678 reduces bioactive GA levels by triggering the synthesis of phasiRNAs from a lncRNA WSGAR (Wheat Seed Germination Associated RNA), thus enhancing seed dormancy (Guo et al., 2018). Notably, in the ncRNA transcriptome profiles of MingXian169, an ideal breeding wheat owing to its high pre-harvest sprouting resistance, differentially expressed miRNAs were identified between germinated and dormant seeds, implying that miRNAs play a pivotal role in the regulatory mechanism of seed germination (Zhang et al., 2021). Furthermore, in barley, a large pool of 24 nt siRNA produced from the promoter region of HvCKX2.1 (CYTOKININ-OXIDASE 2.1), which is dependent on the RNA-directed DNA methylation (RdDM) pathway under drought stress and silencing the mRNA of HvCKX2.1, was found to control seed germination timing (Surdonja et al., 2017). Although there is a negative correlation between seed dormancy and seed germination, they are crucial for yield storage and propagation, respectively. The formation and mutual transition of seed dormancy and germination involves complicated mechanisms at the molecular and cellular levels, among which sRNAs act as the indispensable regulators. In fact, in the near future, the exploitation of more roles of sRNAs in seed programs may assist agricultural production. 3.2 Root architecture establishment Unlike animals, plants are sessile organisms that rely on their roots for anchorage and absorption of water and nutrients. Plant root organs of different species exhibit metabolic plasticity in response to the ambient environment (Coudert et al., 2010; Lombardi et al., 2021; Svolacchia et al., 2020). Root morphology includes several parameters regarding root system architecture and additional organs, such as primary root/crown root, lateral root branching, and root hair, which are constantly formed throughout the plant life. Root morphology differs greatly among plant species (especially between monocots and eudicots) (Dastidar et al., 2012; Hochholdinger & Zimmermann, 2008; Rich & Watt, 2013), and is regulated by both external and internal factors. Hundreds of coding proteins and non-coding RNAs participate in the complex regulatory network of root development. Herein, we summarize the role of sRNAs in regulating the establishment of root morphology in food crops. Small RNAs as emerging regulators of agricultural traits of food crops 75 The control of root growth by the plant hormone auxin is well documented, and the components of auxin signaling are known to be conserved throughout land plants (Chapman & Estelle, 2009; Roychoudhry & Kepinski, 2021). Numerous studies have revealed the mechanism by which miRNAs directly target the modules of the auxin signaling pathway to modulate root growth in food crops. For example, in rice, earlier research has shown that OsmiR167d regulates root elongation by inhibiting OsARF12 (Qi et al., 2012). Besides, the conserved miRNA/target modules involved in auxin signaling are known to exist in different species of rice and barley, and auxin receptors TIR1/AFBs (TRANSPORT INHIBITOR1/ AUXIN-SIGNALLING F-BOXs) are targeted by miR393 to modulate the growth of primary root and crown root (Bai et al., 2017; Bian et al., 2012). In soybean, a leguminous plant, the GmmiR167-directed regulation of the auxin response factors GmARF8a and GmARF8b were found to promote lateral root branching (Wang et al., 2015a), while potato StmiR160a/b was found to cleave the mRNA of StARF10 and StARF16, affecting plant root architecture (Yang et al., 2021b). Converging distinct miRNAs to regulate root development is an emerging consensus, evidenced by miR393 suppressing the expression of miR390 and restricting rice lateral root growth under stress (Lu et al., 2018). NACs (NAC DOMAIN CONTAINING PROTEINs) are a class of plant-specific transcription factors targeted by conserved miR164 in several food crops. In maize and wheat, the regulation of miR164 and NAC1 is involved in the formation of lateral root and crown root, respectively (Li et al., 2012; Li et al., 2021c), and the induction of StmiR164 by osmotic stress in potato results in a decrease in StNAC262 expression and limits the number of lateral roots (Zhang et al., 2018a). miRNAs are able to target more than one target, wheat TamiR164 targets a non-conserved TaPSK5 (PHYTOSULFOKINE) to regulate the root growth (Geng et al., 2020). In addition to the conserved miRNA-target modules, some species-specific modules have also been reported, and crosstalk exists among these regulatory pathways. In rice, the OsmiR156OsSPL3/12 (SQUAMOSA PROMOTER BINDING PROTEIN-LIKE3/ 12) module regulates crown root development by activating downstream OsMADS50 (MIKC-TYPE MADS BOX 50) (Shao et al., 2019), while OsmiR444a guides mRNA cleavage of OsMADS23, OsMADS27a, OsMADS27b, and OsMADS57 to regulate nitrate-dependent root growth (Yan et al., 2014). In maize, the miR165/166-RLD1/2 (ROLLED1/2) module regulates maize root development and is simultaneously affected by the LBL1 (LEAFBLADELESS 1)-mediated tasiR-ARF-ZmARF2/3 76 Plant Small RNA in Food Crops module (Gautam et al., 2021). LBL1 is a homolog of Arabidopsis SGS3, implying that tasiRNAs derived from maize TAS loci have a potentially regulatory role in root establishment. In addition to the discovery of numerous miRNAs and the insights gained into their roles in root development regulation, there have been a few reports on other sRNAs that may also play potential roles in the processes of root establishment. Ma et al. constructed rice sRNA libraries and found that a large pool of sRNAs were highly accumulated in the root tips or in the whole roots, which support our understanding of the molecular mechanisms of root growth (Ma et al., 2013). The long-distance movement of sRNAs from the shoot to the root also provides a new perspective of the mechanisms underlying root development. Soybean has been used as a suitable grafting plant for research on the movement of sRNAs, with 100 shooteroot mobile miRNAs and 32 shooteroot mobile phasiRNAs being predominantly produced in shoots and transported to roots, many of which have been found to enable the cleavage of mRNA targets or phasiRNA precursors in roots (Li et al., 2021b). An optimal architecture is an essential factor in plant growth and provides resistance to adverse environmental conditions. Elucidation of sRNAs that potentially exhibit regulatory functions in food crop root growth will improve our understanding of food crop root architecture manipulation, providing plant breeders with more opportunities for the development of elite food crops in the future. 3.3 Shoot architecture The Green Revolution in the 1960s led to a rapid increase in the yields of major staple grain crops (wheat and rice) to address the food demand of an increasingly large global population. This was possible due to changes in plant shoot architecture, especially plant height. Plant architecture is an important determinant of crop yield that is controlled by a complicated combination of traits, including plant height, branch/tiller number, branch/ tiller angle, and leaf angle (Sakamoto & Matsuoka, 2004; Sasaki et al., 2002). In recent decades, findings on the formation of shoot architecture by several major hormones (e.g. auxin, cytokinin, and strigolactones) and some marker genes, as well as the molecular and genetic regulation networks involved in controlling food crop shoot architecture have gradually become clear (Wang et al., 2018). Notably, sRNAs play a vital role in the complex network, wherein a number of miRNAs affects shoot architecture by inhibiting their target genes. Small RNAs as emerging regulators of agricultural traits of food crops 77 Rice branches are of two types: shoot branches (tillers) during the vegetative stage, and panicle branches that emerge in the reproductive stage. In recent years, miRNAs have been reported as modulators to control rice tillering and panicle branches. For example, in rice, OsmiR156, OsmiR529, and OsmiR535 promote tiller number by directing the transcript cleavage of OsSPL genes (Jiao et al., 2010; Sun et al., 2019a; Yan et al., 2021). The positive role of miR156 in positively controlling tillering/ branch is also conserved in maize, wheat, soybean, and potato (Bhogale et al., 2014; Chuck et al., 2010; Liu et al., 2017; Sun et al., 2019b). OsmiR397 and OsmiR408 promote panicle branching and improve rice yield by cleaving the transcripts of target genes OsLAC (LACCASE) and OsUCL8 (UCLACYANIN-LIKE 8), respectively (Zhang et al., 2013; Zhang et al., 2017a). In contrast to the miRNAs positively regulating branching, there are also negative regulators, especially that the same miRNA could play opposite roles in tillering and panicle branches. In spite of promoting tiller number, overexpression of rice OsmiR156, OsmiR529, and OsmiR535 results in decreased panicle branches (Dai et al., 2018; Jiao et al., 2010; Sun et al., 2019a; Wang et al., 2015b; Yan et al., 2021). OsmiR396 targets OsGRFs (GROWTH-REGULATING FACTORS) and negatively regulates panicle branching, and OsmiR164b overexpression decreases the panicle branch number and yield via cleaving OsNAC2 transcript (Diao et al., 2018; Gao et al., 2015; Jiang et al., 2018; Liebsch & Palatnik, 2020). Overaccumulation of OsmiR172 reduces panicle branching with no effect on tillering in rice, similar function of HvmiR172 is observed in spikelet development in barley (Brown & Bregitzer, 2011; Wang et al., 2015b). Interestingly, the same miRNA may also cause opposite phenotypes in different plant species. For example, overexpression of miR171 results in increased tiller number in rice, which in turn is decreased in barley under short day condition. (Curaba et al., 2013; Fan et al., 2015). The miRNA-mediated auxin signaling cascades participate in shaping shoot architecture. Overexpression of OsmiR393 promotes rice tillering by reducing the expression of OsTIR1 and OsAFB2, whereas OsmiR167 overexpression in transgenic lines represses the tiller number by reducing the transcripts of four OsARFs. OsARF18 is targeted by miR160 in rice, and the overexpressing cleavage-resistant lines of OsARF18 show lower tiller numbers than the wild type (Huang et al., 2016; Li et al., 2016b; Liu et al., 2012; Xia et al., 2012). Additionally, OsmiR444a represses the tiller number by reducing the transcripts of OsMADS57, which is involved in 78 Plant Small RNA in Food Crops strigolactone signaling, and OsmiR319 negatively affects the tiller number by targeting OsTCP21 (TEOSINTE BRANCHED/CYCLOIDEA/PCF 21) and OsGAMYB (GIBBERELLIN- AND ABSCISIC ACIDREGULATED MYB) (Guo et al., 2013; Wang et al., 2021b). In addition to miRNAs, Xu et al. reported that siRNAs derived from the canonical RdDM pathway could repress rice tillering due to DNA methylation in the promoters of OsMIR156d/j and D14 (DWARF14), which reinforces the underlying mechanism of other sRNAs in the regulation of tiller/branch, except for miRNAs (Xu et al., 2020). Decrease in plant height leads to improvements in lodging resistance, especially in grain crops near the harvest stage. Lodging not only reduces yield, but also increases the cost of production by affecting the efficiency of machine harvesting. Evidence indicates that miRNAs play an important role in regulating the height of food crops. The well-studied miR156-SPLs module has been found to markedly decrease plant height in rice and wheat, but not soybean (Dai et al., 2018; Liu et al., 2017; Sun et al., 2019b), indicating that the roles of miR156 in controlling plant height may differ between monocots and dicots. The effects of OsmiR529 overexpression in rice lines in repressing plant height by cleaving SPLs transcripts is similar to that of OsmiR156. Furthermore, OsmiR160 and OsmiR167 negatively regulate rice height through OsARFs, which are involved in the auxin signaling pathway (Huang et al., 2016; Li et al., 2020; Yan et al., 2021). Transcription of OsMIR159 is repressed by brassinosteroid (BR), and the OsmiR159 knockdown lines show increased expression level of its target genes OsGAMYB and OsGAMYBL1 (GAMYB-LIKE1), simultaneously exhibiting short plant height (Gao et al., 2018; Zhao et al., 2017). In contrast, OsMIR396 expression is induced by BR and overexpression of rice OsmiR396d results in semi-dwarf plants (Tang et al., 2018). These results imply that miRNAs cooperate with the components of plant hormone signaling to fine-tune plant height. A group of transgenic short tandem target mimic (STTM) lines that silencing 35 miRNA families in rice were generated and several lines displayed dwarf phenotype in the plant height and increased tiller number, such as STTM398, STTM171, STTM172, STTM159, STTM160, and STTM166, conversely, STTM441 transgenic lines were taller than the wild-type, and STTM156 exhibited fewer tillers (Zhang et al., 2017b). Although the underlying mechanism of some miRNAs in the modulation of agronomic traits has yet to be fully elucidated, STTM technology could be an effective tool for the screening of promising miRNA targets for other food crops. Small RNAs as emerging regulators of agricultural traits of food crops 79 The leaf and tiller/branch angle determine the space occupied by plant shoots above ground. Different angles of leaf and tiller/branch are correlated with light capture for photosynthesis, thus affecting food crop yield (Tian et al., 2019). In rice, targets of OsmiR393, OsmiR396, and OsmiR397 control leaf angle by indirectly altering BR homeostasis (Tang et al., 2018; Zhang et al., 2013, 2015), whereas OsmiR1848 directly affects BR biosynthesis to modulate leaf angle through its target gene OsCYP51G3 (CYTOCHROME P450 SUBFAMILY 51 G3) (Xia et al., 2015). Increased expression of OsLC4 (LEAF INCLINATION 4) in the Osmir394 mutant results in the enlarged leaf angle (Qu et al., 2019). Additionally, the loss of most 24 nt siRNAs in rice Osdcl3a mutants has been found to trigger alterations in important agricultural traits, including dwarfism and larger flag leaf angle, indicating the indispensable function of OsDCL3a-dependent 24 nt siRNA in maintaining normal agricultural traits (Wei et al., 2014). To date, rice has been used as a model crop for sRNA-related plant architecture research based on successful rice transformation systems and research methods. In the future, researchers will have access to an increasing number of food crops as models for the study of sRNA function, screening out sRNA targets for use in optimizing the shoot architecture of food crops. 3.4 Phase transition and flowering time Among the processes during plant life cycle, the transition from vegetative to reproductive growth, also known as flowering time, is crucial for reproductive success and subsequent yield harvest. For sessile plants, the successful completion of this transition is usually exposed to both endogenous factors and exogenous stimuli, eventually establishing a complex genetic regulatory network that converges on a set of floral pathway integrators to control plant flowers appropriately over time (Teotia & Tang, 2015). FT (FLOWERING LOCUS T), SOC1 (SUPRESSOR OF OVEREXPRESSION OF CONSTANS1), FLC (FLOWERING LOCUS C), and LFY (LEAFY) constitute the core nexus in the control of flowering time (Abe et al., 2005; Bastow et al., 2004; Lee et al., 2010; Moyroud et al., 2010). Five main distinct regulatory pathways that regulate flowering have been identified: the vernalization pathway, the autonomous pathway, the photoperiod pathway, the GA-mediated pathway, and the age pathway (Kim et al., 2009; Teotia & Tang, 2015; Yamaguchi & Abe, 2012). Vernalization and autonomous direct flowering by silencing FLC, a repressor that delays flowering by inhibiting the activity of floral pathway 80 Plant Small RNA in Food Crops integrators (FT and SOC1) (Bastow et al., 2004; Cheng et al., 2017). Under long-day condition, the photoperiod pathway triggers the activation of GIGANTEA (GI) and CONSTANS (CO) to induce FT production in leaves, followed by the translocation of FT to shoot apical meristem (SAM) via phloem; eventually, together with FD and SOC1, FT promotes flowering by regulating downstream factors in the meristem (Kobayashi & Weigel, 2007). The GA-mediated pathway stimulates flowering, which is mainly involved in the modulation of the activation of floral integrators SOC1, LFY, and FT in the inflorescence, floral meristems, and leaves, respectively (Mutasa-Göttgens & Hedden, 2009). In the age pathway, sRNAs predominantly participate in the flowering control pathway, regulated by two key miRNAs (miR156 and miR172). The differential expression of miR156 and miR172 with plant age affects floral integrators directly or indirectly by negatively regulating their own sets of target genes. High expression levels of miR156 are observed in young seedlings, which subsequently decline with age. The opposite pattern of expression occurs in miR172, with a low level during the juvenile phase, and subsequently rising before entering floral development. This pathway is conserved across species (Teotia & Tang, 2015). In rice leaves, the abundance of mature OsmiR156 is gradually decreased over the course of development, and is even hardly detectable in old leaves (Yang et al., 2019). Continuously high levels of OsmiR156b and OsmiR156h result in delayed flowering in rice (Xie et al., 2006). In wheat seedlings, TaMIR156 overexpression markedly reduces the mRNA level of SPL2, triggering serious delays in the vegetative phase transitions, as well as booting and flowering (Jian et al., 2017). Similarly, overexpression of soybean GmmiR156b has been found to prolong flowering time by negatively regulating GmSPLs under long-day and natural conditions (Cao et al., 2015). Similarly, overexpression of CG1 (CORNGRASS 1), which contains a tandem ZmmiR156b and ZmmiR156c loci, prolongs juvenility and delays flowering time in maize (Chuck et al. 2007, 2011). Additionally, the comparative genome-wide phylogenetic and expression analyses of SPL genes in potato indicated that the StmiR156-StSPLs module may control flowering (Kavas et al., 2017). In contrast to the expression pattern of OsmiR156 in rice leaves, accumulation of OsmiR172 was observed in older leaves (Zhu et al., 2009), and overexpression of rice OsmiR172 was found to induce flowering by suppressing two AP2(APETALA 2)-like genes, OsIDS1 (INDETERMINATE SPIKELET1) and SNB (SUPERNUMERARY BRACT), which negatively regulate the expression of Ehd1 Small RNAs as emerging regulators of agricultural traits of food crops 81 (EARLY HEADING DATE 1) and florigens (Lee et al., 2014). The same regulatory role of miR172-AP2 exists in maize, barley, and potato. An AP2-related gene GL15(GLOSSY 15) is targeted and negatively regulated by ZmmiR172 to facilitate flowering in maize (Lauter et al., 2005). In barley, HvmiR172 promotes flowering by repressing another AP2-like gene CLY1 (CLEISTOGAMY 1) (Nair et al., 2010). Overexpression of StmiR172 has been shown to downregulate RAP1 (RELATED TO APETALA2 1) in potato and promote flowering (Martin et al., 2009). Intriguingly, the monocot-specific OsmiR528 has been found as an agemodulating miRNA in rice and promotes flowering by targeting OsRFI2 (RED AND FAR-RED INSENSITIVE 2) (Yang et al., 2019). In addition to miR156-SPLs and miR172-AP2, other miRNAs are also reported to control flowering time. Overexpression of OsmiR159 in rice is responsible for delayed head formation, accompanied by a decrease in the transcript levels of OsGAMYB and OsGAMYBL (Tsuji et al., 2006). Upregulation of miR171 in rice and barley results in a prolonged vegetative phase and delayed heading date, possibly by activating the miR156 pathway to inhibit downstream flowering integrators (Curaba et al., 2013; Fan et al., 2015). OsmiR393 overexpression rice plants show an early flowering phenotype through its targets OsTIR1 and OsAFB2, which are involved in the auxin pathway (Xia et al., 2012). A recent study report that knockdown of OsmiR168 by a target mimic (MIM168) shortens flowering time in rice, due to the reduced OsmiR164 and accordingly increased expression level of OsNAC11 (Wang et al., 2021a). Additionally, in wheat, both overexpression of TamiR408 and knockdown of target TaTOC1(TIMING OF CAB EXPRESSION 1) promoted early flowering (Zhao et al., 2016). The mechanism underlying the regulation of phase transition/flowering time by miRNAs in Arabidopsis and several crops is well documented, especially in terms of the relationship between miRNA-target and floral integrators. The intricate regulatory network of flowering is supported not only by the mutual influence between miRNAs, but also by the miRNAtarget module participating in other flowering pathways that are simultaneously affected by environmental cues. However, in addition to miRNAs, other types of sRNAs in the control of flowering time have yet to be fully elucidated. Fortunately, rapid advances in deep sequencing technologies are likely to accelerate researches to elucidate the functions of diverse sRNAs, thereby increasing the number of new strategies for food crop breeding by fine-tuning flowering time. 82 Plant Small RNA in Food Crops 3.5 Reproductive organ development and fertility In recent decades, scientists have made efforts to uncover the genetic network underlying the development of flower organs using genetic, biochemical, and genomic approaches. The detailed regulatory mechanisms of floral meristem formation, floral patterning, floral organ specification, floral organ development, and floral meristem termination have been described at length in reviews (Guo et al., 2015; Thomson & Wellmer, 2019; Yoshida & Nagato, 2011). In the present review, we focus on the findings of how sRNAs contribute to the reproductive organ development and fertility in food crops. The molecular dissection of crop reproductive development has been a popular research field, wherein a clearly defined regulatory network of crop fertility development will establish a research foundation and help breeders achieve their goals. A large pool of sRNAs is produced during flower organ development in both monocots and dicots, implying the importance of these molecules in plant reproductive development (Ding et al., 2016; Johnson et al., 2009). miRNAs are indispensable regulators during reproductive organ development in plants. For example, miR172 plays roles in the diversity of floral organ morphology, and the miR172-AP2 regulatory module is conserved in most plant species, including rice, maize, wheat, and barley. Evidences have been shown that overexpression of rice OsmiR172b results in abnormal flower organs, such as multiple layers of lemma and palea, twisted lemma (Zhu et al., 2009). The loss-function mutant of two AP2-like genes, ZmIDS1 and ZmSID1 (SISTER OF INDETERMINATE SPIKELET 1), targeted by ZmmiR172, leads to defective flowers with fewer tassel branches in maize (Chuck et al., 2008). Besides, overexpression of wheat TamiR172 induces elongated spikes and non-free-threshing grains (Debernardi et al., 2017), while the suppression of HvCLY1 mRNA cleavage mediated by HvmiRNA172 results in cleistogamy in barley, which is caused by the atrophy of lodicules (Nair et al., 2010). miR159 is conserved across plant species. In rice, the severity of defective flower phenotypes depends on the abundance of mature OsmiR159, the mild phenotype is flowers bearing shrunken and whitened anthers, while severe phenotypes include distorted palea and lemma, and small flowers (Tsuji et al., 2006). Upregulation of miR171 results in abnormal reproductive organs in rice and barley by cleaving their targets OsHAMs (HAIRY MERISTEM) and HvSCL (SCARECROW-LIKE), respectively (Curaba et al., 2013; Fan et al., 2015). In addition, overexpressed OsmiR396d Small RNAs as emerging regulators of agricultural traits of food crops 83 transgenic lines exhibit malformed flower phenotypes in rice, including open husks, long sterile lemmas, and altered floral organ morphology, resulted from repression of OsmiR396d targets JMJD2 family OsJMJ706 (JUMONJI C GENE 706) and OsCR4 (CRINKLY-4 RECEPTOR-LIKE KINASE) (Liu et al., 2014). Numerous studies of sRNAs involved in crop fertility have been conducted, which is attributed to advances in sequencing technology. In rice, OsmiR408 and OsmiR528 impact pollen intine formation by directly targeting the same gene family, but different members, of OsUCLs proteins, such as OsUCL8 and OsUCL23, respectively (Zhang et al., 2018c, 2020a). Overexpression lines of OsLAC13, a target of OsmiR397, exhibit semisterility (Yu et al., 2017b), while rice STTM1428 transgenic lines show sterile pollen at the pollen maturation stage (Zhang et al., 2017b). Overexpression of OsmiR159 results in defective pollen development, possibly via repression of OsMYB genes; however, the regulatory mechanism remains unclear (Tsuji et al., 2006). In maize, Jiang et al. reported the potential post-transcriptional regulation of ZmABCG26 (ATP-BINDING CASSETTE TRANSPORTER G 26) by ZmmiR164h-5p to control male sterility by affecting lipid metabolism in anthers (Jiang et al., 2021). The construction and analysis of sRNA libraries of rice anthers from photoperiod- and thermo-sensitive genic male sterile germplasm indicated that other miRNAs, such as OsmiR156, OsmiR5488, and OsmiR399, may also be involved in the regulation of male sterility (Sun et al., 2021). The mutation of maize ZmDCL1, a key enzyme required for miRNA biogenesis, results in male sterility, implying that some miRNAs are involved in male fertility (Field et al., 2016). However, the detailed regulatory mechanisms will need to be elucidated in the future. In addition to miRNAs, the roles of phasiRNAs in flower organ development are gradually being uncovered, according to the rice germplasm of photoperiod-sensitive male sterility (PSMS), which is caused by a single nucleotide polymorphism in PMS1T (PHOTOPHERIODSENSETIVE GENIC MALE STERILITY 1T) loci nearby the OsmiR2118 recognition site. Fan et al. found that OsmiR2118 triggers the production of 21 nt phasiRNAs to maintain male fertility through targeting a long-non-coding RNA PMS1T (Fan et al., 2016). Subsequently, several independent research teams found that OsmiR2118 directly affect the abundance of 21 nt phasiRNAs in male rice germ cells, respectively, assuring male fertility by balancing gene expression (Araki et al., 2020; Jiang et al., 2020; Yang et al., 2021a; Zhang et al., 2020b). These findings 84 Plant Small RNA in Food Crops strongly support the hypothesis that 21 nt phasiRNAs play vital functions in food crop reproductive organ fertility, although experimental data have only been reported in rice. In addition to direct proof, indirect evidence exists that other sRNAs regulate fertility. AGO proteins are the core components of the RISC complexes and are required for sRNA functions. The loss of function of rice and maize AGOs induces male sterility, accompanied by changes in sRNAs, including miRNAs, siRNAs, and phasiRNAs, indicating that these sRNAs are likely to mediate pollen fertility (Das et al., 2020; Li et al., 2021a). NAT-siRNAs are a class of sRNAs in eukaryotes that have rarely been researched in terms of flowering fertility. In Arabidopsis, male gametophytic KPL (KOKOPELLI) transcripts pair with ARI14 (ARIADNE14) to generate the sperm-specific NAT-siRNAs, which facilitate gametophyte formation and double fertilization (Ron et al., 2010), suggesting that NAT-siRNAs perform regulatory functions in food crop fertilization. In recent decades, numerous hybrid germplasm lines have been established based on our understanding of male sterility in rice and maize, which have been used to address global food crises. In particular, the system of photoperiod- and thermo-sensitive male sterility has been used to further promote two-line hybrid rice breeding. Findings on these distinct regulatory pathways and successful cases provide valuable information for the development of other food crops in future works. 3.6 Size and yield of seeds/tubers in food crops Seed weight is an important agricultural trait that contributes to the yield of food crops. Its determinants include seed length, width, and filling. The mechanism underlying the determination of final seed size is explained by cell proliferation and expansion (Xing & Zhang, 2010). Potato tubers are storage organs for starch and other nutrients, as well as a key component in potato reproduction. Potatoes are also the first highest-produced non-cereal food crop and ranks fourth after wheat, maize, and rice in global crop production (Dahal et al., 2019). In recent years, sRNAs have been investigated for their ability to control agricultural traits, including seed weight. Rice has been used as a model crop to investigate the regulatory factors affecting seed weight. It is well known that phytohormones control plant growth, including seed development. miRNAs adjust seed size by involving the auxin and BR pathways, which are exemplified in following cases: OsmiR167a positively regulates seed length and width in rice through the direct and indirect Small RNAs as emerging regulators of agricultural traits of food crops 85 silencing of OsARF6 and OsAUX1(AUXIN1), respectively (Qiao et al., 2021). Unexpectedly, the seed size of mir167a mutant was found to be opposite to that of STTM167 (Peng et al., 2018), which may be due to the use of different rice ecotypes. Therefore, the real seed trait underlying the loss of function of miR167 needs to be confirmed in future studies. In addition, a reduction of OsmiR160 or overexpression of its target OsARF18 contributes to smaller seed size as compared with wild type (Huang et al., 2016; Wang et al., 2017). Similarly, the seeds from overexpressing OsmiR393 transgenic plants are smaller in size (Bian et al., 2012). A mutation of OsGRF4, which results in resistance to OsmiR396mediated cleavage and elevated OsGRF4 expression, leads to larger and the increased number of cells, thereby enhancing grain weight and yield, indicating that OsmiR396 negatively regulates seed size (Hu et al., 2015; Li et al., 2016a). This conclusion is further supported by recent studies of mir396e/mir396f double mutant (Miao et al., 2020; Zhang et al., 2020c). OsGSK2 (GLYCOGEN SYNTHASE KINASE 2) is a type of phosphatase in the BR response pathway. OsGSK2 represses the transcription activation of OsGRF4, implying that BR mediates the module of miR396-OsGRF4 (Che et al., 2015). Rice-specific OsmiR1848 targets OsCYP51G3, an obtusifoliol 1,4-a-demethylase gene which mediates phytosterol and BR biosynthesis. Overexpression of OsmiR1848 causes decreased transcript level of OsCYP51G3 and reduced seed length, which is accompanied by a decline in the concentrations of phytosterol and BR (Xia et al., 2015). Numerous miRNAs participate in the modulation of seed size and yield. Seed width and yield-related QTL, GW8 (OsSPL16), has been mapped and found to positively regulate rice grain cell proliferation. Notably, OsSPL16 is targeted by OsmiR156 and may be simultaneously regulated by OsmiR535, OsmiR529a-5p, and OsmiR529b according to the conserved target site of these miRNAs (Peng et al., 2019; Sun et al., 2019a; Wang et al., 2012; Yan et al., 2021) and similar results were found in soybean that GmmiR156b overexpression resulted in the increase in yield per plant by the positive impact of seed size (Sun et al., 2019b). OsmiR397 overexpressing transgenic lines have been observed to show increased seed length, width, and thickness (Zhang et al., 2013; Zhong et al., 2020). Overaccumulated OsmiR408 increases seed length and width and 1000-grain weight by repression OsUCL8 (Zhang et al., 2017a). In contrast, a lower 1000-grain weight is observed in OsmiR5144-3p overexpression and OsPDIL1;1-RNAi (PROTEIN DISULFIDE ISOMERASE LIKE 1;1RNA INTERFERENCE) transgenic lines (Xia et al., 2018). STTM1432 86 Plant Small RNA in Food Crops or overexpression of OsmiR1432-resistant target OsACOT13 (ACYLCOA THIOESTERASE 13) has been found to significantly improve grain weight by enhancing the grain filling rate, resulting in an increased grain yield (Zhao et al., 2019). Furthermore, the knockdown of OsmiR159, OsmiR166 and OsmiR398 in rice STTM159, STTM166 and STTM398 transgenic lines, respectively, both result in small seed size and low seed weight (Gao et al., 2018; Peng et al., 2018; Zhang et al., 2017b, 2018b; Zhao et al., 2017). In potato, the inverse expression pattern of StmiR156 and StmiR172 from juvenile to adult developmental stages was observed in aerial shoots but not in stolons, and both were reported to accumulate in stolons during tuber swelling as a graft-transmissible signal through the phloem. Overexpression of StmiR156 in potato results in a small tuber and a reduced tuber yield by targeting StSPLs. However, overexpression of StmiR172 accelerates tuberization and trigger tuber formation by downregulating StRAP1 (Bhogale et al., 2014; Martin et al., 2009), suggesting that StmiR156 and StmiR172 exert an additional function in the underground organs of potato. As discussed above, numerous studies on miRNAs involved in seed shaping have been conducted in rice, with few reports focusing on other food crops. Reasons include the completion of high-quality rice genome assembly and an excellent transformation system. The biological process of seed formation is the penultimate step for governing yields, implying the importance of seed traits. Future studies should take advantage of advances in high-throughput sequencing technologies to focus on other miRNAs and types of sRNAs and further elucidate the regulatory mechanisms underlying seed size. 4. Conclusion and future perspectives The widespread occurrence of sRNAs in most developmental processes of food crop growth indicates their role as crucial regulators of agricultural traits. In this review, we summarized the distribution and significance of diverse types of sRNAs that modulate important agricultural traits in major food crops (See Table 4.1). Amounts of plant miRNAs have been reported since two decades ago, subsequently, numerous researches reported the roles of miRNAs in different plant species, expanding our knowledge of gene regulatory networks at the post-transcriptional level. Advances in sRNA deep-sequencing technologies have accelerated research into other sRNAs whose transcripts are derived from non-coding loci. Table 4.1 Summary of small RNAs and their targets in regulation of agronomic traits of six major food crops. Different kinds of small RNAs and their targets are listed in table above that have been verified in experiments. All the abbreviations are descripted in main text. Small RNA Targets Characteristics osa-miR156 IPA, OsSPL3, OsSPL12, other OsSPLs SPL (TF) osa-miR159 OsGAMYB, OsGAMYBL MYB (TF) osa-miR160 OsARF18 ARF (TF) osa-miR164 OsNAC2, OsNAC11 NAC (TF) Agricultural traits and references Rice (Oryza sativa) 87 Continued Small RNAs as emerging regulators of agricultural traits of food crops Seed dormancy/germination, crown root development, tillering, plant height, panicle branch, flower timing, seed size (Jiao et al., 2010; Miao et al., 2019; Shao et al., 2019; Dai et al., 2018; Wang et al., 2012; Xie et al., 2006) Plant height, flower timing, floral organ development/ fertility, seed size (Gao et al., 2018; Tsuji et al., 2006; Zhang et al., 2017b; Zhao et al., 2017) Tillering, plant height, seed size (Huang et al., 2016; Wang et al., 2017) Seed dormancy/germination, flower timing, panicle branch (Jiang et al., 2018; Wang et al., 2021a) 88 Table 4.1 Summary of small RNAs and their targets in regulation of agronomic traits of six major food crops.dcont'd Targets Characteristics osa-miR166 OsHB4 osa-miR167 OsARF12, OsARF6, other OsARFs HD-ZIP III (Class III homeodomain-leucine zipper) ARF (TF) osa-miR168 OsAGO1 AGO osa-miR171 OsHAM GRAS (TF) osa-miR172 OsIDS1, SNB AP2-like (TF) osa-miR319 OsTCP21, OsGAMYB TCP (TF), MYB (TF) Agricultural traits and references Plant height, seed size (Zhang et al., 2018b) Root elongation, plant height, tillering, seed size (Li et al., 2020; Liu et al., 2012; Qi et al., 2012; Qiao et al., 2021) Seed dormancy/germination, flower timing (Wang et al., 2021a; Zhou et al., 2020) Tillering, plant height, flower timing, floral organ development/fertility (Fan et al., 2015) Plant height, flower timing, panicle branch, floral organ development/fertility (Lee et al., 2014; Wang et al., 2015b; Zhu et al., 2009) Tillering (Wang et al., 2021b) Plant Small RNA in Food Crops Small RNA OsTAS3a lncRNA osa-miR393 OsTIR1, OsAFB2 Auxin-signaling F-Box protein osa-miR394 osa-miR396 OsLC4 OsGRF4, OsGRF6, OsGRF10 F-box protein GRFs family protein (TF) osa-miR397 OsLAC Laccase-like protein Lateral root growth (Lu et al., 2018) Seed dormancy/germination, primary/crown root development, tillering, leaf angle, flower timing, seed size (Bian et al., 2012; Guo et al., 2016; Lu et al., 2018; Xia et al., 2012) Leaf angle (Qu et al., 2019) Plant height, leaf angle, panicle branch, floral organ development/fertility, seed size (Che et al., 2015; Diao et al., 2018; Gao et al., 2015; Li et al., 2016a; Hu et al., 2015; Miao et al., 2020; Liebsch & Palatnik, 2020; Liu et al., 2014; Tang et al., 2018; Zhang et al., 2020c) Leaf angle, panicle branch, floral organ development/ fertility, seed size (Yu et al., 2017b; Zhang et al., 2013; Zhong et al., 2020) 89 Continued Small RNAs as emerging regulators of agricultural traits of food crops osa-miR390 Targets Characteristics osa-miR398 CSD1, CSD2 osa-miR399 OsPHO2 osa-miR408 OsUCL8 Cu/Zn-superoxide dismutases Protein containing a ubiquitin-conjugating domain Uclacyanin like protein osa-miR441 No identified No identified osa-miR528 OsRFI2, OsUCL23 osa-miR444 OsMADS23, OsMADS27, OsMADS57 C3HC4-type zinc finger protein (TF), Uclacyanin like protein MIKC-type MADS box protein (TF) osa-miR529 OsSPL2, OsSPL7, OsSPL14, OsSPL16, OsSPL17, OsSPL18 SPL (TF) osa-miR535 OsSPL7, OsSPL12, OsSPL16 SPL (TF) Agricultural traits and references Plant height, seed size (Zhang et al., 2017b) Male fertility (Sun et al., 2021) Panicle branch, male fertility, seed size (Zhang et al., 2018b,c) Plant height (Zhang et al., 2017b) Flower timing, male fertility (Yang et al., 2019; Zhang et al., 2020a) Root development, tillering (Guo et al., 2013; Yan et al., 2014) Plant height, tillering, panicle branch, seed size (Peng et al., 2019; Yan et al., 2021) Plant height, tillering, panicle branch, seed size (Sun et al., 2019a) Plant Small RNA in Food Crops Small RNA 90 Table 4.1 Summary of small RNAs and their targets in regulation of agronomic traits of six major food crops.dcont'd No identified No identified osa-miR1432 osa-miR1848 OsACOT13 OsCYP51G3 osa-miR5144 osa-miR5488 OsPDIL1;1 4CL-3 (predicted) osa-miR2118/21-nt phasiRNA PMS1T Acyl-CoA thioesterase Cytochrome P450 subfamily protein Protein disulfide isomerase 4 Coumarate: coenzyme A ligase lncRNA OsNRPD1a/b-dependent 24nt siRNA OsDCL3a-dependent 24-nt siRNA OsAGO18-dependent small RNA OsMIR156d/j D14 microRNA a/b hydrolase TE Transposable elements No identified No identified zma-miR156 tga1, tsh4 SPL (TF) zma-miR159 ZmMYB33, ZmMYB101 MYB (TF) Male fertility (Zhang et al., 2017b) Seed size (Zhao et al., 2019) Leaf angle, seed size (Xia et al., 2015) Seed size (Xia et al., 2018) Male fertility (Sun et al., 2021) Male fertility (Araki et al., 2020; Fan et al., 2016; Jiang et al., 2020; Zhang et al., 2020b) Tillering (Xu et al., 2020) Plant height, leaf angle (Wei et al., 2014) Male fertility (Das et al., 2020) Maize (Zea mays) Flower timing, floral organ development (Chuck et al. 2007, 2010, 2011) Seed dormancy/germination (Liu et al., 2020) 91 Continued Small RNAs as emerging regulators of agricultural traits of food crops osa-miR1428 Targets Characteristics zma-miR160 ZmARF10 ARF (TF) zma-miR164 ZmNAC1, ZmABCG26 NAC (TF), ATP-binding cassette transporter zma-miR165,zma-miR166 RLD1/2 HD-ZIP III (TF) zma-miR172 GL15, mSID1, mIDS1 AP2-like (TF) LBL1-dependent tasiRNA ZmARF2/3 ARF (TF) ZmAGO5c-dependent small RNA ZmDCL1-dependent microRNA No identified No identified No identified No identified TaSPL3/17, TaSPL2 SPL (TF) Agricultural traits and references Seed dormancy/germination (Liu et al., 2020) Lateral/crown root development, male fertility (Jiang et al., 2021; Li et al., 2012) Primary/lateral/crown root development (Gautam et al., 2021) Flower timing, floral organ development (Lauter et al., 2005) Primary/lateral/crown root development (Gautam et al., 2021) Male fertility (Li et al., 2021a) Male fertility (Field et al., 2016) Wheat (Triticum aestivum) tae-miR156 Tillering, plant height, flower timing (Jian et al., 2017; Liu et al., 2017) Plant Small RNA in Food Crops Small RNA 92 Table 4.1 Summary of small RNAs and their targets in regulation of agronomic traits of six major food crops.dcont'd TaNAC1, TaPSK5 NAC (TF) and A gene encoding a phytosulfokine precursor AP2-like (TF) tae-miR172 Q tae-miR408 TaTOC1 tae-miR9678/phasiRNAs WSGAR Clock-associated PRR (PSEUDO-RESPONSE REGULATOR) (TF) lncRNA hvu-miR171 HvSCL GRFs family protein (TF) hvu-miR172 HvCLY1 AP2-like (TF) RdDM-dependent 24-nt siRNA HvCKX2.1 Cytokinin oxidase GmSPL3, GmSPL9, other GmSPLs SPL (TF) Lateral/crown root development (Geng et al., 2020; Li et al., 2021c) Floral organ development (Debernardi et al., 2017) Flower timing (Zhao et al., 2016) Seed dormancy/germination (Guo et al., 2018) Barley (Hordeum vulgare) Tillering, flower timing, floral organ development (Curaba et al., 2013) Flower timing, panicle branch, floral organ development (Brown et al., 2011; Nair et al., 2010) Seed dormancy/germination (Surdonja et al., 2017) Soybean (Glycine max) gma-miR156 Shoot branch, flower timing, seed size (Cao et al., 2015; Sun et al., 2019b) 93 Continued Small RNAs as emerging regulators of agricultural traits of food crops tae-miR164 Targets Characteristics gma-miR167 GmARF8a/b ARF (TF) PhasiRNAs (produced in shoots) mRNA phasiRNA precursors lncRNA stu-miR156 StSPL3, StSPL6, StSPL9, StSPL13, StLG1(LIGULELESS1) SPL (TF) stu-miR160 StARF10, StARF16 ARF (TF) stu-miR164 StNAC262 NAC (TF) stu-miR172 RAP1 AP2-like (TF) Agricultural traits and references Lateral root growth (Wang et al., 2015a) Root development (potential) (Li et al., 2021b) Potato (Solanum tuberosum) Shoot branch, flower timing (potential), tubers size (Bhogale et al., 2014; Kavas et al., 2017) Root development (Yang et al., 2021b) Lateral root growth (Zhang et al., 2018a) Flower timing, tubers size (Martin et al., 2009) Plant Small RNA in Food Crops Small RNA 94 Table 4.1 Summary of small RNAs and their targets in regulation of agronomic traits of six major food crops.dcont'd Small RNAs as emerging regulators of agricultural traits of food crops 95 Heterochromatic siRNAs comprise RdDM pathway-derived 24 nt sRNAs, which confer gene expression homeostasis through the action of DNA methylation. Literatures on the contribution of siRNA in food crops are scarce and further studies on the functions of siRNAs in the regulation of agricultural traits are necessary. An understanding of the appearance and function of abundant phasiRNAs in the reproductive organs of food crops will enhance our knowledge of sRNAs. Although this type of research is at the initial stage, it is worth noting that the study of unknown regulatory mechanisms controlled by phasiRNAs is valuable. A better understanding of the underlying mechanisms of sRNAs in food crops will not only broaden our understanding of sRNAs in general but also guide breeders in establishing their desired agricultural traits. Two excellent cases were derived from genetic variations in rice we discussed above: one is the IPA1 quantitative trait locus, a point mutation in OsSPL14 that disrupts the cleavage site of OsmiR156, generating an “ideal” rice plant with increased lodging resistance and enhanced grain yield; the other is a single nucleotide polymorphism in PMS1T near the OsmiR2118 target site, linked directly to photoperiod-sensitive male sterility, to which germplasm was applied for the two-line hybrid rice breeding. To date, the rapid development of genome editing by the CRISPR-Cas system has enabled precise genetic manipulation across food crop species and the creation of germplasms with beneficial traits. According to our knowledge of miRNAetarget modules and the components important to the production of sRNAs, the CRISPR-Cas system will enable the expansion of the range of sRNA applications in food crop breeding. Undoubtedly, the identification, validation, and functional analysis of an increasing number of sRNAs in food crops, with the assistance of advanced breeding technologies, will provide additional strategies to safeguard global food security. References Abe, M., Kobayashi, Y., Yamamoto, S., Daimon, Y., Yamaguchi, A., Ikeda, Y., Ichinoki, H., Notaguchi, M., Goto, K., & Araki, T. (2005). FD, a bZIP protein mediating signals from the floral pathway integrator FT at the shoot apex. Science, 309(5737), 1052e1056. https://doi.org/10.1126/science.1115983 Araki, S., Le, N. T., Koizumi, K., Villar-Briones, A., Nonomura, K. I., Endo, M., Inoue, H., Saze, H., & Komiya, R. (2020). miR2118-dependent U-rich phasiRNA production in rice anther wall development. Nature Communications, 11(1). https:// doi.org/10.1038/s41467-020-16637-3 Axtell, M. J. (2013). Classification and comparison of small RNAs from plants. Annual Review of Plant Biology, 64, 137e159. https://doi.org/10.1146/annurev-arplant050312-120043 96 Plant Small RNA in Food Crops Bai, B., Bian, H., Zeng, Z., Hou, N., Shi, B., Wang, J., Zhu, M., & Han, N. (2017). MiR393-Mediated auxin signaling regulation is involved in root elongation inhibition in response to toxic aluminum stress in barley. Plant and Cell Physiology, 58(3), 426e439. https://doi.org/10.1093/pcp/pcw211 Bastow, R., Mylne, J. S., Lister, C., Lippman, Z., Martienssen, R. A., & Dean, C. (2004). Vernalization requires epigenetic silencing of FLC by histone methylation. Nature, 427(6970), 164e167. https://doi.org/10.1038/nature02269 Bhogale, S., Mahajan, A. S., Natarajan, B., Rajabhoj, M., Thulasiram, H. V., & Banerjee, A. K. (2014). MicroRNA156: A potential graft-transmissible microrna that modulates plant architecture and tuberization in Solanum tuberosum ssp. andigena. Plant Physiology, 164(2), 1011e1027. https://doi.org/10.1104/pp.113.230714 Bian, H., Xie, Y., Guo, F., Han, N., Ma, S., Zeng, Z., Wang, J., Yang, Y., & Zhu, M. (2012). Distinctive expression patterns and roles of the miRNA393/TIR1 homolog module in regulating flag leaf inclination and primary and crown root growth in rice (Oryza sativa). New Phytologist, 196(1), 149e161. https://doi.org/10.1111/j.14698137.2012.04248.x Bologna, N. G., Iselin, R., Abriata, L. A., Sarazin, A., Pumplin, N., Jay, F., Grentzinger, T., Dal Peraro, M., & Voinnet, O. (2018). Nucleo-cytosolic shuttling of ARGONAUTE1 prompts a revised model of the plant MicroRNA pathway. Molecular Cell, 69(4), 709e719.e5. https://doi.org/10.1016/j.molcel.2018.01.007 Brown, R. H., & Bregitzer, P. (2011). A Ds insertional mutant of a barley miR172 gene results in indeterminate spikelet development. Crop Science, 51(4), 1664e1672. https:// doi.org/10.2135/cropsci2010.09.0532 Cao, D., Li, Y., Wang, J., Nan, H., Wang, Y., Lu, S., Jiang, Q., Li, X., Shi, D., Fang, C., Yuan, X., Zhao, X., Li, X., Liu, B., & Kong, F. (2015). GmmiR156b overexpression delays flowering time in soybean. Plant Molecular Biology, 89(4e5), 353e363. https:// doi.org/10.1007/s11103-015-0371-5 Chapman, E. J., & Estelle, M. (2009). Mechanism of auxin-regulated gene expression in plants. Annual Review of Genetics, 43, 265e285. https://doi.org/10.1146/annurevgenet-102108-134148 Chen, X. (2009). Small RNAs and their roles in plant development. Annual Review of Cell and Developmental Biology, 25, 21e44. https://doi.org/10.1146/annurev.cellbio. 042308.113417 Cheng, J. Z., Zhou, Y. P., Lv, T. X., Xie, C. P., & Tian, C. E. (2017). Research progress on the autonomous flowering time pathway in Arabidopsis. Physiology and Molecular Biology of Plants, 23(3), 477e485. https://doi.org/10.1007/s12298-017-0458-3 Che, R., Tong, H., Shi, B., Liu, Y., Fang, S., Liu, D., Xiao, Y., Hu, B., Liu, L., Wang, H., Zhao, M., & Chu, C. (2015). Control of grain size and rice yield by GL2-mediated brassinosteroid responses. Nature Plants, 1. https://doi.org/10.1038/nplants.2015.195 Chuck, G., Cigan, A. M., Saeteurn, K., & Hake, S. (2007). The heterochronic maize mutant Corngrass1 results from overexpression of a tandem microRNA. Nature Genetics, 39(4), 544e549. https://doi.org/10.1038/ng2001 Chuck, G., Meeley, R., & Hake, S. (2008). Floral meristem initiation and meristem cell fate are regulated by the maize AP2 genes ids1 and sid1. Development, 135(18), 3013e3019. https://doi.org/10.1242/dev.024273 Chuck, G. S., Tobias, C., Sun, L., Kraemer, F., Li, C., Dibble, D., Arora, R., Bragg, J. N., Vogel, J. P., Singh, S., Simmons, B. A., Pauly, M., & Hake, S. (2011). Overexpression of the maize Corngrass1 microRNA prevents flowering, improves digestibility, and increases starch content of switchgrass. Proceedings of the National Academy of Sciences of the United States of America, 108(42), 17550e17555. https://doi.org/10.1073/ pnas.1113971108 Small RNAs as emerging regulators of agricultural traits of food crops 97 Chuck, G., Whipple, C., Jackson, D., & Hake, S. (2010). The maize SBP-box transcription factor encoded by tasselsheath4 regulates bract development and the establishment of meristem boundaries. Development, 137(8), 1243e1250. https://doi.org/10.1242/ dev.048348 Coudert, Y., Périn, C., Courtois, B., Khong, N. G., & Gantet, P. (2010). Genetic control of root development in rice, the model cereal. Trends in Plant Science, 15(4), 219e226. https://doi.org/10.1016/j.tplants.2010.01.008 Curaba, J., Talbot, M., Li, Z., & Helliwell, C. (2013). Over-expression of microRNA171 affects phase transitions and floral meristem determinancy in barley. BMC Plant Biology, 13(1). https://doi.org/10.1186/1471-2229-13-6 Dahal, K., Li, X. Q., Tai, H., Creelman, A., & Bizimungu, B. (2019). Improving potato stress tolerance and tuber yield under a climate change scenario e a current overview. Frontiers of Plant Science, 10. https://doi.org/10.3389/fpls.2019.00563 Dai, Z., Wang, J., Yang, X., Lu, H., Miao, X., & Shi, Z. (2018). Modulation of plant architecture by the miR156f-OsSPL7-OsGH3.8 pathway in rice. Journal of Experimental Botany, 69(21), 5117e5130. https://doi.org/10.1093/jxb/ery273 Das, S., Swetha, C., Pachamuthu, K., Nair, A., & Shivaprasad, P. V. (2020). Loss of function of Oryza sativa Argonaute 18 induces male sterility and reduction in phased small RNAs. Plant Reproduction, 33(1), 59e73. https://doi.org/10.1007/s00497-020-00386-w Dastidar, M. G., Jouannet, V., & Maizel, A. (2012). Root branching: Mechanisms, robustness, and plasticity. Wiley Interdisciplinary Reviews: Developmental Biology, 1(3), 329e343. https://doi.org/10.1002/wdev.17 Debernardi, J. M., Lin, H., Chuck, G., Faris, J. D., & Dubcovsky, J. (2017). microRNA172 plays a crucial role in wheat spike morphogenesis and grain threshability. Development (Cambridge), 144(11), 1966e1975. https://doi.org/10.1242/dev.146399 Diao, Z., Yu, M., Bu, S., Duan, Y., Zhang, L., & Wu, W. (2018). Functional characterization of OsmiR396a in rice (Oryza sativa L.). Plant Growth Regulation, 85(3), 351e361. https://doi.org/10.1007/s10725-018-0406-4 Ding, X., Li, J., Zhang, H., He, T., Han, S., Li, Y., Yang, S., & Gai, J. (2016). Identification of miRNAs and their targets by high-throughput sequencing and degradome analysis in cytoplasmic male-sterile line NJCMS1A and its maintainer NJCMS1B of soybean. BMC Genomics, 17(1). https://doi.org/10.1186/s12864-015-2352-0 Erdmann, R. M., & Picard, C. L. (2020). RNA-directed DNA methylation. PLoS Genetics, 16(10). https://doi.org/10.1371/journal.pgen.1009034 Fan, T., Li, X., Yang, W., Xia, K., Ouyang, J., & Zhang, M. (2015). Rice osa-miR171c mediates phase change from vegetative to reproductive development and shoot apical meristem maintenance by repressing four OsHAM transcription factors. PLoS One, 10(5). https://doi.org/10.1371/journal.pone.0125833 Fan, Y., Yang, J., Mathioni, S. M., Yu, J., Shen, J., Yang, X., Wang, L., Zhang, Q., Cai, Z., Xu, C., Li, X., Xiao, J., Meyers, B. C., & Zhang, Q. (2016). PMS1T, producing Phased small-interfering RNAs, regulates photoperiod-sensitive male sterility in rice. Proceedings of the National Academy of Sciences of the United States of America, 113(52), 15144e15149. https://doi.org/10.1073/pnas.1619159114 Fei, Q., Xia, R., & Meyers, B. C. (2013). Phased, secondary, small interfering RNAs in posttranscriptional regulatory networks. The Plant Cell Online, 25(7), 2400e2415. https://doi.org/10.1105/tpc.113.114652 Field, S., & Thompson, B. (2016). Analysis of the maize dicer-like1 mutant, fuzzy tassel, implicates MicroRNAs in anther maturation and dehiscence. PLoS One, 11(1). https:// doi.org/10.1371/journal.pone.0146534 Gao, J., Chen, H., Yang, H., He, Y., Tian, Z., & Li, J. (2018). A brassinosteroid responsive miRNA-target module regulates gibberellin biosynthesis and plant development. New Phytologist, 220(2), 488e501. https://doi.org/10.1111/nph.15331 98 Plant Small RNA in Food Crops Gao, F., Wang, K., Liu, Y., Chen, Y., Chen, P., Shi, Z., Luo, J., Jiang, D., Fan, F., Zhu, Y., & Li, S. (2015). Blocking miR396 increases rice yield by shaping inflorescence architecture. Nature Plants, 1. https://doi.org/10.1038/nplants.2015.196 Gautam, V., Singh, A., Yadav, S., Singh, S., Kumar, P., Das, S. S., & Sarkar, A. K. (2021). Conserved LBL1-ta-siRNA and miR165/166-RLD1/2 modules regulate root development in maize. Development (Cambridge), 148(1). https://doi.org/10.1242/ dev.190033 Geng, Y., Jian, C., Xu, W., Liu, H., Hao, C., Hou, J., Liu, H., Zhang, X., & Li, T. (2020). miR164-targeted TaPSK5 encodes a phytosulfokine precursor that regulates root growth and yield traits in common wheat (Triticum aestivum L.). Plant Molecular Biology, 104(6), 615e628. https://doi.org/10.1007/s11103-020-01064-1 Gratten, J., & Visscher, P. M. (2016). Genetic pleiotropy in complex traits and diseases: Implications for genomic medicine. Genome Medicine, 8(1). https://doi.org/10.1186/ s13073-016-0332-x Guo, F., Han, N., Xie, Y., Fang, K., Yang, Y., Zhu, M., Wang, J., & Bian, H. (2016). The miR393a/target module regulates seed germination and seedling establishment under submergence in rice (Oryza sativa L.). Plant, Cell and Environment, 39(10), 2288e2302. https://doi.org/10.1111/pce.12781 Guo, G., Liu, X., Sun, F., Cao, J., Huo, N., Wuda, B., Xin, M., Hu, Z., Du, J., Xia, R., Rossi, V., Peng, H., Ni, Z., Sun, Q., & Yao, Y. (2018). Wheat miR9678 affects seed germination by generating phased siRNAs and modulating abscisic acid/gibberellin signaling. The Plant Cell Online, 30(4), 796e814. https://doi.org/10.1105/ tpc.17.00842 Guo, S., Sun, B., Looi, L.-S., Xu, Y., Gan, E.-S., Huang, J., & Ito, T. (2015). Co-ordination of flower development through epigenetic regulation in two model species: Rice and Arabidopsis. Plant and Cell Physiology, 56(5), 830e842. https://doi.org/10.1093/pcp/ pcv037 Guo, S., Xu, Y., Liu, H., Mao, Z., Zhang, C., Ma, Y., Zhang, Q., Meng, Z., & Chong, K. (2013). The interaction between OsMADS57 and OsTB1 modulates rice tillering via DWARF14. Nature Communications, 4. https://doi.org/10.1038/ncomms2542 Hochholdinger, F., & Zimmermann, R. (2008). Conserved and diverse mechanisms in root development. Current Opinion in Plant Biology, 11(1), 70e74. https://doi.org/10.1016/ j.pbi.2007.10.002 Huang, J., Li, Z., & Zhao, D. (2016). Deregulation of the OsmiR160 target gene OsARF18 causes growth and developmental defects with an alteration of auxin signaling in rice. Scientific Reports, 6. https://doi.org/10.1038/srep29938 Hu, J., Wang, Y., Fang, Y., Zeng, L., Xu, J., Yu, H., Shi, Z., Pan, J., Zhang, D., Kang, S., Zhu, L., Dong, G., Guo, L., Zeng, D., Zhang, G., Xie, L., Xiong, G., Li, J., & Qian, Q. (2015). A rare allele of GS2 enhances grain size and grain yield in rice. Molecular Plant, 8(10), 1455e1465. https://doi.org/10.1016/j.molp.2015.07.002 Jiang, D., Chen, W., Dong, J., Li, J., Yang, F., Wu, Z., Zhou, H., Wang, W., & Zhuang, C. (2018). Overexpression of miR164b-resistant OsNAC2 improves plant architecture and grain yield in rice. Journal of Experimental Botany, 69(7), 1533e1543. https://doi.org/ 10.1093/jxb/ery017 Jiang, P., Lian, B., Liu, C., Fu, Z., Shen, Y., Cheng, Z., & Qi, Y. (2020). 21-nt phasiRNAs direct target mRNA cleavage in rice male germ cells. Nature Communications, 11(1). https://doi.org/10.1038/s41467-020-19034-y Jiang, Y., Li, Z., Liu, X., Zhu, T., Xie, K., Hou, Q., Yan, T., Niu, C., Zhang, S., Yang, M., Xie, R., Wang, J., Li, J., An, X., & Wan, X. (2021). ZmFAR1 and ZmABCG26 regulated by microRNA are essential for lipid metabolism in maize anther. International Journal of Molecular Sciences, 22(15), 7916. https://doi.org/10.3390/ijms22157916 Small RNAs as emerging regulators of agricultural traits of food crops 99 Jian, C., Han, R., Chi, Q., Wang, S., Ma, M., Liu, X., & Zhao, H. (2017). Virus-based MicroRNA silencing and overexpressing in common wheat (Triticum aestivum L.). Frontiers of Plant Science, 8. https://doi.org/10.3389/fpls.2017.00500 Jiao, Y., Wang, Y., Xue, D., Wang, J., Yan, M., Liu, G., Dong, G., Zeng, D., Lu, Z., Zhu, X., Qian, Q., & Li, J. (2010). Regulation of OsSPL14 by OsmiR156 defines ideal plant architecture in rice. Nature Genetics, 42(6), 541e544. https://doi.org/10.1038/ ng.591 Johnson, C., Kasprzewska, A., Tennessen, K., Fernandes, J., Nan, G. L., Walbot, V., Sundaresan, V., Vance, V., & Bowman, L. H. (2009). Clusters and superclusters of phased small RNAs in the developing inflorescence of rice. Genome Research, 19(8), 1429e1440. https://doi.org/10.1101/gr.089854.108 Kavas, M., Kızıldogan, A. K., & Abanoz, B. (2017). Comparative genome-wide phylogenetic and expression analysis of SBP genes from potato (Solanum tuberosum). Computational Biology and Chemistry, 67, 131e140. https://doi.org/10.1016/ j.compbiolchem.2017.01.001 Kim, D. H., Doyle, M. R., Sung, S., & Amasino, R. M. (2009). Vernalization: Winter and the timing of flowering in plants. Annual Review of Cell and Developmental Biology, 25, 277e299. https://doi.org/10.1146/annurev.cellbio.042308.113411 Kobayashi, Y., & Weigel, D. (2007). Move on up, it’s time for change - mobile signals controlling photoperiod-dependent flowering. Genes & Development, 21(19), 2371e2384. https://doi.org/10.1101/gad.1589007 Komiya, R. (2017). Biogenesis of diverse plant phasiRNAs involves an miRNA-trigger and Dicer-processing. Journal of Plant Research, 130(1), 17e23. https://doi.org/10.1007/ s10265-016-0878-0 Komiya, R., Ohyanagi, H., Niihama, M., Watanabe, T., Nakano, M., Kurata, N., & Nonomura, K. I. (2014). Rice germline-specific Argonaute MEL1 protein binds to phasiRNAs generated from more than 700 lincRNAs. The Plant Journal, 78(3), 385e397. https://doi.org/10.1111/tpj.12483 Lauter, N., Kampani, A., Carlson, S., Goebel, M., & Moose, S. P. (2005). microRNA172 down-regulates glossy15 to promote vegetative phase change in maize. Proceedings of the National Academy of Sciences of the United States of America, 102(26), 9412e9417. https:// doi.org/10.1073/pnas.0503927102 Lee, J., & Lee, I. (2010). Regulation and function of SOC1, a flowering pathway integrator. Journal of Experimental Botany, 61(9), 2247e2254. https://doi.org/10.1093/jxb/erq098 Lee, Y. S., Lee, D. Y., Cho, L. H., & An, G. (2014). Rice miR172 induces flowering by suppressing OsIDS1 and SNB, two AP2 genes that negatively regulate expression of Ehd1 and florigens. Rice, 7(1). https://doi.org/10.1186/s12284-014-0031-4 Liebsch, D., & Palatnik, J. F. (2020). MicroRNA miR396, GRF transcription factors and GIF co-regulators: A conserved plant growth regulatory module with potential for breeding and biotechnology. Current Opinion in Plant Biology, 53, 31e42. https:// doi.org/10.1016/j.pbi.2019.09.008 Li, S., Gao, F., Xie, K., Zeng, X., Cao, Y., Zeng, J., He, Z., Ren, Y., Li, W., Deng, Q., Wang, S., Zheng, A., Zhu, J., Liu, H., Wang, L., & Li, P. (2016a). The OsmiR396cOsGRF4-OsGIF1 regulatory module determines grain size and yield in rice. Plant Biotechnology Journal, 14(11), 2134e2146. https://doi.org/10.1111/pbi.12569 Li, X., Xia, K., Liang, Z., Chen, K., Gao, C., & Zhang, M. (2016b). MicroRNA393 is involved in nitrogen-promoted rice tillering through regulation of auxin signal transduction in axillary buds. Scientific Reports, 6. https://doi.org/10.1038/srep32158 Li, Q., Gent, J. I., Zynda, G., Song, J., Makarevitch, I., Hirsch, C. D., Hirsch, C. N., Dawe, R. K., Madzima, T. F., McGinnis, K. M., Lisch, D., Schmitz, R. J., Vaughn, M. W., & Springer, N. M. (2015). RNA-directed DNA methylation enforces boundaries between heterochromatin and euchromatin in the maize genome. 100 Plant Small RNA in Food Crops Proceedings of the National Academy of Sciences of the United States of America, 112(47), 14728e14733. https://doi.org/10.1073/pnas.1514680112 Li, J., Guo, G., Guo, W., Guo, G., Tong, D., Ni, Z., Sun, Q., & Yao, Y. (2012). miRNA164-directed cleavage of ZmNAC1 confers lateral root development in maize (Zea mays L.). BMC Plant Biology, 12. https://doi.org/10.1186/1471-2229-12-220 Li, Y., Huang, Y., Pan, L., Zhao, Y., Huang, W., & Jin, W. (2021a). Male sterile 28 encodes an ARGONAUTE family protein essential for male fertility in maize. Chromosome Research, 29(2), 189e201. https://doi.org/10.1007/s10577-021-09653-6 Li, Y., Li, J., Chen, Z., Wei, Y., Qi, Y., & Wu, C. (2020). OsmiR167a-targeted auxin response factors modulate tiller angle via fine-tuning auxin distribution in rice. Plant Biotechnology Journal, 18(10), 2015e2026. https://doi.org/10.1111/pbi.13360 Li, S., Wang, X., Xu, W., Liu, T., Cai, C., Chen, L., Clark, C. B., & Ma, J. (2021b). Unidirectional movement of small RNAs from shoots to roots in interspecific heterografts. Nature Plants, 7(1), 50e59. https://doi.org/10.1038/s41477-020-00829-2 Li, J., Yang, Z., Yu, B., Liu, J., & Chen, X. (2005). Methylation protects miRNAs and siRNAs from a 30 -end uridylation activity in Arabidopsis. Current Biology, 15(16), 1501e1507. https://doi.org/10.1016/j.cub.2005.07.029 Li, J., Zhang, H., Zhu, J., Shen, Y., Zeng, N., Liu, S., Wang, H., Wang, J., & Zhan, X. (2021c). Role of miR164 in the growth of wheat new adventitious roots exposed to phenanthrene. Environmental Pollution, 284, 117204. https://doi.org/10.1016/ j.envpol.2021.117204 Liu, J., Cheng, X., Liu, P., & Sun, J. (2017). miR156-targeted SBP-box transcription factors interact with DWARF53 to regulate teosinte branched1 and barren STALK1 expression in bread wheat. Plant Physiology, 174(3), 1931e1948. https://doi.org/10.1104/ pp.17.00445 Liu, H., Guo, S., Xu, Y., Li, C., Zhang, Z., Zhang, D., Xu, S., Zhang, C., & Chong, K. (2014). OsmiR396d-regulated OsGRFs function in floral organogenesis in rice through binding to their targets OsJMJ706 and OsCR4. Plant Physiology, 165(1), 160e174. https://doi.org/10.1104/pp.114.235564 Liu, J., Guo, X., Zhai, T., Shu, A., Zhao, L., Liu, Z., & Zhang, S. (2020). Genome-wide identification and characterization of microRNAs responding to ABA and GA in maize embryos during seed germination. Plant Biology, 22(5), 949e957. https://doi.org/ 10.1111/plb.13142 Liu, H., Jia, S., Shen, D., Liu, J., Li, J., Zhao, H., Han, S., & Wang, Y. (2012). Four AUXIN RESPONSE FACTOR genes downregulated by microRNA167 are associated with growth and development in Oryza sativa. Functional Plant Biology, 39(9), 736e744. https://doi.org/10.1071/FP12106 Lloyd, J. P., Tsai, Z. T. Y., Sowers, R. P., Panchy, N. L., & Shiu, S. H. (2018). A modelbased approach for identifying functional intergenic transcribed regions and noncoding RNAs. Molecular Biology and Evolution, 35(6), 1422e1436. https://doi.org/10.1093/ molbev/msy035 Lombardi, M., De Gara, L., & Loreto, F. (2021). Determinants of root system architecture for future-ready, stress-resilient crops. Physiologia Plantarum, 172(4), 2090e2097. https://doi.org/10.1111/ppl.13439 Lu, Y., Feng, Z., Liu, X., Bian, L., Xie, H., Zhang, C., Mysore, K. S., & Liang, J. (2018). MiR393 and miR390 synergistically regulate lateral root growth in rice under different conditions. BMC Plant Biology, 18(1). https://doi.org/10.1186/s12870-018-1488-x  urczak, M., González-Schain, N. D., & Martin, A., Adam, H., Díaz-Mendoza, M., Z Suárez-López, P. (2009). Graft-transmissible induction of potato tuberization by the microRNA miR172. Development, 136(17), 2873e2881. https://doi.org/10.1242/ dev.031658 Small RNAs as emerging regulators of agricultural traits of food crops 101 Ma, X., Shao, C., Wang, H., Jin, Y., & Meng, Y. (2013). Construction of small RNAmediated gene regulatory networks in the roots of rice (Oryza sativa). BMC Genomics, 14. https://doi.org/10.1186/1471-2164-14-510 Miao, C., Wang, D., He, R., Liu, S., & Zhu, J. K. (2020). Mutations in MIR396e and MIR396f increase grain size and modulate shoot architecture in rice. Plant Biotechnology Journal, 18(2), 491e501. Miao, C., Wang, Z., Zhang, L., Yao, J., Hua, K., Liu, X., Shi, H., & Zhu, J. K. (2019). The grain yield modulator miR156 regulates seed dormancy through the gibberellin pathway in rice. Nature Communications, 10(1). https://doi.org/10.1038/s41467-01911830-5 Moyroud, E., Kusters, E., Monniaux, M., Koes, R., & Parcy, F. (2010). LEAFY blossoms. Trends in Plant Science, 15(6), 346e352. https://doi.org/10.1016/j.tplants.2010.03.007 Mutasa-Göttgens, E., & Hedden, P. (2009). Gibberellin as a factor in floral regulatory networks. Journal of Experimental Botany, 60(7), 1979e1989. https://doi.org/10.1093/ jxb/erp040 Nair, S. K., Wang, N., Turuspekov, Y., Pourkheirandish, M., Sinsuwongwat, S., Chen, G., Sameri, M., Tagiri, A., Honda, I., Watanabe, Y., Kanamori, H., Wicker, T., Stein, N., Nagamura, Y., Matsumoto, T., & Komatsuda, T. (2010). Cleistogamous flowering in barley arises from the suppression of microRNA-guided HvAP2 mRNA cleavage. Proceedings of the National Academy of Sciences of the United States of America, 107(1), 490e495. https://doi.org/10.1073/pnas.0909097107 Penfield, S. (2017). Seed dormancy and germination. Current Biology, 27(17), R874eR878. https://doi.org/10.1016/j.cub.2017.05.050 Peng, T., Qiao, M., Liu, H., Teotia, S., Zhang, Z., Zhao, Y., Wang, B., Zhao, D., Shi, L., Zhang, C., Le, B., Rogers, K., Gunasekara, C., Duan, H., Gu, Y., Tian, L., Nie, J., Qi, J., Meng, F., … Tang, G. (2018). A resource for inactivation of MicroRNAs using short tandem target mimic technology in model and crop plants. Molecular Plant, 11(11), 1400e1417. https://doi.org/10.1016/j.molp.2018.09.003 Peng, T., Teotia, S., Tang, G., & Zhao, Q. (2019). MicroRNAs meet with quantitative trait loci: Small powerful players in regulating quantitative yield traits in rice. Wiley Interdisciplinary Reviews: RNA, 10(6). https://doi.org/10.1002/wrna.1556 Qiao, J., Jiang, H., Lin, Y., Shang, L., Wang, M., Li, D., Fu, X., Geisler, M., Qi, Y., Gao, Z., & Qian, Q. (2021). A novel miR167a-OsARF6-OsAUX3 module regulates grain length and weight in rice. Molecular Plant, 14(10), 1683e1698. https://doi.org/ 10.1016/j.molp.2021.06.023 Qi, Y., Wang, S., Shen, C., Zhang, S., Chen, Y., Xu, Y., Liu, Y., Wu, Y., & Jiang, D. (2012). OsARF12, a transcription activator on auxin response gene, regulates root elongation and affects iron accumulation in rice (Oryza sativa). New Phytologist, 193(1), 109e120. https://doi.org/10.1111/j.1469-8137.2011.03910.x Qu, L., Lin, L. B., & Xue, H. W. (2019). Rice miR394 suppresses leaf inclination through targeting an F-box gene, LEAF INCLINATION 4. Journal of Integrative Plant Biology, 61(4), 406e416. https://doi.org/10.1111/jipb.12713 Rai, M. I., Alam, M., Lightfoot, D. A., Gurha, P., & Afzal, A. J. (2019). Classification and experimental identification of plant long non-coding RNAs. Genomics, 111(5), 997e1005. https://doi.org/10.1016/j.ygeno.2018.04.014 Ray, D. K., Mueller, N. D., West, P. C., & Foley, J. A. (2013). Yield trends are insufficient to double global crop production by 2050. PLoS One, 8(6). https://doi.org/10.1371/ journal.pone.0066428 Rich, S. M., & Watt, M. (2013). Soil conditions and cereal root system architecture: Review and considerations for linking Darwin and Weaver. Journal of Experimental Botany, 64(5), 1193e1208. https://doi.org/10.1093/jxb/ert043 102 Plant Small RNA in Food Crops Ron, M., Saez, M. A., Williams, L. E., Fletcher, J. C., & McCormick, S. (2010). Proper regulation of a sperm-specific cis-nat-siRNA is essential for double fertilization in Arabidopsis. Genes & Development, 24(10), 1010e1021. https://doi.org/10.1101/ gad.1882810 Roychoudhry, S., & Kepinski, S. (2021). Auxin in root development. Cold Spring Harbor perspectives in biology. Sakamoto, T., & Matsuoka, M. (2004). Generating high-yielding varieties by genetic manipulation of plant architecture. Current Opinion in Biotechnology, 15(2), 144e147. https://doi.org/10.1016/j.copbio.2004.02.003 Sasaki, A., Ashikari, M., Ueguchi-Tanaka, M., Itoh, H., Nishimura, A., Swapan, D., Ishiyama, K., Saito, T., Kobayashi, M., Khush, G. S., Kitano, H., & Matsuoka, M. (2002). A mutant gibberellin-synthesis gene in rice. Nature, 416(6882), 701e702. https://doi.org/10.1038/416701a Shao, Y., Zhou, H. Z., Wu, Y., Zhang, H., Lin, J., Jiang, X., He, Q., Zhu, J., Li, Y., Yu, H., & Maoa, C. (2019). OsSPL3, an SBP-domain protein, regulates crown root development in rice. The Plant Cell Online, 31(6), 1257e1275. https://doi.org/ 10.1105/tpc.19.00038 Shu, K., Liu, X. D., Xie, Q., & He, Z. H. (2016). Two faces of one seed: Hormonal regulation of dormancy and germination. Molecular Plant, 9(1), 34e45. https://doi.org/ 10.1016/j.molp.2015.08.010 Shu, K., Meng, Y. J., Shuai, H. W., Liu, W. G., Du, J. B., Liu, J., & Yang, W. Y. (2015). Dormancy and germination: How does the crop seed decide? Plant Biology, 17(6), 1104e1112. https://doi.org/10.1111/plb.12356 Sun, Y., Mui, Z., Liu, X., Yim, A., Qin, H., Wong, F.-L., Chan, T.-F., Yiu, S.-M., Lam, H.-M., & Lim, B. (2016). Comparison of small RNA profiles of Glycine max and Glycine soja at early developmental stages. International Journal of Molecular Sciences, 17(12), 2043. https://doi.org/10.3390/ijms17122043 Sun, M., Shen, Y., Li, H., Yang, J., Cai, X., Zheng, G., Zhu, Y., Jia, B., & Sun, X. (2019a). The multiple roles of OsmiR535 in modulating plant height, panicle branching and grain shape. Plant Science, 283, 60e69. https://doi.org/10.1016/j.plantsci.2019.02.002 Sun, Z., Su, C., Yun, J., Jiang, Q., Wang, L., Wang, Y., Cao, D., Zhao, F., Zhao, Q., Zhang, M., Zhou, B., Zhang, L., Kong, F., Liu, B., Tong, Y., & Li, X. (2019b). Genetic improvement of the shoot architecture and yield in soya bean plants via the manipulation of GmmiR156b. Plant Biotechnology Journal, 17(1), 50e62. https://doi.org/ 10.1111/pbi.12946 Sun, Y., Xiong, X., Wang, Q., Zhu, L., Wang, L., He, Y., & Zeng, H. (2021). Integrated analysis of small RNA, transcriptome, and degradome sequencing reveals the MiR156, MiR5488 and MiR399 are involved in the regulation of male sterility in PTGMS rice. International Journal of Molecular Sciences, 22(5), 2260. https://doi.org/10.3390/ ijms22052260 Surdonja, K., Eggert, K., Hajirezaei, M. R., Harshavardhan, V. T., Seiler, C., von Wirén, N., Sreenivasulu, N., & Kuhlmann, M. (2017). Increase of dna methylation at the hvckx2.1 promoter by terminal drought stress in barley. Epigenomes, 1(2). https:// doi.org/10.3390/epigenomes1020009 Svolacchia, N., Salvi, E., & Sabatini, S. (2020). Arabidopsis primary root growth: Let it grow, can’t hold it back anymore! Current Opinion in Plant Biology, 57, 133e141. https://doi.org/10.1016/j.pbi.2020.08.005 Takeda, S., & Matsuoka, M. (2008). Genetic approaches to crop improvement: Responding to environmental and population changes. Nature Reviews Genetics, 9(6), 444e457. https://doi.org/10.1038/nrg2342 Tang, J., & Chu, C. (2017). MicroRNAs in crop improvement: Fine-tuners for complex traits. Nature Plants, 3. https://doi.org/10.1038/nplants.2017.77 Small RNAs as emerging regulators of agricultural traits of food crops 103 Tang, Y., Liu, H., Guo, S., Wang, B., Li, Z., Chong, K., & Xu, Y. (2018). OsmiR396d affects gibberellin and brassinosteroid signaling to regulate plant architecture in rice. Plant Physiology, 176(1), 946e959. https://doi.org/10.1104/pp.17.00964 Teotia, S., & Tang, G. (2015). To bloom or not to bloom: Role of micrornas in plant flowering. Molecular Plant, 8(3), 359e377. https://doi.org/10.1016/j.molp.2014.12.018 Thody, J., Folkes, L., & Moulton, V. (2020). NATpare: A pipeline for high-throughput prediction and functional analysis of nat-siRNAs. Nucleic Acids Research, 48(12), 6481e6490. https://doi.org/10.1093/nar/gkaa448 Thomson, B., & Wellmer, F. (2019). Molecular regulation of flower development, Vol 131. Current topics in developmental biology (pp. 185e210). Academic Press Inc. https:// doi.org/10.1016/bs.ctdb.2018.11.007 Tian, J., Wang, C., Xia, J., Wu, L., Xu, G., Wu, W., Li, D., Qin, W., Han, X., Chen, Q., Jin, W., & Tian, F. (2019). Teosinte ligule allele narrows plant architecture and enhances high-density maize yields. Science, 365(6454), 658e664. https://doi.org/ 10.1126/science.aax5482 Tognacca, R. S., & Botto, J. F. (2021). Post-transcriptional regulation of seed dormancy and germination: Current understanding and future directions. Plant Communications, 2(4). https://doi.org/10.1016/j.xplc.2021.100169 Tsuji, H., Aya, K., Ueguchi-Tanaka, M., Shimada, Y., Nakazono, M., Watanabe, R., Nishizawa, N. K., Gomi, K., Shimada, A., Kitano, H., Ashikari, M., & Matsuoka, M. (2006). GAMYB controls different sets of genes and is differentially regulated by microRNA in aleurone cells and anthers. The Plant Journal, 47(3), 427e444. https:// doi.org/10.1111/j.1365-313X.2006.02795.x Varshney, R. K., Hoisington, D. A., & Tyagi, A. K. (2006). Advances in cereal genomics and applications in crop breeding. Trends in Biotechnology, 24(11), 490e499. https:// doi.org/10.1016/j.tibtech.2006.08.006 Voinnet, O. (2009). Origin, biogenesis, and activity of plant MicroRNAs. Cell, 136(4), 669e687. https://doi.org/10.1016/j.cell.2009.01.046 Wang, Y., Li, K., Chen, L., Zou, Y., Liu, H., Tian, Y., Li, D., Wang, R., Zhao, F., Ferguson, B. J., Gresshoff, P. M., & Li, X. (2015a). MicroRNA167-Directed regulation of the auxin response factors GmARF8a and GmARF8b is required for soybean nodulation and lateral root development. Plant Physiology, 168(3), 984e999. https:// doi.org/10.1104/pp.15.00265 Wang, L., Sun, S., Jin, J., Fu, D., Yang, X., Weng, X., Xu, C., Li, X., Xiao, J., & Zhang, Q. (2015b). Coordinated regulation of vegetative and reproductive branching in rice. Proceedings of the National Academy of Sciences of the United States of America, 112(50), 15504e15509. https://doi.org/10.1073/pnas.1521949112 Wang, H., Li, Y., Chern, M., Zhu, Y., Zhang, L. L., Lu, J. H., Li, X. P., Dang, W. Q., Ma, X. C., Yang, Z. R., Yao, S. Z., Zhao, Z. X., Fan, J., Huang, Y. Y., Zhang, J. W., Pu, M., Wang, J., He, M., Li, W. T., … Wang, W. M. (2021a). Suppression of rice miR168 improves yield, flowering time and immunity. Nature Plants, 7(2), 129e136. https://doi.org/10.1038/s41477-021-00852-x Wang, R., Yang, X., Guo, S., Wang, Z., Zhang, Z., & Fang, Z. (2021b). MiR319-targeted OsTCP21 and OsGAmyb regulate tillering and grain yield in rice. Journal of Integrative Plant Biology, 63(7), 1260e1272. https://doi.org/10.1111/jipb.13097 Wang, L., Liu, H., Li, D., & Chen, H. (2011). Identification and characterization of maize microRNAs involved in the very early stage of seed germination. BMC Genomics, 12. https://doi.org/10.1186/1471-2164-12-154 Wang, B., Smith, S. M., & Li, J. (2018). Genetic regulation of shoot architecture. Annual Review of Plant Biology, 69, 437e468. https://doi.org/10.1146/annurev-arplant042817-040422 104 Plant Small RNA in Food Crops Wang, M., Wu, H. J., Fang, J., Chu, C., & Wang, X. J. (2017). A long noncoding RNA involved in rice reproductive development by negatively regulating osa-miR160. Science Bulletin, 62(7), 470e475. https://doi.org/10.1016/j.scib.2017.03.013 Wang, S., Wu, K., Yuan, Q., Liu, X., Liu, Z., Lin, X., Zeng, R., Zhu, H., Dong, G., Qian, Q., Zhang, G., & Fu, X. (2012). Control of grain size, shape and quality by OsSPL16 in rice. Nature Genetics, 44(8), 950e954. https://doi.org/10.1038/ng.2327 Wei, L., Gu, L., Song, X., Cui, X., Lu, Z., Zhou, M., Wang, L., Hu, F., Zhai, J., Meyers, B. C., & Cao, X. (2014). Dicer-like 3 produces transposable element-associated 24-nt siRNAs that control agricultural traits in rice. Proceedings of the National Academy of Sciences of the United States of America, 111(10), 3877e3882. https://doi.org/10.1073/ pnas.1318131111 Wendte, J. M., & Pikaard, C. S. (2017). The RNAs of RNA-directed DNA methylation. Biochimica et Biophysica Acta - Gene Regulatory Mechanisms, 1860(1), 140e148. https:// doi.org/10.1016/j.bbagrm.2016.08.004 Wierzbicki, A. T., Cocklin, R., Mayampurath, A., Lister, R., Jordan Rowley, M., Gregory, B. D., Ecker, J. R., Tang, H., & Pikaard, C. S. (2012). Spatial and functional relationships among Pol V-associated loci, Pol IV-dependent siRNAs, and cytosine methylation in the Arabidopsis epigenome. Genes & Development, 26(16), 1825e1836. https://doi.org/10.1101/gad.197772.112 Wilusz, J. E., Sunwoo, H., & Spector, D. L. (2009). Long noncoding RNAs: Functional surprises from the RNA world. Genes & Development, 23(13), 1494e1504. https:// doi.org/10.1101/gad.1800909 Xia, K., Ou, X., Tang, H., Wang, R., Wu, P., Jia, Y., Wei, X., Xu, X., Kang, S. H., Kim, S. K., & Zhang, M. (2015). Rice microRNA osa-miR1848 targets the obtusifoliol 14a-demethylase gene OsCYP51G3 and mediates the biosynthesis of phytosterols and brassinosteroids during development and in response to stress. New Phytologist, 208(3), 790e802. https://doi.org/10.1111/nph.13513 Xia, K., Wang, R., Ou, X., Fang, Z., Tian, C., Duan, J., Wang, Y., Zhang, M., & Zhang, B. (2012). OsTIR1 and OsAFB2 downregulation via OsmiR393 overexpression leads to more tillers, early flowering and less tolerance to salt and drought in rice. PLoS One, 7(1), e30039. https://doi.org/10.1371/journal.pone.0030039 Xia, K., Zeng, X., Jiao, Z., Li, M., Xu, W., Nong, Q., Mo, H., Cheng, T., & Zhang, M. (2018). Formation of protein disulfide bonds catalyzed by OsPDIL1;1 is mediated by MicroRNA5144-3p in rice. Plant and Cell Physiology, 59(2), 331e342. https://doi.org/ 10.1093/pcp/pcx189 Xie, D., Chen, M., Niu, J., Wang, L., Li, Y., Fang, X., Li, P., & Qi, Y. (2021). Phase separation of SERRATE drives dicing body assembly and promotes miRNA processing in Arabidopsis. Nature Cell Biology, 23(1), 32e39. https://doi.org/10.1038/s41556-02000606-5 Xie, K., Wu, C., & Xiong, L. (2006). Genomic organization, differential expression, and interaction of SQUAMOSA promoter-binding-like transcription factors and microRNA156 in rice. Plant Physiology, 142(1), 280e293. https://doi.org/10.1104/ pp.106.084475 Xing, Y., & Zhang, Q. (2010). Genetic and molecular bases of rice yield. Annual Review of Plant Biology, 61, 421e442. https://doi.org/10.1146/annurev-arplant-042809-112209 Xu, L., Yuan, K., Yuan, M., Meng, X., Chen, M., Wu, J., Li, J., & Qi, Y. (2020). Regulation of rice tillering by RNA-directed DNA methylation at miniature invertedrepeat transposable elements. Molecular Plant, 13(6), 851e863. https://doi.org/10.1016/ j.molp.2020.02.009 Yamaguchi, A., & Abe, M. (2012). Regulation of reproductive development by non-coding RNA in Arabidopsis: To flower or not to flower. Journal of Plant Research, 125(6), 693e704. https://doi.org/10.1007/s10265-012-0513-7 Small RNAs as emerging regulators of agricultural traits of food crops 105 Yan, A., & Chen, Z. (2020). The control of seed dormancy and germination by temperature, light and nitrate. The Botanical Review, 86(1), 39e75. https://doi.org/10.1007/ s12229-020-09220-4 Yang, R., Li, P., Mei, H., Wang, D., Sun, J., Yang, C., Hao, L., Cao, S., Chu, C., Hu, S., Song, X., & Cao, X. (2019). Fine-tuning of MiR528 accumulation modulates flowering time in rice. Molecular Plant, 12(8), 1103e1113. https://doi.org/10.1016/ j.molp.2019.04.009 Yang, X., You, C., Wang, X., Gao, L., Mo, B., Liu, L., & Chen, X. (2021a). Widespread occurrence of microRNA-mediated target cleavage on membrane-bound polysomes. Genome Biology, 22(1). https://doi.org/10.1186/s13059-020-02242-6 Yang, J., Zhang, N., Zhang, J., Jin, X., Zhu, X., Ma, R., Li, S., Lui, S., Yue, Y., & Si, H. (2021b). Knockdown of MicroRNA160a/b by STTM leads to root architecture changes via auxin signaling in Solanum tuberosum. Plant Physiology and Biochemistry, 166, 939e949. https://doi.org/10.1016/j.plaphy.2021.06.051 Yan, Y., Wang, H., Hamera, S., Chen, X., & Fang, R. (2014). MiR444a has multiple functions in the rice nitrate-signaling pathway. The Plant Journal, 78(1), 44e55. https:// doi.org/10.1111/tpj.12446 Yan, Y., Wei, M., Li, Y., Tao, H., Wu, H., Chen, Z., Li, C., & Xu, J. H. (2021). MiR529a controls plant height, tiller number, panicle architecture and grain size by regulating SPL target genes in rice (Oryza sativa L.). Plant Science, 302. https://doi.org/10.1016/ j.plantsci.2020.110728 Yoshida, H., & Nagato, Y. (2011). Flower development in rice. Journal of Experimental Botany, 62(14), 4719e4730. https://doi.org/10.1093/jxb/err272 Yu, Y., Jia, T., & Chen, X. (2017a). The ‘how’ and ‘where’ of plant microRNAs. New Phytologist, 216(4), 1002e1017. https://doi.org/10.1111/nph.14834 Yu, Y., Li, Q. F., Zhang, J. P., Zhang, F., Zhou, Y. F., Feng, Y. Z., Chen, Y. Q., & Zhang, Y. C. (2017b). Laccase-13 regulates seed setting rate by affecting hydrogen peroxide dynamics and mitochondrial integrity in rice. Frontiers of Plant Science, 8. https://doi.org/10.3389/fpls.2017.01324 Yu, Y., Zhang, Y., Chen, X., & Chen, Y. (2019). Plant noncoding RNAs: Hidden players in development and stress responses. Annual Review of Cell and Developmental Biology, 35(1), 407e431. https://doi.org/10.1146/annurev-cellbio-100818-125218 Zhang, M., Cui, G., Bai, X., Ye, Z., Zhang, S., Xie, K., Sun, F., Zhang, C., & Xi, Y. (2021). Regulatory network of preharvest sprouting resistance revealed by integrative analysis of mRNA, noncoding RNA, and DNA methylation in wheat. Journal of Agricultural and Food Chemistry, 69(13), 4018e4035. https://doi.org/10.1021/ acs.jafc.1c00050 Zhang, Y. C., He, R. R., Lian, J. P., Zhou, Y. F., Zhang, F., Li, Q. F., Yu, Y., Feng, Y. Z., Yang, Y. W., Lei, M. Q., He, H., Zhang, Z., & Chen, Y. Q. (2020a). OsmiR528 regulates rice-pollen intine formation by targeting an uclacyanin to influence flavonoid metabolism. Proceedings of the National Academy of Sciences of the United States of America, 117(1), 727e732. https://doi.org/10.1073/pnas.1810968117 Zhang, Y. C., Lei, M. Q., Zhou, Y. F., Yang, Y. W., Lian, J. P., Yu, Y., Feng, Y. Z., Zhou, K. R., He, R. R., He, H., Zhang, Z., Yang, J. H., & Chen, Y. Q. (2020b). Reproductive phasiRNAs regulate reprogramming of gene expression and meiotic progression in rice. Nature Communications, 11(1). https://doi.org/10.1038/s41467-02019922-3 Zhang, S., Wang, S., Xu, Y., Yu, C., Shen, C., Qian, Q., Geisler, M., Jiang, D. A., & Qi, Y. (2015). The auxin response factor, OsARF19, controls rice leaf angles through positively regulating OsGH3-5 and OsBRI1. Plant, Cell and Environment, 38(4), 638e654. https://doi.org/10.1111/pce.12397 106 Plant Small RNA in Food Crops Zhang, J. P., Yu, Y., Feng, Y. Z., Zhou, Y. F., Zhang, F., Yang, Y. W., Lei, M. Q., Zhang, Y. C., & Chen, Y. Q. (2017a). MiR408 regulates grain yield and photosynthesis via a phytocyanin protein. Plant Physiology, 175(3), 1175e1185. https://doi.org/ 10.1104/pp.17.01169 Zhang, H., Zhang, J., Yan, J., Gou, F., Mao, Y., Tang, G., Botella, J. R., & Zhu, J. K. (2017b). Short tandem target mimic rice lines uncover functions of miRNAs in regulating important agronomic traits. Proceedings of the National Academy of Sciences of the United States of America, 114(20), 5277e5282. https://doi.org/10.1073/ pnas.1703752114 Zhang, Y. C., Yu, Y., Wang, C. Y., Li, Z. Y., Liu, Q., Xu, J., Liao, J. Y., Wang, X. J., Qu, L. H., Chen, F., Xin, P., Yan, C., Chu, J., Li, H. Q., & Chen, Y. Q. (2013). Overexpression of microRNA OsmiR397 improves rice yield by increasing grain size and promoting panicle branching. Nature Biotechnology, 31(9), 848e852. https:// doi.org/10.1038/nbt.2646 Zhang, L., Yao, L., Zhang, N., Yang, J., Zhu, X., Tang, X., Calderón-Urrea, A., & Si, H. (2018a). Lateral root development in potato is mediated by Stu-mi164 regulation of NAC transcription factor. Frontiers of Plant Science, 9. https://doi.org/10.3389/ fpls.2018.00383 Zhang, J., Zhang, H., Srivastava, A. K., Pan, Y., Bai, J., Fang, J., Shi, H., & Zhu, J. K. (2018b). Knockdown of rice microRNA166 confers drought resistance by causing leaf rolling and altering stem xylem development. Plant Physiology, 176(3), 2082e2094. https://doi.org/10.1104/pp.17.01432 Zhang, F., Zhang, Y. C., Zhang, J. P., Yu, Y., Zhou, Y. F., Feng, Y. Z., Yang, Y. W., Lei, M. Q., He, H., Lian, J. P., & Chen, Y. Q. (2018c). Rice UCL8, a plantacyanin gene targeted by miR408, regulates fertility by controlling pollen tube germination and growth. Rice, 11(1). https://doi.org/10.1186/s12284-018-0253-y Zhang, J., Zhou, Z., Bai, J., Tao, X., Wang, L., Zhang, H., & Zhu, J. K. (2020c). Disruption of MIR396e and MIR396f improves rice yield under nitrogen-deficient conditions. National Science Review, 7(1), 102e112. https://doi.org/10.1093/nsr/nwz142 Zhao, X. Y., Hong, P., Wu, J. Y., Chen, X. B., Ye, X. G., Pan, Y. Y., Wang, J., & Zhang, X. S. (2016). The tae-miR408-mediated control of taTOC1 genes transcription is required for the regulation of heading time in wheat. Plant Physiology, 170(3), 1578e1594. https://doi.org/10.1104/pp.15.01216 Zhao, Y. F., Peng, T., Sun, H. Z., Teotia, S., Wen, H. L., Du, Y. X., Zhang, J., Li, J. Z., Tang, G. L., Xue, H. W., & Zhao, Q. Z. (2019). miR1432-OsACOT (Acyl-CoA thioesterase) module determines grain yield via enhancing grain filling rate in rice. Plant Biotechnology Journal, 17(4), 712e723. https://doi.org/10.1111/pbi.13009 Zhao, Y., Wen, H., Teotia, S., Du, Y., Zhang, J., Li, J., Sun, H., Tang, G., Peng, T., & Zhao, Q. (2017). Suppression of microRNA159 impacts multiple agronomic traits in rice (Oryza sativa L.). BMC Plant Biology, 17(1). https://doi.org/10.1186/s12870-017-1171-7 Zhong, J., He, W., Peng, Z., Zhang, H., Li, F., & Yao, J. (2020). A putative AGO protein, OsAGO17, positively regulates grain size and grain weight through OsmiR397b in rice. Plant Biotechnology Journal, 18(4), 916e928. https://doi.org/10.1111/pbi.13256 Zhou, M., Palanca, A. M. S., & Law, J. A. (2018). Locus-specific control of the de novo DNA methylation pathway in Arabidopsis by the CLASSY family. Nature Genetics, 50(6), 865e873. https://doi.org/10.1038/s41588-018-0115-y Zhou, Y., Zhou, S., Wang, L., Wu, D., Cheng, H., Du, X., Mao, D., Zhang, C., & Jiang, X. (2020). miR164c and miR168a regulate seed vigor in rice. Journal of Integrative Plant Biology, 62(4), 470e486. https://doi.org/10.1111/jipb.12792 Zhu, Q. H., Upadhyaya, N. M., Gubler, F., & Helliwell, C. A. (2009). Over-expression of miR172 causes loss of spikelet determinacy and floral organ abnormalities in rice (Oryza sativa). BMC Plant Biology, 9. https://doi.org/10.1186/1471-2229-9-149 SECTION 2 Small RNA from food crops: mechanism and regulation 107 This page intentionally left blank CHAPTER 5 Exploring small RNA in food crops: techniques and approaches Saurabh Chaudhary School of Biosciences, Cardiff University, Cardiff, United Kingdom 1. Introduction Small RNAs (sRNAs) emerged as one of the potential molecular targets for improving food crops (Chaudhary et al., 2021; Kamthan et al., 2015, 2016; Summanwar et al., 2020). The class of sRNAs belongs to the small (1830bp) non-coding RNA that regulates the gene transcriptional and translation activities in plants and animals (Storz, 2002). In the last two decades, various plant-specific sRNAs have been discovered and continue to be found as technologies, origins, and applications of sRNAs progress. Depending upon the function and biogenesis, plant sRNAs are categorized into two (i) hairpin-derived sRNA (hpsRNA) such as microRNA (miRNA) (Reinhart et al., 2002); (ii) small interfering RNA (siRNA) (Hamilton & Baulcombe, 1999) such as trans-acting siRNA (ta-siRNA), natural antisense transcript siRNA (nat-siRNA), and heterochromatin siRNA (hc-siRNA) (Morgado, 2020). In plants, sRNAs perform multiple functions, including stress response, immunity, DNA repair and genome stability at different development and growth stages (Jin, 2008; Slotkin et al., 2009; Sunkar et al., 2007; Wei et al., 2012). Among all, RNA interference (RNAi) or RNA silencing is the most studied and understood mechanism where miRNA or siRNA transcriptionally or posttranscriptionally degrades the messenger RNA (mRNA) and stops translation. In RNAi, a sequence-specific small (double-stranded RNA) dsRNA (miRNA or siRNA) modifies the expression of an endogenous gene, which is considered the most potent approach of genetic engineering to improve the food crop varieties (Frizzi & Huang, 2010; Sabu & Nadiya, 2020). In plants, the RNAi technique has been utilized for fundamental discoveries in the model system, including Arabidopsis thaliana (Beris et al., 2022; Gao et al., 2018; Guan et al., 2013; Huang et al., 2006), and applied Plant Small RNA in Food Crops ISBN 978-0-323-91722-3 https://doi.org/10.1016/B978-0-323-91722-3.00016-6 © 2023 Elsevier Inc. All rights reserved. 109 110 Plant Small RNA in Food Crops in a variety of food crops such as potato (Andersson et al., 2006), tomato (Ammara et al., 2015; Sun et al., 2012; Vu et al., 2013), rice (Ahmed et al., 2017; Khandagale et al., 2020; Zhao et al., 2020), Wheat (Fu et al., 2007; Marín-Sanz et al., 2022; Wilkinson et al., 2021), and many others. One can explore the RNAi technique and its implementation in food crops somewhere in the current book series on “Small RNA in food crops”. In the present chapter, the potential techniques and approaches which can be utilized to explore the sRNAs in food crops are discussed systematically and in detail. The emergence of high-throughput next-generation sequencing (HTNGS) in the 1990s and its advancement during the last three decades opens a new horizon for identifying and analyzing sRNAs in food crop research. A large amount of HT-NGS-based RNA-sequencing (RNASeq) and sRNA-sequencing (sRNA-Seq) data is generated every day, which not only has the potential to explore only conserved but also can be utilized for the discovery of novel sRNA in food crops. In addition to the sRNA-seq, the development and availability of bioinformatics tools to analyze a large amount of NGS data efficiently further enhanced the discovery of novel RNA molecules, including sRNA (Narayan & Kumar, 2022). Moreover, tissue and condition-dependent RNA-seq data from wide food crop varieties available in the public domain could be utilized for the in-silico exploration of sRNA using various bioinformatics tools (Mohideen et al., 2022). 2. High throughput next generation small RNA sequencing The workflow for the HT-NGS-based sRNA-Seq includes three significant steps: (i) extraction of sRNA from the plant tissues; (ii) library preparation and sequencing of sRNAs; and (iii) bioinformatics analysis of sRNA-Seq data. 2.1 Extraction of the small RNA population Acquiring high-quality sRNAs from the desired plant tissues is the first and most crucial step in HT-NGS-based sRNA-Seq analysis. However, the high content of secondary metabolites, phenolics, and bioactive compounds in food crops hinders the extraction of sRNA from them. Therefore, different crops required standardization of the protocol for sRNA extraction accordingly. Several species-specific protocols for sRNA extraction are Exploring small RNA in food crops: techniques and approaches 111 available in the literature, and one can choose the desired protocol for their respective food crop (Accerbi et al., 2010; Rosas-Cárdenas et al., 2011). The most common strategy to extract sRNA from the plant tissue is to precipitate Low Molecular Weight (LMW) RNAs from the total RNA extract using lithium chloride (LiCl), polyethylene glycol (PEG), sodium salts, or alcohol. However, secondary metabolites, phenolic compounds, and carbohydrates make it challenging to extract intact total RNA and depend upon the different food crops and tissues of interest. Additionally, there are protocols available to extract direct sRNA population from the plant tissues. 2.1.1 Recovery of small RNA from the total RNA extract Several methods are available to recover the sRNA population from the total RNA extract in the food crops. In general, the protocol to yield highquality total RNA from specific food crops and tissue include (i) RNA isolation reagents such as cetyl trimethyl ammonium bromide (CTAB) (Jaakola et al., 2001; Porebski et al., 1997) and guanidinium thiocyanate (GITC) (Chomczynski & Sacchi, 1987), (ii) commercially available TRI Reagent®, TRIzol®, or plant RNA extraction kits and (iii) guanidinium free methods. However, occasional modifications such as extra chloroform and high salt to the total RNA extraction protocol are introduced to recover the sRNA population (Accerbi et al., 2010). The flow diagram in Fig. 5.1A outlines the significant steps in recovering the sRNA population from the total RNA extract. After harvesting the desired tissues such as leaf, stem, root, or fruit from the food crop of interest, they can be snap freeze in the liquid nitrogen (liq-N2) or collected in RNAlater to store at 80 C deep freezer for future use. The tissues are further crushed in liq-N2, for total RNA extraction using one of the methods mentioned above. Depending upon the quality of RNA, modifications can be introduced in the protocols. For instance, the high-quality total RNA can proceed further for recovery of the sRNA population; however, in the case of low-quality total RNA or yield, modifications such as extra chloroform, acid-phenol or high salt concentrations are required in the lysis buffer Fig. 5.1A. Once a high quality and yield of total RNA are achieved, the sRNA population can recover as described below. The most common method to isolate the sRNA population from total RNA is separating the LMW RNAs (sRNAs) from the high molecular weight (HMW) by reprecipitation of the total RNA with LiCl or PEG (Smith & Eamens, 2012). The total RNA extracted by methods mentioned 112 Plant Small RNA in Food Crops Figure 5.1 The flow chart showing generalized methodology for isolation of small RNA (sRNA) population from different plants, including food crops, by (A) recovery of small RNA (sRNA) population from the total RNA (Accerbi et al., 2010; Smith & Eamens, 2012), and (B) direct isolation of sRNA (Rosas-Cárdenas et al., 2011). (A) Total RNA can be extracted from the frozen pulverized plant tissue using cetyl trimethyl ammonium bromide (CTAB) and guanidinium thiocyanate (GITC), (ii) commercially available TRI Reagent®, TRIzol®, or plant RNA extraction kits or (iii) guanidinium free methods. To enhance the quality and yield of total RNA and sRNA population, extra chloroform, high salt concentration, and/or phenol are required. Once the good quality and yield of total RNA are achieved, reprecipitation using lithium chloride (LiCl) or polyethene glycol (PEG) can be achieved. (B) Direct isolation of sRNA population from different plants and tissues can be performed using lithium chloride (LiCl) buffer containing ethylenediaminetetraacetic acid (EDTA), TRIS hydrochloride (tris-HCl), sodium dodecyl sulfate (SDS), and phenol (pH ¼ 8.0). The flow chart is designed and created using the https://biorender.com/app. above can be reprecipitated with 8 M LiCl followed by centrifuge and isopropanol precipitation of supernatant. Wash the final pellet containing LMW RNAs with 70% ethanol and dissolve it in nuclease-free dH2O. The Exploring small RNA in food crops: techniques and approaches 113 quality and quantity of the dissolved pellet containing the LMW RNA, including miRNA and siRNA, could be accessed with polyacrylamide gel electrophoresis and Bioanalyzer. 2.1.2 Direct isolation of small RNAs Several protocols for isolating sRNAs from food crops and other plant species are available. Most of these protocols, as explained in the above section, involve separating LMW RNA (sRNA) from the total RNA. However, the high concentration of bioactive compounds and secondary metabolites in food crops hinders in good quality and yield of total RNA. Therefore, a method to isolate sRNA by omitting the step of total RNA is required in food crops (Fig. 5.1B) (Rosas-Cárdenas et al., 2011). The protocol described by Rosas-Cárdenas (Rosas-Cárdenas et al., 2011) can be utilized to isolate sRNA in a number of plant species, including food crops. The flow diagram in Fig. 5.1B shows the steps used in the direct isolation of sRNA. Briefly, the pulverized plant tissues are dissolved in LiCl buffer (LiCl, EDTA, and Tris HCL) and phenol (pH ¼ 8.0). The nucleic acids, including the LMW sRNA population, can be separated using chloroform:isoamyl alcohol (C:I) in the ratio of 24:1 in the supernatant phase. The supernatant is further treated with NaCl and PEG on ice for 30 min. In the second round of separation, phenol:chloroform:isoamyl (P:C:I) alcohol is used in the ratio of 25:24:1, followed by centrifugation. After centrifuging, NaCl and PEG are added to the sRNA population in the supernatant, which is further reprecipitated overnight at 20 C using absolute ethanol and 3 M sodium acetate. Next, centrifuge the overnight precipitated samples at 4 C with a maximum speed of 10 min. The pellet, after removing the supernatant, is washed three times with 70% ethanol and finally dissolved in nuclease-free water (NFeH2O). The final sRNA population can be assessed for quality and quantity assurance using a bioanalyzer. 2.2 Small RNA library preparation and sequencing The first sRNA-Seq using HT-NGS was performed in 2005 for the discovery of several types of sRNAs in the Arabidopsis thaliana (Lu et al., 2005), which later was adopted in many other species, including food crops. HTNGS opens a whole new horizon of possibilities to explore the sRNA population in a cell, tissue, and condition-dependent manner in food crops. HT-NGS-based sRNA discovery could further be utilized for transcript abundance and understanding of the chromatin modifications of crucial 114 Plant Small RNA in Food Crops genes, and genetic engineering to enhance the production of stress resistance and high-yield food crop varieties. After high-quality extraction of the sRNA population by the methods mentioned in the previous section, the following steps are to prepare a sRNA-Seq library and sequencing using HT-NGS platforms. In the sRNA-Seq library preparation, because of the small size of sRNAs, primer binding sites are introduced for reverse transcription (RT) and amplification, by ligation. However, ligation steps were identified as the primary source of bias in the sRNA-Seq and its data analysis (Fuchs et al., 2015; Raabe et al., 2014). Additionally, amplification of variable size and secondary structure molecules in the sRNA population is considered another source of biases affecting the downstream analysis of sRNA-Seq data. To overcome these biases, efforts have been made to develop different library preparation approaches for sRNA-Seq. Several commercial kits with improved technology are available to prepare a cDNA library for sRNASeq (Table 5.1) (Benesova et al., 2021). One of the technologies includes high definition (HD) adapters to reduce the ligation biases in sRNA libraries for Illumina platforms which are preferred sRNA-Seq because of its ability to generate short nucleic acid fragments (Sorefan et al., 2012). Additionally, Epicenter Biotechnologies introduced RecJ exonuclease, which removes the excessive 30 -adapter in the sRNA library preparation to prevent 50 -30 adapters ligations (Pease, 2011). The protocol explained here, based on the Illumina Truseq 2.0 kit and HD adapters (replacement for original adapters in Illumina Trueseq 2.0 kit), can be utilized for preparing a high-quality biased free sRNA-Seq library from different food crops (Xu et al., 2015). In the HT-NGS-based sRNA-Seq library preparation (Fig 5.2), the main steps are (i) HD adapter ligation at 30 end, (ii) removal of excessive 30 HD adapters, (iii) 50 HD adapter ligation, (iv) cDNA synthesis, and (v) PCR amplification and size selection. The sRNA-Seq library preparation using HD adapters starts with approximately 300 ng of the sRNA. During the sRNA-Seq library preparation, the 30 and 50 HD adapters are ligated to the 30 and 50 end of sRNA, through T4 RNA ligase 2 (T4 Rnal2) and T4 RNA ligase 1 (T4 Rnal1) enzymes respectively. The sequence to synthesize the 30 HD adapter is [50 eNNNNTGGAATTCTCGGGTGCCAAGG (20 30 ddC)-30 ], and the 50 HD adapter is (50 -/5AmMC6/GUUCAGAGUUCUACAGUCCGACGAUCNNNN-30 ] (Xu et al., 2015). Exploring small RNA in food crops: techniques and approaches 115 Table 5.1 Commercially available high throughput small RNA analysis techniques. Product name Company name Technology used Reference FirePlex miRNA assays Abcam, UK NA nCounter miRNA expression panels NanoString, USA Hybridization without NGS Hybridization without NGS HTG EdgeSeq miRNA whole transcriptome assay SMARTer smRNA-seq kit HTG molecular diagnostics, USA Takara bio., Japan Dennis et al. (2015) NA Hybridization probe-based sequencing Polyadenylation and template switching Polyadenylation and template switching UMI NA UMI NA BarberánSoler et al. (2018) NA CATS small RNA-seq kit Diagenode, Belgium TrueQuant SmallRNA seq kit QIAseq miRNA library kit RealSeq-AC kit GenXPro GmbH, Germany Qiagen, Germany Somagenics, USA Single adapter ligation and circularization RealSeq-biofluids kit Somagenics, USA SMARTer microRNAseq kit Takara bio., Japan NEXTflex small RNA sequencing kit PerkinElmer, USA Single adapter ligation and circularization Single adapter ligation and circularization Randomized adapters ScriptMiner library preparation technology for small RNA CleanTag small RNA library prep kit Cambio ltd., UK Two adapter ligations TriLink BioTechnologies, USA Two adapter ligations NA NA NA BaranGale et al. (2015) Pease (2011) Shore et al. (2016) Continued 116 Plant Small RNA in Food Crops Table 5.1 Commercially available high throughput small RNA analysis techniques.dcont'd Product name Company name Technology used Reference NEBNext multiplex small RNA library prep kit TailorMix miRNA sample preparation kit TrueSeq small RNA library prep kit Small RNA library prep kit Small RNA-Seq library prep kit New england biolabs, USA Two adapter ligations NA SeqMatic, USA Two adapter ligations Two adapter ligations Two adapter ligations Two adapter ligations NA Illumina, USA Norgen biotek corp., Canada Lexogen, Austria NA NA NA Adopted from Benesova, S., Kubista, M., & Valihrach, L. (2021). Small rna-sequencing: Approaches and considerations for mirna analysis. Diagnostics, 11, 1e19. https://doi.org/10.3390/diagnostics11060964. 2.2.1 Adapter ligation at 30 end of small RNA For the addition of a 30 HD adapter to the sRNA, and to minimize the intramolecular circularization, a truncated T4 Rnl2 K227Q mutant enzyme can be used (Viollet et al., 2011). The 30 HD adapters are first phosphorylated with T4 polynucleotide kinase (NEB), followed by adenylation with Mth RNA ligase (NEB) enzyme. For the 30 adapter ligation, a reaction is set up including sRNA and phosphorylated and pre-adenylated 30 HD adapters as follows: 2 mL 10 T4 Rnl2 buffer; 0.75 mL RNaseOUT (40 U/mL); 1 mL Truncated T4 Rnl2 K227Q (200U/mL); 3.25 mL NFeH2O; and 4 mL 50% PEG 800. The total 11 mL reaction is incubated at 26 C for 2e3 h. The 30 end ligated reaction can further be cleaned using an RNA clean and concentration kit by Zymo Research. 2.2.2 Excessive 30 high-definition adapter removal The excessive 30 HD adapters can be removed by deadenylating and treated with RecJ exonuclease enzymes in the following two-step reaction. Step 1: 1.6 mL 10 deadenylase buffer; 0.8 mL 100 mM DTT; 0.5 mL RNAaseOut (40 U/mL); and 1 mL 50 deadenylase (10U/mL). Incubate the reaction mixture at 30 C for 30 min and stop the reaction using 25 mM EDTA. Step 2: Add 2 mL 0.5 M Tris-HCl (pH 9.0), 7 mL 50 mM MgCl2 and 1 mL of RecJ exonuclease to the reaction product of Step 1. Mix the reaction mixture well and incubate at 37 C for 30 min. Exploring small RNA in food crops: techniques and approaches 117 Figure 5.2 Overview of the preparation of sRNA-Seq library for high throughput nextgeneration sequencing (HT-NGS) using high definition (HD) Illumina adapters. (i) 30 adapter ligation step includes the addition of excessive phosphorylated and preadenylated 30 high definition (HD) adapters to the sRNA population. The ligation of 30 HD adapters is carried out by truncated T4 RNA ligase 2 (T4 Rnl2) enzyme. (ii) Excessive 30 HD adapters can be removed using an exonuclease enzyme. (iii) 50 adapter ligation includes the 30 adapter-ligated sRNA and 50 HD adapter in the presence of T4 RNA ligase 1 (T4 Rnl1) enzyme. This will generate a single-stranded ditagged sRNA population. (iv) In the next step, the complementary DNA (cDNA) strand of the single-stranded di-tagged sRNA is synthesized using a reverse transcription reaction. (v) Finally, PCR amplification is performed using the DNA polymerase enzyme and size selection is done by beads or polyacrylamide gel (PAGE) to get sRNA-Seq library. The flow chart is designed and created using the https:// biorender.com/app. 2.2.3 Adapter ligation at 50 end of 30 adapter-ligated small RNA To the reaction mixture in the previous step, add 1 mL of denatured 20 mM 50 HD adapter. Add subsequently the following to the reaction mixture: 1 mL 10 T4 Rnl1 buffer; 10 mM ATP; 50 adapter (20 mM); 1 mL T4 Rnl1; and 7 mL 50% PEG 8000. Mix well the reaction mixture and incubate at 26 C for 2e3 h. The 50 end ligated reaction can further be cleaned using an RNA clean and concentration kit by Zymo Research. 118 Plant Small RNA in Food Crops 2.2.4 cDNA strand synthesis for di-tagged small RNA The following reaction can be set up for the synthesis of the cDNA strand of di-tagged sRNA: 30 mL di-tagged sRNA from the previous step; 4 mL 10 MMLV reverse transcription buffer; 2 mL 10 mM dNTP; 2 mL 100 mM DTT; 1 mL 20 mM Reverse transcription primer; and 1 mL highperformance MMLV reverse transcriptase (RT). Mix the reaction mixture well and incubate at 37 C for 20 min, followed by deactivation of RT enzyme at 85 C for 15 min. The cDNA library can be stored at 20 C till further use. 2.2.5 PCR amplification and size selection of small RNA library For the final step of amplification, the following PCR reaction can be set up: 4 mL cDNA; 9.3 mL NFeH2O; 0.5 mL 10 mM dNTP; 4 mL 5 high fidelity Phusion buffer; 1 mL Illumina RP-1 primer (10 mM); 1 mL Illumina index primer (10 mM); and 0.2 mL Phusion DNA polymerase (2U/mL). Mix the PCR reaction well and spin down the reaction mixture to set up the following PCR program: initial denaturation at 98 C for 30 s; 10e15 cycle of 98 C for 10 s; 55 C for 30 s; 72 C for 15 s; followed by a final extension at 72 C for 10 min; and 4 C hold. After the final amplification, the size selection for the sRNA-Seq library can be made using SPRI beads or PAGE electrophoresis. The final sRNA-Seq library is sequenced using Illumina platforms depending upon the size and coverage required for the library. 2.3 Computational analysis of next generation small RNA sequencing data The advent of HT-NGS-based sRNA-Seq made it feasible to explore the entire population of sRNA produced in a plant, cell, or tissue, under any developmental, or experimental conditions. Moreover, due to the diverse functions of sRNA in food crops, sRNA-Seq data started to emerge in several food crop species. Since plants can produce millions of sRNA involved in diverse pathways and functions, and collectively captured in a single sRNA-Seq experiment, it is quite a tedious task to manage all the HT-NGS data generated for the sRNA population. However, the continuous efforts of computational biologists to develop bioinformatics and statistical tools enable us to convert the enormous raw sequencing data into a biological sense. In the current section, the primary strategy (Fig. 5.3) is discussed that can be used for the bioinformatics analysis of sRNA-Seq data generated for food crops by HT-NGS platforms. The detailed Exploring small RNA in food crops: techniques and approaches 119 Figure 5.3 Systematic workflow for the computational analysis of the sRNA-Seq data generated through high throughput next generation sequencing (HT-NGS). The flow chart is designed and created using the https://biorender.com/app. command lines and computational tools are reviewed recently by Garg and Varshney (2022), and in the current chapter, the command lines are modified from the same study. For different computational tools for plant sRNA detection and characterization from sRNA-Seq data, one can also refer to the review by Morgado and Johannes (2018). Unlike the analysis of RNA-Seq data, the sRNA-Seq analysis involved the step of mapping and removing ribosomal RNA (rRNA), transfer RNA (tRNA), small nuclear RNA (snRNA), and small nucleolar RNA (snoRNA) (Garg & Varshney, 2022). However, the strategy for filtering low-quality, adapter reads, and mapping of sRNA-Seq data remains like in RNA-Seq data analysis. The main steps in sRNA-Seq data analysis include (Fig. 5.3): (i) quality control of raw reads, (ii) mapping of high-quality reads to the reference sequences, (iii) prediction of precursor RNA and secondary structure, (iv) differential expression of sRNA, and (v) prediction of sRNA target genes (Garg & Varshney, 2022). To follow the strategy discussed here, a 64-bit Linux operating workstation with a minimum of 4 GB RAM is required. 120 Plant Small RNA in Food Crops 2.4 Pre-processing of sRNA-Seq data The first step in sRNA-Seq data analysis is the quality control of the raw reads generated from the HT-NGS. The raw sequence reads, usually in fastq format, are processed for the quality check with the “fastQC” tools (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) followed by the removal of adapters, low-quality reads using the java-based “trimmomatic” program (Bolger et al., 2014). The general command line for the single end reads is: > java -jar trimmomatic-0.39.jar SE -phred 33 input.fq.gz clean.fq.gz ILLUMINACLIP:illumina.fa:2:30:10 SLIDINGWINDOW:10:20 The “illumina.fa” in the command line used is the default adapter sequence used in the Illumina sequencing. One can use these default adapter sequences in the command line with the sequence of Illumina HD adapter sequences as described in the sRNA-Seq library preparation section. Once the adapter sequences are removed, the next step is to remove the reads with poly-A tail and too long too short sequences. This can be done using the “Cutadapt” tool (https://github.com/marcelm/cutadapt), with the following command line: > cutadapt -a “A{20}” -m 18 -M 34 -o clean.polyAremoved.fq.gz clean.fq.gz The -m and -M represents the minimum and maximum read length to be considered. The clean reads with no poly-A tail in fastq format are further converted to fasta format using the following Linux-based command and FASTX-Toolkit (Garg & Varshney, 2022). > cat clean.polyAremoved.fq | paste - - - - | sed ‘s/^@/>/g’ | cut -f 1,2 | tr ‘t’ ‘n’>HQ-reads.fa > fastx_collapser -i HQ-reads.fa -o Unique-HQ-reads.fa > sed -i ‘s/-/_x/’ Unique-HQ-reads.fa The final step in the pre-processing of sRNA-Seq data includes the removal of rRNA, tRNA, snRNA, and soRNA sequences. The mapping of non-coding RNA can be performed using the “Bowtie” tool (http:// bowtie-bio.sourceforge.net/index.shtml), and the reference Rfam database (Kalvari et al., 2018). The reference sequences are first converted to the index file using “-bowtie-build” followed by the alignment using “-bowtie” command lines, respectively. > bowtie-build rfam.fa rfam-index > bowtie rfam-index -f Unique-HQ-reads.fa -S Unique-HQ-.ncRNA.sam eun HQ-filtered.fa Exploring small RNA in food crops: techniques and approaches 121 Once the pre-processing of raw sequencing data is done and HQ filtered sequence read file is achieved, one can proceed to the next step of identification of conserved or known sRNA. 2.5 Identification of conserved or known sRNA To identify the conserved and known sRNA in the sRNA-Seq, a reference database such as miRBase (Kozomara et al., 2019) for miRNA is required. The reference miRNA sequence “miRbase.fa” file can be downloaded from the miRBase, and indexing can be performed using the “-bowtiebuild” command. After indexing, the alignment can be performed to identify the unique aligned sequences of conserved or known miRNA. > bowtie-build mirbase.fa mirbase-index > bowtie mirbase-index -n 2 -f HQ-filtered.fa -S Conserved.sam –un NovelPrediction.fa The alignment file “Conserved.sam” in SAM format and the unmapped reads “NovelPrediction.fa” in fasta format will further be processed for prediction of secondary structure and novel miRNA identification, respectively. 2.6 Identification of novel sRNA To identify the novel sRNA (in the current case, miRNA), the reference genome of the species is required to be downloaded from the respective database. The prediction of novel sRNA can be carried out using “miRDeep-P”, a Perl-script-based package (Yang & Li, 2011). The reference genome (genome.fa) first index using the command: > bowtie-build genome.fa genome-index The unmapped reads (NovelPrediction.fa) from the bowtie alignment in the previous section now align to the reference genome using the command line: > bowtie genome-index -n 0 -f NovelPrediction.fa eS NovelPrediction.sam The alignment file in the SAM format is further converted to blast format using the script “convert_SAM_to_blast.pl”, of miRDeep-P package, as explained in earlier in the review by Garg and Varshney (Garg & Varshney, 2022). > perl miRDeep-P/convert_SAM_to_blast.pl NovelPrediction.sam NovelPrediction.fa genome.fa > genome.bst 122 Plant Small RNA in Food Crops Next, filtering of multiple aligned reads can be performed using the command: > perl miRDeep-P/filter_alignments.pl genome.bst -c 15 > genomefiltered.bst To discard the reads aligned with features such as exons and CDS regions, the reference annotation file can be downloaded for a particular species of interest from public domains such as NCBI (https://www.ncbi. nlm.nih.gov/). The annotation file, along with the blast format file generated in the previous command, is used as: > perl miRDeep-P/overlap.pl genome-filtered.bst genome.gff -b > genome-filtered-overlapCDS > perl miRDeep-P/alignedselected.pl genome-filtered.bst -g genomefiltered-overlapCDS > genome-filtered-overlapCDS.bst Convert the filtered file generated in the previous step into fasta format using: > perl miRDeep-P/filter_alignments.pl genome-filtered-overlapCDS.bst -b NovelPrediction.fa > genome-filter-CDS-filtered.fa To extract the potential precursor sequences from the reference genome and the filtered aligned reads, the following command line can be used: > perl miRDeep-P/excise_candidate.pl genome.fa genome-filter-CDSfiltered.fa 250 > genome-filter-CDS-precursors.fa The secondary structure of the potential precursor RNA can be predicted using RNAfold function of ViennaRNA package (https://www.tbi. univie.ac.at/RNA/). > cat genome-filter-CDS-precursors.fa | RNAfold –noPS > genomefilter-CDS-structures Nest the filtered sequences mapped to the precursor sequences to generate miRNA signature with the following command lines: > bowtie-build -f genome-filter-CDS-precursors.fa genome-filter-CDSprecursors-index > bowtie genome-filter-CDS-precursors-index -f genome-filter-CDSfiltered.fa -S genome-filter-CDS-precursors.sam > perl miRDeep-P/convert_SAM_to_blast.pl genome-filter-CDSprecursors.sam genome-filter-CDS-filtered.fa genome-filter-CDSprecursors.fa > genome-filter-CDS-precursors.bst > sort þ3 -25 genome-filter-CDS-precursors.bst > miRNA-signatures To predict the novel miRNA, the final step is to combine the miRNA signatures with the RNAfold predicted structure in previous sections. For that, the following commands can be used: Exploring small RNA in food crops: techniques and approaches 123 > perl miRDeep-P/miRDP.pl miRNA-signatures genome-filter-CDSstructures -y > predicted-miRNA > samtools faidx genome.fa > genome. fa.fai > perl miRDeep-P/rm_redundant_meet_plant.pl genome.fa.fai genomefilter-CDS-precursors.fa predicted-miRNA predicted-novel-miRNA The final output of the command lines is the list of sequences of novel sRNA (miRNA in our case) predicted in the analysis. 2.7 Prediction of sRNA target genes To understand the regulatory function of sRNA conserved or novel predicted in the sRNA-Seq analysis, the identification of their target is a crucial step. Unlike animals, plant sRNA has a perfect or nearly perfect complementary structure with their targets. Several user-friendly tools have been developed to predict the targets of sRNA in plants. Therefore for food crops as well, one can choose according to the need from the following sRNA target predictor: (i) TAPIR (Bonnet et al., 2010), (ii) psRobot (Wu et al., 2012), (iii) comTAR (Chorostecki & Palatnik, 2014), and (iv) psRNATarget (Dai & Zhao, 2011). 2.8 Differential expression of sRNA from sRNA-Seq data For the differential expression, sRNA analysis across different conditions or samples can be performed using tools and steps like RNA-Seq analysis. The alignment files generated using a short read aligner such as bowtie, are quantified using samtools. The quantification files can be passed to Rstudio, and the expression pattern can be generated using the program DESeq2 (Love et al., 2014). Several R-studio-based packages are available to generate heatmaps and expression patterns of differentially expressed sRNA predicted in DESeq2. 3. Future perspectives The advancement in the HT-NGS-based sRNA-Seq has accelerated food crop research in the recent past. However, techniques and approaches require to explore sRNA in food crops have challenges in every step. For instance, the high content of bioactive compounds in food crops hinders the extraction of good quality sRNA population, which is the most crucial for HT-NGS-based sRNA-Seq. Biases in library preparation and analysis of 124 Plant Small RNA in Food Crops the enormous amount of data also require a streamlined strategy for a better understanding of the regulatory functions of sRNA in food crops. Although the current chapter compiles most of the steps required to explore food crops in a systematic way, continuous efforts need to be made to utilize these methods and tools for the betterment of food crop varieties in the future. Acknowledgment I acknowledge all the researchers for understanding sRNA biology and developing related protocols for benefiting the scientific community. I am thankful to all the authors cited in the chapter. My special thanks to the authors of two excellent reviews by Morgado and Johannes (2018), and Garg and Varshney (2022) for providing details of computational analysis of sRNA-Seq data. References Accerbi, M., Schmidt, S. A., De Paoli, E., Park, S., Jeong, D. H., & Green, P. J. (2010). Methods for isolation of total RNA to recover miRNAs and other small RNAs from diverse species. Methods in Molecular Biology, 592, 31e50. https://doi.org/10.1007/9781-60327-005-2_3 Ahmed, M. M. S., Bian, S., Wang, M., Zhao, J., Zhang, B., Liu, Q., Zhang, C., Tang, S., Gu, M., & Yu, H. (2017). RNAi-mediated resistance to rice black-streaked dwarf virus in transgenic rice. Transgenic Research, 26, 197e207. https://doi.org/10.1007/s11248016-9999-4 Ammara, U. E., Mansoor, S., Saeed, M., Amin, I., Briddon, R. W., & Al-Sadi, A. M. (2015). RNA interference-based resistance in transgenic tomato plants against Tomato yellow leaf curl virus-Oman (TYLCV-OM) and its associated betasatellite. Virology Journal, 12. https://doi.org/10.1186/s12985-015-0263-y Andersson, M., Melander, M., Pojmark, P., Larsson, H., Bülow, L., & Hofvander, P. (2006). Targeted gene suppression by RNA interference: An efficient method for production of high-amylose potato lines. Journal of Biotechnology, 123, 137e148. https://doi.org/ 10.1016/j.jbiotec.2005.11.001 Baran-Gale, J., Lisa Kurtz, C., Erdos, M. R., Sison, C., Young, A., Fannin, E. E., Chines, P. S., & Sethupathy, P. (2015). Addressing bias in small RNA library preparation for sequencing: A new protocol recovers microRNAs that evade capture by current methods. Frontiers in Genetics, 6, 1e9. https://doi.org/10.3389/ fgene.2015.00352 Barberán-Soler, S., Vo, J. M., Hogans, R. E., Dallas, A., Johnston, B. H., & Kazakov, S. A. (2018). Decreasing miRNA sequencing bias using a single adapter and circularization approach. Genome Biology, 19, 1e9. https://doi.org/10.1186/s13059-018-1488-z Benesova, S., Kubista, M., & Valihrach, L. (2021). Small rna-sequencing: Approaches and considerations for mirna analysis. Diagnostics, 11, 1e19. https://doi.org/10.3390/ diagnostics11060964 Beris, D., Podia, V., Dervisi, I., Kapolas, G., Isaioglou, I., Tsamadou, V., Pikoula, L., Rovoli, M., Vallianou, A., Roussis, A., Milioni, D., Giannoutsou, H., & Haralampidis, K. (2022). RNAi silencing of the Arabidopsis thaliana ULCS1 gene Exploring small RNA in food crops: techniques and approaches 125 results in pleiotropic phenotypes during plant growth and development. International Journal of Developmental Biology. https://doi.org/10.1387/ijdb.210114kh Bolger, A. M., Lohse, M., & Usadel, B. (2014). Trimmomatic: A flexible trimmer for illumina sequence data. Bioinformatics. https://doi.org/10.1093/bioinformatics/btu170 Bonnet, E., He, Y., Billiau, K., & van de Peer, Y. (2010). TAPIR, a web server for the prediction of plant microRNA targets, including target mimics. Bioinformatics, 26, 1566e1568. https://doi.org/10.1093/bioinformatics/btq233 Chaudhary, S., Grover, A., & Sharma, P. C. (2021). MicroRNAs: Potential targets for developing stress-tolerant crops. Life, 11, 1e21. https://doi.org/10.3390/life11040289 Chomczynski, P., & Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analytical Biochemistry, 162, 156e159. https://doi.org/10.1016/0003-2697(87)90021-2 Chorostecki, U., & Palatnik, J. F. (2014). ComTAR: A web tool for the prediction and characterization of conserved microRNA targets in plants. Bioinformatics, 30, 2066e2067. https://doi.org/10.1093/bioinformatics/btu147 Dai, X., & Zhao, P. X. (2011). PsRNATarget: A plant small RNA target analysis server. Nucleic Acids Research. https://doi.org/10.1093/nar/gkr319 Dennis, D., Rhodes, M., & Maclean, K. (2015). Targeted miRNA discovery and validationusing the nCounter® platform. NanoString technol. | WHITE pap. | nCounter PanCancer immune profiling panel 1e8. Frizzi, A., & Huang, S. (2010). Tapping RNA silencing pathways for plant biotechnology. Plant Biotechnology Journal.. https://doi.org/10.1111/j.1467-7652.2010.00505.x Fuchs, R. T., Sun, Z., Zhuang, F., & Robb, G. B. (2015). Bias in ligation-based small RNA sequencing library construction is determined by adaptor and RNA structure. PLoS One, 10, 1e24. https://doi.org/10.1371/journal.pone.0126049 Fu, D., Uauy, C., Blechl, A., & Dubcovsky, J. (2007). RNA interference for wheat functional gene analysis. Transgenic Research, 16, 689e701. https://doi.org/10.1007/s11248007-9150-7 Gao, H., Yang, M., Yang, H., Qin, Y., Zhu, B., Xu, G., Xie, C., Wu, D., Zhang, X., Li, W., Yan, J., Song, S., Qi, T., Ding, S. W., & Xie, D. (2018). Arabidopsis ENOR3 regulates RNAi-mediated antiviral defense. Journal of Genetics and Genomics, 45, 33e40. https://doi.org/10.1016/j.jgg.2017.11.005 Garg, V., & Varshney, R. K. (2022). Analysis of small RNA sequencing data in plants. Methods in Molecular Biology, 2443, 497e509. https://doi.org/10.1007/978-1-07162067-0_26 Guan, Q., Lu, X., Zeng, H., Zhang, Y., & Zhu, J. (2013). Heat stress induction of miR 398 triggers a regulatory loop that is critical for thermotolerance in Arabidopsis. The Plant Journal, 74, 840e851. https://doi.org/10.1111/tpj.12169 Hamilton, A. J., & Baulcombe, D. C. (1999). A species of small antisense RNA in posttranscriptional gene silencing in plants. Science (80-. ), 286, 950e952. https://doi.org/ 10.1126/science.286.5441.950 Huang, G., Allen, R., Davis, E. L., Baum, T. J., & Hussey, R. S. (2006). Engineering broad root-knot resistance in transgenic plants by RNAi silencing of a conserved and essential root-knot nematode parasitism gene. Proceedings of the National Academy of Sciences of the United States of America, 103, 14302e14306. https://doi.org/10.1073/pnas.0604698103 Jaakola, L., Pirttilä, A. M., Halonen, M., & Hohtola, A. (2001). Isolation of high quality RNA from bilberry (Vaccinium myrtillus L.) fruit. Applied Biochemistry and Biotechnology Part B Molecular Biotechnology -, 19, 201e203. https://doi.org/10.1385/MB:19:2:201 Jin, H. (2008). Endogenous small RNAs and antibacterial immunity in plants. FEBS Letters, 582, 2679e2684. https://doi.org/10.1016/j.febslet.2008.06.053 126 Plant Small RNA in Food Crops Kalvari, I., Nawrocki, E. P., Argasinska, J., Quinones-Olvera, N., Finn, R. D., Bateman, A., & Petrov, A. I. (2018). Non-coding RNA analysis using the rfam database. Current Protocols in Bioinformatics., 62, 1e44. https://doi.org/10.1002/cpbi.51 Kamthan, A., Chaudhuri, A., Kamthan, M., & Datta, A. (2015). Small RNAs in plants: Recent development and application for crop improvement. Frontiers of Plant Science, 6, 1e17. https://doi.org/10.3389/fpls.2015.00208 Kamthan, A., Chaudhuri, A., Kamthan, M., & Datta, A. (2016). Genetically modified (GM) crops: Milestones and new advances in crop improvement. Theoretical and Applied Genetics. https://doi.org/10.1007/s00122-016-2747-6 Khandagale, K. S., Chavhan, R., & Nadaf, A. B. (2020). RNAi-mediated down regulation of BADH2 gene for expression of 2-acetyl-1-pyrroline in non-scented indica rice IR64 (Oryza sativa L.). 3 Biotech, 10, 1e9. https://doi.org/10.1007/s13205-020-2131-8 Kozomara, A., Birgaoanu, M., & Griffiths-Jones, S. (2019). MiRBase: From microRNA sequences to function. Nucleic Acids Research. https://doi.org/10.1093/nar/gky1141 Love, M. I., Huber, W., & Anders, S. (2014). Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biology, 15, 1e21. https://doi.org/ 10.1186/s13059-014-0550-8 Lu, C., Tej, S. S., Luo, S., Haudenschild, C. D., Meyers, B. C., & Green, P. J. (2005). Genetics: Elucidation of the small RNA component of the transcriptome. Science (80-. ), 309, 1567e1569. https://doi.org/10.1126/science.1114112 Marín-Sanz, M., Iehisa, J. C. M., & Barro, F. (2022). New transcriptomic insights in two RNAi wheat lines with the gliadins strongly down-regulated by two endosperm specific promoters. Crop Journal, 10, 194e203. https://doi.org/10.1016/j.cj.2021.04.009 Mohideen, H. S., Sonawala, K., & Ghosh, S. (2022). In-silico identification of small RNAs : A tiny silent tool against agriculture pest, bioinformatics in agriculture. INC. https://doi.org/ 10.1016/B978-0-323-89778-5.00002-7 Morgado, L. (2020). Diversity and types of small RNA, plant small RNA. Elsevier Inc. https:// doi.org/10.1016/b978-0-12-817112-7.00002-x Morgado, L., & Johannes, F. (2018). Computational tools for plant small RNA detection and categorization. Briefings in Bioinformatics, 20, 1181e1192. https://doi.org/10.1093/ bib/bbx136 Narayan, A., & Kumar, S. (2022). Identification of novel RNAs in plants with the help of next-generation sequencing technologies. Bioinformatics in Agriculture. INC. https:// doi.org/10.1016/B978-0-323-89778-5.00018-0 Pease, J. (2011). Small-RNA sequencing libraries with greatly reduced adaptor-dimer background. Nature Methods, 8, iiieiv. https://doi.org/10.1038/nmeth.f.336 Porebski, S., Bailey, L. G., & Baum, B. R. (1997). Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components. Plant Molecular Biology Reporter, 15, 8e15. https://doi.org/10.1007/BF02772108 Raabe, C. A., Tang, T. H., Brosius, J., & Rozhdestvensky, T. S. (2014). Biases in small RNA deep sequencing data. Nucleic Acids Research, 42, 1414e1426. https://doi.org/ 10.1093/nar/gkt1021 Reinhart, B. J., Weinstein, E. G., Rhoades, M. W., Bartel, B., & Bartel, D. P. (2002). MicroRNAs in plants. Genes & Development. https://doi.org/10.1101/gad.1004402 Rosas-Cárdenas, F. de F., Durán-Figueroa, N., Vielle-Calzada, J. P., Cruz-Hernández, A., Marsch-Martínez, N., & de Folter, S. (2011). A simple and efficient method for isolating small RNAs from different plant species. Plant Methods, 7, 1e7. https://doi.org/ 10.1186/1746-4811-7-4 Sabu, K. K., & Nadiya, F. (2020). Small RNA manipulation in plants: Techniques and recent developments, Plant Small RNA. Elsevier Inc. https://doi.org/10.1016/b978-0-12817112-7.00018-3 Exploring small RNA in food crops: techniques and approaches 127 Shore, S., Henderson, J. M., Lebedev, A., Salcedo, M. P., Zon, G., McCaffrey, A. P., Paul, N., & Hogrefe, R. I. (2016). Small RNA library preparation method for nextgeneration sequencing using chemical modifications to prevent adapter dimer formation. PLoS One, 11, 1e26. https://doi.org/10.1371/journal.pone.0167009 Slotkin, R. K., Vaughn, M., Borges, F., Tanurdzic, M., Becker, J. D., Feijó, J. A., & Martienssen, R. A. (2009). Epigenetic reprogramming and small RNA silencing of transposable elements in pollen. Cell, 136, 461e472. https://doi.org/10.1016/ j.cell.2008.12.038 Smith, N. A., & Eamens, A. L. (2012). Isolation and detection of small RNAs from plant tissues. Methods in Molecular Biology, 894, 155e172. https://doi.org/10.1007/978-161779-882-5_11 Sorefan, K., Pais, H., Hall, A. E., Kozomara, A., Griffiths-Jones, S., Moulton, V., & Dalmay, T. (2012). Reducing ligation bias of small RNAs in libraries for next generation sequencing. Silence, 3, 1. https://doi.org/10.1186/1758-907X-3-4 Storz, G. (2002). An expanding universe of noncoding RNAs. Science (80-. ), 296, 1260e1263. https://doi.org/10.1126/science.1072249 Summanwar, A., Basu, U., Rahman, H., & Kav, N. N. V. (2020). Non-coding RNAs as emerging targets for crop improvement. Plant Science, 297, 110521. https://doi.org/ 10.1016/j.plantsci.2020.110521 Sunkar, R., Chinnusamy, V., Zhu, J., & Zhu, J. K. (2007). Small RNAs as big players in plant abiotic stress responses and nutrient deprivation. Trends in Plant Science. https:// doi.org/10.1016/j.tplants.2007.05.001 Sun, G., Stewart, C. N., Xiao, P., & Zhang, B. (2012). MicroRNA expression analysis in the cellulosic biofuel crop switchgrass (panicum virgatum) under abiotic stress. PLoS One. https://doi.org/10.1371/journal.pone.0032017 Viollet, S., Fuchs, R. T., Munafo, D. B., Zhuang, F., & Robb, G. B. (2011). T4 RNA Ligase 2 truncated active site mutants: Improved tools for RNA analysis. BMC Biotechnology, 11, 1e14. https://doi.org/10.1186/1472-6750-11-72 Vu, T. Van, Roy Choudhury, N., & Mukherjee, S. K. (2013). Transgenic tomato plants expressing artificial microRNAs for silencing the pre-coat and coat proteins of a begomovirus, Tomato leaf curl New Delhi virus, show tolerance to virus infection. Virus Research, 172, 35e45. https://doi.org/10.1016/j.virusres.2012.12.008 Wei, W., Ba, Z., Gao, M., Wu, Y., Ma, Y., Amiard, S., White, C. I., Danielsen, J. M. R., Yang, Y. G., & Qi, Y. (2012). A role for small RNAs in DNA double-strand break repair. Cell, 149, 101e112. https://doi.org/10.1016/j.cell.2012.03.002 Wilkinson, M. D., Kosik, O., Halsey, K., Walpole, H., Evans, J., Wood, A. J., Ward, J. L., Mitchell, R. A. C., Lovegrove, A., & Shewry, P. R. (2021). RNAi suppression of xylan synthase genes in wheat starchy endosperm. PLoS One, 16, 1e19. https://doi.org/ 10.1371/journal.pone.0256350 Wu, H. J., Ma, Y. K., Chen, T., Wang, M., & Wang, X. J. (2012). PsRobot: A web-based plant small RNA meta-analysis toolbox. Nucleic Acids Research, 40, 22e28. https:// doi.org/10.1093/nar/gks554 Xu, P., Billmeier, M., Mohorianu, I., Green, D., Fraser, W. D., & Dalmay, T. (2015). An improved protocol for small RNA library construction using High Definition adapters. Methods Next Generation Seqencing., 2, 1e10. https://doi.org/10.1515/mngs-2015-0001 Yang, X., & Li, L. (2011). miRDeep-P: A computational tool for analyzing the microRNA transcriptome in plants. Bioinformatics, 27, 2614e2615. https://doi.org/10.1093/bioinformatics/btr430 Zhao, Q., Ye, Y., Han, Z., Zhou, L., Guan, X., Pan, G., Asad, M. A. U., & Cheng, F. (2020). SSIIIa-RNAi suppression associated changes in rice grain quality and starch biosynthesis metabolism in response to high temperature. Plant Science, 294. https:// doi.org/10.1016/j.plantsci.2020.110443 This page intentionally left blank CHAPTER 6 Plant small RNAs: biogenesis, mechanistic functions and applications S.V. Ramesha, *, S. Rajeshb, * and T. Radhamanib a ICAR-Central Plantation Crops Research Institute, Kasaragod, Kerala, India; bCentre for Plant Molecular Biology and Biotechnology, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India 1. Introduction Non-coding RNAs (ncRNAs) constitute a greater proportion of transcribed RNAs in any eukaryotic genome since only 2 % of transcribed RNA in a eukaryotic genome is translated into proteins (Yu et al., 2019c). These untranslated RNA molecules constitute multiple variants of non-coding RNAs (both small and long non-coding RNAs) which are generally transcribed from the genomic regions, once considered to be genetically silent, including intergenic regions, pseudogenes, repetitive sequences and transposable elements. The plant ncRNAs are classified into small (18e30 nucleotides (nt), medium (31e200 nt) and long ncRNAs (greater than 200 nt). Small RNAs repertoire of plants has grown multifold since the discovery of 22-nt long RNA molecule with a regulatory role in post-embryonic development of C. elegans. The various classes of small RNAs are identified principally based on their mode of biogenesis or precursor RNAs and mode of action. The three main small RNA players of importance are microRNAs (miRNAs), small interfering RNA (siRNAs) and phased siRNAs (phasiRNAs) (Yu et al., 2019a,b,c). The diverse yet vital roles of plant small RNAs especially miRNAs in various developmental processes such as growth, stress response, temporal transitions, etc are delineated (D’Ario et al., 2017; Martinez & Köhler, 2017; Tang & Chu, 2017). * S.V. Ramesh and S. Rajesh authors have contributed equally. Plant Small RNA in Food Crops ISBN 978-0-323-91722-3 https://doi.org/10.1016/B978-0-323-91722-3.00012-9 © 2023 Elsevier Inc. All rights reserved. 129 130 Plant Small RNA in Food Crops 2. Small RNA biogenesis and mode of action In general, small interfering RNAs (siRNAs) are formed from dsRNA molecules whereas one of the widely studied and the abundant small RNA component of plant system, miRNAs, are generated from single stranded mRNA precursor molecule with self-complementary hairpin RNA (hpRNA) loop. miRNAs are encoded by MIR genes that are generally located in intergenic regions. miRNAs are transcribed due to the action of DNA-dependent RNA polymerase II (Pol II) producing primary miRNA transcript (pri-miRNA) which is further 50 capped and 30 polyadenylated. The self complementary hpRNA precursor molecule is acted upon by the Dicer-Like (DCL) proteins. DCL proteins are characterized with multiple functional units including RNAse III and helicase domains so as to recognize and dice the dsRNA at specific position (Margis et al., 2006). The number of DCL genes in an organism varies as rice genome harbors 5 different DCL genes whereas Arabidopsis have 4 genes. Moreover the specificity of DCL genes for small RNA class depends on the RNA sequence and secondary structural features of RNA molecules. Other RNA binding proteins such as HYPONASTIC LEAVES1 (HYL1), SERRATE (SE), TOUGH (TGH), and DAWDLE (DDL) are known to be involved in processing of small RNAs along with DCL. Thus, primary miRNA (primiRNA) precursor is sequentially processed into a precursor miRNA and ultimately into a miRNA:miRNA* duplex. Further the stability of the processed small RNA molecule is determined by the activities of methyl transferase HUA ENHANCER 1 (HEN1) which methylates the 30 terminal ribose of the small RNA. This activity ensures that the affinity of small RNA to SMALL RNA DEGRADING ENZYME 1 (SDN1), a protein involved in small RNA turnover, remains low. Also, HEN1 methylated small RNA is precluded from interacting with a protein HEN1 SUPPRESSOR 1 (HESO1) that adds polyuridine residues to the small RNA molecule. Thus, overall HEN1 methylation ensures the stability of the small RNA molecule. Besides HESO1, Arabidopsis URIDYLYLTRANSFERASE 1 (URT1) has been demonstrated to be involved in terminal uridyltransferase activity. Uridylation of miRNAs by terminal uridylyltransferases (TUTases such as HESO1 and URT1) triggers degradation of such small RNAs (Zuber et al., 2018). Endogenous small RNAs warrant the action of DNA-dependent RNA polymerase IV (Pol IV) for transcription. Additionally some non-coding transcripts of Pol II origin recruit and direct Pol IV to various genomic Plant small RNAs: biogenesis, mechanistic functions and applications 131 regions in order to direct siRNA biosynthesis. Unlike the miRNAs which are directly acted upon by the DCL proteins, siRNAs (especially tasiRNAstransacting siRNAs) require the action of RNA-dependent RNA polymerases (RdRs) prior to the activity of DCLs. miRNAs are involved in the biogenesis of tasiRNAs, since the action of RdRs is preceded by the activity of miRNA-mediated AGO led cleavage of tasiRNA precursors. All kinds of small RNAs (miRNAs and endogenous siRNAs) are exported to cytoplasm by the protein HASTY 1 (HST1). These small RNAs in the cytoplasm are recruited into a ribonucleoprotein complex called RNA inducing Silencing Complex (RISC) which comprises various kinds of AGOs and other ancillary proteins necessary for silencing. RISC targets the specific RNA molecule in the cytosolic pool based on the nucleotide sequence complementarity with the recruited small RNA. Hence, small RNA-sequence complementarity is utilized by the protein machinery to target specific RNA molecule for downregulation of gene expression. Recent studies divulge that miRNAs are matured, methylated and loaded onto AGO-1 within the nucleus and are transported to cytosol as miRNA:AGO1 complex in a CRM1(EXPO1)/NES-dependent manner (Bologna et al., 2018). Further, the indispensable role of plant TREX-2 complex in co-ordinating the transcription, processing and export of miRNAs was also uncovered (Zhang et al., 2020). Nevertheless, the presence of unloaded miRNAs in cytoplasm suggests that a little proportion of miRISC assembly may occur in cytoplasm (Dalmadi et al., 2019). Multiple variants of AGOs exist in plants as Arabidopsis genome harbors ten different AGO homologs with their function ranging from sequencespecific cleavage of target RNA molecule, inhibition of translational machinery and directing chromatin silencing (Rogers & Chen, 2013; Bologna et al., 2018; Dalmadi et al., 2019). Small RNAs of plants are broadly classified into two categories namely miRNAs and siRNAs depending on the process of biogenesis (Axtell, 2013). Functionally small RNAs form an integral component of two distinct pathways viz., RISC pathway and RNA-directed DNA methylation (RdDM) pathway (Aufsatz et al., 2002; Zilberman et al., 2004). RISC pathway involves post-transcriptional silencing of gene expression wherein small RNAs bind to cognate mRNAs due to sequence homology (complementation in base pairing) and cause sequence dependent cleavage of target mRNA. Alternatively this base-pairing results in inhibition of translation causing post-transcriptional gene silencing. On the other hand, RdDM pathway is an epigenetic mode of gene regulation where small 132 Plant Small RNA in Food Crops RNAs direct genomic DNA methylation causing silencing of gene expression at the transcriptional level (Chen, 2009). 3. Plant small RNAs in response to stress Plants being sessile have adopted sophisticated molecular mechanisms to tide over external stressors. Among the various gene regulatory mechanisms which plants adopt in the wake of stresses such as drought, salinity, elevated or low temperature, nutrient deprivation conditions, transcriptional control of gene expression has been very well investigated. The roles of plant transcription factors (TFs) in modulating the expression of battery of genes, through its interaction with cis-acting DNA elements, have been documented. However, with the advent of the phenomenon of RNA silencing, small RNAs and their pivotal regulatory roles in the wake external stressors have led to the characterization of molecular machinery orchestrating posttranscriptional gene regulation. Improvements in molecular biology techniques such as have led to the identification of stress-related miRNAs and cognate gene transcripts that are regulated by responsive miRNAs (Axtell, 2013). Small RNAs play a pivotal role in the manifestation of abiotic or biotic stress tolerance. Analysis of conserved nature of small RNAs in general and miRNAs in particular in plants have identified 24 miRNA families and couple of transacting siRNAs (tasiRNAs) that are found in most of the plant species suggesting the conserved nature of functioning of small RNAs in genetic regulation (Chen et al., 2018). Several of the well characterized and conserved miRNAs, tasiRNAs are known to target transcription factors (TFs). For instance, the well characterized miR156 targets SQUAMOSA promoter binding-like (SPL) transcription factors to govern phase transition and trichome development (Wang et al., 2011; Yu et al., 2010) whereas miR172 cleaves APETALA 2 (AP2) TFs to regulate traits such as flowering and organ identity (Aukerman & Sakai, 2003; Jung et al., 2007; Zhao et al., 2007b). miR164 regulates the expression of NAC TFs (NAM, ATAF, and CUC). Furthermore, multiple layers of miRNA-mediated gene regulation is documented in plants as miR390 targets transacting siRNA 3 (TAS3) to induce the production of tasiRNAs which in turn affect the accumulation of major TFs, Auxin response factors (ARFs). ARFs are known to influence multiple traits such as leaf polarity, fruit development and lateral development of root (Hao et al., 2015; Marin et al., 2010a,b; Ren et al., 2017). In addition, the target genes of conserved miRNAs are shown to exhibit Plant small RNAs: biogenesis, mechanistic functions and applications 133 common functions for eg. miR172 mediated regulation of AP2 TFs is involved in flower development of diverse species such as rice and kiwifruit (Varkonyi-Gasic et al., 2012). The important developmental functions such as phase transition and trichome development mediated by miR156 induced regulation of SPL TFs is found in diverse speciwes such as Arabidopsis, tomato and even perennial trees (Wang et al., 2009, 2011; Yu et al., 2010; Zhang et al., 2011). Besides, these conserved small RNAs, some species-specific miRNAs have been demonstrated to induce specific plant phenotypes (Akagi et al., 2014; Moxon et al., 2008). 4. Small RNAs of field crops Small RNAs has been demonstrated to play multi-various roles in several crops particularly, growth and development, signaling pathways and homeostasis and response to biotic and abiotic stresses (Sun, 2012; Xie et al., 2014; Zhang & Wang, 2015). miRNAs has been subjected to reprogramming the cell metabolism in the event of microbe-associated molecular pattern (MAMP) molecules during pathogenesis in plants (DjamiTchatchou & Dubery, 2015). The sequences of miRNAs are identified in diverse organisms are documented in miRBase version 22.1 (http:// www.mirbase.org). A summary of functional role ascribed to miRNAs identified in field crop plants is presented in Table 6.1. 4.1 Rice (Oryza sativa) Rice is a staple food for half the world’s population and its production is largely affected by several biotic and abiotic factors. miRNAs playing various roles viz., plant growth and development, disease resistance, transport and metabolic functions has been reported in rice (Sunkar et al., 2005; Wang et al., 2004; Zhao et al., 2007a). Over expression of miR397 down regulated the L-ascorbate oxidase levels leading to increased grain size and panicle branching in rice (Zhang et al., 2013). Expression profiling analysis has revealed the role of rice miRNAs in ovule development and programmed cell death (Wu et al., 2017). miR529a has been reported to be regulating the SPL target genes, OsSPL2, OsSPL16, OsSPL17 and SPL18 in rice and control the plant height, tiller number, panicle architecture and grain size (Yan et al., 2021). Role of miRNAs in implicating abiotic stress tolerance is reported by Sunkar et al. (2007). miRNA-mediated regulation of signaling pathway during short and prolonged chromium stress in rice has been reported Table 6.1 Functional role of miRNAs in field crops. miRNA Target genes Functional involvement References miR393 Auxin receptor gene (TIR1 and AFB2) Drought response High tillering and early flowering miR820 DRM2 miR167 ARF transcription factors Response to salt, high temperature Cold-stress miR397 l-ascorbate oxidase miR319 miR1848 Transcription factors PCF5-like, PCF6-like Transport Inhibitor Response1 (OsTIR1) and Auxin signaling f-box 2 (OsAFB2) TATA box binding protein associated factor (TAF), growth regulating factor protein, Cytochrome P450 51G3 Zhou et al. (2010) Jiao et al. (2010) Xia et al. (2012a) Sharma et al. (2015) Jeong et al. (2011) Jeong et al. (2011) Yang et al. (2013) Zhao et al. (2019) miR1861 OsSBDCP1 Root elongation, leaf development and stress responses Plant development via regulation phytosterol biosynthesis Grain filling miR156a OsSPL2dSBP Regulatory pathways osa-miR393 osa-miR396c Heat stress response and adaptation Cold, Salt and drought responsive miRNA Salt and drought stress responsive miRNA Gao et al. (2010) Xia et al. (2015) Teng et al. (2021) Kansal et al. (2021) Plant Small RNA in Food Crops Rice (Oryza sativa) 134 Plant species Maize (Zea mays) Barley (Hordeum vulgare) Soybean (Glycine max) Development Endosperm development Ear development miR167 ARF transcription factors Stress response miR396 miR169 Growth factor NF-YA transcription factors Drought stress response miR397, miR437 miR395 l-ascorbate oxidase ATP sulfurylase genes Development Abiotic stress miR1435/miR51812 miR156d Squamosa binding protein miR396d Growth factor miR399b miR164 Phosphatase transporter ARF transcription factors miR169 miR160 miR396e NFYA3 ARF GRF3 and GRF5 miR2118, miR171c, miR156, miR160 miR164, miR166 miR172, miR396 TCP Squamosa binding protein ARF transcription factors Growth factor Ion transportation Development, drought stress Seed development Cell differentiation Drought stress response Lateral root and leaf development Drought Tolerance Heavy metal stress Resistance against bean pyralid larvae infestation Soybean cyst nematode Seed development Li et al. (2012) Gu et al. (2013) Ding et al. (2013) Sheng et al. (2015) Sheng et al. (2015) Han et al. (2013) Curaba et al. (2012) Shuzuo et al. (2012) Deng et al. (2015) Gupta et al. (2021) Song et al. (2011) Continued 135 Squamosa binding protein ARF transcription factors NAC1 (NAM, ATAF, CUC) Transcription factors Plant small RNAs: biogenesis, mechanistic functions and applications Wheat (Triticum aestivum) miR156 miR160 miR164 Cowpea (Vigna unguiculata) Peanut (Arachis hypogaea) miRNA Target genes miR160 miR166 Functional involvement References Drought stress BarreraFigueroa et al. (2011) Shui et al. (2013) miR159 miR167 ARF transcription factors Enhanced drought tolerance miR169 miR319 Growth factor miR390, miR393 miR396, miR403 miR156b,f Metabolic pathways of physiological changes associated with Drought stress Multicystatin gene Shui et al. (2013) miR156 Squamosa binding protein Protein degradation/ drought stress/keep cellular proteins Peanut growth and development Lipid and protein accumulation Disease resistance Drought stress Zhang et al. (2017) miR159, miR171 miR159, miR396 miR156, miR157 miR169, miR166 miR2111 miR482 miR160, miR164 miR167 miR156 ahy-miR3508 Auxin response factors Lipid transfer protein MYB TF, RLKs Lipid transfer protein NAC transcription factor TIR-NBS-LRR resistance protein Calcium-dependent protein kinase SPLs Pectin esterase gene Defense response against A. flavus infection Pod development Nodule functions Chi et al. (2011) Zhao et al. (2010) Zhao et al. (2015) Li et al. (2016) Figueredo et al. (2020) Plant Small RNA in Food Crops Plant species 136 Table 6.1 Functional role of miRNAs in field crops.dcont'd Disease resistant proteins, auxin responsive proteins, squamosa promoter binding like proteins, cotransporter protein, transposable element genes, NAD(P) binding protein and topoisomerase II Metabolic, biosynthetic, degradation and signaling pathways Ram et al. (2019) miR156e Squamosa promoter binding proteinlike (SPL) TCP4 Phenylpropanoid biosynthesis, linoleic acid metabolism, and cutin, suberine, and wax biosynthesis SBP/SPL transcription factors Seed expansion. Ma et al. (2018) miR319d_Lþ1R-2 novel miR_416 and novel miR_73 Sugarcane (Saccharum sp.) miR156 miR159 miR169 MYB protein HAP12-CCAAT-box transcription factors miR398 Serine/threonine kinase-like miR164 miR399 miR319 miR159 miR395 NAC transcription factors Inorganic pyrophosphatase 2 Myb transcription factor (GAMyb, TC94752) and TCP (PCF6, TC111376) MYB gene SULTR2; 1 miR156 SPL Seed vigor Drought Tolerance Development, stress response Development Salt stress tolerance Salt stress tolerance/ metabolism Drought stress response Drought stress response Cold stress Drought stress Al stress Zanca et al. (2010) CarnavaleBottino et al. (2013) Ferreira et al. (2012) Thiebaut et al. (2012) Lin et al. (2014) Capaldi et al. (2015) Li et al. (2017) 137 Leaf abscission during the maturity time Ren et al. (2021) Plant small RNAs: biogenesis, mechanistic functions and applications miR156, miR166, miR167, miR319, miR398, miR399, miR482 and miR1507 Continued Plant species Target genes Functional involvement References miR396 GRF miR393 Auxin signaling pathway Cold stress Drought and salinity stress Salinity stress Salinity stress Salinity stress miR160 ARF transcription factors Squamosa Binding Protein (SBP) SPL9 SPX-domain containing protein (SDP) Auxin response factor 17 (ARF17) Yang et al. (2018) Mazalmazraei et al. (2021) miR390 miR408 miR529 miR827 miR156 SBP/SPL transcription factors miR169 NFY miR398 miR170/171 Selenium binding protein GRAS domain transcription factors Development, increased biomass metabolism Development, drought response Transportation Development miR395 ATP, APS1 and Sultr1 miR396 Growth-regulating factor miR397/398/408 Laccase miR399 UBC24 enzyme Al stress Development, low Su response Development, stress response Response to Cu deficiency Phosphate deficiency Silva et al. (2021) Katiyar et al. (2012) Maheswari et al. (2019) Du et al. (2010) Zhang et al. (2011) Katiyar et al. (2012) Katiyar et al. (2012) Plant Small RNA in Food Crops Sorghum (Sorghum bicolor) miRNA 138 Table 6.1 Functional role of miRNAs in field crops.dcont'd Cotton (Gossypium hirsutum) SBP/SPL transcription factors miR172 AP2, SPL3 miR319 MYB protein miR396 Callose synthase Development, stress response Flower development, phase change Controlled leaf development Cotton fiber development miR167a ARF transcription factors Salt stress tolerance miR395 miR397a/b miR399a miR395 miR398 novel_miR_57 novel_miR_58 miRNVL5 APS1 Laccase UBC24 enzyme NAC domain transcription factor Sulfate transporter Nuclear transcription factor Y subunit A-1, putative isoform 1 Bacterial-induced lipoxygenase GhCHR miR2950 miR167b miR447 miR319 TUB auxin response factor 17 heat shock 70 protein (HSC70). TCP4 miR2950 ORFs C1, C4, V1 Salt and drought stresses Disease resistance Salt stress Cotton fiber initiation Development Short and thick fibers on the ovular surface. Leaf curl disease resistance Wang and Wang (2015) Zhang et al. (2007) Yin et al. (2012) Wang et al. (2013) Wang et al. (2013) Zhang et al. (2015b) Gao et al. (2016) Wang et al. (2017) Continued 139 Akmal et al. (2017) Plant small RNAs: biogenesis, mechanistic functions and applications miR156 Tobacco (Nicotiana tabacum) miRNA Target genes Functional involvement References miR160a_A05 ARF17 Fiber architecture Liu et al. (2019) miRNA ghr-miR414c GhFSD1(antioxidase gene) Salinity stress miRna-23 miR156 Xyloglucan endotransglucosylase/ hydrolase SPL miR160/167 ARF transcription factors miR164 miR169 miR171 miR172 NAC transcription factors NFY GRAS domain transcription factors AP2, SPL3 MiR319 miR393 MYB protein ARF and AFB miR166 miR399 miR408 Leucine-rich (LRR) repeat family 4-Coumarate-coenzyme A ligase miR395 Sulfur transporter genes miR159 miR160 MYB101 and MYB33 transcripts ARF transcription factors Anther development in CMS cotton Development, stress response Development, stress response Lateral root development Development, drought Development, growth Development, stress response Development Development, stress response Disease resistance Stress response Response to wounding and topping Salinity and drought stresses Drought stress Drought tolerance Wang et al. (2019) Li et al. (2022) Guo et al. (2011) Frazier et al. (2010) Guo et al. (2011) Tang et al. (2012) Frazier et al. (2011) Yin et al. (2015) Plant Small RNA in Food Crops Plant species 140 Table 6.1 Functional role of miRNAs in field crops.dcont'd CSD1 and CSD2 Cr stress tolerance Bukhari et al. (2015) miR169 Cd stress He et al. (2016) (nta-miR393a-5p CCAAT-binding transcription factor subunit B Transport inhibitor response 1 Abiotic stress tolerance nt_miR91 RR9 Potyvirus resistance miR159 GAMYB bra-miR398 miR156 miR156 BracCSD1 BracSPL2 SPL Pathogen defense Response Heat stress Khan et al. (2020) Xiao et al. (2022) Zheng et al. (2020) Yu et al. (2012) miR156 miR160 miR164 miR2926 SPL2 ARF17 NAC1 Transcription factors Heat stress miR160 Auxin response factors Silique length miR394 miR319 miR1885 Leaf curling responsiveness (AtLCR) TCPs PHAS loci residing within NBSLRR genes Leaf size regulation Leaf development Disease resistance Seed maturation Abiotic stress tolerance Huang et al. (2013) Bhardwaj et al. (2014) Singh et al. (2017) Chen et al. (2018) Karamat et al. (2021) Regmi et al. (2021) Plant small RNAs: biogenesis, mechanistic functions and applications Brassica miR398 141 142 Plant Small RNA in Food Crops (Dubey et al., 2020). Rice miRNAs target the plant homeodomain (PHD) finger proteins and play role in plant’s response to salinity stress. OsPHD2, OsPHD35 and OsPHD11 have been targeted by putative miRNAs, athmiRf10010-akr, ath-miRf10110-akr, osa-miR1857-3p, osa-miRf10863akr, and osa-miRf11806-akr (Waziri et al., 2020). Differentially expressed lncRNAs, TCONS_00,008,914 and TCONS_00,008,749 were identified as putative target mimics of reported miRNAs of rice (Jain et al., 2021). Overexpression of the miR7695 showed improved disease resistance to rice blast disease (Campo et al., 2013). A map-based cloning of a QTL conferring immunity to bacterial blight pathogen Xanthomonas oryzae pv. Oryzae (Xoo) has been reported. Expression of NBS8R, an NB-ARC protein and its expression has regulated Osa-miR1876 which is inducible by non-TAL effector XopQ through DNA methylation. This proves to be a useful tool in rice breeding for BLB resistance (Jiang et al., 2020). Seven rice miRNAs were predicted to play vital role in silencing of the Rice Yellow Mottle Virus (RYMV) genome based on computational approaches (Jabbar et al., 2019). 4.2 Maize (Zea mays) Maize is a major millet crop used widely for food, feed and fuel purposes world over. miRNAs in maize, miR156, miR160, miR166, miR167, miR169 were characterized based on its potent target genes with the functional involvement in plant growth and development and its response to biotic stresses (Mica et al., 2006). Maize miRNA, miR164 is reported to play regulatory role in development and stress by modulating the ZmNAC1 TF (Li et al., 2012) and also in endosperm development (Gu et al., 2013). Differentially expressed miRNAs, belonging to 11 miRNA families such as miR159a, miR164a, miR171c, miR398a etc., exhibited functional participation in ear development of the crop (Li et al., 2015). Maize kernel development involves regulation of gene expression at transcriptional and post transcriptional levels, especially the miRNA genes viz., miR159, miR164, miR166, miR171, miR390, miR399, and miR529 families that has demonstrated role of grain development, whereas, miR167 and miR528 families has been reported to be involved in stress response during nutrient storage (Li et al., 2016). Role of maize miRNA and the target regulation in response to hormone depletion and light exposure has been experimented during the somatic embryogenesis in maize. Among the eight miRNAs validated, the expression levels of miR528, miR408, and Plant small RNAs: biogenesis, mechanistic functions and applications 143 miR398 are altered the most (Chávez-Hernández et al., 2015). Small ncRNAs and transacting siRNAs (Tasi-RNAs) play key role in regulating gene expression that control plant development and response to biotic and abiotic factors. Regulatory mechanism of miRNAs in response to Pi deficiency in maize is reported. miR390-directed tasiRNAs, that belong to TAS3 gene family were also identified (Gupta et al., 2017). miRNAs are involved in regulatory role in submergence, drought, and alternated stress in maize. miR159a, miR166b, miR167c, and miR169c were downregulated by submergence in maize and its close relative teosinte. miR156k and miR164e are downregulated in maize under drought stress situations (Sepúlveda-García et al., 2020). During chilling stress in maize, miRNAs regulate the leaf size thereby helps in sustaining the redox homeostasis of cell (Aydinoglu, 2020). Significant differential expression of miRNAs, down-regulation of miR159, miR160, miR166, miR319, and miR396 and up-regulation of miRNA, miR393 were observed under Cd stress (Gao et al., 2019). miR168 and miR528 genes were upregulated and miR159, miR397 and miR827 were down regulated during the course of infection which shows regulatory role of miRNAs in plants response to Sugarcane mosaic virus infection (SCMV) infection in maize (Xia et al., 2018). 4.3 Wheat (Triticum aestivum L.) Wheat along with rice and maize forms the core of three major cereals produced throughout the world and has important nutritional attributes that can assure food security. Novel miRNAs involved in plant development (miR397, miR437), abiotic stress response (miR395d, miR1435), disease resistance (miR1436, miR1439, miR5067, miR5205), metabolic pathways (miR774, miR1126), and ion transportation (miR5181, miR5175) were identified by EST analysis and their potential target genes predicted (Han et al., 2013). miRNAs that regulate target genes encoding Fbox/Kelch-repeat proteins, ubiquitin carrier protein, BTB/POZ domaincontaining proteins and the transcription factors involved in plant growth and stress responses were reported (Pandey et al., 2013). Abiotic stress responsive miRNAs, Ta-miR1122, Ta-miR1117, TamiR1134 and TamiR113, Ta-miR5653, Ta-miR855, Ta-miR819k, Ta-miR3708, and Ta-miR5156 were identified in wheat (Sun et al., 2014; Shi et al., 2018). Based on the de novo assembly and homology-based approaches, 132 mature miRNAs and 37 novel miRNAs were identified in wheat and these 144 Plant Small RNA in Food Crops miRNA are validated and reported to be heat responsive in nature (Kumar et al., 2015). miR430 has been reported to manipulate the expression of target genes under heat stress and aids in development of climate-smart wheat crop in future (Kumar et al., 2017). Functional miRNAs, miR156, miR164, miR1432, miR398, and miR397 were differentially expressed in immature and mature embryos playing a key role in embryogenic callus formation and somatic embryogenesis in wheat (Chu et al., 2016). Analysis of miRNA in T. aestivum identified about 89 miRNA family members, 68 of known and 21 novel ones which are differentially expressed under K-deficiency condition. The target genes of 11 differentially expressed miRNAs (novel_17, miRNA319, miRNA531, miRNA9773, miRNA9670-3p, miRNA398, miRNA159a, miRNA9778, miRNA408, miRNA9776, and miRNA1133) were found to be associated with tolerance of wheat plants to potassium deficiency. These miRNA genes are enriched in functional categories attributed to essential roles played by these miRNAs on energy and secondary metabolism under potassium deprivation in wheat (Zhao et al., 2020). 16 miRNAs and 22 lncRNAs responsive to Lr-28 mediated leaf rust resistance in wheat were identified using RNAseq data. Among them, three lncRNAs possessed binding sites identical to the targets for miRNAs (Jain et al., 2020). 4.4 Barley (Hordeum vulgare L.) Barley is an important cereal crop produced and consumed for its food and feed values. In addition to wheat, barley has been widely used as a model crop for genetics and breeding studies (Sreenivasulu et al., 2008). About 12 drought stress-responsive miRNAs that participates in gene expression, metabolism, signaling and transportation and their target genes involved in drought tolerance is reported (Qiu et al., 2020; Zare et al., 2019). High throughput sequencing of barley resulted in identification and characterization of 133 novel and 126 highly conserved miRNAs that were categorized into 58 miRNA families. miR-n026 and miR-n028 are characterized to play key role in drought, salt and/or other environmental stress responses (Shuzuo et al., 2012). In barley, 152 salinity stress responsive miRNAs were identified, out of which 142 are conserved and 10 are novel. Degradome sequencing was employed to identify the target genes of these barley miRNAs, in which 86 and 37 target gene families were involved in metabolic process and response to stimulus, respectively (Deng et al., 2015). Plant small RNAs: biogenesis, mechanistic functions and applications 145 Cold deacclimation is a key factor which allows plants to survive in winter by loss of freezing tolerance thereby initiating plant growth. In barley, a total of 36 known and 267 novel miRNAs were identified that are differentially expressed during cold deacclimation. The putative targets of the novel miRNAs were genes that encode phytohormones, C-repeat binding factor (CBF), osmoprotectant, antioxidant activity and flower development (Li et al., 2020). MicroRNAs, miR156, miR159, miR166, miR167, miR171 and miR393 that regulate target genes including SPL, MYB, HD-Zip, ARF, GRAS and TIR in barley are reported (Yu et al., 2019a). About 525 miRNAs including 198 known and 327 novel members were identified. Among these, 31 miRNAs belonging to 17 families, five (miR166a, miR166a-3p, miR167b-5p, miR172b-3p and miR390), four (MIR159a, miR160a, miR172b-5p and miR393) and three (miR156a, miR156d and miR171a-3p) miRNAs were responsive to aluminum, cadmium and salt stress, respectively (Kuang et al., 2021). miRNAs of barley also play role are metabolic sensors of nutrients. Out of 13 barley microRNAs identified, 2 microRNAs are nitrogen excess responsive in nature (Grabowska et al., 2020). MicroRNA-based markers serve as tool to monitor the response of barley to soil compaction. Molecular markers corresponding to dehydratation stress-responsive barley miRNAs (hvu-miR156, and hvu-miR408) and nutrition-sensitive markers (hvu-miR399 and hvu-miR827) were used to analyze barley in response to dehydratation and nutrition stress (Razna et al., 2020). In an analysis of miRNAs associated with Barley yellow dwarf virus (BYDV) infection, 73 miRNAs were differentially expressed and miRNA10778 was specifically upregulated. This miRNA is identified as that belongs Rf01280 (snoR14 family of RNAs) with H box and ACA box (H/ACA) domains (Jarasova et al., 2020). Barley leaf stripe (BLS) is a serious fungal disease that affects production and quality of grains in Tibetan hulless barley. miRNA profiles before and after BLS infection was compared. About 10 candidate target genes, CYP450 genes, a RGA gene, a LIN gene, a SAM gene, a PSD gene, and a NDB gene; the three transcription factor genes from the WRKY family were regulated by eight miRNAs (hvumiR168-3p, hvu-miR171-5p, hvu-miR159b, hvu-miR156a, hvu-novel91, hvu-novel-46, hvu-novel-52, and hvu-novel-11 (Yao et al., 2021). Barley whole grain is used to ameliorate hyperlipidemia through modulating AMPK/SREBP-1c/FAS pathway, cecal microbiota and related miRNAs. The miRNA levels of miRNA-122, miRNA-33, miRNA-34a, and miRNA-206 were downregulated triggering the alleviation of 146 Plant Small RNA in Food Crops dyslipidemia (Deng, He, et al., 2020). Similarly, whole grain of highland barley exhibited a hypoglycemic effect by regulation of IRS-1/PI3K/Akt pathway and related miRNAs affecting the target gene expression of G6PC, PEPCK, and FOXO1 mRNAs and p-GSK3b protein inhibiting hepatic gluconeogenesis and pronounced improved glycogen synthesis thereby alleviating insulin resistance (Deng, Guo, et al., 2020). 4.5 Sorghum [Sorghum bicolor (L.) Moench] Sorghum is a cereal crop popular for its nutritional values and used as food, feed and raw material for production of starch, biofuel and alcohol (Mutegi et al., 2010; Prasad et al., 2008). miRNAs gains attention for improvement in biomass accumulation and plant stress tolerance. Currently there are about 205 precursors and 241 mature miRNA available in miRbase 22.1. Zhang et al. (2011) in an attempt to understand the post transcriptional gene regulation in sorghum, identified 29 miRNA families and the targets for the miRNAs are predicted. Laccase is a key regulator of plant lignin biosynthesis and sweet sorghum [Sorghum bicolor (L.) Moench] being an ideal feedstock considered for the ethanol production has limitation with lignin that affects the production efficiency. Seven SbLACs genes were predicted to be potential candidates for the sbi-miRNA targets. SbLAC8 was found to be possibly targeted by sbi-miRNA164a, b, d, and e (Wang et al., 2017a). Small RNA profiling performed for identification of miRNA target molecules during the meiotic and post-meiotic stages of anthers was performed with a view to engineer male fertility in sorghum. Out of 262 miRNAs, 58 miRNAs were differentially expressed and the target identification resulted in 5622 miRNA/target modules (Dhaka et al., 2020). Comparative expression analysis of miRNAs and their targets revealed 226 conserved and novel miRNAs and about 475 targets predicted in sweet sorghum. miRNAs expression patterns and target genes showed variation during transitions of the developmental stages of the crop (Gyawali et al., 2021). Differentially expression analysis of miRNA in the stems and leaves of sorghum during sugar accumulation was studied by high-throughput sequencing. mir-271 and mir 431 specifically expressed in the stems and leaves, respectively during the dough stage (Yu et al., 2015). miRNAs are integral part of gene regulatory networks and thus helps plant adapt to stress conditions. miR160a, miR164 and miR390 levels were upregulated under both heat and/or drought stress situations (Puli et al., Plant small RNAs: biogenesis, mechanistic functions and applications 147 2021). In case of sweet sorghum, the comparative analysis of sRNAs, degradome deciphered the role of miRNAs, sbi-miR169p/q, sbimiR171g/j, sbi-miR172a/c/d, sbi-miR172e, sbi-miR319a/b, sbimiR396a/b, miR408, sbi-miR5384, sbi-miR5565e and nov_23 in Cd accumulation and tolerance (Jia et al., 2021). Genome-scale identification and tissue specific expression analysis of the LEA genes in Sorghum resulted in identification of 15 miRNAs that targets about 25 SbLEAs which play role in development, abiotic stress tolerance. Among them, Sbi-miR6225, sbi-miR437x, sbi-miR5568, and sbi-miR6220 appeared to be most common miRNAs that participate in cleavage and translation (Nagaraju et al., 2019). Plant NUCLEAR FACTOR Y (NFeY) is reported to be involved in plants adaptation to several abiotic stresses and also in plant development. These proteins bind to the CCAAT box region in the promoter of the target genes and regulate gene expressions. miR169 identified was reported to be involved in post transcriptional regulation and interestingly all the in silico predicted targets for the miRNAs showed its involvement in translation and cleavage events (Maheswari et al., 2019). For identification of the immune responses of sorghum to anthracnose infection, genome-wide miRNA profiles of resistant and susceptible sorghum genotypes were studied. Out of 75 miRNAs, 36 novel miRNAs that are differentially expressed and the corresponding 149 target genes were identified (Fu et al., 2020). 4.6 Sugarcane (Saccharum sp.) Sugarcane is a major energy efficient crop valued for the sugar and feedstock. The polyploidy nature of the crop and its genomic peculiarities makes the crop improvement approaches more complex for the agronomically important and economic traits like yield, sugar content and plants resistance to biotic and abiotic stresses (Swapna & Kumar, 2017). Sugarcane is a commercial crop valued for its sugar and ethanol production (Azevedo et al., 2011). Sugarcane has a complex polyploid genome and its miRNA sequences share homology with sorghum miRNAs (Zanca et al., 2010), this makes sorghum as reference genome for sugarcane in genomic analysis. About 19 sugarcane miRNAs and its 46 potential targets were identified that has important role in several biological processes. miRNAs involved in sucrose accumulation of sugarcane (Saccharum species hybrid) has been reported (Banerjee et al., 2022; Wang et al., 2022). About 148 Plant Small RNA in Food Crops 23 miRNAs that regulate 50 target genes were identified and modulation of gene expression by these miRNA were associated with lesser and slower accumulation of sucrose in sugarcane. These miRNAs target the genes involved in stress tolerance, hormone signaling, phosphate homeostasis, hydrolases, transport, cellulose metabolism. Genome-wide identification of miRNAs in sugarcane (Saccharum officinarum L.) revealed miR5384 and some miRNA-mRNA modules, miR156-SPL, miR408-LAC, miR319TPR2 and miR396-GRF which regulates the leaf abscission in sugarcane during the maturity time (LI et al., 2017). Sugarcane miRNAs are involved in gene expression regulation during abiotic stresses. Salt stress responsive miRNAs were identified and miRNA, miR528 that regulate putative laccase gene is reported (Carnavale Bottino et al., 2013). Sugarcane miRNAs affecting the growth and development, especially reduction in the internodes, shoot branching and higher degree of leaf senescence under drought stress by targeting the transcription factors and genes that govern tolerance to oxidative stress and cell modification is reported (Gentile et al., 2015; Qiu et al., 2019). A drought stress responsive miR399 has been identified based on in silico approach (Ferreira et al., 2012). Sugarcane although originated from the tropics, primarily grown in the sub-tropical areas and is often exposed to cold stress. Out of 412 sugarcane miRNAs, 62 were found to show differential expression under cold stress, among which 34 were upregulated and 28 being downregulated. miR156 in plant response to cold stress has been validated in sugarcane (Yang et al., 2017). miRNAs plays role in plant tolerance to heavy metals by modulating gene expression of target genes. miR395, a miRNA involved aluminum detoxification was identified and miR160, miR167, miR6225-5p showed protective role through lateral root formation and conferred tolerance to the genotype under aluminum stress (Silva et al., 2021). Gene expression regulation by the miRNAs during the host-pathogen interaction between sugarcane and the red rot pathogen, Colletotrichum falcatum has been reported (Nandakumar et al., 2021). A total of 80 miRNAs and their putative targets included transcription factors, membrane-bound proteins, receptor proteins, lignin biosynthesis, signaling, transporter, mitochondrial proteins, ER proteins, defense-related and stress response proteins. miRNAs targeting the sugarcane bacilliform virus (SCBV) was identified through the in silico analysis in sugarcane (S. officinarum L.). Comprehensive analysis of miRNAs, revealed Plant small RNAs: biogenesis, mechanistic functions and applications 149 14 potential candidate miRNAs that are involved in silencing of SCBV with consensus of the three algorithms predicting the hybridization site of sof-miR159e at the common locus 5534 (Ashraf et al., 2022). 4.7 Soybean (Glycine max L.) Soybean is a wonder crop and a rich source of oil and protein making it amenable for food and feed. Extensive research work has been carried out on this crop for understanding the role of several genes involved in growth and development and cell functions. miRNAs in soybean has been characterized and its targets for gene regulation predicted. 55 families of miRNAs including 20 conserved and 35 novel ones were identified (Subramanian et al., 2008). Currently 684 soybean miRNA sequences are deposited in miRbase 22.1. Analysis of the regulatory network of miRNAs in soybean revealed functions during the seed development process (Song et al., 2011). Small RNAs regulate gene expression in the roots of soybean under water deficit stress (Kulcheski et al., 2011) and play regulatory roles in response to aluminum stress (Qiao-Ying et al., 2012; Huang et al., 2013). Similarly, miRNAs as key regulators in response to pathogen infections in seedlings by Asian Soybean rust (Kulcheski et al., 2011) and root rot disease caused by Phytophthora sojae are reported (Jingi et al., 2011). The vulnerability of yellow mosaic virus (YMV) and Mungbean yellow mosaic Indian virus (MYMIV) to the miRNAs targeting its genome has been reported. miRNAs derived from G. max, and Glycine soja displayed 63 and 18 potential targets on the begomovirus genomes (Ramesh et al., 2016). Further expressional changes of soybean derived miRNAs and its role in repressing the expression of MYMIV-derived transcript encoding viral movement protein was proven (Ramesh et al., 2017). MicroRNA-encoded peptide, miPEP172c treatment in soybean has mimicked the miR172c overexpression leading to regulation of symbiotic phenotypes during the process of nodulation at both phenotypic and molecular levels (Yadav et al., 2021). A detailed review on miRNAs of soybean in general and stress-responsive miRNAs have been explored and its regulatory role in various biological processes presented (Gupta et al., 2021; Ramesh et al., 2019). Noncoding RNAs, especially the miRNAs and lncRNAs are key regulators of gene functions. These noncoding RNAs confers salinity stress tolerance in soybean by arm switching of miR166 m and interactions of miRNA:lncRNAs (Li et al., 2022). 150 Plant Small RNA in Food Crops 4.8 Cowpea (Vigna unguiculata L.) Cowpea is an important tropical food grain legume crop raised in semi-arid and arid tropical regions and serves as food and nutritious feed for livestock. Cowpea plants adapt to drought stress conditions and have been widely used in genetic studies for understanding the stress response. miRNAs involved in drought stress responses has been reported and its mechanism described (Barrera-Figueroa et al., 2011; Zhou et al., 2010). About 47 miRNAs from 13 miRNA families identified in Cowpea using in silico prediction strategies and its potential target genes involved in the growth and development, metabolism, and other physiological processes predicted (Lu & Yang, 2010). About 15 potential target genes of cowpea were predicted and identified as TFs and out of these, seven V. unguiculata miRNAs were upregulated in the root tissues under salt stress conditions (Paul et al., 2011). The drought stress associated miRNAs identified include miR156, miR160, miR162, miR164, miR166, miR159, miR167, miR169, miR171, miR319, miR390, miR393, miR396 miR403 and miR482. Among these, miR169 confers drought tolerance by altering the expression of orthologs of nuclear factor Y TF (Barrera-Figueroa et al., 2011). miRNA regulated program cell death due to Tospo viral infection leading to necrosis and premature senescence has been reported in V. unguiculata (Permar et al., 2014). The homology-based miRNA identification in the cowpea genome detected 617 mature miRNA which were classified into 89 miRNA families. In response to challenge by the cowpea severe mosaic virus (CPSMV), the miRNAs and the Argonaute genes, AGO 2 and AGO4 showed differential expression patterns indicating their role in cowpea defense (Martins et al., 2020). 4.9 Peanut (Arachis hypogaea L.) Peanut (also known as groundnut) is an important oilseed crop widely cultivated world over. Research on miRNA using next generation sequencing platform Solexa sequencing resulted in identification of 14 novel (miRn1 to miRn14) and 75 conserved miRNAs (miR156 to miR894, etc.) in peanut (Zhao et al., 2010). The targets of these conserved miRNAs were predicted, which include GRAS (gibberellic-acid insensitive, repressor of gai, and scarecrow) family of TFs, NAC1 TF, nuclear TF Y subunit, proteins involved in translation, auxin signaling F-box 3 protein, transport inhibitor response 1, resveratrol synthase, basic blue copper Plant small RNAs: biogenesis, mechanistic functions and applications 151 protein, endonuclease, growth regulator factor 5, protein kinase, and proteins responsive to disease resistance. Chi et al. (2011) identified 25 novel and 126 conserved miRNAs in peanut that belonged to 33 miRNA families and its target genes involved in plant metabolism and environmental stress response. miR156, miR159, miR171, and miR14 families targeted genes involved in amino acid, fatty acid and lipid metabolism suggesting the key role played by miRNAs in peanut. Role of miRNAs in bacterial wilt disease of peanut caused by Ralstonia solanacearum is demonstrated (Zhao et al., 2015). Several miRNAs were upregulated during the infection response including miR156/157, miR159, miR162, miR166, miR169, miR396, miR482, miR530, miR3516, miR894, miR1511, miR2118, miR2199, miR3513 whereas miR397, miR408, miR1508, miR2111, miR3515, miR3522, miR4144 were downregulated. The targets for these miRNAs predicted which includes defense response genes like aquaporin, MYB, GRAS family of TFs, lipid transfer protein, auxin response factors, hypersensitive-induced response protein, leucine-rich repeat (LRR) receptor-like serine/ threonine-protein kinase and MLP-like protein. miRNA analysis was performed in the Arachis duranensis during gynophorogenesis at different development stages, A1, A2, and A3 corresponding to 5, 10, and 20 days of gynophore development, respectively. 266 known and 357 novel miRNAs were obtained and their targets were predicted (Shen et al., 2016). High-throughput sequencing approaches used to explore the role of miRNAs in peanut embryogenesis and early pod development led to identification of 70 known and 24 novel miRNA families. These miRNAs are demonstrated to be peanut-specific and expressed during early pod development (Gao et al., 2017). About 116 differentially expressed miRNA were identified through miRNA profiling and the functional analysis revealed their target genes like SPL, B3 domain transcription factors, KCS, PLC to have important role during the process of seed development (Sui et al., 2019). Comparative genomics strategy have aided in identification of miRNAs and their corresponding functions. About 34 conserved miRNAs, belonging to 17 miRNA families were identified from the A. hypogaea EST and Genome Survey Sequences (GSS). Functional annotation of the identified targets for the miRNAs revealed their regulatory roles in important major biological and metabolic processes (Rajendiran et al., 2019). 152 Plant Small RNA in Food Crops miRNAs involved in functional processes of nodulation is studied in peanut. 32 differentially expressed miRNAs precursors in nodules compared to roots were identified. Among these, 20 miRNAs belonging to 14 conserved miRNAs families including 12 A. hypogaea-specific miRNAs were identified. Three miRNAs, ahy-miR399, ahy-miR159 and ahymiR3508 were validated by qPCR and demonstrated to have regulatory role in nodule functionality (Figueredo et al., 2020). Genome-wide identification of auxin response factor (ARF) gene family and the overexpression of miR160 and its target ARF18 showed protective role played by miRNAs under salt stress in peanut (Tang et al., 2022). 4.10 Sunflower (Helianthus annuus) Sunflower is one among the major oilseed crop recognized for its beauty and as an source of edible vegetable oil and has other uses as healthy snack and nutritious ingredient to many foods. However, there are only limited reports of miRNAs of sunflower identified and listed in the public repositories. Inter-species homologs of the miRNA precursors in Helianthus expressed sequence tags (ESTs) were searched and resulted in identification of 61 novel miRNAs from 34 families. Out of the 61 novel miRNAs, 20 belongs to H. tuberosus, 17 miRNAs to H. annus, 8 from H. ciliaris, 5 from H. exilis, four each from H. argophyllous, H. petiolaris each and 3 are from H. paradoxus. Prediction of the targets of identified miRNAs revealed their regulatory role in growth and development, pathway signaling, transport and stress resistant functions (Barozai et al., 2012). In sunflower, about seven conserved miRNAs, miR160, miR167, miR172, miR398, miR403, miR426 and miR842 associated with abiotic stress factors like heat, drought, salt and cadmium stresses were characterized. Expression analysis of the miRNAs and their targets revealed their regulatory role in key pathways such as auxin and ethylene phyto hormonal signaling, RNA mediated gene silencing and DNA methylation processes. miR172 and miR403 is assumed to play major role in epigenetic responses to various stress in a tissue-specific manner (Ebrahimi Khaksefidi et al., 2015). Regulatory mechanisms governing lipid metabolism in high oleic acid seeds of sunflower are explored. High-throughput sequencing of differentially expressing mRNAs, lncRNAs and miRNAs of sunflower seed RNA samples resulted in identification of 15,652, 123,888 and 98 novel mRNAs, lncRNAs, and miRNAs, respectively (Liu et al., 2021). Plant small RNAs: biogenesis, mechanistic functions and applications 153 4.11 Cotton (Gossypium hirsutum L.) Cotton is a most important fiber crop among the economic crops that meets the demands of natural textile industries and edible oil. Cotton is a model crop for the geneticists to understand plant polyploidization and biosynthesis of cell wall and cellulose (Wang et al., 2012b). Zhang et al. (2007) has identified and characterized 158 miRNAs in cotton. The role of miR396, miR414, and miR782 in regulating callose synthase, fiber protein Fb23 and fiber quinone oxidoreductase in cotton fiber differentiation and development were demonstrated. The target genes of cotton miRNAs are predicted and are associated with biological and metabolic processes like fiber initiation and development, embryogenesis, floral development, and responses to biotic and environmental stresses (Abdurakhmonov et al., 2008). MicroRNAs have been reported in regulating the development of male sterility in Upland cotton. 77 known and 256 novel miRNAs belonging to 54 and 141 miRNA families were identified. Analysis of the transcriptome revealed 232 target genes for these miRNAs regulating pollen germination, cellular development, cell death and sexual reproduction (Nie et al., 2018). miRNAs also play key role in conferring tolerance to abiotic stresses. About 17 cotton miRNAs belonging to eight families that are responsive to salinity stress were identified (Yin et al., 2012). Analysis of the comparative miRNAome of drought sensitive and resistant cotton revealed conserved and novel miRNAs (Boopathi et al., 2016). Role of miR398 as a key regulator of Cu homeostasis and mediator in temperature stress response is identified. Under low copper availability, this miRNA reduces the allocation of Cu to chloroplastic copper/zinc superoxide dismutases (Wang et al., 2016). About 140 miRNA families and 58 novel miRNAs and their potential targets were identified that were involved in growth and development of cotton (Zhang et al., 2015b). miRNA-target pair, miR164-NAC100 was found to be responsive to Verticillium dahliae infection and has modulated the expression of downstream genes with CGTA-box thus influencing plant disease resistance (Hu et al., 2020). In planta analysis of the role of Gossypium arboreum-encoded miRNAs on the genome of Cotton leaf curl Multan virus (CLCuMuV) and Cotton leaf curl Multan betasatellite (CLCuMB) was investigated. Function validation of miR398 and miR2950 by overexpression in G. hirsutum var. HS6 plants showed virus resistance monitored through inoculation of viruliferous 154 Plant Small RNA in Food Crops whitefly (Bemisia tabaci) insect vectors under protected conditions (Akmal et al., 2017). RNAi-mediated virus control strategy was deployed to validate the role of these miRNA in suppression of virus (Akhter & Khan, 2018). miRNAs also play role in heavy metal-related stress responses by altering the changes in ultra structure of chloroplast, induction of cell membrane damage, growth inhibition and photosynthesis. Expression analysis of miRNAs, miR159, miR162, miR167, miR395, miR396, miR156, miR398, miR399, miR414, miR833a, and miR5658 and their targets in both leaves and roots of cotton plants showed altered expression pattern due to lead induced stress compared to control (He et al., 2014). miRNA-target pairs, miR159-MYB, miR319-TCP4 and miR167ARF8 showed inverse expression patterns suggesting their role in regulatory role in response to Meloidogyne infection in cotton (Pan et al., 2019). In another experiment, a total of 266 miRNAs, including 193 known and 73 novel miRNAs, were identified by deep sequencing technology (Cai et al., 2021). Potassium deficiency significantly affects the seedling photosynthesis and respiration thus reducing growth and development through an miRNAmediated mechanism. 20 miRNAs, miR160, miR164, miR165, miR166, miR167, miR 169, miR171, miR390, miR393, miR 396, miR 847, miR857, miR156, miR162, miR 172, miR 319, miR 395, miR778, miR 399, and miR 827 and their corresponding target genes, ARF10, NAC1, HD ZiP, HD ZiP, NFYA, ARF3, TIR1, bHLH74, LACCASE7, SPL3, AP2, respectively were identified and characterized for their reported association with plant root development and homeostasis of nitrogen or phosphorus (Fontana et al., 2020). About 54 miRNAs were compared between the fiberless mutant Xu142-fL and its wild type in cotton. In wild type, 26 and 48 miRNAs were involved in cotton fiber initiation and primary wall synthesis and secondary wall thickening, respectively. These miRNAs target 723 genes that include transcription factors such as LRR, MYB and ARF (Sun et al., 2017). 4.12 Tobacco plant (Nicotiana tabacum) Tobacco is an economically important non-food crop valued for its use as fumigatory and masticatory uses. Also, it serves as a model plant by for the molecular biologists to study fundamental biological processes (Chen et al., 2017). Extensive research on genomics of the crop led to availability of draft Plant small RNAs: biogenesis, mechanistic functions and applications 155 genome of tobacco (Sierro et al., 2014) in public domains for transcriptomic and genomic studies. Frazier et al. (2010) reported 259 tobacco miRNAs belonging to 65 families and predicted 1225 miRNA targets that regulate the genes, like TFs, DNA replication proteins and enzymes involved in growth and development. In flue cured tobacco, topping is an agronomic practice that aids in removal of the flowering head and young leaves thereby helps in transition of plant from reproductive to vegetative phase thus aiding more foliage yield of tobacco. About 136 conserved and 126 novel miRNAs belonging to 32 and 77 miRNA families were differentially expressed (Guo et al., 2011). Similarly, another study detected the expression of 100 known miRNAs from 27 families and 59 novel tobacco-specific miRNAs belonging to 38 families in roots and leaves (Tang et al., 2012). Role of miRNAs in regulating nicotine biosynthesis in tobacco has been investigated (Jin et al., 2020; Li et al., 2015). miRX27 that targets quinolinate phosphoribosyl transferase-2 encoding a quinolinate phosphoribosyl transferase involved in nicotine biosynthesis and catabolism pathways has been reported. An RNA-seq technique used to analyze the mRNA and sRNA of tobacco plants under salt and alkali stress revealed 33 differentially expressed miRNAs that are involved in ion channel, aquaporin and antioxidant activities. About 15 and 22 miRNAs that were specifically expressed in response to salt (NaCl) and alkali (NaHCO3), respectively were identified (Xu et al., 2019). Potato virus Y (PVY) is a globally and economically important pathogen of potato, tobacco, tomato and other staple crops and caused significant yield losses and reductions in quality. About 81 differentially expressed miRNAs from 29 families were identified. Also the virus-derived siRNAs targeting translationally controlled tumor protein (NtTCTP) of tobacco mediating PYV resistance has been demonstrated (Guo et al., 2017). The regulatory mechanism of PVY resistance in tobacco has been reported through Potyvirus-related transcriptome profiling of mRNA and miRNA and showed enhanced resistance to Potyvirus by regulation of photosynthesis, phytohormone and ROS scavenger ability (Xiao et al., 2022). 5. Small RNAs of horticultural crops Initially the focus of research on small RNAs has been on model plants (eg. Arabidopsis) and field crops (rice, wheat etc). Nevertheless, with the advent 156 Plant Small RNA in Food Crops of next generation sequencing (NGS) techniques the diversity and functional roles, including gene regulatory functions of small RNAs of horticultural crops have received a great impetus (Table 6.2) (Fig. 6.1). Unraveling of genome sequences of many horticultural crops have greatly aided in this process of characterization of miRNAs. Small RNAs have been characterized in diverse horticultural crops such as flower crops (Chrysanthemum) (Zhang et al., 2015a); vegetables namely tomato (Moxon et al., 2008), Brassica rapa L. ssp. pekinensis (Wang et al., 2012a), fruits crops such as apple (Xia et al., 2012b), banana (Bi et al., 2015), citrus (Wu et al., 2015), pear (Niu et al., 2016) persimmon (Luo et al., 2015), among others. Analysis of miRNAs of diverse plant species suggests that conserved miRNAs such as miR156, miR162, miR164, miR166, and miR172 are found in horticultural species apple, kiwi fruit and pear (Guo et al., 2020). However, some miRNAs are specific to group of plants e.g. miR528 is a core regulator of reactive oxygen species (ROS) homeostasis is found in monocots (Zhu et al., 2020). On the other hand, PHAS-derived phasiRNAs, and tasiRNAs (TAS1 and TAS2) derived due to the activity of miR173 are specific to Arabidopsis (Felippes & Weigel, 2009). Similarly, miR390 and miR828 are known to cleave TAS3 and TAS4, respectively to yield ARF-tasiRNAs, which is involved in flower and fruit development and MYB-tasiRNAs controlling the secondary metabolite synthetic pathway namely phenylpropanoid pathway with profound implications for novel horticultural and consumer traits such as flavor, pigmentation, and texture (Allan & Espley, 2018; Fahlgren et al., 2006; Luo et al., 2012). In citrus a species-specific miR3954 is involved in the generation of phasiRNAs that has serious implications for flowering time (Liu et al., 2017). 6. Cross-kingdom transfer of small RNAs The effectors of RNA-based gene silencing ie) small RNAs (siRNAs and miRNAs) are mobile and non-cell-autonomous. This movement of small RNAs occurs not only between cells of an organism but also between two different interacting organisms in a phenomenon called cross-kingdom transfer (Weiberg et al., 2014). The plant-fungus interactions have been influenced by the bidirectional transmission of small RNAs between the interacting partners. In order to achieve effective host colonization, fungus delivers proteins, toxins and RNA molecules to suppress the immunity of host plants (Weiberg et al., 2013). For instance B. cinerara exports small RNA molecules into host and disturb the innate immunity pathways. Table 6.2 Salient developmental functions of miRNAs characterized in horticultural crops. miRNA Target transcripts Developmental role(s) References Apple miR172 AP2 Fruit size reduction miR169a, mir160e, miR167bg ARF TFs mdm-siR277-1 and mdm-siR277-2 (siRNAs) produced from MdhpRNA277 miR156 5 R-genes Resistance to fire blight disease Leaf spot resistance Yao et al. (2015) Kaja et al. (2015) Zhang et al. (2018) miR 397 miR 171 miR528 HSP 70 HSP 90 Polyphenol oxidase mac-novmiR20 ARF OGI MeGI, a homeodomain TF Banana Caucasian persimmon (Diospyros lotus) SPL2-like/33 Anthocyanin accumulation during the light Thermo tolerance ROS metabolism regulation Fruit development and ripening Sex determination Yang et al. (2019) Vidya et al. (2018) Zhu et al. (2020) Lakhwani et al. (2020) Akagi et al. (2014) Continued Plant small RNAs: biogenesis, mechanistic functions and applications Crop 157 158 Table 6.2 Salient developmental functions of miRNAs characterized in horticultural crops.dcont'd miRNA Target transcripts Developmental role(s) References Citrus sinensis miR3954 Regulation of flowering Liu et al. (2017) Grape vine miR159, miR319 NAC TF and noncoding RNA transcripts (lncRNAs, Cs1 g09600 and Cs1 g09635) MYB TF Han et al. (2014) Kiwi fruit miRNA159 (VvmiR159a, VvmiR159b, VvmiR159c) miR164 Vegetative to reproductive phase transition and stress responsive Flowering, seedless grapes development Fruit development Fragaria vesca Poncirus trifoliata miR399 Prunus persica (peach) and Rose Pyrus bretschneideri Radish miR172 ptr-miR396b GAMYBTF AdNAC6 and AdNAC7 PHO2 Fruit quality improvement Wang et al. (2018a) Wang et al. (2020) Wang et al. (2017b) Zhang et al. (2016a) Aminocyclopropane-1carboxylic acid oxidase (ACO) AP2-type ortholog in rosaceae TARGET OF EAT (TOE) Cold tolerance Commercially valued double flowers development Gattolin et al. (2018) Pbr-miR397a laccase (LAC) 494 known miRNAs and 220 novel miRNAs e Reduced lignin and stone cell number in fruit Development of tap roots Xue et al. (2019) Sun et al. (2015b) Plant Small RNA in Food Crops Crop Tomato Fruit ripening miR172 AP2a miR160 SlyARF10 miR168 miR396 SlAGO1A and SlAGO1B, SlAGO1s SlMYB7-like and SlMYB48-like SlARF2A and SlARF2B SlGRF Altered fruit shape, carotenoid accumulation and orange ripe fruits Alteration of leaf and fruit morphology Phase transition, leaf epinasty and fruit development Regulation of anthocyanin biosynthesis Affects fruit ripening miR171 SlGRAS24 miR159 SIGAMYB1/2 miR390 SlARF2 miR393 TIR1-like genes (CsTIR1 and CsAFB2) miR482 NBSeLRRs miR858 miR390 Larger flowers, sepals and fruits Advancement of phase transition and flowering, Fruit set and development, plant architecture Affects fruit set Regulation of lateral root formation, flower senescence, high frequency of pear-shaped immature green fruit Pleotropic effects of leaf abnormalities, seed germination and parthenocarpy Resistance to P. infestans Chen et al. (2015a) Karlova et al. (2011) Hendelman et al. (2012) Xian et al. (2014) Jia et al. (2015) Hao et al. (2015) Cao et al. (2016) Huang et al. (2017) Da Silva et al. (2017) Ren et al. (2017) Xu et al. (2017a,b) Jiang et al. (2018) Continued 159 LeSPL-CNR Plant small RNAs: biogenesis, mechanistic functions and applications miR157 160 Table 6.2 Salient developmental functions of miRNAs characterized in horticultural crops.dcont'd Crop Target transcripts Developmental role(s) References miR1917 SlCTR4 splice variants (SlCTR4sv) Ethylene biosynthesis Wang et al. (2018b) Strawberry 24-nt siRNA Fruit ripening Potato miR172 DNA methylation via RdDM pathway RAP1 Cheng et al. (2018) Martin et al. (2009) Bhogale et al. (2014) miR156 miR473 miR475 miR160 Sweet potato StSPL3, StSPL6, StSPL9, StSPL13, and StLIGULELESS1 Serine-threonine kinase-like Thioredoxin StARF10 Novel_8, Novel_9, Novel_105, miR156d-3p, miR160a-5p, miR162a-3p, miR172b-3p and miR398a-5p miR156, miR162 SPL TF miR167 ARF TF miR160, miR164, miR166, miR398 ARF TF, NAC TF Multiple respective targets Tuberization Regulation of potato development Growth and development Din et al. (2014) SAR against Phytophthora infestans Drought and heat tolerance Natarajan et al. (2018) Öztürk Gökçe et al. (2021) Storage root initiation and development Development of stamen Sun et al. (2015a) Sun et al. (2015a) Sun et al. (2015a) Root development including storage organs Plant Small RNA in Food Crops miRNA Tea e Chilling stress tolerance Xie et al. (2017) ib-miR156 SPL TF Anthocyanin biosynthesis IbmiR162 IbDCL1 miR2111 IbFBK Chilling and heat stress response Response to wounding miR390a-3p and miR159a Auxin-responsive protein IAA14 and cytokinin dehydrogenase 6 Multiple TFs He et al. (2019) Yu et al. (2020) Weng et al. (2020) Tang et al. (2020) miR156, miR157, miR159, miR160, miR414, mir473 mir172, miR319, miR395, miR396, miR397 61 known and 471 novel miRNAs CsmiR156 Multiple TFs and growth factors TFs, genes involved in hormone biosynthesis, and cellular signaling SPL miR7814 miR5264 csn-miR164a and csn-miR168 CHS1 ANR2 beta-glucosidase Storage root formation Stress and development responses Starch metabolism Storage root development Regulation of catechins biosynthesis Response to Colletotrichum gloeosporioides Patanun et al. (2013) Chen et al. (2015b) Tang et al. (2020) Fan et al. (2015) Ping et al. (2018) Jeyaraj et al. (2019) 161 Continued Plant small RNAs: biogenesis, mechanistic functions and applications Cassava 190 known microRNAs (miRNAs) and 191 novel miRNAs miRNA Target transcripts Developmental role(s) References Coconut mir2673 TFs Auxin signaling miR156, miR164, miR166, miR167, miR169, miR171, miR172, miR394, miR397, miR408, miR444, miR535, miR827, miR1134 and miR2118 110 conserved, 47 novel miRNAs transcript of squamosa promoter binding-like protein, allene oxide synthase In vitro embryogenesis Viveka and Moossab (2016) Sabana et al. (2018) car-MIR167a2-5p 16 miRNAs (C. arabica) and 20 (C. canephora) Coffee ARF8 e Coffee e Regulate somatic embryogenesis e Hormonal and stress responses Sabana et al. (2020) Chaves et al. (2015) Plant Small RNA in Food Crops Crop 162 Table 6.2 Salient developmental functions of miRNAs characterized in horticultural crops.dcont'd Plant small RNAs: biogenesis, mechanistic functions and applications 163 Figure 6.1 The diverse role of small RNAs (siRNAs and miRNAs) in various developmental phases of horticultural crops. (Source: Wang et al., 2020, https://doi.org/10.1080/ 07352689.2020.1741923) Similarly, plant RNA silencing machinery could recognize the dsRNAs originating from phytopathogenic viruses thereby process them to create virus derived siRNAs which ultimately enhances the anti-viral immunity of host plants (Duan et al., 2012). This phenomenon has helped in devising a strategy called virus induced gene silencing (VIGS) an important plant functional genomics tool with profound basic and applied value in plant science research (Dunoyer & Voinnet, 2005). Alternatively, host plants also transfer key small RNA molecules to fungal system during the infection process so as to sabotage the pathogenicity of microorganisms in a process called Host induced gene silencing (HIGS). Intriguingly analysis of small RNAs involved in trans-kingdom transfer revealed that host miRNAs viz., miR166 and miR159 t target Ca2þ-dependent cysteine protease and isotrichodermin C-15 hydroxylase are indeed pathogenicity determinants of fungal pathogen Verticillium dahlia (Zhang et al., 2016b). In this context, host-pathogen interactions and adaptation of host toward fungal pathogen could not be explained only based on genetic processes such as horizontal gene transfer, elaborate chromosomal rearrangements but also warrants the inclusion of cross-kingdom sRNA transfer. On application front, both HIGS and VIGS have been utilized as an innovative molecular tool to develop pathogenic resistance in crop plants. However, a major demerit of both these technologies is the requirement of an efficient plant transformation protocol and consequences biosafety and allied issued associated with genetically modified crops. Hence, altogether a novel approach of direct, exogenous application of dsRNAs targeting the pests, pathogens have been designed and successfully utilized to manage biotic stresses of plants. Though this technology is still in its infancy, appropriate dsRNA 164 Plant Small RNA in Food Crops delivery agents have been developed to aid the induction of plant resistance against invading pathogens (Mitter et al., 2017). 7. Small RNAs in the food and nutritional security Small RNAs–based genetic manipulations have been considered as an important tool to develop crops with desirable traits. Two alternate approaches are followed while utilizing miRNA-based transgenics in crop improvement. In order to suppress the activity of a miRNA, overexpression of miRNA-resistant targets or alternatively a short tandem target mimics (STTM) to arrest the activity of miRNAs are utilized (DjamiTchatchou et al., 2017). On the other hand, miRNA backbones are utilized to develop artificial miRNAs (amiRNAs)-based gene silencing approaches to develop crop tolerance to abiotic and biotic stresses especially for virus resistance (Qu et al., 2007). Molecular understanding of cellular role of miRNAs has opened up avenues to develop stress tolerant crops to maximize yields. Transgenic rice over-expressing miR398-resistant forms of Cu-SOD or Zn-SOD conferred tolerance to salinity and water deficit stress (Lu et al., 2011). Similarly, improved drought tolerant tomato plants were obtained by over expressing a drought responsive miR169 (Zhang et al., 2011). Instances of manipulation of miRNAs for imparting biotic stress tolerance in crops are also available. Transgenic over-expression of Osa-miR7696 imparts blast disease resistance to rice (Campo et al., 2013), and over-expression of miR393 conferred bacterial resistance (Navarro et al., 2006). Studies have demonstrated the indispensable role of plant miRNAs in normal growth and developmental processes of field and horticultural crops (Tables 6.1 and 6.2). Hence, miRNA-based crop improvement strategies have potential applications in improving the plant or economic part biomass, improving plant architecture, enhancing the grain yield, increased fruit size and improved shelf life etc. Involvement of OsmiR397 in enhancing the grain size and promoting panicle branching (Zhang et al., 2013), maize miR172 and its regulation of APETALA-like gene glossy15 in phase transition (Lauter et al., 2005), miR156 in increasing the shape and number of leaves, decreased apical dominance, but enhanced biomass (Xie et al., 2012) miR390 in lateral root development are some of the instances which can be exploited for development of crops with desired growth features. Further, the natural variation in sequence features of miRNAs and their target transcripts has greater implications for modern molecular breeding. Plant small RNAs: biogenesis, mechanistic functions and applications 165 SNPs in the miRNAs and cognate targets could affect their biogenesis, miRNA-RNA interaction, altered transcriptional patterns with profound impacts for plant inflorescence architecture, domestication [(SNP in miR172-binding site in the AP2-like domestication gene Q) (Houston et al., 2013) SNP at the osa-miR156-targeting site in OsSPL14 (Jiao et al., 2010), grain size (Duan et al., 2015) are valuable resources for improving the phenology of crops. Expression profiling of miRNAs in hybrids have delineated the potential contributions of miRNA-mediated regulatory networks to heterosis. Direct functional relevance of specific miRNAs in yield improvement such as the role of miR396 in rice three-line hybrid Yuetai-A/9311 (Gao et al., 2015) warrants exploration of more such connectivity between miRNAs and its influence on heterosis. It would greatly aid in development of molecular breeding techniques to harness miRNAs for heterosis breeding. 8. Concluding remarks From the investigations described above it is apparent that miRNAs or small RNAs are not only involved in suppression of target gene expression but are involved in complex gene regulatory mechanisms. Our improved comprehension of the functioning of small RNAs broadens their biological significance and offer novel tools for modifying agriculturally important traits. It is important to acknowledge the fact that despite the improved understanding of functioning of conserved miRNAs, molecular biology of species or tissuespecific miRNAs warrants more attention for their translational use in crop breeding. Similarly, small RNA-based Investigations in model plants such as Arabidopsis, Medicago etc warrant translational studies in food, fruit, fiber and tree crops of economic importance. In addition, the importance of point mutations in miRNAs and specific target genes underscores the utilization of genome editing technologies (CRISPR/Cas technology) to engineer precise nucleotide or base replacement at appropriate target loci which would usher in an era of miRNA-based crop improvement strategies. References Abdurakhmonov, I. Y., Devor, E. J., Buriev, Z. T., Huang, L. Y., Makamov, A., Shermatov, S. E., et al. (2008). Small RNA regulation of ovule development in the cotton plant G. hirsutum L. BMC Plant Biology, 8, 93. Akagi, T., Henry, I. M., Tao, R., & Comai, L. (2014). A Ychromosome-encoded small RNA acts as a sex determinant in persimmons. Science, 346, 646e650. 166 Plant Small RNA in Food Crops Akhter, Y., & Khan, J. A. (2018). Genome wide identification of cotton (Gossypium hirsutum)-encoded microRNA targets against Cotton leaf curl Burewala virus. Gene, 638, 60e65. Akmal, M., Baig, M. S., & Khan, J. A. (2017). Suppression of cotton leaf curl disease symptoms in Gossypium hirsutum through over expression of host-encoded miRNAs. Journal of Biotechnology, 263, 21e29. Allan, A. C., & Espley, R. V. (2018). MYBs drive novel consumer traits in fruits and vegetables. Trends. Plant Science, 23, 693e705. Ashraf, M. A., Feng, X., Hu, X., Ashraf, F., Shen, L., Iqbal, M. S., & Zhang, S. (2022). In silico identification of sugarcane (Saccharum officinarum L.) genome encoded microRNAs targeting sugarcane bacilliform virus. PLoS One, 17(1), e0261807. Aufsatz, W., Mette, M. F., Van Der Winden, J., Matzke, A. J., & Matzke, M. (2002). RNAdirected DNA methylation in Arabidopsis. Proceedings of the National Academy of Sciences, 99(Suppl. 4), 16499e16506. Aukerman, M. J., & Sakai, H. (2003). Regulation of flowering time and floral organ identity by a microRNA and its APETALA2-like target genes. The Plant Cell Online, 15, 2730e2741. Axtell, M. J. (2013). Classification and comparison of small RNAs from plants. Annual Review of Plant Biology, 64, 137e159. Aydinoglu, F. (2020). Elucidating the regulatory roles of microRNAs in maize (Zea mays L.) leaf growth response to chilling stress. Planta, 251(2), 1e15. Azevedo, R. A., Carvalho, R. F., Cia, M. C., & Gratão, P. L. (2011). Sugarcane under pressure: An overview of biochemical and physiological studies of abiotic stress. Tropical Plant Biology, 4, 42e51. https://doi.org/10.1007/s12042-011-9067-4 Banerjee, N., Kumar, S., Singh, A., Annadurai, A., & Thirugnanasambandam, P. P. (2022). Identification of microRNAs involved in sucrose accumulation in sugarcane (Saccharum species hybrid). Plant Gene, 29, 100352. https://doi.org/10.1016/ j.plgene.2022.100352 Barozai, M. Y. K., Baloch, I. A., & Din, M. (2012). Identification of MicroRNAs and their targets in Helianthus. Molecular Biology Reports, 39(3), 2523e2532. Barrera-Figueroa, B. E., Gao, L., Diop, N. N., Wu, Z., Ehlers, J. D., Roberts, P. A., … Liu, R. (2011). Identification and comparative analysis of drought-associated microRNAs in two cowpea genotypes. BMC Plant Biology, 11(1), 1e11. Bhardwaj, A. R., Joshi, G., Pandey, R., Kukreja, B., Goel, S., Jagannath, A., Kumar, A., Katiyar-Agarwal, S., & Agarwal, M. (2014). A genome-wide perspective of miRNAome in response to high temperature, salinity and drought stresses in Brassica juncea (Czern) L. PloS one, 9(3), e92456. Bhogale, S., Mahajan, A. S., Natarajan, B., Rajabhoj, M., Thulasiram, H. V., & Banerjee, A. K. (2014). MicroRNA156: A potential graft-transmissible microRNA that modulates plant architecture and tuberization in Solanum tuberosum ssp. andigena. Plant Physiology, 164(2), 1011e1027. Bi, F., Meng, X., Ma, C., & Yi, G. (2015). Identification of miRNAs involved in fruit ripening in Cavendish bananas by deep sequencing. BMC Genomics, 16, 776. Bologna, N. G., Iselin, R., Abriata, L. A., et al. (2018). Nucleo-cytosolic shuttling of ARGONAUTE1 prompts a revised model of the plant microrna pathway. Molecular Cell, 69(4), 709e719e5. Boopathi, M. N., Sathish, S., Kavitha, P., Dachinamoorthy, P., & Ravikesavan, R. (2016). Comparative miRNAome analysis revealed numerous conserved and novel drought responsive miRNAs in cotton (Gossypium spp.). Cotton Genomics and Genetics, 7. Bukhari, S. A. H., Shang, S., Zhang, M., Zheng, W., Zhang, G., Wang, T. Z., … Wu, F. (2015). Genome-wide identification of chromium stress-responsive micro RNAs and their target genes in tobacco (Nicotiana tabacum) roots. Environmental Toxicology and Chemistry, 34(11), 2573e2582. Cai, C., Li, C., Sun, R., Zhang, B., Nichols, R. L., Hake, K. D., & Pan, X. (2021). Small RNA and degradome deep sequencing reveals important roles of microRNAs in cotton Plant small RNAs: biogenesis, mechanistic functions and applications 167 (Gossypium hirsutum L.) response to root-knot nematode Meloidogyne incognita infection. Genomics, 113(3), 1146e1156. Campo, S., Peris-Peris, C., Siré, C., Moreno, A. B., Donaire, L., Zytnicki, M., … San Segundo, B. (2013). Identification of a novel micro RNA (mi RNA) from rice that targets an alternatively spliced transcript of the N ramp6 (N atural resistance-associated macrophage protein 6) gene involved in pathogen resistance. New Phytologist, 199(1), 212e227. Cao, D., Wang, J., Ju, Z., Liu, Q., Li, S., Tian, H., Fu, D., Zhu, H., Luo, Y., & Zhu, B. (2016). Regulations on growth and development in tomato cotyledon, flower and fruit via destruction of miR396 with short tandem target mimic. Plant Science, 247, 1e12. Capaldi, F. R., Gratão, P. L., Reis, A. R., Lima, L. W., & Azevedo, R. A. (2015). Sulfur metabolism and stress defense responses in plants. Tropical Plant Biology, 8(3), 60e73. Carnavale Bottino, M., Rosario, S., Grativol, C., Thiebaut, F., Rojas, C. A., & Farrineli, L. (2013). High-throughput sequencing of small RNA transcriptome reveals salt stress regulated microRNAs in sugarcane. PLoS One, 8, e59423. Chaves, S. S., Fernandes-Brum, C. N., Silva, G. F. F., Ferrara-Barbosa, B. C., Paiva, L. V., Nogueira, F. T. S., … Chalfun-Junior, A. (2015). New insights on Coffea miRNAs: Features and evolutionary conservation. Applied Biochemistry and Biotechnology, 177(4), 879e908. Chávez-Hernández, E. C., Alejandri-Ramírez, N. D., Juárez-González, V. T., & Dinkova, T. D. (2015). Maize miRNA and target regulation in response to hormone depletion and light exposure during somatic embryogenesis. Frontiers of Plant Science, 6, 555. Chen, X. (2009). Small RNAs and their roles in plant development. Annual Review of Cell and Developmental Biology, 25, 21e44. Cheng, J., Niu, Q., Zhang, B., Chen, K., Yang, R., Zhu, J.-K., Zhang, Y., & Lang, Z. (2018). Downregulation of RdDM during strawberry fruit ripening. Genome Biology, 19, 212. Chen, W., Kong, J., Lai, T., Manning, K., Wu, C., et al. (2015a). Tuning LeSPL-CNR expression by SlymiR157 affects tomato fruit ripening. Scientific Reports, 5, 7852. Chen, Q., Li, M., Zhang, Z., Tie, W., Chen, X., Jin, L., … Zhou, H. (2017). Integrated mRNA and microRNA analysis identifies genes and small miRNA molecules associated with transcriptional and post-transcriptional-level responses to both drought stress and re-watering treatment in tobacco. BMC Genomics, 18(1), 1e16. Chen, L., Meng, J., & Luan, Y. (2019). miR1916 plays a role as a negative regulator in drought stress resistance in tomato and tobacco. Biochemical and Biophysical Research Communications, 508(2), 597e602. Chen, X., Xia, J., Xia, Z., Zhang, H., Zeng, C., Lu, C., et al. (2015b). Potential functions of microRNAs in starch metabolism and development revealed by miRNA transcriptome profiling of cassava cultivars and their wild progenitor. BMC Plant Biology, 15, 33. https://doi.org/10.1186/s12870-014-0355-7 Chen, C., Zeng, Z., Liu, Z., & Xia, R. (2018). Small RNAs, emerging regulators critical for the development of horticultural traits. Horticultural Research, 5, 63. Chi, X., Yang, Q., Chen, X., Wang, J., Pan, L., Chen, M., et al. (2011). Identification and characterization of microRNAs from peanut (Arachis hypogaea L.) by high-throughput sequencing. PLoS One, 6(11), Article e27530. https://doi.org/10.1371/ journal.pone.0027530 Chu, Z., Chen, J., Xu, H., Dong, Z., Chen, F., & Cui, D. (2016). Identification and comparative analysis of microRNA in wheat (Triticum aestivum L.) callus derived from mature and immature embryos during in vitro culture. Frontiers of Plant Science, 7, 1302. Curaba, J., Spriggs, A., Taylor, J., Li, Z., & Helliwell, C. (2012). miRNA regulation in the early development of barley seed. BMC Plant Biology, 12, 120. Da Silva, E. M. E., Silva, G. F. F. E., Bidoia, D. B., Da Silva Azevedo, M., De Jesus, F. A., Pino, L. E., Peres, L. E. P., Carrera, E., L_opez D_ıaz, I., & Nogueira, F. T. S. (2017). 168 Plant Small RNA in Food Crops microRNA159-targeted SlGAMYB transcription factors are required for fruit set in tomato. The Plant Journal, 92, 95e109. Dalmadi, Á., Gyula, P., Balint, J., et al. (2019). AGO-unbound cytosolic pool of mature miRNAs in plant cells reveals a novel regulatory step at AGO1 loading. Nucleic Acids Research, 47(18), 9803e9817. D’Ario, M., Griffiths-Jones, S., & Kim, M. (2017). Small RNAs: Big impact on plant development. Trends in Plant Science, 22, 1056e1068. Deng, N., Guo, R., Zheng, B., Li, T., & Liu, R. H. (2020). IRS-1/PI3K/Akt pathway and miRNAs are involved in whole grain highland barley (Hordeum vulgare L.) ameliorating hyperglycemia of db/db mice. Food & Function, 11(11), 9535e9546. Deng, N., He, Z., Guo, R., Zheng, B., Li, T., & Liu, R. H. (2020). Highland barley whole grain (Hordeum vulgare L.) ameliorates hyperlipidemia by modulating cecal microbiota, miRNAs, and AMPK pathways in leptin receptor-deficient db/db mice. Journal of Agricultural and Food Chemistry, 68(42), 11735e11746. Deng, P., Wang, L., Cui, L., Feng, K., Liu, F., Du, X., … Weining, S. (2015). Global identification of microRNAs and their targets in barley under salinity stress. PLoS One, 10(9), e0137990. Dhaka, N., Sharma, S., Vashisht, I., Kandpal, M., Sharma, M. K., & Sharma, R. (2020). Small RNA profiling from meiotic and post-meiotic anthers reveals prospective miRNA-target modules for engineering male fertility in sorghum. Genomics, 112(2), 1598e1610. Din, M., Barozai, M. Y. K., & Baloch, I. A. (2014). Identification and functional analysis of new conserved microRNAs and their targets in potato (Solanum tuberosum L.). Turkish Journal of Botany, 38, 1199e1213. Ding, H., Gao, J., Luo, M., Peng, H., Lin, H., Yuan, G., et al. (2013). Identification and functional analysis of miRNAs in developing kernels of a viviparous mutant in maize kernel. The Crop Journal, 1, 115e126. https://doi.org/10.1016/j.cj.2013.07.013 Djami-Tchatchou, A. T., & Dubery, I. A. (2015). Lipopolysaccharide perception leads to dynamic alterations in the microtranscriptomes of Arabidopsis thaliana cells and leaf tissues. BMC Plant Biology, 15, 79. Djami-Tchatchou, A. T., Sanan-Mishra, N., Ntushelo, K., & Dubery, I. A. (2017). Functional roles of microRNAs in agronomically important plantsdpotential as targets for crop improvement and protection. Frontiers of Plant Science, 8, 378. Duan, P., et al. (2015). Regulation of OsGRF4 by OsmiR396 controls grain size and yield in rice. Nature Plants, 2, 15203. Duan, C. G., Wang, C. H., & Guo, H. S. (2012). Application of RNAa silencing to plant disease resistance. Silence, 3, 5. https://doi.org/10.1186/1758-907X-3-5 Dubey, S., Saxena, S., Chauhan, A. S., Mathur, P., Rani, V., & Chakrabaroty, D. (2020). Identification and expression analysis of conserved microRNAs during short and prolonged chromium stress in rice (Oryza sativa). Environmental Science and Pollution Research, 27(1), 380e390. Dunoyer, P., & Voinnet, O. (2005). The complex interplay between plant and host RNAsilencing pathways. Current Opinion in Plant Biology, 8, 415e423. Du, J. F., Wu, Y. J., Fang, X. F., Xia, C. J., Liang, Z., & Heng, T. S. (2010). Prediction of sorghum miRNAs and their targets with computational methods. Chinese Science Bulletin, 55, 1263e1270. Ebrahimi Khaksefidi, R., Mirlohi, S., Khalaji, F., Fakhari, Z., Shiran, B., Fallahi, H., … Ebrahimie, E. (2015). Differential expression of seven conserved microRNAs in response to abiotic stress and their regulatory network in Helianthus annuus. Frontiers of Plant Science, 6, 741. Fahlgren, N., Montgomery, T. A., Howell, M. D., Allen, E., Dvorak, S. K., Alexander, A. L., & Carrington, J. C. (2006). Regulation of AUXIN RESPONSE Plant small RNAs: biogenesis, mechanistic functions and applications 169 FACTOR3 by TAS3 ta-siRNA affects developmental timing and patterning in Arabidopsis. Current Biology, 16, 939e944. Fan, K., Fan, D., Ding, Z., Su, Y., & Wang, X. (2015). Cs-miR156 is involved in the nitrogen form regulation of catechins accumulation in tea plant (Camellia sinensis L.). Plant Physiology and Biochemistry, 97, 350e360. Felippes, F. F., & Weigel, D. (2009). Triggering the formation of tasiRNAs in Arabidopsis thaliana: The role of microRNAs miR173. EMBO Reports, 10, 264e270. Ferreira, T. H., Gentile, A., Vilela, R. D., Costa, G. G., Dias, L. I., & Endres, L. (2012). microRNAs associated with drought response in the bioenergy crop sugarcane (Saccharum spp.). PLoS One, 7, e46703. Figueredo, M. S., Formey, D., Rodríguez, J., Ibáñez, F., Hernández, G., & Fabra, A. (2020). Identification of miRNAs linked to peanut nodule functional processes. Journal of Biosciences, 45(1), 1e7. Fontana, J. E., Wang, G., Sun, R., Xue, H., Li, Q., Liu, J., … Pan, X. (2020). Impact of potassium deficiency on cotton growth, development and potential microRNAmediated mechanism. Plant Physiology and Biochemistry, 153, 72e80. Frazier, T. P., Sun, G., Burklew, C. E., & Zhang, B. (2011). Salt and drought stresses induce the aberrant expression of microRNA genes in tobacco. Molecular Biotechnology, 49(2), 159e165. Frazier, T. P., Xie, F., Freistaedter, A., Burklew, C. E., & Zhang, B. (2010). Identification and characterization of microRNAs and their target genes in tobacco (Nicotiana tabacum). Planta, 232, 1289e1308. Fu, F., Girma, G., & Mengiste, T. (2020). Global mRNA and microRNA expression dynamics in response to anthracnose infection in sorghum. BMC Genomics, 21(1), 1e16. Gao, P., Bai, X., Yang, L., Lv, D. K., Li, Y., Cai, H., et al. (2010). Over-expression of osaMIR396c decreases salt and alkali stress tolerance. Planta, 231, 991e1001. https:// doi.org/10.1007/s00425-010-1104-2 Gao, J., Luo, M., Peng, H., Chen, F., & Li, W. (2019). Characterization of cadmium responsive MicroRNAs and their target genes in maize (Zea mays) roots. BMC Molecular Biology, 20(1), 1e9. Gao, F., Wang, K., Liu, Y., Chen, Y., Chen, P., Shi, Z., … Li, S. (2015). Blocking miR396 increases rice yield by shaping inflorescence architecture. Nature Plants, 2(1), 1e9. Gao, C., Wang, P., Zhao, S., Zhao, C., Xia, H., Hou, L., … Wang, X. (2017). Small RNA profiling and degradome analysis reveal regulation of microRNA in peanut embryogenesis and early pod development. BMC Genomics, 18(1), 1e18. Gao, S., Yang, L., Zeng, H. Q., Zhou, Z. S., Yang, Z. M., Li, H., … Zhang, B. (2016). A cotton miRNA is involved in regulation of plant response to salt stress. Scientific Reports, 6(1), 1e14. Gattolin, S., Cirilli, M., Pacheco, I., Ciacciulli, A., Da Silva Linge, C., Mauroux, J.-B., Lambert, P., Cammarata, E., Bassi, D., Pascal, T., & Rossini, L. (2018). Deletion of the miR172 target site in a TOE-type gene is a strong candidate variant for dominant double-flower trait in Rosaceae. The Plant Journal, 96, 358e371. Gentile, A., Dias, L. I., Mattos, R. S., Ferreira, T. H., & Menossi, M. (2015). MicroRNAs and drought responses in sugarcane. Frontiers of Plant Science, 6, 58. Grabowska, A., Smoczynska, A., Bielewicz, D., Pacak, A., Jarmolowski, A., & Szweykowska-Kulinska, Z. (2020). Barley microRNAs as metabolic sensors for soil nitrogen availability. Plant Science, 299, 110608. Gu, Y., Liu, Y., Zhang, J., Liu, H., Hu, Y., Du, H., et al. (2013). Identification and characterization of microRNAs in the developing maize endosperm. Genomics, 102, 472e478. Guo, Y., Jia, M. A., Yang, Y., Zhan, L., Cheng, X., Cai, J., … Shamsi, I. H. (2017). Integrated analysis of tobacco miRNA and mRNA expression profiles under PVY infection provides insight into tobacco-PVY interactions. Scientific Reports, 7(1), 1e10. Guo, H., Kan, Y., & Liu, W. (2011). Differential expression of miRNAs in response to topping in flue-cured tobacco (Nicotiana tabacum) roots. PLoS One, 6, e28565. 170 Plant Small RNA in Food Crops Guo, Z., Kuang, Z., Wang, Y., Zhao, Y., Tao, Y., Cheng, C., Yang, J., Lu, X., Hao, C., Wang, T., Cao, X., Wei, J., Li, L., & Yang, X. (2020). PmiREN: A comprehensive encyclopedia of plant miRNAs. Nucleic Acids Research, 48. Gupta, S., Kumari, M., Kumar, H., & Varadwaj, P. K. (2017). Genome-wide analysis of miRNAs and Tasi-RNAs in Zea mays in response to phosphate deficiency. Functional & Integrative Genomics, 17(2), 335e351. Gupta, K., Mishra, S. K., Gupta, S., Pandey, S., Panigrahi, J., & Wani, S. H. (2021). Functional role of miRNAs: Key players in soybean improvement. Phyton, 90(5), 1339. Gyawali, B., Barozai, M. Y. K., & Aziz, A. N. (2021). Comparative expression analysis of microRNAs and their targets in emerging bio-fuel crop sweet sorghum (Sorghum bicolor L.). Plant Gene, 26, 100274. Han, J., Fang, J., Wang, C., Yin, Y., Sun, X., Leng, X., et al. (2014). Grapevine microRNAs responsive to exogenous Gibberellin. BMC Genomics, 15, 111. https://doi.org/ 10.1186/1471-2164-15-111 Han, J., Kong, M. L., Xie, H., Sun, Q. P., Nan, Z. J., Zhang, Q. Z., et al. (2013). Identification of miRNAs and their targets in wheat (Triticum aestivum L.) by EST analysis. Genetics and Molecular Research, 12, 3793e3805. Hao, Y., Hu, G., Breitel, D., Liu, M., Mila, I., Frasse, P., Fu, Y., Aharoni, A., Bouzayen, M., & Zouine, M. (2015). Auxin response factor SlARF2 is an essential component of the regulatory mechanism controlling fruit ripening in tomato. PLoS Genetics, 11, e1005649. Hendelman, A., Buxdorf, K., Stav, R., Kravchik, M., & Arazi, T. (2012). Inhibition of lamina outgrowth following Solanum lycopersicum AUXIN RESPONSE FACTOR 10 (SlARF10) derepression. Plant Molecular Biology, 78, 561e576. He, L., Tang, R., Shi, X., Wang, W., Cao, Q., Liu, X., … Jia, X. (2019). Uncovering anthocyanin biosynthesis related microRNAs and their target genes by small RNA and degradome sequencing in tuberous roots of sweetpotato. BMC Plant Biology, 19(1), 1e19. He, X., Zheng, W., Cao, F., & Wu, F. (2016). Identification and comparative analysis of the microRNA transcriptome in roots of two contrasting tobacco genotypes in response to cadmium stress. Scientific Reports, 6(1), 1e14. He, Q., Zhu, S., & Zhang, B. (2014). MicroRNAetarget gene responses to lead-induced stress in cotton (Gossypium hirsutum L.). Functional & Integrative Genomics, 14(3), 507e515. Houston, K., McKim, S. M., Comadran, J., Bonar, N., Druka, I., Uzrek, N., … Waugh, R. (2013). Variation in the interaction between alleles of HvAPETALA2 and microRNA172 determines the density of grains on the barley inflorescence. Proceedings of the National Academy of Sciences, 110(41), 16675e16680. Huang, D., Koh, C., Feurtado, J. A., Tsang, E. W., & Cutler, A. J. (2013). MicroRNAs and their putative targets in Brassica napus seed maturation. BMC Genomics, 14, 140. https://doi.org/10.1186/1471-2164-14-140 Huang, W., Peng, S., Xian, Z., Lin, D., Hu, G., Yang, L., Ren, M., & Li, Z. (2017). Overexpression of a tomato miR171 target gene SlGRAS24 impacts multiple agronomical traits via regulating gibberellin and auxin homeostasis. Plant Biotechnology J., 15, 472e488. Hu, G., Lei, Y., Liu, J., Hao, M., Zhang, Z., Tang, Y., … Wu, J. (2020). The ghr-miR164 and GhNAC100 modulate cotton plant resistance against Verticillium dahlia. Plant Science, 293, 110438. Jabbar, B., Iqbal, M. S., Batcho, A. A., Nasir, I. A., Rashid, B., Husnain, T., & Henry, R. J. (2019). Target prediction of candidate miRNAs from Oryza sativa for silencing the RYMV genome. Computational Biology and Chemistry, 83, 107127. Plant small RNAs: biogenesis, mechanistic functions and applications 171 Jain, P., Hussian, S., Nishad, J., Dubey, H., Bisht, D. S., Sharma, T. R., & Mondal, T. K. (2021). Identification and functional prediction of long non-coding RNAs of rice (Oryza sativa L.) at reproductive stage under salinity stress. Molecular Biology Reports, 48(3), 2261e2271. Jain, N., Sinha, N., Krishna, H., Singh, P. K., Gautam, T., Prasad, P., & Gupta, P. K. (2020). A study of MiRNAs and lncRNAs during Lr28-mediated resistance against leaf rust in wheat (Triticum aestivum L.). Physiological and Molecular Plant Pathology, 112, 101552. Jarosová, J., Singh, K., Chrpová, J., & Kundu, J. K. (2020). Analysis of small RNAs of barley genotypes associated with resistance to Barley Yellow Dwarf Virus. Plants, 9(1), 60. Jeong, D. H., Park, S., Zhai, J., Gurazada, S. G., De Paoli, E., Meyers, B. C., et al. (2011). Massive analysis of rice small RNAs: Mechanistic implications of regulated microRNAs and variants for differential target RNA cleavage. The Plant Cell Online, 23, 4185e4207. Jeyaraj, A., Wang, X., Wang, S., Liu, S., Zhang, R., Wu, A., & Wei, C. (2019). Identification of regulatory networks of microRNAs and their targets in response to Colletotrichum gloeosporioides in tea plant (Camellia sinensis L.). Frontiers of Plant Science, 1096. Jia, W., Lin, K., Lou, T., Feng, J., Lv, S., Jiang, P., … Li, Y. (2021). Comparative analysis of sRNAs, degradome and transcriptomics in sweet sorghum reveals the regulatory roles of miRNAs in Cd accumulation and tolerance. Planta, 254(1), 1e18. Jiang, G., Liu, D., Yin, D., Zhou, Z., Shi, Y., Li, C., … Zhai, W. (2020). A rice NBS-ARC gene conferring quantitative resistance to bacterial blight is regulated by a pathogen effector-inducible miRNA. Molecular Plant, 13(12), 1752e1767. Jiang, N., Meng, J., Cui, J., Sun, G., & Luan, Y. (2018). Function identification of miR482b, a negative regulator during tomato resistance to Phytophthora infestans. Horticultural Research, 5, 9. Jiao, Y., Wang, Y., Xue, D., Wang, J., Yan, M., Liu, G., Dong, G., Zeng, D., Lu, Z., Zhu, X., & Qian, Q. (2010). Regulation of OsSPL14 by OsmiR156 defines ideal plant architecture in rice. Nature Genetics, 42(6), 541e544. Jia, X., Shen, J., Liu, H., Li, F., Ding, N., Gao, C., Pattanaik, S., Patra, B., Li, R., & Yuan, L. (2015). Small tandem target mimic-mediated blockage of microRNA858 induces anthocyanin accumulation in tomato. Planta, 242, 283e293. Jingi, W. A. N. G., Liu, C. Y., Zhang, L. W., Wang, J. L., Hu, G. H., Ding, J. J., & Chen, Q. S. (2011). MicroRNAs involved in the pathogenesis of phytophthora root rot of soybean (Glycine max). Agricultural Sciences in China, 10(8), 1159e1167. Jin, J., Xu, Y., Lu, P., Chen, Q., Liu, P., Wang, J., … Cao, P. (2020). Degradome, small RNAs and transcriptome sequencing of a high-nicotine cultivated tobacco uncovers miRNA’s function in nicotine biosynthesis. Scientific Reports, 10(1), 1e11. Jung, J. H., Seo, Y. H., Seo, P. J., Reyes, J. L., Yun, J., Chua, N. H., & Park, C. M. (2007). The GIGANTEA-regulated microRNA172 mediates photoperiodic flowering independent of CONSTANS in Arabidopsis. The Plant Cell Online, 19, 2736e2748. Kaja, E., Szczesniak, M. W., Jensen, P. J., Axtell, M. J., McNellis, T., & Makałowska, I. (2015). Identification of apple miRNAs and their potential role in fire blight resistance. Tree Genetics & Genomes, 11, 812. https://doi.org/10.1007/s11295-014-0812-3 Kansal, S., Panwar, V., Mutum, R. D., & Raghuvanshi, S. (2021). Investigations on regulation of MicroRNAs in rice reveal [Ca2þ] cyt signal transduction regulated microRNAs. Frontiers of Plant Science, 2239. Karamat, U., Sun, X., Li, N., & Zhao, J. (2021). Genetic regulators of leaf size in Brassica crops. Horticulture Research, 8, 91. https://doi.org/10.1038/s41438-021-00526-x Karlova, R., Rosin, F. M., Busscher-Lange, J., Parapunova, V., Do, P. T., Fernie, A. R., Fraser, P. D., Baxter, C., Angenent, G. C., & De Maagd, R. A. (2011). Transcriptome and metabolite profiling show that APETALA2a is a major regulator of tomato fruit ripening. The Plant Cell Online, 23, 923e941. Katiyar, A., Smita, S., Chinnusamy, V., Pandey, D. M., & Bansal, K. (2012). Identification of miRNAs in sorghum by using bioinformatics approach. Plant signaling & behavior, 7(2), 246e259. 172 Plant Small RNA in Food Crops Khan, A., Shrestha, A., Shaju, M., Panigrahi, K. C., & Dey, N. (2020). Identification of miRNA targets by AtFT overexpression in tobacco. Plant Molecular Biology Reporter, 38(1), 48e61. Kuang, L., Yu, J., Shen, Q., Fu, L., & Wu, L. (2021). Identification of microRNAs responding to aluminium, cadmium and salt stresses in barley roots. Plants, 10(12), 2754. Kulcheski, F. R., de Oliveira, L. F. V., Molina, L. G., Almerão, M. P., Rodrigues, F. A., Marcolino, J., et al. (2011). Identification of novel soybean microRNAs involved in abiotic and biotic stresses. BMC Genomics, 12, 307. Kumar, M., Kumar, R. R., Goswami, S., Verma, P., Rai, R. D., Chinnusamy, V., & Praveen, S. (2017). miR430: the novel heat-responsive microRNA identified from miRNome analysis in wheat (Triticum aestivum L.). Indian Journal of Plant Physiology, 22(4), 566e576. Kumar, R. R., Pathak, H., Sharma, S. K., Kala, Y. K., Nirjal, M. K., Singh, G. P., … Rai, R. D. (2015). Novel and conserved heat-responsive microRNAs in wheat (Triticum aestivum L.). Functional & Integrative Genomics, 15(3), 323e348. Lakhwani, D., Pandey, A., Sharma, D., Asif, M. H., & Trivedi, P. K. (2020). Novel microRNAs regulating ripening-associated processes in banana fruit. Plant Growth Regulation, 90(2), 223e235. Lauter, N., Kampani, A., Carlson, S., Goebel, M., & Moose, S. P. (2005). MicroRNA172 down-regulates glossy15 to promote vegetative phase change in maize. Proceedings of the National Academy of Sciences of the United States of America, 102, 9412e9417. https:// doi.org/10.1073/pnas.0503927102 Li, J., Guo, G., Guo, W., Guo, G., Tong, D., Ni, Z., … Yao, Y. (2012). miRNA164directed cleavage of ZmNAC1 confers lateral root development in maize (Zea mays L.). BMC Plant Biology, 12(1), 1e14. Li, M., Liang, Z., He, S., Zeng, Y., Jing, Y., Fang, W., … Tan, F. (2017). Genome-wide identification of leaf abscission associated microRNAs in sugarcane (Saccharum officinarum L.). BMC Genomics, 18(1), 1e14. Li, D., Liu, Z., Gao, L., Wang, L., Gao, M., Jiao, Z., … Kan, Y. (2016). Genome-wide identification and characterization of microRNAs in developing grains of Zea mays L. PLoS One, 11(4), 0153168. Lin, S., Chen, T., Qin, X., Wu, H., Khan, M. A., & Lin, W. (2014). Identification of microrna families expressed in sugarcane leaves subjected to drought stress and the targets thereof. Pakistan Journal of Agricultural Sciences, 51(4). Li, W., Chen, Y., Ye, M., Lu, H., Wang, D., & Chen, Q. (2020). Evolutionary history of the C-repeat binding factor/dehydration-responsive element-binding 1 (CBF/DREB1) protein family in 43 plant species and characterization of CBF/DREB1 proteins in Solanum tuberosum. BMC Evolutionary Biology, 20, 142. https://doi.org/10.1186/ s12862-020-01710-8 Li, Y., Nie, T., Zhang, M., Zhang, X., Shahzad, K., Guo, L., Qi, T., Tang, H., Wang, H., Qiao, X., & Feng, J. (2022). Integrated analysis of small RNA, transcriptome and degradome sequencing reveals that micro-RNAs regulate anther development in CMS cotton. Industrial Crops and Products, 176, 114422. Li, C., Nong, W., Zhao, S., Lin, X., Xie, Y., Cheung, M. Y., … Lam, H. M. (2022). Differential microRNA expression, microRNA arm switching, and microRNA: Long noncoding RNA interaction in response to salinity stress in soybean. BMC Genomics, 23(1), 1e13. Liu, G., Liu, J., Pei, W., Li, X., Wang, N., Ma, J., Zang, X., Zhang, J., Yu, S., Wu, M., & Yu, J. (2019). Analysis of the MIR160 gene family and the role of MIR160a_A05 in regulating fiber length in cotton. Planta, 250(6), 2147e2158. Liu, Y., Ke, L., Wu, G., Xu, Y., Wu, X., Xia, R., Deng, X., & Xu, Q. (2017). miR3954 is a trigger of phasiRNAs that affects flowering time in citrus. The Plant Journal, 92, 263e275. Plant small RNAs: biogenesis, mechanistic functions and applications 173 Liu, Y., Zhou, F., Huang, X., Wang, W., Zhang, S., & Feng, F. (2021). Identification and integrated analysis of mRNAs, lncRNAs, and microRNAs of developing seeds in high oleic acid sunflower (Helianthus annuus L.). Acta Physiologiae Plantarum, 43(6), 1e13. Li, F., Wang, W., Zhao, N., Xiao, B., Cao, P., Wu, X., … Fan, L. (2015). Regulation of nicotine biosynthesis by an endogenous target mimicry of microRNA in tobacco. Plant Physiology, 169(2), 1062e1071. Lu, Y. Z., Feng, Z., Bian, L. Y., Xie, H., & Liang, J. S. (2011). miR398 regulation in rice of the responses to abiotic and biotic stresses depends on CSD1 and CSD2 expression. Functional Plant Biology, 38, 44e53. https://doi.org/10.1071/FP10178 Luo, Q. J., Mittal, A., Jia, F., & Rock, C. D. (2012). An autoregulatory feedback loop involving PAP1 and TAS4 in response to sugars in Arabidopsis. Plant Molecular Biology, 80, 117e129. Luo, Y., Zhang, X., Luo, Z., Zhang, Q., & Liu, J. (2015). Identification and characterization of microRNAs from Chinese pollination constant non-astringent persimmon using high-throughput sequencing. BMC Plant Biology, 15, 11. Lu, Y., & Yang, X. (2010). Computational identification of novel microRNAs and their targets in Vigna unguiculata. Comparative and Functional Genomics, 128297. Maheshwari, P., Kummari, D., Palakolanu, S. R., Nagasai Tejaswi, U., Nagaraju, M., Rajasheker, G., … Kavi Kishor, P. B. (2019). Genome-wide identification and expression profile analysis of nuclear factor Y family genes in Sorghum bicolor L. (Moench). PLoS One, 14(9), e0222203. Margis, R., Fusaro, A. F., Smith, N. A., Curtin, S. J., Watson, J. M., Finnegan, E. J., & Waterhouse, P. M. (2006). The evolution and diversification of Dicers in plants. FEBS Letters, 580(10), 2442e2450. Marin, E., Jouannet, V., Herz, A., Lokerse, A. S., Weijers, D., Vaucheret, H., et al. (2010b). MiR390, ArabidopsisTAS3 tasiRNAs, and their auxin response factor targets define an autoregulatory network quantitatively regulating lateral root growth. The Plant Cell Online, 22, 1104e1117. https://doi.org/10.1105/tpc.109.072553 Marin, E., Jouannet, V., Herz, A., Lokerse, A. S., Weijers, D., Vaucheret, H., Nussaume, L., Crespi, M. D., & Maizel, A. (2010a). miR390, Arabidopsis TAS3 tasiRNAs, and their AUXIN RESPONSE FACTOR targets define an autoregulatory network quantitatively regulating lateral root growth. The Plant Cell Online, 22, 1104e1117. Martin, A., Adam, H., Díaz-Mendoza, M., Zurczak, M., González-Schain, N. D., & Suárez-López, P. (2009). Graft-transmissible induction of potato tuberization by the microRNA miR172. Development, 136(17), 2873e2881. Martinez, G., & Köhler, C. (2017). Role of small RNAs in epigenetic reprogramming during plant sexual reproduction. Current Opinion in Plant Biology, 36, 22e28. Martins, T. F., Souza, P. F., Alves, M. S., Silva, F. D. A., Arantes, M. R., Vasconcelos, I. M., & Oliveira, J. T. (2020). Identification, characterization, and expression analysis of cowpea (Vigna unguiculata [L.] Walp.) miRNAs in response to cowpea severe mosaic virus (CPSMV) challenge. Plant Cell Reports, 39(8), 1061e1078. Mazalmazraei, T., Mehdikhanlou, K., & Ahmadi, D. N. (2021). Comparative analysis of differentially expressed miRNAs in leaves of three sugarcane (Saacharum officinarum L.) cultivars during salinity stress. Ma, X., Zhang, X., Zhao, K., Li, F., Li, K., Ning, L., … Yin, D. (2018). Small RNA and degradome deep sequencing reveals the roles of microRNAs in seed expansion in peanut (Arachis hypogaea L.). Frontiers of Plant Science, 9, 349. Mica, E., Gianfranceschi, L., & Pe, M. E. (2006). Characterization of five microRNA families in maize. Journal of Experimental Botany, 57, 2601e2612. Mitter, N., Worrall, E. A., Robinson, K. E., Li, P., Jain, R. G., Taochy, C., … Xu, Z. P. (2017). Clay nanosheets for topical delivery of RNAi for sustained protection against plant viruses. Nature Plants, 3(2), 1e10. Moxon, S., Jing, R., Szittya, G., Schwach, F., Pilcher, R. L. R., Moulton, V., & Dalmay, T. (2008). Deep sequencing of tomato short RNAs identifies microRNAs targeting genes involved in fruit ripening. Genome Research, 18(10), 1602e1609. 174 Plant Small RNA in Food Crops Mutegi, E., Sagnard, F., Muraya, M., Kanyenji, B., Rono, B., Mwongera, C., … Labuschagne, M. (2010). Ecogeographical distribution of wild, weedy and cultivated sorghum bicolor (L.) Moench in Kenya: Implications for conservation and crop-to-wild gene flow. Genetic Resources and Crop Evolution, 57(2), 243e253. Nagaraju, M., Kumar, S. A., Reddy, P. S., Kumar, A., Rao, D. M., & Kavi Kishor, P. B. (2019). Genome-scale identification, classification, and tissue specific expression analysis of late embryogenesis abundant (LEA) genes under abiotic stress conditions in Sorghum bicolor L. PLoS One, 14(1), 0209980. Nandakumar, M., Malathi, P., Sundar, A. R., Rajadurai, C. P., Philip, M., & Viswanathan, R. (2021). Role of miRNAs in the hostepathogen interaction between sugarcane and Colletotrichum falcatum, the red rot pathogen. Plant Cell Reports, 40(5), 851e870. Natarajan, B., Kalsi, H. S., Godbole, P., Malankar, N., Thiagarayaselvam, A., Siddappa, S., Thulasiram, H. V., Chakrabarti, S. K., & Banerjee, A. K. (2018). MiRNA160 is associated with local defense and systemic acquired resistance against Phytophthora infestans infection in potato. Journal of Experimental Botany, 69(8), 2023e2036. Navarro, L., Dunoyer, P., Jay, F., Arnold, B., Dharmasiri, N., Estelle, M., et al. (2006). A plant miRNA contributes to antibacterial resistance by repressing auxin signaling. Science, 312, 436e439. Nie, H., Wang, Y., Su, Y., & Hua, J. (2018). Exploration of miRNAs and target genes of cytoplasmic male sterility line in cotton during flower bud development. Functional & Integrative Genomics, 18(4), 457e476. Niu, Q., Li, J., Cai, D., Qian, M., Jia, H., Bai, S., Hussain, S., Liu, G., Teng, Y., & Zheng, X. (2016). Dormancy-associated MADS-box genes and microRNAs jointly control dormancy transition in pear (Pyrus pyrifolia white pear group) flower bud. EXBOTJ, 67, 239e257. Öztürk Gökçe, Z. N., Aksoy, E., Bakhsh, A., Demirel, U., Çalıskan, S., & Çalıskan, M. E. (2021). Combined drought and heat stresses trigger different sets of miRNAs in contrasting potato cultivars. Functional & Integrative Genomics, 21(3), 489e502. Pandey, B., Gupta, O. P., Pandey, D. M., Sharma, I., & Sharma, P. (2013). Identification of new microRNA and their targets in wheat using computational approach. Plant Signaling & Behavior, 8, e23932. Pan, X., Nichols, R. L., Li, C., & Zhang, B. (2019). MicroRNA-target gene responses to root knot nematode (Meloidogyne incognita) infection in cotton (Gossypium hirsutum L.). Genomics, 111(3), 383e390. Patanun, O., Lertpanyasampatha, M., Sojikul, P., Viboonjun, U., & Narangajavana, J. (2013). Computational identification of microRNAs and their targets in cassava (Manihot esculenta Crantz.). Molecular Biotechnology, 53(3), 257e269. Paul, S., Kundu, A., & Pal, A. (2011). Identification and validation of conserved microRNAs along with their differential expression in roots of Vigna unguiculata grown under salt stress. Plant Cell, Tissue and Organ Culture, 105(2), 233e242. Permar, V., Singh, A., Pandey, V., Alatar, A. A., Faisal, M., Jain, R. K., & Praveen, S. (2014). Tospo viral infection instigates necrosis and premature senescence by micro RNA controlled programmed cell death in Vigna unguiculata. Physiological and Molecular Plant Pathology, 88, 77e84. Ping, S. U. N., ZHANG, Z. L., ZHU, Q. F., ZHANG, G. Y., Xiang, P., LIN, Y. L., … LIN, J. K. (2018). Identification of miRNAs and target genes regulating catechin biosynthesis in tea (Camellia sinensis). Journal of Integrative Agriculture, 17(5), 1154e1164. Prasad, P. V. V., Pisipati, S. R., Mutava, R. N., & Tuinstra, M. R. (2008). Sensitivity of grain sorghum to high temperature stress during reproductive development. Crop Science, 48, 1911e1917. Puli, C. O. R., Zheng, Y., Li, Y. F., Jagadeeswaran, G., Suo, A., Jiang, B., … Sunkar, R. (2021). MicroRNA profiles in Sorghum exposed to individual drought or heat or their combination. Journal of Plant Biochemistry and Biotechnology, 30(4), 848e861. Plant small RNAs: biogenesis, mechanistic functions and applications 175 Qiao-Ying, Z., Yang, C. Y., Ma, Q. B., Li, X. P., Dong, W. W., & Nian, H. (2012). Identification of wild soybean miRNAs and their target genes responsive to aluminum stress. BMC Plant Biology, 12, 182. Qiu, L., Chen, R., Fan, Y., Huang, X., Luo, H., Xiong, F., … Li, Y. (2019). Integrated mRNA and small RNA sequencing reveals microRNA regulatory network associated with internode elongation in sugarcane (Saccharum officinarum L.). BMC Genomics, 20(1), 1e14. Qiu, C. W., Liu, L., Feng, X., Hao, P. F., He, X., Cao, F., & Wu, F. (2020). Genome-wide identification and characterization of drought stress responsive microRNAs in Tibetan wild barley. International Journal of Molecular Sciences, 21(8), 2795. Qu, J., Ye, J., & Fang, R. X. (2007). Artificial microRNA-mediated virus resistance in plants. Journal of Virology, 81, 6690e6699. Rajendiran, A., Vijayakumar, S., & Pan, A. (2019). Exploring microRNAs, target mRNAs and their functions in leguminous plant Arachis hypogaea. MicroRNA, 8(2), 135e146. Ramesh, S. V., Chouhan, B. S., Kumar, G., Praveen, S., & Chand, S. (2017). Expression dynamics of Glycine max (L.) Merrill microRNAs (miRNAs) and their targets during Mungbean yellow mosaic India virus (MYMIV) infection. Physiological and Molecular Plant Pathology, 100, 13e22. Ramesh, S. V., Govindasamy, V., Rajesh, M. K., Sabana, A. A., & Praveen, S. (2019). Stress-responsive miRNAome of Glycine max (L.) Merrill: Molecular insights and way forward. Planta, 249(5), 1267e1284. Ramesh, S. V., Gupta, G. K., & Husain, S. M. (2016). Soybean (Glycine max) microRNAs display proclivity to repress begomovirus genomes. Current Science, 424e428. Ram, M. K., Mukherjee, K., & Pandey, D. M. (2019). Identification of miRNA, their targets and miPEPs in peanut (Arachis hypogaea L.). Computational Biology and Chemistry, 83, 107100. Razná, K., Rataj, V., Macák, M., & Galambosová, J. (2020). MicroRNA-based markers as a tool to monitor the barley (Hordeum vulgare L.) response to soil compaction. Acta Fytotechnica et Zootechnica, 23(3). Regmi, R., Newman, T. E., Kamphuis, L. G., & Derbyshire, M. C. (2021). Identification of Brassica napus small RNAs responsive to infection by a necrotrophic pathogen. BMC Plant Biology, 21(366). https://doi.org/10.1186/s12870-021-03148-6 Ren, Z., Liu, R., Gu, W., & Dong, X. (2017). The Solanum lycopersicum auxin response factor SlARF2 participates in regulating lateral root formation and flower organ senescence. Plant Science, 256, 103e111. Ren, J., Zhang, H., Shi, X., Ai, X., Dong, J., Zhao, X., Zhong, C., Jiang, C., Wang, J., & Yu, H. (2021). Genome-wide identification of key candidate microRNAs and target genes associated with peanut drought tolerance. DNA and Cell Biology, 40(2), 373e383. Rogers, K., & Chen, X. (2013). Biogenesis, turnover, and mode of action of plant microRNAs. The Plant Cell Online, 25, 2383e2399. Sabana, A. A., Antony, G., Rahul, C. U., & Rajesh, M. K. (2018). In silico identification of microRNAs and their targets associated with coconut embryogenic calli. Agri Gene, 7, 59e65. Sabana, A. A., Rajesh, M. K., & Antony, G. (2020). Dynamic changes in the expression pattern of miRNAs and associated target genes during coconut somatic embryogenesis. Planta, 251(4), 1e18. Sepúlveda-García, E. B., Pulido-Barajas, J. F., Huerta-Heredia, A. A., Peña-Castro, J. M., Liu, R., & Barrera-Figueroa, B. E. (2020). Differential expression of maize and teosinte microRNAs under submergence, drought, and alternated stress. Plants, 9(10), 1367. Sharma, N., Tripathi, A., & Sanan-Mishra, N. (2015). Profiling the expression domains of a rice-specific microRNA under stress. Frontiers of Plant Science, 6, 333. Sheng, L., Chai, W., Gong, X., Zhou, L., Cai, R., Li, X., et al. (2015). Identification and characterization of novel maize miRNAs involved in different genetic background. International Journal of Biological Sciences, 11, 781e793. https://doi.org/10.7150/ ijbs.11619 176 Plant Small RNA in Food Crops Shen, Y., Liu, Y. H., Zhang, X. J., Sha, Q., & Chen, Z. D. (2016). Gynophore miRNA analysis at different developmental stages in Arachis duranensis. Genetics and Molecular Research, 10. Shi, G. Q., Fu, J. Y., Rong, L. J., Zhang, P. Y., Guo, C. J., & Kai, X. I. A. O. (2018). TaMIR1119, a miRNA family member of wheat (Triticum aestivum), is essential in the regulation of plant drought tolerance. Journal of Integrative Agriculture, 17(11), 2369e2378. Shui, X. R., Chen, Z. W., & Li, J. X. (2013). MicroRNA prediction and its function in regulating drought-related genes in cowpea. Plant Science, 210, 25e35. Shuzuo, L. V., Nie, X., Le, W., Du, X., Biradar, S. S., Jia, X., et al. (2012). Identification and characterization of microRNAs from barley (Hordeum vulgare L.) by highthroughput sequencing. International Journal of Biological Sciences, 13, 2973e2984. Sierro, N., Battey, J. N. D., Ouadi, S., Bakaher, N., Bovet, L., Willig, A., et al. (2014). The tobacco genome sequence and its comparison with those of tomato and potato. Nature Communications, 5, 3833. Silva, J. D. O. L., Silva, R. G. D., Nogueira, L. D. F., & Zingaretti, S. M. (2021). MicroRNAs regulate tolerance mechanisms in sugarcane (Saccharum spp.) under aluminum stress. Crop Breeding and Applied Biotechnology, 21. Singh, I., Smita, S., Mishra, D. C., Kumar, S., Singh, B. K., & Rai, A. (2017). Abiotic stress responsive miRNA-target network and related markers (SNP, SSR) in Brassica juncea. Frontiers in Plant Science, 8, 1943. Song, Q. X., Liu, Y. F., Hu, X. Y., Zhang, W. K., Ma, B., Chen, S. Y., et al. (2011). Identification of miRNAs and their target genes in developing soybean seeds by deep sequencing. BMC Plant Biology, 11, 5. Sreenivasulu, N., Graner, A., & andWobus, U. (2008). Barley genomics: An overview. International Journal of Plant Genomics, 2008, 486258. Subramanian, S., Fu, Y., Sunkar, R., Barbazuk, W. B., Zhu, J. K., & Yu, O. (2008). Novel and nodulation-regulated microRNAs in soybean roots. BMC Genomics, 9, 160. Sui, J., Qiao, L., Li, E., Zhu, H., Wang, X., Zhao, C., … Yu, S. (2019). Transcriptome and miRNA profiling of a hydroxyproline-tolerant peanut mutant with higher grain size and oil contents. International Journal of Agriculture and Biology, 22(6), 1331e1337. Sun, G. (2012). MicroRNAs and their diverse functions in plants. Plant Molecular Biology, 80(1), 17e36. Sun, R., Guo, T., Cobb, J., Wang, Q., & Zhang, B. (2015a). Role of microRNAs during flower and storage root development in sweet potato. Plant Molecular Biology Reporter, 33(6), 1731e1739. Sun, F., Guo, G., Du, J., Guo, W., Peng, H., Ni, Z., et al. (2014). Whole-genome discovery of miRNAs and their targets in wheat (Triticum aestivum L.). BMC Plant Biology, 14, 142. Sunkar, R., Girke, T., Jain, P. K., & Zhu, J. K. (2005). Cloning and characterization of microRNA from rice. The Plant Cell Online, 17, 1397e1411. https://doi.org/10.1105/ tpc.105.031682 Sun, R., Li, C., Zhang, J., Li, F., Ma, L., Tan, Y., … Zhang, B. (2017). Differential expression of microRNAs during fiber development between fuzzless-lintless mutant and its wild-type allotetraploid cotton. Scientific Reports, 7(1), 1e10. Sun, X., Xu, L., Wang, Y., Yu, R., Zhu, X., Luo, X., Gong, Y., Wang, R., Limera, C., Zhang, K., & Liu, L. (2015). Identification of novel and salt-responsive miRNAs to explore miRNA-mediated regulatory network of salt stress response in radish (Raphanus sativus L.). BMC Genomics, 16(1), 1e16. Sun, Y., Qiu, Y., Zhang, X., Chen, X., Shen, D., Wang, H., & Li, X. (2015b). Genomewide identification of microRNAs associated with taproot development in radish (Raphanus sativus L.). Gene, 569(1), 118e126. Sunkar, R., Chinnusamy, V., Zhu, J., & Zhu, J. K. (2007). Small RNAs as big players in plant abiotic stress responses and nutrient deprivation. Trends in Plant Science, 12(7), 301e309. Plant small RNAs: biogenesis, mechanistic functions and applications 177 Swapna, M., & Kumar, S. (2017). MicroRNAs and their regulatory role in sugarcane. Frontiers of Plant Science, 8, 997. Tang, C., Han, R., Zhou, Z., Yang, Y., Zhu, M., Xu, T., Wang, A., Li, Z., & Dong, T. (2020). Identification of candidate miRNAs related in storage root development of sweet potato by high throughput sequencing. Journal of Plant Physiology, 251, 153224. https://doi.org/10.1016/j.jplph.2020.153224 Tang, J., & Chu, C. (2017). MicroRNAs in crop improvement: Fine-tuners for complex traits. Nature Plants, 3(7), 1e11. Tang, Y., Du, G., Xiang, J., Hu, C., Li, X., Wang, W., … Sui, J. (2022). Genome-wide identification of auxin response factor (ARF) gene family and the miR160-ARF18mediated response to salt stress in peanut (Arachis hypogaea L.). Genomics, 114(1), 171e184. Tang, C., Han, R., Zhou, Z., Yang, Y., Zhu, M., Xu, T., Wang, A., Li, Z., & Dong, T. (2020). Identification of candidate miRNAs related in storage root development of sweet potato by high throughput sequencing. Journal of Plant Physiology, 251, 153224. Tang, S., Wang, Y., Li, Z., Gui, Y., Xiao, B., Xie, J., et al. (2012). Identification of wounding and topping responsive small RNAs in tobacco (Nicotiana tabacum). BMC Plant Biology, 12, 28. Teng, Z., Chen, Y., Yuan, Y., Peng, Y., Yi, Y., Yu, H., … Ye, N. (2021). Identification of microRNAs regulating grain filling of rice inferior spikelets in response to moderate soil drying post-anthesis. The Crop Journal, 10(4), 962e971. https://doi.org/10.1016/ j.cj.2021.11.004 Thiebaut, F., Rojas, C. A., Almeida, K. L., Grativol, C., Domiciano, G. C., Lamb, C. R. C., … Ferreira, P. C. (2012). Regulation of miR319 during cold stress in sugarcane. Plant, Cell and Environment, 35(3), 502e512. Varkonyi-Gasic, E., Lough, R. H., Moss, S. M. A., Wu, R., & Hellens, R. P. (2012). Kiwifruit floral gene APETALA2 is alternatively spliced and accumulates in aberrant indeterminate flowers in the absence of miR172. Plant Molecular Biology, 78, 417e429. Vidya, S. M., Ravishankar, K. V., & Laxman, R. H. (2018). Genome wide analysis of heat responsive microRNAs in banana during acquired thermo tolerance. Journal of Horticultural Sciences, 13(1), 61e71. Viveka, A. T., & Moossab, F. (2016). Identification of novel micro RNAs and their targets in Cocos nuciferaea bioinformatics approach. Bioscience Biotechnology Research Communications, 9(3), 481e488. Wang, H., & Wang, H. (2015). The miR156/SPL module, a regulatory hub and versatile toolbox, gears up crops for enhanced agronomic traits. Molecular Plant, 8(5), 677e688. Wang, J. W., Czech, B., & Weigel, D. (2009). miR156-regulated SPL transcription factors define an endogenous flowering pathway in Arabidopsis thaliana. Cell, 138, 738e749. Wang, J., Feng, J., Jia, W., Fan, P., Bao, H., Li, S., & Li, Y. (2017a). Genome-wide identification of Sorghum bicolor laccases reveals potential targets for lignin modification. Frontiers of Plant Science, 8, 714. Wang, C., Jogaiah, S., Zhang, W., Abdelrahman, M., & Fang, J. G. (2018a). Spatiotemporal expression of miRNA159 family members and their GAMYB target gene during the modulation of gibberellin-induced grapevine parthenocarpy. Journal of Experimental Botany, 69(15), 3639e3650. Wang, M., Li, A. M., Liao, F., Qin, C. X., Chen, Z. L., Zhou, L., Li, Y. R., Li, X. F., Lakshmanan, P., & Huang, D. L. (2022). Control of sucrose accumulation in sugarcane (Saccharum spp. hybrids) involves miRNA-mediated regulation of genes and transcription factors associated with sugar metabolism. GCB Bioenergy, 14(2), 173e191. Wang, F., Li, L., Liu, L., Li, H., Zhang, Y., Yao, Y., Ni, Z., & Gao, J. (2012a). Highthroughput sequencing discovery of conserved and novel microRNAs in Chinese cabbage (Brassica rapa L. ssp. pekinensis). Molecular Genetics and Genomics, 287, 555e563. Wang, W., Liu, D., Chen, D., Cheng, Y., Zhang, X., Song, L., … Shen, F. (2019). MicroRNA414c affects salt tolerance of cotton by regulating reactive oxygen species metabolism under salinity stress. RNA Biology, 16(3), 362e375. 178 Plant Small RNA in Food Crops Wang, Q., Liu, N., Yang, X., Tu, L., & Zhang, X. (2016). Small RNA-mediated responses to low-and high-temperature stresses in cotton. Scientific Reports, 6(1), 1e14. Wang, J. W., Park, M. Y., Wang, L. J., Koo, Y., Chen, X. Y., Weigel, D., & Poethig, R. S. (2011). miRNA control of vegetative phase change in trees. PLoS Genetics, 7, e1002012. Wang, M., Wang, Q., & Wang, B. (2012b). Identification and characterization of microRNAs in Asiatic cotton (Gossypium arboreum L.). PLoS One, 7(4), e33696. Wang, W. Q., Wang, J., Wu, Y. Y., Li, D. W., Allan, A. C., & Yin, X. R. (2020). Genome-wide analysis of coding and non-coding RNA reveals a conserved miR164NAC regulatory pathway for fruit ripening. New Phytologist, 225, 1618e1634. Wang, M., Wang, Q., & Zhang, B. (2013). Response of miRNAs and their targets to salt and drought stresses in cotton (Gossypium hirsutum L.). Gene, 530(1), 26e32. Wang, Y., Zhang, J., Cui, W., Guan, C., Mao, W., & Zhang, Z. (2017b). Improvement in fruit quality by overexpressing miR399a in woodland strawberry. Journal of Agricultural and Food Chemistry, 65, 7361e7370. Wang, J. F., Zhou, H., Chen, Y. Q., Luo, Q. J., & Qu, L. H. (2004). Identification of 20 microRNAs from Oryza sativa. Nucleic Acids Research, 32, 1688e1695. https://doi.org/ 10.1093/nar/gkh332 Wang, Y., Zou, W., Xiao, Y., Cheng, L., Liu, Y., Gao, S., Shi, Z., Jiang, Y., Qi, M., Xu, T., & Li, T. (2018b). MicroRNA1917 targets CTR4 splice variants to regulate ethylene responses in tomato. Journal of Experimental Botany, 69, 1011e1025. Waziri, A., Singh, D. K., Sharma, T., Chatterjee, S., & Purty, R. S. (2020). Genome-wide analysis of PHD finger gene family and identification of potential miRNA and their PHD finger gene specific targets in Oryza sativa indica. Non-Coding RNA Research, 5(4), 191e200. Weiberg, A., Wang, M., Bellinger, M., & Jin, H. (2014). Small RNAs: A new paradigm in plant-microbe interactions. Annual Review of Phytopathology, 52, 495e516. Weiberg, A., Wang, M., Lin, F. M., Zhao, H., Zhang, Z., Kaloshian, I., Huang, H. D., & Jin, H. (2013). Fungal small RNAs suppress plant immunity by hijacking host RNA interference pathways. Science, 342, 118e123. Weng, S. T., Kuo, Y. W., King, Y. C., Lin, H. H., Tu, P. Y., Tung, K. S., & Jeng, S. T. (2020). Regulation of micoRNA2111 and its target IbFBK in sweet potato on wounding. Plant Science, 292, 110391. Wu, X. M., Kou, S. J., Liu, Y. L., Fang, Y. N., Xu, Q., & Guo, W. W. (2015). Genomewide analysis of small RNAs in nonembryogenic and embryogenic tissues of citrus: microRNA-and siRNA-mediated transcript cleavage involved in somatic embryogenesis. Plant Biotechnology Journal, 13, 383e394. Wu, Y., Yang, L., Yu, M., & Wang, J. (2017). Identification and expression analysis of microRNAs during ovule development in rice (Oryza sativa) by deep sequencing. Plant Cell Reports, 36(11), 1815e1827. Xian, Z., Huang, W., Yang, Y., Tang, N., Zhang, C., Ren, M., & Li, Z. (2014). miR168 influences phase transition, leaf epinasty, and fruit development via SlAGO1s in tomato. Journal of Experimental Botany, 65, 6655e6666. Xiao, Q., Cui, G., Chen, Y., Zhou, X., Deng, B., Huang, P., … Zhao, T. (2022). Combined analysis of mRNA and miRNA transcriptomes reveals the regulatory mechanism of PVY resistance in tobacco. Industrial Crops and Products, 176, 114322. Xia, K., Ou, X., Tang, H., Wang, R., Wu, P., Jia, Y., Wei, X., Xu, X., Kang, S. H., Kim, S. K., & Zhang, M. (2015). Rice microRNA osa-miR1848 targets the obtusifoliol 14a-demethylase gene Os CYP 51G3 and mediates the biosynthesis of phytosterols and brassinosteroids during development and in response to stress. New Phytologist, 208(3), 790e802. Xia, K., Wang, R., Ou, X., Fang, Z., Tian, C., Duan, J., et al. (2012a). OsTIR1 and OsAFB2 down regulation via OsmiR393 overexpression leads to more tillers, early flowering and less tolerance to salt and drought in rice. PLoS One, 7, e30039. Plant small RNAs: biogenesis, mechanistic functions and applications 179 Xia, Z., Zhao, Z., Li, M., Chen, L., Jiao, Z., Wu, Y., … Fan, Z. (2018). Identification of miRNAs and their targets in maize in response to Sugarcane mosaic virus infection. Plant Physiology and Biochemistry, 125, 143e152. Xia, R., Zhu, H., An, Y. Q., Beers, E. P., & Liu, Z. (2012b). Apple miRNAs and tasiRNAs with novel regulatory networks. Genome Biology, 13, R47. Xie, K., Shen, J., Hou, X., Yao, J., Li, X., Xiao, J., & Xiong, L. (2012). Gradual increase of miR156 regulates temporal expression changes of numerous genes during leaf development in rice. Plant physiology, 158(3), 1382e1394. Xie, F., Stewart, C. N., Jr., Taki, F. A., He, Q., Liu, H., & Zhang, B. (2014). Highthroughput deep sequencing shows that microRNAs play important roles in switchgrass responses to drought and salinity stress. Plant Biotechnology Journal, 12, 354e366. Xie, Z., Wang, A., Li, H., Yu, J., Jiang, J., Tang, Z., Ma, D., Zhang, B., Han, Y., & Li, Z. (2017). High throughput deep sequencing reveals the important roles of microRNAs during sweetpotato storage at chilling temperature. Scientific Reports, 7(1), 16578. https://doi.org/10.1038/s41598-017-16871-8 Xu, J., Chen, Q., Liu, P., Jia, W., Chen, Z., & Xu, Z. (2019). Integration of mRNA and miRNA analysis reveals the molecular mechanism underlying salt and alkali stress tolerance in tobacco. International Journal of Molecular Sciences, 20(10), 2391. Xu, J., Li, J., Cui, L., Zhang, T., Wu, Z., Zhu, P. Y., Meng, Y. J., Zhang, K. J., Yu, X. Q., Lou, Q. F., & Chen, J. F. (2017a). New insights into the roles of cucumber TIR1 homologs and miR393 in regulating fruit/seed set development and leaf morphogenesis. BMC Plant Biology, 17(1), 1e14. Xu, J., Li, J., Cui, L., Zhang, T., Wu, Z., Zhu, P.-Y., Meng, Y.-J., Zhang, K.-J., Yu, X.-Q., Lou, Q.-F., & Chen, J.-F. (2017b). New insights into the roles of cucumber TIR1 homologs and miR393 in regulating fruit/seed set development and leaf morphogenesis. BMC Plant Biology, 17, 130. Xue, C., Yao, J. L., Qin, M. F., Zhang, M. Y., Allan, A. C., Wang, D. F., & Wu, J. (2019). PbrmiR397a regulates lignifications during stone cell development in pear fruit. Plant Biotechnology Journal, 17, 103e117. Yadav, A., Sanyal, I., Rai, S. P., & Lata, C. (2021). An overview on miRNA-encoded peptides in plant biology research. Genomics, 113(4), 2385e2391. Yang, C., Li, D., Mao, D., Liu, X., Ji, C., Li, X., et al. (2013). Overexpression of microRNA319 impacts leaf morphogenesis and leads to enhanced cold tolerance in rice (Oryza sativa L.). Plant, Cell and Environment, 36, 2207e2218. https://doi.org/10.1111/ pce.12130 Yang, T., Ma, H., Zhang, J., Wu, T., Song, T., Tian, J., & Yao, Y. (2019). Systematic identification of long noncoding RNAs expressed during light-induced anthocyanin accumulation in apple fruit. The Plant Journal, 100, 572e590. Yang, Y., Yu, Q., Yang, Y., Su, Y., Ahmad, W., Guo, J., Gao, S., Xu, L., & Que, Y. (2018). Identification of cold-related miRNAs in sugarcane by small RNA sequencing and functional analysis of a cold inducible ScmiR393 to cold stress. Environmental and Experimental Botany, 155, 464e476. Yang, Y., Zhang, X., Su, Y., Zou, J., Wang, Z., Xu, L., & Que, Y. (2017). miRNA alteration is an important mechanism in sugarcane response to low-temperature environment. BMC Genomics, 18(1), 1e18. Yan, Y., Wei, M., Li, Y., Tao, H., Wu, H., Chen, Z., … Xu, J. H. (2021). MiR529a controls plant height, tillernumber, panicle architecture and grain size by regulating SPL target genes in rice (Oryza sativa L.). PlantScience, 302, 110728. Yao, X., Wang, Y., Yao, Y., Bai, Y., Wu, K., & Qiao, Y. (2021). Identification microRNAs and target genes in Tibetan hulless barley to BLS infection. Agronomy Journal, 113(3), 2273e2292. Yao, J.-L., Xu, J., Cornille, A., Tomes, S., Karunairetnam, S., Luo, Z., Bassett, H., Whitworth, C., Rees-George, J., Ranatunga, C., Snirc, A., Crowhurst, R., de Silva, N., Warren, B., Deng, C., Kumar, S., Chagn_e, D., Bus, V. G. M., Volz, R. K., … 180 Plant Small RNA in Food Crops Gleave, A. P. (2015). A microRNA allele that emerged prior to apple domestication may underlie fruit size evolution. The Plant Journal, 84, 417e427. Yin, Z. J., Li, Y., Yu, J. W., Liu, Y. D., Li, C. H., Han, X. L., et al. (2012). Difference in miRNA expression profiles between two cotton cultivars with distinct salt sensitivity. Molecular Biology Reports, 39, 4961e4970. Yin, F., Qin, C., Gao, J., Liu, M., Luo, X., Zhang, W., … Pan, G. (2015). Genome-wide identification and analysis of drought-responsive genes and microRNAs in tobacco. International Journal of Molecular Sciences, 16(3), 5714e5740. Yu, N., Cai, W. J., Wang, S., Shan, C. M., Wang, L. J., & Chen, X. Y. (2010). Temporal control of trichome distribution by microRNA156-targeted SPL genes in Arabidopsis thaliana. The Plant Cell Online, 22, 2322e2335. Yu, H., Cong, L., Zhu, Z., Wang, C., Zou, J., Tao, C., … Lu, X. (2015). Identification of differentially expressed microRNA in the stems and leaves during sugar accumulation in sweet sorghum. Gene, 571(2), 221e230. Yu, Y., Ni, Z., Wang, Y., Wan, H., Hu, Z., Jiang, Q., … Zhang, H. (2019b). Overexpression of soybean miR169c confers increased drought stress sensitivity in transgenic Arabidopsis thaliana. Plant Science, 285, 68e78. Yu, J., Su, D., Yang, D., Dong, T., Tang, Z., Li, H., & Zhang, B. (2020). Chilling and heat stress-induced physiological changes and microRNA-related mechanism in sweetpotato (Ipomoea batatas L.). Frontiers of Plant Science, 11, 687. Yu, X., Wang, H., Lu, Y., de Ruiter, M., Cariaso, M., Prins, M., et al. (2012). Identification of conserved and novel microRNAs that are responsive to heat stress in Brassica rapa. Journal of Experimental Botany, 63, 1025e1038. Yu, J., Wu, L., Fu, L., Shen, Q., Kuang, L., Wu, D., & Zhang, G. (2019a). Genotypic difference of cadmium tolerance and the associated microRNAs in wild and cultivated barley. Plant Growth Regulation, 87(3), 389e401. Yu, Y., Zhang, Y., Chen, X., & Chen, Y. (2019c). Plant noncoding RNAs: Hidden players in development and stress responses. Annual Review of Cell and Developmental Biology, 35, 407e431. Zanca, A. S., Vicentini, R., Ortiz-Morea, F. A., Bem, L. E. D., da Silva, M. J., Vincentz, M., et al. (2010). Identification and expression analysis of microRNAs and targets in the biofuel crop sugarcane. BMC Plant Biology, 10, 260. Zare, S., Nazarian-Firouzabadi, F., Ismaili, A., & Pakniyat, H. (2019). Identification of miRNAs and evaluation of candidate genes expression profile associated with drought stress in barley. Plant Gene, 20, 100205. Zhang, F., Dong, W., Huang, L., Song, A., Wang, H., Fang, W., Chen, F., & Teng, N. (2015a). Identification of microRNAs and their targets associated with embryo abortion during chrysanthemum cross breeding via highthrough put sequencing. PLoS One, 10, e0124371. Zhang, T., Hu, S., Yan, C., Li, C., Zhao, X., Wan, S., & Shan, S. (2017). Mining, identification and function analysis of microRNAs and target genes in peanut (Arachis hypogaea L.). Plant Physiology and Biochemistry, 111, 85e96. Zhang, Q., Ma, C., Zhang, Y., Gu, Z., Li, W., Duan, X., Wang, S., Hao, L., Wang, Y., Wang, S., & Li, T. (2018). A single-nucleotide polymorphism in the promoter of a hairpin RNA contributes to Alternaria alternata leaf spot resistance in apple (Malus X domestica). The Plant Cell Online, 30, 1924e1942. Zhang, B., & Wang, Q. (2015). MicroRNA-based biotechnology for plant improvement. Journal of Cellular Physiology, 230, 1e15. Zhang, Y., Wang, W., Chen, J., Liu, J., Xia, M., & Shen, F. (2015b). Identification of miRNAs and their targets in cotton inoculated with Verticillium dahliae by highthroughput sequencing and degradome analysis. International Journal of Molecular Sciences, 16, 14749e14768. Zhang, B., Wang, Q., Wang, K., Pan, X., Liu, F., Guo, T., Cobb, G. P., & Anderson, T. A. (2007). Identification of cotton microRNAs and their targets. Gene, 397(1-2), 26e37. Plant small RNAs: biogenesis, mechanistic functions and applications 181 Zhang, X., Wang, W., Wang, M., Zhang, H. Y., & Liu, J. H. (2016a). The miR396b of Poncirus trifoliata functions in cold tolerance by regulating ACC Oxidase gene expression and modulating ethyleneepolyamine homeostasis. Plant and Cell Physiology, 57, 1865e1878. Zhao, J., Yuan, S., Zhou, M., Yuan, N., Li, Z., Hu, Q., Bethea, F. G., Jr., Liu, H., Li, S., & Luo, H. (2019). Transgenic creeping bentgrass overexpressing Osa-miR393a exhibits altered plant development and improved multiple stress tolerance. Plant Biotechnology Journal, 17(1), 233e251. Zhang, B., You, C., Zhang, Y., et al. (2020). Linking key steps of microRNA biogenesis by TREX-2 and the nuclear pore complex in Arabidopsis. Nature Plants, 6(8), 957e969. Zhang, Y. C., Yu, Y., Wang, C. Y., Li, Z. Y., Liu, Q., Xu, J., … Chen, Y. Q. (2013). Overexpression of microRNA OsmiR397 improves rice yield by increasing grain size and promoting panicle branching. Nature Biotechnology, 31(9), 848. Zhang, T., Zhao, Y. L., Zhao, J. H., Wang, S., Jin, Y., Chen, Z. Q., Fang, Y. Y., Hua, C. L., Ding, S. W., & Guo, H. S. (2016b). Cotton plants export microRNAs to inhibit virulence gene expression in a fungalpathogen. Nature Plants, 2, 16153. Zhang, X., Zou, Z., Gong, P., Zhang, J., Ziaf, K., Li, H., … Ye, Z. (2011). Overexpression of microRNA169 confers enhanced drought tolerance to tomato. Biotechnology Letters, 33(2), 403e409. Zhao, B., Liang, R., Ge, L., Li, W., Xiao, H., Lin, H., et al. (2007a). Identification of drought-induced microRNAs in rice. Biochemical and Biophysical Research Communications, 354, 585e590. Zhao, L., Kim, Y., Dinh, T. T., & Chen, X. (2007b). miR172 regulates stem cell fate and defines the inner boundary of APETALA3 and PISTILLATA expression domain in Arabidopsis floral meristems. The Plant Journal, 51, 840e849. Zhao, C., Xia, H., Cao, T., Yang, Y., Zhao, S., Hou, L., et al. (2015). Small RNA and degradome deep sequencing reveals peanut microRNA roles in response to pathogen infection. Plant Molecular Biology Reporter, 33, 1013e1029. Zhao, C. Z., Xia, H., Frazier, T. P., Yao, Y. Y., Bi, Y. P., Li, A. Q., et al. (2010). Deep sequencing identifies novel and conserved microRNAs in peanuts (Arachis hypogaea L.). BMC Plant Biology, 10, 3. Zhao, Y., Xu, K., Liu, G., Li, S., Zhao, S., Liu, X., … Xiao, K. (2020). Global identification and characterization of miRNA family members responsive to potassium deprivation in wheat (Triticum aestivum L.). Scientific Reports, 10(1), 1e13. Zheng, Z., Wang, N., Jalajakumari, M., Blackman, L., Shen, E., Verma, S., … Millar, A. A. (2020). miR159 represses a constitutive pathogen defense response in tobacco. Plant Physiology, 182(4), 2182e2198. Zhou, L., Liu, Y., Liu, Z., Kong, D., Duan, M., & Luo, L. (2010). Genome-wide identification and analysis of drought-responsive microRNAs in Oryza sativa. Journal of Experimental Botany, 61, 4157e4168. Zhu, H., Chen, C., Zeng, J., Yun, Z., Liu, Y., Qu, H., Jiang, Y., Duan, X., & Xia, R. (2020). MicroRNA528, a hub regulator modulating ROS homeostasis via targeting of a diverse set of genes encoding copper-containing proteins in monocots. New Phytologist, 225, 385e399. Zilberman, D., Cao, X., Johansen, L. K., Xie, Z., Carrington, J. C., & Jacobsen, S. E. (2004). Role of Arabidopsis ARGONAUTE4 in RNA-directed DNA methylation triggered by inverted repeats. Current Biology, 14(13), 1214e1220. Zuber, H., Scheer, H., Joly, A. C., & Gagliardi, D. (2018). Respective contributions of URT1 and HESO1 to the uridylation of 50 fragments produced from RISC-cleaved mRNAs. Frontiers of Plant Science, 1438. This page intentionally left blank CHAPTER 7 RNAi based approaches for abiotic and biotic stresses tolerance of crops Neha Patwaa, b, Om Prakash Guptaa, Vanita Pandeya and Anita Yadavb a b ICAR-Indian Institute of Wheat and Barley Research, Karnal, Haryana, India; Department of Biotechnology, Kurukshetra University, Kurukshetra, Haryana, India 1. Introduction Plants provide all of humanity’s essential necessities, including food, fodder, and shelter, as well as additional products derived from plants such as timber, gum, resin, fiber, oil, colors, pharmaceutically related secondary metabolites, medications, and fossil fuels, directly or indirectly. Abiotic, biotic stressors and climatic factors affect the growth, development, yield, and plant products in the face of the worldwide shortage of arable land, water resources, and climate change (Mohr et al., 2020). Currently, the world population size is approx. 7.9 billion by January 2021 and it is expected to rise by 9.7 billion by 2050 according to UN Reports. As the world’s population grows, so does the need for plants, resulting in future food security, malnutrition, and famine (Godfray et al., 2010). A combination of new contemporary breeding, molecular genetics, recombinant DNA, and biotechnology approaches based on genomics and proteomics will be required to improve crop production (Rabuma et al., 2022; Gupta et al., 2022a). By using these novel techniques new crop varieties showing resistance to various diseases and stress-tolerant lines are produced for higher yield (Tester & Langridge, 2010). The development and growth of the plant, pathogen defense, and environmental challenges are all regulated by RNAi (Gupta et al., 2014a,b). This is a sequence-specific mechanism that interferes with or suppresses the function of a gene. The small RNA is produced inside the nucleus, and maturation takes place in the cytoplasm of the plant cell by activating the RNAi machinery. It reduces the expression of target genes by blocking the process of protein synthesis. Fire and Mello discovered RNAi in nematodes in 1998 and Nobel Prize was awarded in Plant Small RNA in Food Crops ISBN 978-0-323-91722-3 https://doi.org/10.1016/B978-0-323-91722-3.00013-0 © 2023 Elsevier Inc. All rights reserved. 183 184 Plant Small RNA in Food Crops 2006. The first miRNA was discovered in Caenorhabditis elegans in 1993 (Lee et al., 1993). Subsequently, other miRNAs have been discovered in plants, human beings, fruit flies, and other species, etc. RNAi is a technique that is more particular and precise in its activity, and it is being evaluated as a potential tool for functional genomics research. It helps in the development of food varieties without compromising other agronomic qualities in the last 15 years. This is more accurate, stable, and efficient as compared to previously used antisense RNA technology. It has also been used as an innovative way of gaining a better knowledge of the fundamentals of plant defense and metabolism (Khalid et al., 2017). Protection of crop against biotic and abiotic factors, increase in the shelf life of fruits and vegetables, improvement of nutritional content, change in plant architecture for better adaptation to environmental conditions, overexpression or removal of secondary metabolites, generation of male sterile lines, and production of seedless fruits are just a several of the desirable traits that have been improved in crops by using RNAi. RNAi has been used successfully in the last two decades to delineate the functional roles of numerous key genes in various plant species. These genes play a role in fiber development, somatic embryogenesis, allergen/toxin elimination, and tolerance to various types of stresses among food crop varieties (Jagtap et al., 2011). Increased agricultural productivity has long been a major goal in the quest to feed the world’s ever-increasing population. Crop diseases provide a significant obstacle to accomplishing these objectives. Among various plant diseases, viral pathogens cause a serious hazard to crops, resulting in a large loss of agricultural productivity (Sharma et al., 2021). The use of small (sRNA) based silencing technology helps in the generation of disease-resistant agricultural varieties by targeting pathogenic genetic controls using the host-induced pathogenic gene silencing mechanism. However, there are public issues and uncertainties about this technology’s usage in modern agriculture, biosafety requirements, and the environmental impact of genetically altered crops, specifically when genes originated from creatures other than plants are employed (Herdt, 2006). The production of transgenic plants and food crops has prompted concerns about potential risks to humans and the environment. The main concern involves transgene migration to other types and wild relatives, which could result in monster crops, a loss of genetic diversity, and ecological disruption. As a result, before releasing transgenic crops for general use, they must undergo extensive testing to identify the hazards and ensure their safety. Transgenic crop development consequently necessitates additional time, money, and RNAi based approaches for abiotic and biotic stresses tolerance of crops 185 skill. As a result, new crop improvement strategies and safe methods must be developed, which may be more acceptable to the general public. sRNAbased silencing mechanism has been caught the interest of scientists who were working in various fields of molecular biology working all over the world. The various researches on RNAi help in the understanding of gene regulation of genes their function and analysis, as well as opening up new avenues for the development of fascinating technology with enormous potential for use in genetic analysis, plant protection, and a variety of other areas related to crop improvement. In the following section, we look at the advances made in the field of RNAi using small RNA (sRNA) for crop development in various types of varieties. In 2002, the miRNA Registry which was later renamed miRBase was established. It contains a comprehensive and detailed database of identified miRNAs, their nomenclature, precursor and mature sequences, and corresponding literature (GriffithsJones et al., 2006). Approximately 48,885 mature miRNAs from 271 species (Kozomara et al., 2019) has been submitted in the current release (v22.1), Arabidopsis thaliana (428 mature miRNAs), Medicago truncatula (756 mature miRNAs), Brachypodium distachyon (525), Oryza sativa (738), Triticum aestivum (125), Zea mays (325) and Solanum lycopersicum (147) (
Se
cic & Kogel, 2021) (Fig. 7.1). Figure 7.1 Schematic representation of the possible role of RNAi in crop development. 186 Plant Small RNA in Food Crops 2. Mechanism and biogenesis of sRNA Different forms of sRNAs are produced via RNAi-related pathways in plants i. e, siRNAs, miRNAs, ta-siRNA, and pha (phased)-siRNAs (Treiber et al., 2019). In this chapter, we studied the general mechanism of small RNA (sRNA) pathways. The different forms of dsRNAs derived from plant endogenous RNA-dependent DNA polymerase, dsRNAs of natural sense and antisense transcripts, and single-stranded RNAs that form hairpin-loop secondary structures are all used to create siRNAs and miRNAs. Dicer-like (DCL) proteins identify and cleave these dsRNA molecules, resulting in siRNAs. The plants have a core set of four types of DCL proteins (DCL1e4) that produce various forms of siRNAs. DCL4 produces 21-nt siRNA that play an important role in trans-acting siRNAs (ta-siRNAs), which are used to modulate gene expression. DCL2 produces 22-nt siRNAs and has been shown to work in tandem with DCL4 in antiviral defense; in the absence of DCL4, DCL2 can produce a large number of viral secondary siRNAs (Martínez et al., 2015). DCL3 produces 24-nt siRNAs that are linked to the silencing of transposons and repetitive elements and have been linked to DNA virus defense (Allen et al., 2005). After the conversion of dsRNA into siRNA duplex by DCLs each duplex is loaded with RISC complex and AGO protein. Furthermore, one of the duplex molecule’s strands is chosen to serve as a guide strand. By WatsonCrick base pairing, the guide strand targets and identifies certain cognate RNAs, resulting in RISC cleavage/slicing of the RNA target (Joga et al., 2016). Plants, like DCLs, have numerous AGOs encoded. AGO2 is one of the AGOs that has been linked to antiviral, antibacterial, and anti-stress defenses via siRNA. AGO proteins generally have three types of conserved domains PAZ, MID and PIWI domains (Swarts et al., 2014). The PAZ domain is present near the ND and binds the 30 end of short RNA (the guide RNA). The MID and PIWI domains are found in the C-terminal domain. A binding pocket exists at the intersection of these two domains, which anchors the guide RNA’s 5 ends. The PIWI domain of the siRNA-target duplex-loaded AGO protein has an RNase-H-like activity and a conserved catalytic site, that cleaves the target RNA. Intronic and independently transcribed pri-miRNAs both are co-transcriptionally processed by Drosha in human cells (Kim & Kim, 2007). Plant MIR genes are similar to protein-coding genes in that their promoters contain the TATAbox motif and other transcription factor binding motifs. The transcription of MIR genes requires general and specific transcription regulators (Megraw et al., 2006). Primary miRNAs (pri-miRNAs) in plants have distinct RNAi based approaches for abiotic and biotic stresses tolerance of crops 187 secondary structures and are first converted into precursor miRNAs (premiRNAs). The pre-miRNA are processed by various protein complexes called Transcription Coupled Export 2 (TREX2), DICER-LIKE 1 (DCL), SERRATE (SE) and double-stranded RNA binding protein called HYPONASTIC-LEAVES1 (HYL1). TREX-2 complex plays an important role in the biogenesis of miRNA including transcription, processing of pre-miRNA, and export of miRNA RISC complex to the cytoplasm by nuclear pore complex. TREX-2 had two core subunits THP1 and SAC3A which interacts and colocalize with RNA polymerase II to promote the transcription of MIR genes in the nucleoplasm (Zhang et al., 2020). After processing of pri-miRNA HEAT SHOCK PROTEIN 90 (HSP90) helps in loading of miRNA into AGO1 (Iki et al., 2010). ENHANCED MIRNA ACTIVITY1 (EMA1) and TRANSPORTIN1 (TRN1) proteins interacts with AGO1 and inhibited or promoted the loading of miRNAs into AGO1. A nuclear export signal in AGO1 allows AGO1emiRNA complexes exported them to the cytoplasm. Certain molecular processes are restricted to either the nucleus or the cytoplasm by the nuclear envelope. Nuclear pore complexes (NPCs) carry mRNAs and small RNAs from the nucleus to the cytoplasm, where they function (Beck & Hurt, 2017). The miRNA is further transported to the cytoplasm by EXORTIN5 (Bologna et al., 2018; Zhang et al., 2020). miRNA duplexes are then loaded into AGO in the RISC complex, and one of the strands in 50 to 30 direction is chosen as the guide strand. The guide strand binds with targeted 30 to 50 RNAs and translationally repressed or degraded the targeted gene (Cui et al., 2017; Stepien et al., 2017; Yu et al., 2017) (Fig. 7.2). 3. Role of small RNA in abiotic stress tolerance 3.1 Heavy metal tolerance Accumulation of non-essential metals such as cadmium, mercury, arsenic at excessive levels can be dangerous for plant growth and development and these metals affect the health of human beings through the food chain (Gielen et al., 2012; Gupta et al., 2014b; Dhiman et al., 2021). Overexpression of miR395 in B. napus results in a reduction of the level of oxidative stress caused by heavy metals and provides tolerance to Cd stress (Zhang et al., 2013a,b). It was studied that miRNAs have a role in Al3þ toxicity and tolerance. They discovered that 18 miRNAs responded after 4 h of Al3þ treatment and that 4 miRNAs from 4 families responded after 4 h and 24 min of Al3þ therapy (Chen et al., 2012). The treatment of Al3þ 188 Plant Small RNA in Food Crops RNA POL II miRNA gene Pri-miRNA NUCLEUS HYL1 Pre -miRNA DCL-1 TREX2 SE EXPORTIN5 RISC COMPLEX siRNA/miRNA duplex CYTOPLASM ARGONAUTE PROTEIN CLEAVAGE OF PASSENGER STRAND 5’ 3’ Guide strand binds to target Guide strand is used to silence the target gene by binding to its complementary 5’ 3’ 3’ 5’ The silencing of target genes inhibits the protein synthesis ×NO PROTEIN SYNTHESIS (No product formation) Figure 7.2 Mechanism and biogenesis of small RNA: miRNA are derived from endogenously ln-RNA and act as a regulator of gene expression. The primary microRNA or (pri-miRNA) are first processed into precursor microRNA or (pre-miRNA) or mature miRNA. TRANSCRIPTION COUPLED EXPORT 2 (TREX2) protein associated with DICERLIKE1 (DCL-1), SERRATE (SE), and (HYPONASTIC LEAVES1 HYL-1) helps in the processing of pri-miRNA. TREX2 also helps in the export of the miRISC complex to the cytoplasm with the help of EXPORTIN5 (EXPO5). The miRNA duplex is loaded into the RISC complex having argonaute (Arg) protein in the cytoplasm and the guide strand in a 50 to 30 direction is selected to silence the target gene in a 30 to 50 direction and the other strand acts as a passenger strand. Table 7.1 List of micro RNA associated with crop development. miRNA/siRNA Target gene Function References Apple (Malus domestica) miR169a, miR160e, miR167b,g, miR168a,b miR164 ARF transcription factors Fire blight resistance Kaja et al. (2015) ARF transcription factor Transcription factors Lateral root and leaf development Development, stress response Starch biosynthesis, metabolism Development metabolism Development, cellular signaling pathways Growth and development Starch accumulation Deng et al. (2015) Din et al. (2014) Metabolism Din et al. (2014) Metabolism Din et al. (2014) Grain size shape and quality Zhang et al. (2013a,b) Barley (Hordeum vulgare) Cassava (Manihot esculenta) Cassava (Manihot esculenta) Coffee (Coffea arabica) miR156, miR159, miR395, miR319, miR171 Coffee (Coffea arabica) miR390 Transcription factors, growth factors GRAS family transcription factors TAS3 Potato (Solanum tuberosum) Potato (Solanum tuberosum) Potato (Solanum tuberosum) Potato (Solanum tuberosum) Rice (Oryza sativa) miR160 Auxin response factor miR172 Auxin response factor miR473 miR475 Serine/threonine kinase Thioredoxin miR397 OsLAC miR157, miR160 miR172, miR396 Patanun et al. (2013) Chen et al. (2015) Chaves et al. (2015) Chaves et al. (2015) Din et al. (2014) 189 Continued RNAi based approaches for abiotic and biotic stresses tolerance of crops Plant 190 Table 7.1 List of micro RNA associated with crop development.dcont'd miRNA/siRNA Target gene Function References Sugarcane (Saccharum) Soybean (Glycine max) miR159 miR156, miR160 Development Seed development Zanca et al. (2010) Song et al. (2011) Soybean (Glycine max) miR164, miR166 Seed development Song et al. (2011) Soybean (Glycine max) Wheat (Triticum aestivum) miR172, miR396 miR397/437 MYB protein Squamosa binding protein ARF transcription factors Growth factors L-ascorbate oxidase Seed development In development process Song et al. (2011) Han et al. (2013) Plant Small RNA in Food Crops Plant RNAi based approaches for abiotic and biotic stresses tolerance of crops 191 for 24 h is responsible for the downregulation of late responsive miRNAs, miR390. Only miR390 has been identified as a regulator of the auxin response factor, which is involved in lateral root growth (Marin et al., 2010). 3.2 Drought stress Drought-responsive miRNAs were studied in the legume M. truncatula. In response to drought stress, upregulation of 22 members of four miRNA families (miR399, miR 2089, miR2111, and miR2118) was observed, and downregulation of 10 members of six miRNA families (miR164, miR169, miR171, miR396, miR398 and miR1510) were observed. Droughtresponsive miRNAs’ known and projected targets were discovered to be engaged in a variety of cellular activities in plants, they play important role in development, transcription, protein degradation, detoxification, nutritional status, and cross adaptability (Wang et al., 2011; Gupta et al., 2014c). In potato plants (Solanum tuberosum), miR171 family members (miR171a, miR171b, and miR171c) have been discovered which show a response to drought stress (Hwang et al., 2011). Trindade et al. (2010) have reported that in water-stressed M. truncatula plants, numerous conserved miRNAs have differential expression levels with miR169 being down-regulated in roots and miR398a/b and miR408 being highly up-regulated in both shoots and roots. By using RNAi farnesyl transferase genes FTA or FTB has been suppressed which is responsible for lower stomatal conductance and thus transpiration, and results in greater crop yield (Wang et al., 2009). 3.3 Heat and cold stress The unique plant thermotolerance mechanism has been discovered, which is important for reproductive organ protection. It was discovered that miR398 leads to downregulation of target genes, the CSD (copper/zinc superoxide dismutase) genes CSD1 and CSD2, and CCS (encodes copper chaperone for both CSD1 and CSD2. The CSD1, CSD2, and CCS mutants are responsible for a higher accumulation of heat stress transcription factors and heat shock protein which causes less flower damage in plants and they show more resistance in comparison to control plants (Guan et al., 2013). Cold stress declines the growth and development of plants. Cold stress promotes the evolutionarily conserved miR319 in sugarcane and rice. Overexpression of miR319 decreases the expression of TEOSINTE BRANCHED1, CYCLOIDEA, (PCNABF) Proliferating cell nuclear antigen-binding factor genes, and improves cold tolerance in both species, implying that miR319 provides tolerance against cold stress. By targeting 192 Plant Small RNA in Food Crops the auxin receptor gene TIR1/AFB, cold-inducible miR393 also favorably controls cold tolerance in switchgrass. Cold tolerance is improved by overexpression of miR393 or mutation of TIR1/AFB, which is enhanced expression of cold-responsive genes (Liu et al., 2017). In wheat (T. aestivum L.), 12 miRNAs were identified that were responsive to heat stress (Xin et al., 2010). In the Rice plant, the two miRNAs, miR393 and miR169, have been discovered which were up-regulated in response to dehydration stress (Zhao et al., 2007). It was studied that in Arabidopsis miR159 was involved in the hormonal signaling and dehydration process (Achard et al., 2004). As a result, miR156 and miR172 expression patterns are revealed to be inversely associated. In rice plants, it was observed that Histone deacetylase genes have different expression patterns and developmental activities as compared to closely related Arabidopsis homologs and that the majority of them are drought and salt tolerant (Hu et al., 2009). 3.4 Phosphate deficiency Abiotic stressors like phosphate deficiency have also been shown to upregulate miR399 and miR2111 (Bari et al., 2006; Pant et al., 2009). In all living species, inorganic phosphorus (Pi) is required for macromolecule production, energy transfer, enzyme activity, and signal transduction. Furthermore, agricultural outputs are frequently hampered by Pi levels. MiR399 was the first miRNA discovered to be upregulated in Pi starvation response in Arabidopsis (Fujii et al., 2005). Under Pi-depleted conditions, the putative ubiquitin-conjugating enzyme PHO2 attaches polyubiquitin to the Pi transporter PHOSPHATE TRANSPORTER 1 (PHT1) and directs PHT1 for destruction (Huang et al., 2013), allowing for optimal Pi absorption. In Arabidopsis and rice, low-Pi stress causes miR399 to downregulate PHO2. PHO2 is downregulated by miR399, which raises PHT1 levels and hence promotes Pi acquisition and translocation (Bari et al., 2006; Fujii et al., 2005; Hu et al., 2011). PvPHR1 positively regulated the genes involved in phosphorus transport, mobilization, and homeostasis. miRNA399 (PvmiR399) from Phaseolus vulgaris is a key component of the PvPHR1 signaling pathway in common bean (ValdésLópez et al., 2008). MiR395 and miR399 are up-regulated in Arabidopsis plants when they are deprived of sulfur and phosphate (Jones-Rhoades & Bartel, 2004). 3.5 Salinity stress The RNAi approach has been effectively used to promote salt tolerance in plants, similarly to other drought and temperature stress (Dutta et al., 2020). RNAi based approaches for abiotic and biotic stresses tolerance of crops 193 Similarly, RNA interfering lines of the gene OsVTC-1 revealed that it is involved in salt tolerance (Qin et al., 2017). OsVTC-1 is a GMPase gene that catalyzes the conversion of D-mannose-1-P to GDP-D-mannose and is involved in cellular and developmental events such as cell division, senescence, root expansion, and blooming (Zhang et al., 2013a,b). OsVTC-1 in the production of ascorbic acid works as a scavenger and eliminates reactive oxygen species (ROSs). Under salt stress, OsVTC-1-RNAi lines demonstrated decreased tolerance and a higher amount of reactive oxygen species (ROSs). Furthermore, decreased grain output in OsVTC-1-RNAi lines revealed that OsVTC-1 genes are involved in supplying resistance in both the vegetative and reproductive phases (Qin et al., 2017). In Arabidopsis AtbZIP24 gene which belongs to the bZIP TFs family, was discovered to be a negative regulator of salt stress tolerance, with the AtbZIP24-RNAi line showing increased resistance to salt stress (Yang et al., 2009). One more main gene peroxisomal biogenesis factor 11 (OsPEX11), was discovered in rice using RNAi technology, and it was observed that OsPEX11 is involved in Naþ/Kþ homeostasis and salt tolerance. OsPEX11-RNAi lines were more sensitive to salt stress, with lower levels of lipid peroxidation, a lower Naþ/Kþ ratio, and poorer antioxidant enzyme activity such as SODs, PODs, and CATs (Table 7.2). 4. Role of small RNA during biotic stress miRNAs, 21e23 nucleotides long, and siRNAs, 21e24 nucleotides long are two important families of small RNA produced from double-stranded RNA (dsRNA) or single-stranded RNA precursor. sRNA was gained widespread recognition as a key signaling molecule for its important role in the development and growth of plants and abiotic and biotic stress responses via post-translational gene silencing (PTGS) since the discovery that dsRNA can cause gene silencing in C. elegans (Pattanayak et al., 2013). Plants must struggle with a variety of diseases, including bacteria, fungus, viruses, and parasites, to survive and reproduce in a harsh environment (Ali et al., 2020). Pattern-triggered immunity refers to the pathogen-associated molecular pattern-induced basal resistance response in plants (PTIs). The resistance protein (R protein) and intracellular nucleotide-binding/leucinerich-repeat (NLR) receptors in plants can detect pathogen effectors and generate a robust resistance response known as effector-triggered immunity. Pathogens release tiny cysteine-rich proteins containing signal peptides during host colonization to modify host defense responses, thereby establishing host colonization (Muhammad et al., 2019). Table 7.2 List of miRNA associated with abiotic stress tolerance in plants. Target gene Function References Arabidopsis Arabidopsis Barley (Hordeum vulgare) Cassava (Manihot esculenta) Coffee (Coffea arabica) miR169 miR398 miR156d Drought tolerance Heat stress tolerance Development, drought stress Drought tolerance Li et al. (2008) Guan et al. (2013) Curaba et al. (2012) miR393 Rice (Oryza sativa) miR5517 Rice(Oryza sativa) Soybean and Arabidopsis Sugarcane (Saccharum) miR319 miR169 NFYA5 CSD1, CSD2, CCS Squamosa binding protein NAC transcription factors Transport inhibitor like protein, DNA binding protein, GPR 1 like protein Target genes that are involved specifically in flower or embryonic development PCF5/PCF8 GmNFYA3 miR164 Sugarcane (Saccharum) miR169 Sugarcane (Saccharum) miR398 Wheat (Triticum aestivum) Wheat (Triticum aestivum) miR395 miR164 miR393 NAC transcription factors HAP12-CCAAT-box transcription factors Serine/threonine kinaselike ATP-sulfurylase genes TRANSPORT INHIBITOR RESPONSE 1 (TIR1) Patanun et al. (2013) Chitin cold, salt stress, and water deprivation Akter et al. (2014) Crucial role in the regulatory network of drought response Cheah et al. (2017) Cold tolerance Drought tolerance Yang et al. (2013) Ni et al. (2013) Drought stress response Salt stress tolerance Ferreira et al. (2012) Salt stress tolerance/ metabolism Abiotic stress Regulate auxin signaling and would thus reduce plant growth under drought stress Carnavale Bottino et al. (2013) Carnavale Bottino et al. (2013) Akdogan et al. (2016) Giusti et al. (2017) Plant Small RNA in Food Crops miRNA/siRNA 194 Plant RNAi based approaches for abiotic and biotic stresses tolerance of crops 195 4.1 Parasitic weeds The parasitic weeds the crop fields in many areas around the world, resulting in significant agricultural yield losses. There are various traditional techniques for controlling parasitic weeds, but they all have drawbacks, hence a biotechnological instrument to manage parasitic weeds is needed. RNAi technology has recently been used to generate weed-resistant plant kinds, according to some researchers. Transgenic tomato plants with the M6PR dsRNA-expression cassette were developed by (Aly et al. 2009). In transgenic tomato plants decrease in the concentration of mannitol and endogenous M6PR mRNA in the tubercles and underground shoots of Orobanche egyoetiace and an increase in the amount of dead Orobanche aegyptiaca tubercles were observed. hpRNA-mediated RNAi resistance to Striga asiatica L. was used to generate a parasitic weed-resistant maize cultivar (Aly et al., 2009). 4.2 Insect and nematode resistance Crop losses and insecticides cost billions of dollars due to insect pests. Insecticide resistance remains a constant danger to farmers, fueling a neverending hunt for alternate pest-control measures (Ferry et al., 2006; Banerjee et al., 2017). These worms act as a vector for transmitting virus diseases thereby responsible for the decline in yield concentration (Ali et al., 2015). Uncontrollable phytoparasitic nematodes cause annual crop losses of roughly US$125 billion. Plants’ resistance against nematodes has been produced by the expression of dsRNA in a host plant against housekeeping or parasitism genes in the root-knot nematode (Gheysen & Vanholme, 2007). Plant-parasitic cyst nematodes cause syncytium (plant root organ) differentiation as a source of nourishment. The syncytium is generated by the re-differentiation and fusing of a large number of root cells. miR396 is involved in phase transition in Arabidopsis (Hewezi et al. 2012). The beginning of the syncytial formation phase is marked by strong downregulation of miR396 in cells that give rise to the syncytium, and the beginning of the maintenance phase is marked by upregulation of miR396 in the mature syncytium when no new cells are absorbed into the syncytium. MiR396 and its growth-regulating factor (GRF) target genes induced a reduction in syncytium size and a halt in worm development. This identified miR396 as a major regulator in root cell reprogramming, indicating that it could be a useful molecular target for parasitic animals looking to manipulate plant cells into a new developmental route. Targeting of parasitism-related genes in place of nematode housekeeping genes showed 196 Plant Small RNA in Food Crops tolerance against multiple nematode species (Huang et al., 2006). Opperman et al., 2008 sequenced the genome of Meloidogyne hapla and discovers the new HD-RNAi targets. Host-induced RNAi has been used to target nematode parasitism genes 3B05, 4G06, 8H07, AND 10A06 of the sugar beet cyst worm (Heterodera schachtii), in the A. thaliana host. No resistance was seen, and the number of mature nematode females was reduced by 23e64% in different RNAi lines (Sindhu et al., 2009). RNAi constructs were used to target four distinct genes against Heterodera glycines (essential soybean cyst nematode) and C. elegans to see if they were effective in reducing Meloidogyne incognita galls in roots of the soybean plant. It was observed that by targeting genes codes for Tyrosine phosphatase (TP) and Mitochondrial stress 70 protein precursor (MSP), two of them constructs capable of minimizing gall formation by 92 and 94.7% (Ibrahim et al., 2011). Plant-nematode interactions are also thought to be mediated by miRNAs. In Arabidopsis, miR161, miR164, miR167a, miR172c, miR396c, miR396a,b, and miR398a were down-regulated in response to infection by the nematode H. schachtii (Khraiwesh et al., 2012). According to a comparative investigation of soybean miRNA profiling, 101 miRNAs from 40 families were sensitive to infection by the soybean cyst nematode (SCN; H. glycines), the most damaging disease in soybean. It was also shown that 20 miRNAs were expressed differently in SCN-resistant and susceptible soybean cultivars (Li et al., 2012). Different miRNAs and sRNAs are produced by nematode which is involved in the development of feeding sites and parasitism (Hewezi et al., 2008). Overexpression of these nematode-induced candidate miRNAs, as well as degradation of their targets, reveals new insights into plant-nematode parasitism, as well as nematode resistance in agricultural plants. Resistance against nematodes has also been accomplished by producing a miRNA, which combines with known miRNA genes of the seed region of a plant-parasitic nematode’s essential gene (parasitism or housekeeping). 4.3 Virus resistance Resistance derived from pathogens is the most effective strategy for combating virus infections in plants among the several options. The PDR idea has aided in the development of virus-resistant plants (Simón-Mateo & García, 2011). Another technique targets numerous areas of a viral gene in tomato plants, resulting in broad-spectrum resistance to topoviruses. This technique is based on using a miRNA construct that is capable of expressing RNAi based approaches for abiotic and biotic stresses tolerance of crops 197 numerous artificial miRNAs (amiRNAs) that target different sections of a viral gene (Bucher et al., 2006). The influence of RNAi which targets the coat protein (CP) gene of the virus, is particularly successful in the development of virus resistance in plants. Some viral coat proteins target RNAimodified virus-resistant plants, such as BNYVV-resistant tobacco (Andika et al., 2005), and PVY-resistant potato (Missiou et al., 2004), PRSV-Wresistant Cucumis melo L. var. cantalupensis cv. Sun Lady (Krubphachaya et al., 2007). According to (Pradeep et al., 2012), introducing inverted repetitions of the Tobacco Streak Virus (TSVs) CP gene may be an effective and dependable technique for establishing TSV resistance in commercially significant crops. Rice Stripe Virus CP gene and diseasespecific protein gene sequences were used to produce an RNAi construct (Zhou et al., 2012). Suyunuo and Guangling xiangjing, two sensitive japonica cultivars, were altered using an RNAi construct to develop resistance to Rice Stripe Disease. After self-fertilization, the homozygous progeny of rice plants expressing RNAi constructs in the T5 and T7 generations were shown to be highly resistant to viral infection, with no morphological or developmental abnormalities. The African cassava mosaic virus (ACMVs) was silenced by RNAi, which results in a 99% decline in Rep transcripts and a 66% reduction in viral DNA (Vanitharani et al. 2003). The siRNA method can only silence ACMV strains that are closely related. RNAimediated resistance was first observed in cassava (M.esculenta) against Cassava Brown Streak Disease (CBSD) (Patil et al., 2011). CBSD was regarded as the most serious threat to cassava farming in East Africa (Pooggin et al., 2003). demonstrated that the DNA of a reproducing virus can also be a target of RNAi using the black gram (Vigna mungo) as a study system. They were able to recover V. mungo from MYMIVs (Mungbean Yellow Mosaic India Virus) infection by using an RNAi technique to silence the gene associated with the bidirectional promoter. RNAi technique has been used to create geminivirus-resistant BGMV-resistant common bean (Bonfim et al., 2007). Many other transgenic plants such as Tomato Yellow Leaf Curl Virus (TYLC)-resistant tomato (Fuentes et al., 2016), Rice Tungro Bacilliform Virus (RTBV)-resistant rice (Tyagi et al., 2008), and Citrus Tristeza Virus (CTV)-resistant Mexican lime (López-García et al., 2010), have improved defense through RNAi-mediated gene silencing. According to (Schwind et al., 2009), employing the hpRNA construct in S. lycopersicum (tomato) against Potato Spindle Tuber Viroid (PSTVd) results in PSTVd-resistant tomato plant varieties. 198 Plant Small RNA in Food Crops 4.4 Bacterial pathogens Bacterial pathogens are among the most serious problems in tomato, soybean, and banana and various type of crop production. Bacterial diseases spread quickly and are difficult to manage; therefore, the only method to avoid bacterial infections is to prevent them. The use of RNAi to improve bacterial resistance in the experimental plant A. thaliana has yielded promising results. In Arabidopsis (Dunoyer et al., 2006), studied the silencing of bacterial genes (iaaM and ipt) reduced the crown gall tumors (Agrobacterium tumefaciens) to nearly zero, implying that crown gall disease resistance engineered in various trees and ornamental plants. Four apple miRNAs (miR169a, miR160e, miR167beg, and miR168a,b) have recently been discovered to be implicated in the resistance to fire blight, a contagious bacterial disease caused by Erwinia amylovora, by targeting stress response proteins (Kaja et al., 2015) (Table 7.1). 4.5 Fungal resistance RNAi has been proved to be an effective technique for generating disease resistance in a variety of agricultural plants. RNAi-mediated silencing of the aricegene OsSSI2 resulted in increased resistance to the blast fungus Magnaporthe grisea and the bacterium Xanthomonas oryzae that causes leaf blight (Jiang et al., 2009). Furthermore, the inhibition of two genes, OsFAD7 and OsFAD8, which are -3 fatty acid desaturase genes, resulted in increased disease resistance against M. grisea in rice (Yara et al., 2007). Due to the decline in the concentration of lignin, RNAi-mediated targeting of genes involved in lignin formation improved soybean resistance to the phytopathogen Sclerotinia sclerotiorum (Peltier et al., 2009). 24 miRNAs were recently discovered to be involved in the response to the fungus Blumeria graminis f. sp. tritici (Bgt) attacking wheat, which causes the fatal illness powdery mildew (Xin et al., 2010). Similarly, several miRNAs have been identified in wheat in response to stem rust infection (Gupta et al., 2012). Furthermore, it was discovered that overexpression of OsmiR397 and downregulation of the corresponding OsLAC target gene increased grain size and promoted panicle branching, increasing yield (Zhang et al., 2013a,b) In reaction to the blast fungus Magnaporthe oryzae, the rice miRNA osa-miR7695 negatively regulates a natural resistance-associated macrophage protein6 (OsNramp6). Overexpression of Osa-miR7696 resulted in improved resistance to rice blast infection (Campo et al., 2013). (Jiao & Peng, 2018) studied that in wheat plant miR1023 targeted the alpha/beta hydrolase gene in F. graminearum and suppressed the invasion of the fungal pathogen (Table 7.3). RNAi based approaches for abiotic and biotic stresses tolerance of crops 199 Table 7.3 Role of small RNA in fungal resistance. Plant Target gene Trait improved References Potato SYR1 Rice OsFAD7 And OsFAD8 OsSSI2 Phytophthora infestans Magnaporthe grisea Magnaporthe grisea Xanthomonas oryzae Eschen-Lippold et al. (2012) Yara et al. (2007) Jiang et al. (2009) Rice 5. Applications of RNAi technology in crop development A new combination of biotechnology techniques like genetic engineering, genomics, proteomics, and plant physiology, will be required to solve all malnutrition and food-related requirement of a large population (Mittler & Blumwald, 2010). The promise of the RNAi technique in crop development has been demonstrated by its contribution to achieving desirable traits by modifying genetic expression. 5.1 Seedless fruit development Phytohormones have been widely known for their role in regulating the transition between blooming, fertilization, and fruiting. Parthenocarpy has the potential to produce vegetables and fruits when pollination fails to occur. Seedlessness has been found in recent research to improve the texture and shelf life of fruits, such as watermelon and eggplant (Pandolfini, 2009). Seeds were found to be the source of fruit deterioration in watermelon. Thus, substituting edible fruit tissue for seeds and seed cavities is useful (Varoquaux et al., 2000) and can be beneficial to consumers, the processing business, and breeding companies. It has been studied that ARF8 of A. thaliana and ARF7 of tomato both have high levels of expression in non-pollinated flowers before being down-regulated after pollination (De Jong et al., 2009). SlARF7 works as a modulator of both auxin and gibberellin responses during tomato fruit set and development. After pollination and fertilization, SlARF7 transcript levels are normally lowered (Vriezen et al., 2008). SlARF7 repression may be released by an RNAi 200 Plant Small RNA in Food Crops method, resulting in partial activation of auxin and GA signaling pathways enforced by SlARF7 independent of pollination and fertilization, and thus in parthenocarpic fruit growth in tomato (S. lycopersicum). As a result, the auxin signaling transduction pathway’s fertilization-dependent stage may be avoided, which may be required to commence cell division activity and boost GA production. 5.2 Enhancement of the shelf life of crop plants Fruits and vegetables are higher vulnerable to spoilage as compared to grains, and this spoilage results in inedible waste. Despite being one of the world’s top producers of fruits and vegetables, India loses roughly 30% of its overall production because of spoiling. As a result, an increase in the shelf life of vegetables and fruits is required as another important agronomic attribute that can reduce the degradation and rotting of vegetables and fruits, hence reducing horticulture loss. The silencing of genes involved in ethylene generation or ripening has enhanced the shelf life of tomatoes. RNAi technology was utilized by Xiong et al. (2005) to extend the shelf life of tomatoes. They used a unit of dsRNA to stop the ACC oxidase gene from expressing in tomato. The rate of ethylene generation in transgenic plants’ ripened fruits and leaves was shown to be greatly reduced, ensuring a longer shelf life for tomatoes. (Meli et al., 2010), on the other hand, identified two ripening-specific N-glycoprotein modifying enzymes, mannosidase (Man) and -D-N-acetylhexosaminidase, in tomato and repressed them. 5.3 Male sterility and fertility The essential quality to assure purity in hybrid plants for hybrid seed production are the establishment of male sterility. Plant scientists utilize a variety of approaches based on conventional and genetic engineering to abort pollens from various crop species. RNAi has been employed in genetic engineering to create male-sterile plant species such as tobacco and tomato (Nizampatnam & Kumar, 2011). Small RNA (osa-smR5864) has been regulated the photoperiod and temperature-regulated male sterility in rice (Zhu & Deng, 2012). In plants mitochondria and plastids MutS HOMOLOG 1 (Msh1) is a nuclear gene product that keeps the genome stable. When Msh1 is suppressed in response to abiotic stressors, it activates a plastidial response that involves non-genetic inheritance and changes a variety of plant metabolic pathways (Xu et al. 2012). RNAi technology has RNAi based approaches for abiotic and biotic stresses tolerance of crops 201 been used to alter Msh1 expression in tobacco and tomato, resulting in mitochondrial DNA rearrangements consistent with naturally occurring cytoplasmic male sterility (Sandhu et al., 2007). The use of these RNAi lines for breeding may have a few downsides, such as the hybrid carrying the construct in the F1 generation, resulting in severe impacts. In terms of fertility, studies shows that mir151 affects the development of rice anthers. Overexpression of miR159 in rice will result in malformed flower development and no pollen in the stamens. In addition overexpression of wheat miR159 in rice or tae-miR159 can also cause male sterility in rice (Wang et al., 2012). Recent studies found that miR1227 and miR2275 may be related to male sterility in wheat and targated the CAF1 (CCR4 AF1) and SMARCA3L3 genes (Sun et al., 2018). 5.4 Biofortification Over two-thirds of the population around the world consumes food that is deficient in various key mineral elements (White & Broadley, 2009; Gupta et al., 2022b; Ibba et al., 2022). The tomato plant has been biofortified with dietary antioxidants and important elements such as Zn, Mg, Cu, Se, Ca, Fe, I, S, P, and others by using RNAi technology (Niggeweg et al., 2004). Molecular pharming or bio-pharming is the process of creating perspective pharmacological substances by applying novel genetic engineering and transgenic techniques such as agroinfiltration, virus infection, and magnification. Various proteins and secondary metabolites have been produced by using molecular pharming. Molecular pharming is used to create a variety of medicines and nutraceuticals (Obembe et al., 2011). The use of RNAi has reduced the sinapate esters to 76% in T3 generation transgenic canola seeds. It suppresed the UDP-Glc:sinapateglucosyltransferase gene activity. The flavor of canola seeds has been enhanced by the elimination of sinapate esters (Hüsken et al., 2005). RNAi technology was used for the development of GluB hairpin RNA which resulted in the LGC-1 (low glutenin content 1) rice variety with reduced glutenin levels, thus providing comfort to kidney patients who are not able to digest gluten (Kusaba et al., 2003). The ingestion of alpha-linolenic acid (18:3) is harmful to both humans and animals. The lowering of alpha-linolenic acid (18:3) improves the flavor and stability of soybean oil while reducing the requirement for hydrogenation (Flores et al., 2008). used the glycinin promoter to create Hairpin RNA for seed-specific silencing of omega-3 fatty acid desaturase (GmFAD3A, GmFAD3B, and GmFAD3C). Sucrose biosynthesis is divided 202 Plant Small RNA in Food Crops into two phases, each of which is catalyzed by an enzyme: sucrosephosphate synthase (SPS), which is then hydrolyzed by another enzyme sucrose phosphatase (SPP) to produce sucrose and Pi inorganic phosphate). In a process known as “cold sweetening,” storing potato tubers at a lower temperature (4 C) results in the accumulation of glucose and fructose. To suppress SPP expression in transgenic potato tubers a CaMV 35S promoterdriven hairpin RNAi construct has been synthesized having part of the coding region of the tobacco NtSPP2 gene (Chen et al., 2008). Suc6P accumulates in RNAi-silenced sucrose phosphatase (SPP) potato tubers after cold storage at 4 C, according to the researchers. They discovered that in SPP-silenced tubers, cold-induced expression of vacuolar invertase (VI) was prevented, resulting in a lower sucrose-to-hexose conversion. VI expression was found to be adversely linked with Suc6P levels. RNAi technology was used for silencing the expression of specific -gliadins, reporting a 55e80% reduction in -gliadins in the bread wheat cultivar “Bobwhite” lines (BW208) and a 33e43% reduction in the “Bobwhite” lines (BW 2003) (Gil-Humanes et al., 2008). Furthermore (Gil-Humanes et al., 2012), found that RNAi-mediated downregulation of gammagliadins in wheat lines resulted in a compensatory impact in the remaining gluten proteins, with no statistically significant changes in overall gliadin content but an increase in glutenin content. As a result, the total protein content of most transgenic lines was modestly raised. 5.5 Allergen and toxin elimination Food allergy is an overactive immune reaction in our bodies caused by allergens found in foods like peanuts, apples, mangoes, etc. As a result, allergen levels in our meals must be reduced or eliminated. Not only that but there is a need to design plants that are devoid of hazardous chemicals, as natural toxins can be found in a wide range of plants that are regularly eaten as food. These hazardous compounds can be dangerous to human health if consumed in large amounts or if they are not handled properly, resulting in food poisoning. The removal of allergens and toxic compounds can be accomplished through RNA interference, which can alter allergen production by altering its metabolic pathway, thereby improving food quality and reducing the risk of food allergy and toxicity. Mal d1, a prominent apple allergen that belongs to the PR10 group of pathogenesisrelated proteins, was inhibited using RNAi (Gilissen et al. 2005) used RNAi-based gene silencing to successfully decrease Mal d1 expression. In RNAi based approaches for abiotic and biotic stresses tolerance of crops 203 crude peanut extract, a 25% drop in Arah2 content was observed by employing RNAi which leads to down-regulation of its expression using its hpRNA construct. (Dodo et al., 2008). Ara h2 is an allergenic protein found in peanuts, out of a total of seven allergenic proteins (Jørgensen et al., 2005). used RNAi to inhibit the biosynthesis of linamarin and lotaustralin by suppressing the cytochrome P450 enzyme, resulting in transgenic cassava (Manihot esculenta) plants with less than 1% cyanogenic glucosides in leaves and 92% reduction in cyanogenic glucosides in tubers. Grass peas are consumed by people in India, Bangladesh, and Ethiopia (Lathyrus sativus). The neurotoxic beta-N-oxalyl-ami-noalanine-L-alanine (BOAA) found in grass peas and chickling peas can cause lathyrism, a paralytic condition (Spencer et al., 1986). BOAA provides immunity to plants in stress conditions and by using RNAi technology level of BOAA is reduced to a suitable concentration can make the crop safe to eat (Angaji et al., 2010). Cotton seeds contain a higher amount of dietary protein, but their poisonous terpenoid component, gossypol, makes them unfit for human ingestion. Cotton stocks with decreased levels of delta-cadinene synthase, a major enzyme in the gossypol biosynthesis pathway, have been created using RNAi without influencing the enzyme’s production in other sections of the plant, where gossypol is vital in avoiding pest damage. Transgenic cotton seeds had a 99% lower gossypol level than wild cotton seeds. It has been studied that silencing of delta-cadinene synthase gene by using RNAi knockdown technology cotton plants has been developed that generated cottonseed with ultra-low gossypol levels (ULGCS) (Sunilkumar et al., 2006). They also discovered that the ULGCS phenotype induced by RNAi exhibited multi-generational stability (Rathore et al., 2012). 5.6 Phenotype change and altered architecture Flowers and ornamental plants have been enhanced since antiquity by adjusting numerous qualities of size, shape, color, appearance, as well as plant architecture. Changes in the architecture of crop plants, such as Dwarf Rice, may also result in increased crop output. As a result, there is a need to improve plant and flower phenotype. Plant architecture is responsible for different patterns of plant growth such as plant height and canopy, leaf size and number, branches, number of flowers, fruit size and shape, root size, and structure. The plant architecture can be modified by using RNAi techniques. It helps in the enhancement of the yield of ornamental, fruit, and crop plants (Dai et al., 2018). The Arabidopsis flowering locus T (FT) and terminal flower I (TFL1) genes, as 204 Plant Small RNA in Food Crops well as orthologs from other species, are well known for balancing and distributing indeterminate and determinate growth. These genes can be altered to improve plant/flower morphology or increase agricultural output (McGarry & Ayre, 2012). The use of RNAi in plants to change their morphology as needed has shown its potential (such as height, inflorescence, branching, and size). The cauliflower mosaic virus 35S promoter has been used for overexpression of the unique maize miRNA gene dubbed Corngrass1 (Cg1), which belongs to the miR156 family. When compared to wild-type stem lignin, transgenics had significantly larger axillary meristem expansion, shorter internode length, and up to a 30% drop in stem lignin concentration (Rubinelli et al., 2013). McGarry and Ayre (2012) recently revealed that the HSPp:FT1/ FT2-RNAi lines produced inflorescences as well, implying that FT1 signaling is adequate for reproductive onset. Various miRNAs have been discovered that are involved in the development and ripening of tomato fruits (Molesini et al., 2012). miR156 is a small RNA that silenced the gene involved in fruit ripening called colorless never ripe. The color of transgenic tomato plants overexpressing sly-miR156 was slightly lighter than wild-type controls, but they could eventually ripen (Zhang et al., 2011). Further, it has been observed that the miR156 overexpression in tomato leads to smaller fruit size, increased leaf numbers, and reduced height and size of leaf implying that reduction in miR156 expression helps in increased yield of the crop (Zhang et al., 2011). Transgenic studies are needed to confirm this. Recent researches show that miR172 regulates fruit ripening and negatively regulates ethylene generation. AP2a is also favorably regulated by CNR, and play important role in fruit development and ripening (Karlova et al., 2013). 6. Conclusion and future prospective With continued population growth and consumption, global food demand is expected to rise. The challenge for agriculture will be rising as agricultural output is sustainably in the future time. By using genetic engineering and metabolic engineering novel techniques high yielding crops will be designed, so that the world can produce more food and assure that it is used more efficiently. According to a WHO report, 2020 malnutrition is a major problem all over the world especially in developing countries and acc to WHO reports India is among countries that miss world nutrition targets set for the year 2025 (WHO, 2020). Chronic food deficits have an effect on approximately 811 million population worldwide as well as 20 % of the population in developing countries. It is necessary to develop high-quality RNAi based approaches for abiotic and biotic stresses tolerance of crops 205 biofortified food crop varieties, fruits, and vegetables by enriching them with nutritionally vital components such as essential minerals, antioxidants, vitamins, fatty acids, and amino acids to assure a healthy diet for a healthy world. Biotic and abiotic stress tolerance is one of the most significant agronomical qualities that agricultural plants should have. The improved or engineered varieties are helpful against food insecurities that arise around the world. RNAi technology plays a major role in crop development to address food security, hunger, and famine issues. Small non-coding RNA research, on the other hand, is opening the way for agricultural advancement and, as a result, enhancing the overall people’s lives. Many unique crops have been produced by using sRNA and microRNA technology including highly biofortified nutrient-rich crops. The genetically modified Arctic apples are on the verge of becoming approved in the United States. The apples were created by gene silencing inhibition of the PPO (polyphenol oxidase) gene, which resulted in apple cultivars that do not brown when sliced. By silencing PPO receptors using RNAi mechanism chlorogenic acid will not be able to convert into a quinone product. Using different advanced molecular biotechnological approaches the function of miRNA can be manipulated for better crop improvement and yield resistance. The ability of biomolecules like glucose, lignin, and fat to create bioactive compounds. Metabolic engineering can also be used to make it easier to synthesize and mass-produce commercially valuable plant products like medicines, pigments, perfumes, volatile oils, and flavors. sRNA can be used in a novel way to reduce enzyme activity, which will alter biochemical reactions and lead to the synthesis of desired chemicals rather than unwanted/toxic molecules. RNAi can reduce photorespiration and thereby increase C3 plant output. Reduction of the flowering period, delayed ripening and senescence, breaking dormancy, production of stress resistance plants, overcoming self-sterility, and other traits could be induced with this knockdown approach. Artificial restriction enzymes have recently been developed that may successfully able to alterations in genes. These are viable alternatives to gene silencing and have a promising future. Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and LAGLIDADG homing endonucleases (LHEs), sometimes known as “meganucleases,” are three types of nucleases that help in crop development along with desired characters or traits. ZFNs and TALENs allow programmed subtle genetic changes by causing breaks in the double strands of DNA (Curtin et al., 2018). TALEs (programmable DNA binding domain) and FokI (cleavage domain) have been fused to create TALENs 206 Plant Small RNA in Food Crops and further by using this stress resistance novel varieties with improved characters and traits can be developed by applying gene editing in the double-stranded DNA (Yasumoto et al., 2020). References Achard, P., Herr, A., Baulcombe, D. C., & Harberd, N. P. (2004). Modulation of floral development by a gibberellin-regulated microRNA. Akdogan, G., Tufekci, E. D., Uranbey, S., & Unver, T. (2016). miRNA-based drought regulation in wheat. Functional & Integrative Genomics, 16(3), 221e233. Akter, A., Islam, M. M., Mondal, S. I., Mahmud, Z., Jewel, N. A., Ferdous, S., … Rahman, M. M. (2014). Computational identification of miRNA and targets from expressed sequence tags of coffee (Coffea arabica). Saudi Journal of Biological Sciences, 21(1), 3e12. Ali, M. A., Abbas, A., Azeem, F., Javed, N., & Bohlmann, H. (2015). Plant-nematode interactions: From genomics to metabolomics. International Journal of Agriculture and Biology, 17(6). Ali, M., Javaid, A., Naqvi, S. H., Batcho, A., Kayani, W. K., Lal, A., … Nwogwugwu, J. O. (2020). Biotic stress-triggered small RNA and RNAi defense responses in plants. Molecular Biology Reports, 47(7), 5511e5522. Allen, E., Xie, Z., Gustafson, A. M., & Carrington, J. C. (2005). microRNA-directed phasing during trans-acting siRNA biogenesis in plants. Cell, 121(2), 207e221. Aly, R., Cholakh, H., Joel, D. M., Leibman, D., Steinitz, B., Zelcer, A., … Gal-On, A. (2009). Gene silencing of mannose 6-phosphate reductase in the parasitic weed Orobanche aegyptiaca through the production of homologous dsRNA sequences in the host plant. Plant Biotechnology Journal, 7(6), 487e498. Andika, I. B., Kondo, H., & Tamada, T. (2005). Evidence that RNA silencing-mediated resistance to Beet necrotic yellow vein virus is less effective in roots than in leaves. Molecular Plant-Microbe Interactions, 18(3), 194e204. Angaji, S., Hedayati, S. S., Hosein, R., Samad, S., Shiravi, S., & Madani, S. (2010). Application of RNA interference in plants. Plant Omics, 3(3), 77e84. Banerjee, S., Banerjee, A., Gill, S. S., Gupta, O. P., Dahuja, A., Jain, P. K., & Sirohi, A. (2017). RNA Interference: A novel source of resistance to combat plant parasitic nematodes. Frontiers in Plant Science, 8, 834. https://doi.org/10.3389/fpls.2017.00834 Bari, R., Datt Pant, B., Stitt, M., & Scheible, W. R. (2006). PHO2, microRNA399, and PHR1 define a phosphate-signaling pathway in plants. Plant Physiology, 141(3), 988e999. Beck, M., & Hurt, E. (2017). The nuclear pore complex: Understanding its function through structural insight. Nature Reviews Molecular Cell Biology, 18(2), 73e89. Bologna, N. G., Iselin, R., Abriata, L. A., Sarazin, A., Pumplin, N., Jay, F., … Voinnet, O. (2018). Nucleo-cytosolic shuttling of ARGONAUTE1 prompts a revised model of the plant microRNA pathway. Molecular Cell, 69(4), 709e719. Bonfim, K., Faria, J. C., Nogueira, E. O., Mendes, É. A., & Aragão, F. J. (2007). RNAimediated resistance to Bean golden mosaic virus in genetically engineered common bean (Phaseolus vulgaris). Molecular Plant-Microbe Interactions, 20(6), 717e726. Bucher, E., Lohuis, D., van Poppel, P. M., Geerts-Dimitriadou, C., Goldbach, R., & Prins, M. (2006). Multiple virus resistance at a high frequency using a single transgene construct. Journal of General Virology, 87(12), 3697e3701. Campo, S., Peris-Peris, C., Siré, C., Moreno, A. B., Donaire, L., Zytnicki, M., … San Segundo, B. (2013). Identification of a novel micro RNA (mi RNA) from rice that targets an alternatively spliced transcript of the N ramp6 (N atural resistance-associated macrophage protein 6) gene involved in pathogen resistance. New Phytologist, 199(1), 212e227. RNAi based approaches for abiotic and biotic stresses tolerance of crops 207 Carnavale Bottino, M., Rosario, S., Grativol, C., Thiebaut, F., Rojas, C. A., Farrineli, L., … Ferreira, P. C. G. (2013). High-throughput sequencing of small RNA transcriptome reveals salt stress regulated microRNAs in sugarcane. PLoS One, 8(3), e59423. Chaves, S. S., Fernandes-Brum, C. N., Silva, G. F. F., Ferrara-Barbosa, B. C., Paiva, L. V., Nogueira, F. T. S., … Chalfun-Junior, A. (2015). New insights on coffea miRNAs: Features and evolutionary conservation. Applied Biochemistry and Biotechnology, 177(4), 879e908. Cheah, B. H., Jadhao, S., Vasudevan, M., Wickneswari, R., & Nadarajah, K. (2017). Identification of functionally important microRNAs from rice inflorescence at heading stage of a qDTY4. 1-QTL bearing near isogenic line under drought conditions. PLoS One, 12(10), e0186382. Chen, S., Hajirezaei, M. R., ZANOR, M. I., Hornyik, C., Debast, S., Lacomme, C., … Boernke, F. (2008). RNA interference-mediated repression of sucrose-phosphatase in transgenic potato tubers (Solanum tuberosum) strongly affects the hexose-to-sucrose ratio upon cold storage with only minor effects on total soluble carbohydrate accumulation. Plant, Cell and Environment, 31(1), 165e176. Chen, L., Wang, T., Zhao, M., Tian, Q., & Zhang, W. H. (2012). Identification of aluminum-responsive microRNAs in Medicago truncatula by genome-wide highthroughput sequencing. Planta, 235(2), 375e386. Chen, X., Xia, J., Xia, Z., Zhang, H., Zeng, C., Lu, C., … Wang, W. (2015). Potential functions of microRNAs in starch metabolism and development revealed by miRNA transcriptome profiling of cassava cultivars and their wild progenitor. BMC Plant Biology, 15(1), 1e11. Cui, Y., Yi, L., Zhao, J. Z., & Jiang, Y. G. (2017). Long noncoding RNA HOXA11-AS functions as miRNA sponge to promote the glioma tumorigenesis through targeting miR-140-5p. DNA and Cell Biology, 36(10), 822e828. Curaba, J., Spriggs, A., Taylor, J., Li, Z., & Helliwell, C. (2012). miRNA regulation in the early development of barley seed. BMC Plant Biology, 12(1), 1e16.
Curtin, S. J., Xiong, Y., Michno, J. M., Campbell, B. W., Stec, A. O., Cermák, T., … Stupar, R. M. (2018). Crispr/cas9 and TALENs generate heritable mutations for genes involved in small rna processing of Glycine max and Medicago truncatula. Plant Biotechnology Journal, 16(6), 1125e1137. Dai, Z., Wang, J., Yang, X., Lu, H., Miao, X., & Shi, Z. (2018). Modulation of plant architecture by the miR156feOsSPL7eOsGH3. 8 pathway in rice. Journal of Experimental Botany, 69(21), 5117e5130. De Jong, M., Mariani, C., & Vriezen, W. H. (2009). The role of auxin and gibberellin in tomato fruit set. Journal of Experimental Botany, 60(5), 1523e1532. Deng, P., Wang, L., Cui, L., Feng, K., Liu, F., Du, X., … Weining, S. (2015). Global identification of microRNAs and their targets in barley under salinity stress. PLoS One, 10(9), e0137990. Dhiman, P., Rajora, N., Bhardwaj, S., Sudhakaran, S., Kumar, A., Raturi, G., Chakraborty, K., Gupta, O. P., Devanna, B. N., Tripathi, D. K., & Deshmukh, R. (2021). Fascinating role of silicon to combat salinity stress in plants: An updated overview. Plant Physiology and Biochemistry, 162, 110e123. Din, M., Barozai, M. Y. K., & Baloch, I. A. (2014). Identification and functional analysis of new conserved microRNAs and their targets in potato (Solanum tuberosum L.). Turkish Journal of Botany, 38(6), 1199e1213. Dodo, H. W., Konan, K. N., Chen, F. C., Egnin, M., & Viquez, O. M. (2008). Alleviating peanut allergy using genetic engineering: The silencing of the immunodominant allergen Ara h 2 leads to its significant reduction and a decrease in peanut allergenicity. Plant Biotechnology Journal, 6(2), 135e145. Dunoyer, P., Himber, C., & Voinnet, O. (2006). Induction, suppression and requirement of RNA silencing pathways in virulent Agrobacterium tumefaciens infections. Nature Genetics, 38(2), 258e263. Dutta, T., Neelapu, N. R. R., Wani, S. H., & Surekha, C. (2020). Salt stress tolerance and small RNA. Plant Small RNA, 191e207. 208 Plant Small RNA in Food Crops Eschen-Lippold, L., Landgraf, R., Smolka, U., Schulze, S., Heilmann, M., Heilmann, I., … Rosahl, S. (2012). Activation of defense against Phytophthora infestans in potato by downregulation of syntaxin gene expression. New Phytologist, 193(4), 985e996. Ferreira, T. H., Gentile, A., Vilela, R. D., Costa, G. G. L., Dias, L. I., Endres, L., & Menossi, M. (2012). microRNAs associated with drought response in the bioenergy crop sugarcane (Saccharum spp.). Ferry, N., Edwards, M. G., Gatehouse, J., Capell, T., Christou, P. A. M. R., & Gatehouse, A. M. R. (2006). Transgenic plants for insect pest control: A forward looking scientific perspective. Transgenic Research, 15(1), 13e19. Flores, T., Karpova, O., Su, X., Zeng, P., Bilyeu, K., Sleper, D. A., … Zhang, Z. J. (2008). Silencing of gm FAD3 gene by siRNA leads to low a-linolenic acids (18: 3) of fad3mutant phenotype in soybean [Glycine max (Merr.)]. Transgenic Research, 17(5), 839e850. Fuentes, A., Carlos, N., Ruiz, Y., Callard, D., Sánchez, Y., Ochagavía, M. E., … Pooggin, M. M. (2016). Field trial and molecular characterization of RNAi-transgenic tomato plants that exhibit resistance to tomato yellow leaf curl geminivirus. Molecular Plant-Microbe Interactions, 29(3), 197e209. Fujii, H., Chiou, T. J., Lin, S. I., Aung, K., & Zhu, J. K. (2005). A miRNA involved in phosphate-starvation response in Arabidopsis. Current Biology, 15(22), 2038e2043. Gheysen, G., & Vanholme, B. (2007). RNAi from plants to nematodes. Trends in Biotechnology, 25(3), 89e92. Gielen, H., Remans, T., Vangronsveld, J., & Cuypers, A. (2012). MicroRNAs in metal stress: Specific roles or secondary responses? International Journal of Molecular Sciences, 13(12), 15826e15847. Gil-Humanes, J., Pistón, F., Giménez, M. J., Martín, A., & Barro, F. (2012). The introgression of RNAi silencing of g-gliadins into commercial lines of bread wheat changes the mixing and technological properties of the dough. Gil-Humanes, J., Pistón, F., Hernando, A., Alvarez, J. B., Shewry, P. R., & Barro, F. (2008). Silencing of g-gliadins by RNA interference (RNAi) in bread wheat. Journal of Cereal Science, 48(3), 565e568. Gilissen, L. J., Bolhaar, S. T., Matos, C. I., Rouwendal, G. J., Boone, M. J., Krens, F. A., … Van Ree, R. (2005). Silencing the major apple allergen Mal d 1 by using the RNA interference approach. The Journal of Allergy and Clinical Immunology, 115(2), 364e369. Giusti, L., Mica, E., Bertolini, E., De Leonardis, A. M., Faccioli, P., Cattivelli, L., & Crosatti, C. (2017). microRNAs differentially modulated in response to heat and drought stress in durum wheat cultivars with contrasting water use efficiency. Functional & Integrative Genomics, 17(2e3), 293e309. Godfray, H. C. J., Beddington, J. R., Crute, I. R., Haddad, L., Lawrence, D., Muir, J. F., … Toulmin, C. (2010). Food security: The challenge of feeding 9 billion people. Science, 327(5967), 812e818. Griffiths-Jones, S., Grocock, R. J., Van Dongen, S., Bateman, A., & Enright, A. J. (2006). miRBase: microRNA sequences, targets and gene nomenclature. Nucleic Acids Research, 34(Suppl. _1), D140eD144. Guan, Q., Lu, X., Zeng, H., Zhang, Y., & Zhu, J. (2013). Heat stress induction of mi R 398 triggers a regulatory loop that is critical for thermotolerance in A rabidopsis. The Plant Journal, 74(5), 840e851. Gupta, O. P., Deshmukh, R., Kumar, A., Singh, S. K., Sharma, P., Ram, S., & Singh, G. P. (2022a). From gene to biomolecular networks: A review of evidences for understanding complex biological function in plants. Current Opinion in Biotechnology, 74, 66e74. https://doi.org/10.1016/j.copbio.2021.10.023 Gupta, O. P., Sharma, P., Gupta, R. K., & Sharma, I. (2014a). Current status on role of miRNAs during plant-fungus interaction. Physiological and Molecular Plant Pathology, 85, 1e7. RNAi based approaches for abiotic and biotic stresses tolerance of crops 209 Gupta, O. P., Sharma, P., Gupta, R. K., & Sharma, I. (2014b). MicroRNA mediated regulation of metal toxicity in plants: Present status and future perspectives. Plant Molecular Biology, 84(1e2), 1e18. https://doi.org/10.1007/s11103-013-0120-6 Gupta, O. P., Meena, N. L., Sharma, I., & Sharma, P. (2014c). Differential regulation of microRNAs in response to osmotic, salt and cold stresses in wheat. Molecular Biology Reports, 41(7), 4623e4629. https://doi.org/10.1007/s11033-014-3333-0 Gupta, O. P., Permar, V., Koundal, V., Singh, U. D., & Praveen, S. (2012). MicroRNA regulated defense responses in Triticum aestivum L. during Puccinia graminis f.sp. tritici infection. Molecular Biology Reports, 39, 817e824. Gupta, O. P., Singh, A. K., Singh, A., Singh, G. P., Bansal, K. C., & Datta, S. K. (2022b). Wheat biofortification: Utilizing natural genetic diversity, genome-wide association mapping, genomic selection, and genome editing technologies. Frontiers in Nutrition, 9, Article 826131. https://doi.org/10.3389/fnut.2022.826131 Han, J., Kong, M. L., Xie, H., Sun, Q. P., Nan, Z. J., Zhang, Q. Z., & Pan, J. B. (2013). Identification of miRNAs and their targets in wheat (Triticum aestivum L.) by EST analysis. Genetics and Molecular Research, 12(3793), 805. Herdt, R. W. (2006). Biotechnology in agriculture. Annual Review of Environment and Resources, 31, 265e295. Hewezi, T., Howe, P., Maier, T. R., & Baum, T. J. (2008). Arabidopsis small RNAs and their targets during cyst nematode parasitism. Molecular Plant-Microbe Interactions, 21(12), 1622e1634. Hewezi, T., Maier, T. R., Nettleton, D., & Baum, T. J. (2012). The Arabidopsis microRNA396-GRF1/GRF3 regulatory module acts as a developmental regulator in the reprogramming of root cells during cyst nematode infection. Plant Physiology, 159(1), 321e335. Huang, G., Allen, R., Davis, E. L., Baum, T. J., & Hussey, R. S. (2006). Engineering broad root-knot resistance in transgenic plants by RNAi silencing of a conserved and essential root-knot nematode parasitism gene. Proceedings of the National Academy of Sciences, 103(39), 14302e14306. Hu, Q., Hollunder, J., Niehl, A., Kørner, C. J., Gereige, D., Windels, D., … Heinlein, M. (2011). Specific impact of tobamovirus infection on the Arabidopsis small RNA profile. PLoS One, 6(5), e19549. Hu, Y., Qin, F., Huang, L., Sun, Q., Li, C., Zhao, Y., & Zhou, D. X. (2009). Rice histone deacetylase genes display specific expression patterns and developmental functions. Biochemical and Biophysical Research Communications, 388(2), 266e271. Hüsken, A., Baumert, A., Strack, D., Becker, H. C., Möllers, C., & Milkowski, C. (2005). Reduction of sinapate ester content in transgenic oilseed rape (Brassica napus) by dsRNAi-based suppression of BnSGT1 gene expression. Molecular Breeding, 16(2), 127e138. Hwang, E. W., Shin, S. J., Yu, B. K., Byun, M. O., & Kwon, H. B. (2011). miR171 family members are involved in drought response in Solanum tuberosum. Journal of Plant Biology, 54(1), 43e48. Ibba, M. I., Gupta, O. P., Govindan, V., Johnson, A. A. T., Brinch-Pedersen, H., Nikolic, M., & Taleon, V. (2022). Editorial: Wheat biofortification to alleviate global malnutrition. Frontiers in Nutrition, 9, Article 1001443. https://doi.org/10.3389/ fnut.2022.1001443 Ibrahim, H. M., Alkharouf, N. W., Meyer, S. L., Aly, M. A., Abd El Kader, Y., Hussein, E. H., & Matthews, B. F. (2011). Post-transcriptional gene silencing of rootknot nematode in transformed soybean roots. Experimental Parasitology, 127(1), 90e99. Iki, T., Yoshikawa, M., Nishikiori, M., Jaudal, M. C., Matsumoto-Yokoyama, E., Mitsuhara, I., … Ishikawa, M. (2010). In vitro assembly of plant RNA-induced silencing complexes facilitated by molecular chaperone HSP90. Molecular Cell, 39(2), 282e291. Jagtap, U. B., Gurav, R. G., & Bapat, V. A. (2011). Role of RNA interference in plant improvement. Naturwissenschaften, 98(6), 473e492. 210 Plant Small RNA in Food Crops Jiang, C. J., Shimono, M., Maeda, S., Inoue, H., Mori, M., Hasegawa, M., … Takatsuji, H. (2009). Suppression of the rice fatty-acid desaturase gene OsSSI2 enhances resistance to blast and leaf blight diseases in rice. Molecular plant-microbe interactions, 22(7), 820e829. Jiao, J., & Peng, D. (2018). Wheat microRNA1023 suppresses invasion of Fusarium graminearum via targeting and silencing FGSG_03101. Journal of Plant Interactions, 13(1), 514e521. Joga, M. R., Zotti, M. J., Smagghe, G., & Christiaens, O. (2016). RNAi efficiency, systemic properties, and novel delivery methods for pest insect control: What we know so far. Frontiers in Physiology, 7, 553. Jones-Rhoades, M. W., & Bartel, D. P. (2004). Computational identification of plant microRNAs and their targets, including a stress-induced miRNA. Molecular Cell, 14(6), 787e799. Jørgensen, K., Bak, S., Busk, P. K., Sørensen, C., Olsen, C. E., Puonti-Kaerlas, J., & Møller, B. L. (2005). Cassava plants with a depleted cyanogenic glucoside content in leaves and tubers. Distribution of cyanogenic glucosides, their site of synthesis and transport, and blockage of the biosynthesis by RNA interference technology. Plant Physiology, 139(1), 363e374. Kaja, E., Szczesniak, M. W., Jensen, P. J., Axtell, M. J., McNellis, T., & Makałowska, I. (2015). Identification of apple miRNAs and their potential role in fire blight resistance. Tree Genetics & Genomes, 11(1), 812. Karlova, R., van Haarst, J. C., Maliepaard, C., van de Geest, H., Bovy, A. G., Lammers, M., … de Maagd, R. A. (2013). Identification of microRNA targets in tomato fruit development using high-throughput sequencing and degradome analysis. Journal of Experimental Botany, 64(7), 1863e1878. Khalid, A., Zhang, Q., Yasir, M., & Li, F. (2017). Small RNA based genetic engineering for plant viral resistance: Application in crop protection. Frontiers in Microbiology, 8, 43. Khraiwesh, B., Zhu, J. K., & Zhu, J. (2012). Role of miRNAs and siRNAs in biotic and abiotic stress responses of plants. Biochimica et Biophysica Acta (BBA)-Gene Regulatory Mechanisms, 1819(2), 137e148. Kim, Y. K., & Kim, V. N. (2007). Processing of intronic microRNAs. The EMBO Journal, 26(3), 775e783. Kozomara, A., Birgaoanu, M., & Griffiths-Jones, S. (2019). miRBase: From microRNA sequences to function. Nucleic Acids Research, 47(D1), D155eD162. Krubphachaya, P., Juricek, M., & Kertbundit, S. (2007). Induction of RNA-mediated resistance to papaya ringspot virus type W. BMB Reports, 40(3), 404e411. Kusaba, M., Miyahara, K., Iida, S., Fukuoka, H., Takano, T., Sassa, H., … Nishio, T. (2003). Low glutelin content1: A dominant mutation that suppresses the glutelin multigene family via RNA silencing in rice. The Plant Cell Online, 15(6), 1455e1467. Lee, R. C., Feinbaum, R. L., & Ambros, V. (1993). The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell, 75(5), 843e854. Liu, Q., Yan, S., Yang, T., Zhang, S., Chen, Y. Q., & Liu, B. (2017). Small RNAs in regulating temperature stress response in plants. Journal of Integrative Plant Biology, 59(11), 774e791. Li, F., Vallabhaneni, R., Yu, J., Rocheford, T., & Wurtzel, E. T. (2008). The maize phytoene synthase gene family: Overlapping roles for carotenogenesis in endosperm, photomorphogenesis, and thermal stress tolerance. Plant Physiology, 147(3), 1334e1346. Li, X., Wang, X., Zhang, S., Liu, D., Duan, Y., & Dong, W. (2012). Identification of soybean microRNAs involved in soybean cyst nematode infection by deep sequencing. PLoS One, 7(6), e39650. López-García, F., Andreu-García, G., Blasco, J., Aleixos, N., & Valiente, J. M. (2010). Automatic detection of skin defects in citrus fruits using a multivariate image analysis approach. Computers and Electronics in Agriculture, 71(2), 189e197. RNAi based approaches for abiotic and biotic stresses tolerance of crops 211 Marin, E., Jouannet, V., Herz, A., Lokerse, A. S., Weijers, D., Vaucheret, H., … Maizel, A. (2010). miR390, Arabidopsis TAS3 tasiRNAs, and their AUXIN RESPONSE FACTOR targets define an autoregulatory network quantitatively regulating lateral root growth. The Plant Cell Online, 22(4), 1104e1117. Martínez de Alba, A. E., Moreno, A. B., Gabriel, M., Mallory, A. C., Christ, A., Bounon, R., … Maizel, A. (2015). In plants, decapping prevents RDR6-dependent production of small interfering RNAs from endogenous mRNAs. Nucleic Acids Research, 43(5), 2902e2913. McGarry, R. C., & Ayre, B. G. (2012). Manipulating plant architecture with members of the CETS gene family. Plant Science, 188, 71e81. Megraw, M., Baev, V., Rusinov, V., Jensen, S. T., Kalantidis, K., & Hatzigeorgiou, A. G. (2006). MicroRNA promoter element discovery in Arabidopsis. RNA, 12(9), 1612e1619. Meli, V. S., Ghosh, S., Prabha, T. N., Chakraborty, N., Chakraborty, S., & Datta, A. (2010). Enhancement of fruit shelf life by suppressing N-glycan processing enzymes. Proceedings of the National Academy of Sciences, 107(6), 2413e2418. Missiou, A., Kalantidis, K., Boutla, A., Tzortzakaki, S., Tabler, M., & Tsagris, M. (2004). Generation of transgenic potato plants highly resistant to potato virus Y (PVY) through RNA silencing. Molecular Breeding, 14(2), 185e197. Mittler, R., & Blumwald, E. (2010). Genetic engineering for modern agriculture: Challenges and perspectives. Annual Review of Plant Biology, 61, 443e462. Mohr, C., Cao, H. X., & Humbeck, K. (2020). Future scope of small RNA technology in crop science. In Plant small RNA (pp. 567e585). Academic Press. Molesini, B., Pii, Y., & Pandolfini, T. (2012). Fruit improvement using intragenesis and artificial microRNA. Trends in Biotechnology, 30(2), 80e88. Muhammad, T., Zhang, F., Zhang, Y., & Liang, Y. (2019). RNA interference: A natural immune system of plants to counteract biotic stressors. Cells, 8(1), 38. Niggeweg, R., Michael, A. J., & Martin, C. (2004). Engineering plants with increased levels of the antioxidant chlorogenic acid. Nature Biotechnology, 22(6), 746e754. Ni, Z., Hu, Z., Jiang, Q., & Zhang, H. (2013). GmNFYA3, a target gene of miR169, is a positive regulator of plant tolerance to drought stress. Plant Molecular Biology, 82(1e2), 113e129. Nizampatnam, N. R., & Kumar, V. D. (2011). Intron hairpin and transitive RNAi mediated silencing of orfH522 transcripts restores male fertility in transgenic male sterile tobacco plants expressing orfH522. Plant Molecular Biology, 76(6), 557e573. Obembe, O. O., Popoola, J. O., Leelavathi, S., & Reddy, S. V. (2011). Advances in plant molecular farming. Biotechnology Advances, 29(2), 210e222. Opperman, C. H., Bird, D. M., Williamson, V. M., Rokhsar, D. S., Burke, M., Cohn, J., … Windham, E. (2008). Sequence and genetic map of Meloidogyne hapla: A compact nematode genome for plant parasitism. Proceedings of the National Academy of Sciences, 105(39), 14802e14807. Pandolfini, T. (2009). Seedless fruit production by hormonal regulation of fruit set. Nutrients, 1(2), 168e177. Pant, B. D., Musialak-Lange, M., Nuc, P., May, P., Buhtz, A., Kehr, J., … Scheible, W. R. (2009). Identification of nutrient-responsive Arabidopsis and rapeseed microRNAs by comprehensive real-time polymerase chain reaction profiling and small RNA sequencing. Plant Physiology, 150(3), 1541e1555. Patanun, O., Lertpanyasampatha, M., Sojikul, P., Viboonjun, U., & Narangajavana, J. (2013). Computational identification of microRNAs and their targets in cassava (Manihot esculenta Crantz.). Molecular Biotechnology, 53(3), 257e269. Patil, B. L., Ogwok, E., Wagaba, H., Mohammed, I. U., Yadav, J. S., Bagewadi, B., … Fauquet, C. M. (2011). RNAi-mediated resistance to diverse isolates belonging to two virus species involved in Cassava brown streak disease. Molecular Plant Pathology, 12(1), 31e41. 212 Plant Small RNA in Food Crops Pattanayak, D., Solanke, A. U., & Kumar, P. A. (2013). Plant RNA interference pathways: Diversity in function, similarity in action. Plant Molecular Biology Reporter, 31(3), 493e506. Peltier, A. J., Hatfield, R. D., & Grau, C. R. (2009). Soybean stem lignin concentration relates to resistance to Sclerotinia sclerotiorum. Plant Disease, 93(2), 149e154. Pooggin, M., Shivaprasad, P. V., Veluthambi, K., & Hohn, T. (2003). RNAi targeting of DNA virus in plants. Nature Biotechnology, 21(2), 131e132. Pradeep, K., Satya, V. K., Selvapriya, M., Vijayasamundeeswari, A., Ladhalakshmi, D., Paranidharan, V., … Velazhahan, R. (2012). Engineering resistance against Tobacco streak virus (TSV) in sunflower and tobacco using RNA interference. Biologia Plantarum, 56(4), 735e741. Qin, T., Zhao, H., Cui, P., Albesher, N., & Xiong, L. (2017). A nucleus-localized long non-coding RNA enhances drought and salt stress tolerance. Plant Physiology, 175(3), 1321e1336. Rabuma, T., Gupta, O. P., & Chhokar, V. (2022). Genome-wide comprehensive analysis of miRNAs and their target genes expressed in resistant and susceptible Capsicum annuum genotypes during Phytophthora capsici infection. Molecular Genetics & Genomics. https:// doi.org/10.1007/s00438-022-01979-y Rathore, K. S., Sundaram, S., Sunilkumar, G., Campbell, L. M., Puckhaber, L., Marcel, S., … Wedegaertner, T. C. (2012). Ultra-low gossypol cottonseed: Generational stability of the seed-specific, RNAi-mediated phenotype and resumption of terpenoid profile following seed germination. Plant Biotechnology Journal, 10(2), 174e183. Rubinelli, P. M., Chuck, G., Li, X., & Meilan, R. (2013). Constitutive expression of the Corngrass1 microRNA in poplar affects plant architecture and stem lignin content and composition. Biomass and Bioenergy, 54, 312e321. Sandhu, A. P. S., Abdelnoor, R. V., & Mackenzie, S. A. (2007). Transgenic induction of mitochondrial rearrangements for cytoplasmic male sterility in crop plants. Proceedings of the National Academy of Sciences, 104(6), 1766e1770. Schwind, N., Zwiebel, M., Itaya, A., Ding, B., WANG, M. B., Krczal, G., & Wassenegger, M. (2009). RNAi-mediated resistance to Potato spindle tuber viroid in transgenic tomato expressing a viroid hairpin RNA construct. Molecular Plant Pathology, 10(4), 459e469.
Se
cic, E., & Kogel, K. H. (2021). Requirements for fungal uptake of dsRNA and gene silencing in RNAi-based crop protection strategies. Current Opinion in Biotechnology, 70, 136e142. Shanker, A. K., & Maheswari, M. (2017). Small RNA and drought tolerance in crop plants. Indian Journal of Plant Physiology, 22(4), 422e433. Sharma, S. K., Gupta, O. P., Pathaw, N., Sharma, D., Maibam, A., Sharma, P., Sanasam, J., Karkute, S. G., Kumar, S., & Bhattacharjee, B. (2021). CRISPR-Cas-Led revolution in diagnosis and management of emerging plant viruses: New avenues toward food and nutritional security. Frontiers in Nutrition, 8, 751512. https://doi.org/10.3389/ fnut.2021.751512 Simón-Mateo, C., & García, J. A. (2011). Antiviral strategies in plants based on RNA silencing. Biochimica et Biophysica Acta (BBA)-Gene Regulatory Mechanisms, 1809(11e12), 722e731. Sindhu, A. S., Maier, T. R., Mitchum, M. G., Hussey, R. S., Davis, E. L., & Baum, T. J. (2009). Effective and specific in planta RNAi in cyst nematodes: Expression interference of four parasitism genes reduces parasitic success. Journal of Experimental Botany, 60(1), 315e324. Song, Q. X., Liu, Y. F., Hu, X. Y., Zhang, W. K., Ma, B., Chen, S. Y., & Zhang, J. S. (2011). Identification of miRNAs and their target genes in developing soybean seeds by deep sequencing. BMC Plant Biology, 11(1), 1e16. RNAi based approaches for abiotic and biotic stresses tolerance of crops 213 Spencer, P., Ludolph, A., Dwivedi, M. P., Roy, D., Hugon, J., & Schaumburg, H. H. (1986). Lathyrism: Evidence for role of the neuroexcitatory aminoacid BOAA. The Lancet, 328(8515), 1066e1067. Stepien, A., Knop, K., Dolata, J., Taube, M., Bajczyk, M., Barciszewska-Pacak, M., … Szweykowska-Kulinska, Z. (2017). Posttranscriptional coordination of splicing and miRNA biogenesis in plants. Wiley Interdisciplinary Reviews: RNA, 8(3), e1403. Sunilkumar, G., Campbell, L. M., Puckhaber, L., Stipanovic, R. D., & Rathore, K. S. (2006). Engineering cottonseed for use in human nutrition by tissue-specific reduction of toxic gossypol. Proceedings of the National Academy of Sciences, 103(48), 18054e18059. Sun, L., Sun, G., Shi, C., & Sun, D. (2018). Transcriptome analysis reveals new microRNAs-mediated pathway involved in anther development in male sterile wheat. BMC Genomics, 19(1), 1e17. Swarts, D. C., Makarova, K., Wang, Y., Nakanishi, K., Ketting, R. F., Koonin, E. V., … Van Der Oost, J. (2014). The evolutionary journey of Argonaute proteins. Nature Structural & Molecular Biology, 21(9), 743e753. Tester, M., & Langridge, P. (2010). Breeding technologies to increase crop production in a changing world. Science, 327(5967), 818e822. Treiber, T., Treiber, N., & Meister, G. (2019). Regulation of microRNA biogenesis and its crosstalk with other cellular pathways. Nature Reviews Molecular Cell Biology, 20(1), 5e20. Trindade, I., Capitão, C., Dalmay, T., Fevereiro, M. P., & Dos Santos, D. M. (2010). miR398 and miR408 are up-regulated in response to water deficit in Medicago truncatula. Planta, 231(3), 705e716. Tyagi, H., Rajasubramaniam, S., Rajam, M. V., & Dasgupta, I. (2008). RNA-interference in rice against Rice tungro bacilliform virus results in its decreased accumulation in inoculated rice plants. Transgenic Research, 17(5), 897e904. VALDÉS-LÓPEZ, O., ARENAS-HUERTERO, C., Ramirez, M., Girard, L., Sanchez, F., Vance, C. P., … Hernandez, G. (2008). Essential role of MYB transcription factor: PvPHR1 and microRNA: PvmiR399 in phosphorus-deficiency signalling in common bean roots. Plant, Cell and Environment, 31(12), 1834e1843. Vanitharani, R., Chellappan, P., & Fauquet, C. M. (2003). Short interfering RNA-mediated interference of gene expression and viral DNA accumulation in cultured plant cells. Proceedings of the National Academy of Sciences, 100(16), 9632e9636. Varoquaux, F., Blanvillain, R., Delseny, M., & Gallois, P. (2000). Less is better: New approaches for seedless fruit production. Trends in Biotechnology, 18(6), 233e242. Vriezen, W. H., Feron, R., Maretto, F., Keijman, J., & Mariani, C. (2008). Changes in tomato ovary transcriptome demonstrate complex hormonal regulation of fruit set. New Phytologist, 177(1), 60e76. Wang, Y., Beaith, M., Chalifoux, M., Ying, J., Uchacz, T., Sarvas, C., … Huang, Y. (2009). Shoot-specific down-regulation of protein farnesyltransferase (a-subunit) for yield protection against drought in canola. Molecular Plant, 2(1), 191e200. Wang, T., Chen, L., Zhao, M., Tian, Q., & Zhang, W. H. (2011). Identification of drought-responsive microRNAs in Medicago truncatula by genome-wide highthroughput sequencing. BMC Genomics, 12(1), 1e11. Wang, Y., Sun, F., Cao, H., Peng, H., Ni, Z., Sun, Q., & Yao, Y. (2012). TamiR159 directed wheat TaGAMYB cleavage and its involvement in anther development and heat response. PLoS One, 7(11), e48445. White, P. J., & Broadley, M. R. (2009). Biofortification of crops with seven mineral elements often lacking in human dietseiron, zinc, copper, calcium, magnesium, selenium and iodine. New Phytologist, 182(1), 49e84. WHO. (2020). https://www.who.int/news-room/fact-sheets/detail/malnutrition. 214 Plant Small RNA in Food Crops Xin, M., Wang, Y., Yao, Y., Xie, C., Peng, H., Ni, Z., & Sun, Q. (2010). Diverse set of microRNAs are responsive to powdery mildew infection and heat stress in wheat (Triticum aestivum L.). BMC Plant Biology, 10(1), 1e11. Xiong, A. S., Yao, Q. H., Peng, R. H., Li, X., Han, P. L., & Fan, H. Q. (2005). Different effects on ACC oxidase gene silencing triggered by RNA interference in transgenic tomato. Plant Cell Reports, 23(9), 639e646. Xu, Y., Huang, L., Fu, S., Wu, J., & Zhou, X. (2012). Population diversity of rice stripe virusderived siRNAs in three different hosts and RNAi-based antiviral immunity in Laodelphgax striatellus. Yang, Q., Chen, Z. Z., Zhou, X. F., Yin, H. B., Li, X., Xin, X. F., … Gong, Z. (2009). Overexpression of SOS (Salt Overly Sensitive) genes increases salt tolerance in transgenic Arabidopsis. Molecular Plant, 2(1), 22e31. Yang, C., Li, D., Mao, D., Liu, X. U. E., Ji, C., Li, X., … Zhu, L. (2013). Overexpression of micro RNA 319 impacts leaf morphogenesis and leads to enhanced cold tolerance in rice (Oryza sativa L.). Plant, Cell and Environment, 36(12), 2207e2218. Yara, A., Yaeno, T., Hasegawa, M., Seto, H., Montillet, J. L., Kusumi, K., … Iba, K. (2007). Disease resistance against Magnaporthe grisea is enhanced in transgenic rice with suppression of u-3 fatty acid desaturases. Plant and Cell Physiology, 48(9), 1263e1274. Yasumoto, S., Sawai, S., Lee, H. J., Mizutani, M., Saito, K., Umemoto, N., & Muranaka, T. (2020). Targeted genome editing in tetraploid potato through transient TALEN expression by Agrobacterium infection. Plant Biotechnology, 20e0525. Yu, Y., Jia, T., & Chen, X. (2017). The ‘how’ and ‘where’ of plant micro RNA s. New Phytologist, 216(4), 1002e1017. Zanca, A. S., Vicentini, R., Ortiz-Morea, F. A., Del Bem, L. E., da Silva, M. J., Vincentz, M., & Nogueira, F. T. (2010). Identification and expression analysis of microRNAs and targets in the biofuel crop sugarcane. BMC Plant Biology, 10(1), 1e13. Zhang, C., Yin, X., Gao, K., Ge, Y., & Cheng, W. (2013a). Non-protein thiols and glutathione S-transferase alleviate Cd stress and reduce root-to-shoot translocation of Cd in rice. Journal of Plant Nutrition and Soil Science, 176(4), 626e633. Zhang, B., You, C., Zhang, Y., Zeng, L., Hu, J., Zhao, M., & Chen, X. (2020). Linking key steps of microRNA biogenesis by TREX-2 and the nuclear pore complex in Arabidopsis. Nature plants, 6(8), 957e969. Zhang, Y. C., Yu, Y., Wang, C. Y., Li, Z. Y., Liu, Q., Xu, J., … Chen, Y. Q. (2013b). Overexpression of microRNA OsmiR397 improves rice yield by increasing grain size and promoting panicle branching. Nature Biotechnology, 31(9), 848. Zhang, X., Zou, Z., Zhang, J., Zhang, Y., Han, Q., Hu, T., … Ye, Z. (2011). Overexpression of sly-miR156a in tomato results in multiple vegetative and reproductive trait alterations and partial phenocopy of the sft mutant. FEBS Letters, 585(2), 435e439. Zhao, B., Liang, R., Ge, L., Li, W., Xiao, H., Lin, H., … Jin, Y. (2007). Identification of drought-induced microRNAs in rice. Biochemical and Biophysical Research Communications, 354(2), 585e590. Zhou, Y., Yuan, Y., Yuan, F., Wang, M., Zhong, H., Gu, M., & Liang, G. (2012). RNAidirected down-regulation of RSV results in increased resistance in rice (Oryza sativa L.). Biotechnology Letters, 34(5), 965e972. Zhu, D., & Deng, X. W. (2012). A non-coding RNA locus mediates environmentconditioned male sterility in rice. Cell Research, 22(5), 791e792. CHAPTER 8 Regulation of morphogenesis and development in food crops: role of small RNA Jayanti Jodder Presidency University, Institute of Health Sciences, Kolkata, India 1. Introduction Small RNAs (sRNAs) are involved in the regulation of development and morphogenesis in plants and can play a critical role in the improvement of crop plants leading to economic success and as an efficient tool for genetic engineering (Chen et al., 2018). Plant development, growth, and morphogenesis, regulated by small RNA (sRNA), were first reported in Arabidopsis in 2002 (Llave et al., 2002; Park et al., 2002; Reinhart et al., 2002). In plants, two types of sRNAs play major regulatory functions, microRNA (miRNA) and small interfering RNA (siRNA). Both of them are involved in regulating several biological processes, development pathways, and stress responses. In cells, miRNAs are originated from the transcription of genes called MIR genes. Early biogenesis of miRNA occurs in the nucleus, they are small (20e24 nucleotide long) non-coding RNAs (Jones-Rhoades et al., 2006). MIR genes are present in clusters in the whole genome of an organism and transcribed into long polycistronic RNAs (Bartel, 2004; Jones-Rhoades et al., 2006), by RNA polymerase II (Pol II), into primary miRNAs, having 50 cap of modified nucleotide and 30 polyadenylated tail (Pantaleo et al., 2010). DICER-LIKE enzyme (DCL1) which belongs to a class of RNase III-like enzymes and other enzymes like hyponastic leaves 1(HYL1), serrate (SE) cleave the primary miRNAs to precursor miRNAs forming a hairpin loop structure which is then cleaved into miRNA:miRNA* duplex by DCL1 (Bartel, 2004; Papp et al., 2003; Peláez et al., 2012). After being methylated by HUA enhancer 1 (HEN1) an S-adenosyl-L-methionine-dependent RNA methyltransferase at their 30 end, the duplexes are transported to the cytoplasm by HASTY, plant homolog ofEXPORTIN-5 (Park et al., 2005; Plant Small RNA in Food Crops ISBN 978-0-323-91722-3 https://doi.org/10.1016/B978-0-323-91722-3.00011-7 © 2023 Elsevier Inc. All rights reserved. 215 216 Plant Small RNA in Food Crops Peláez et al., 2012; Sun et al., 2012). These double-stranded miRNAs are directed to an RNA-induced silencing complex (RISC), which carries ARGONAUTE (AGO) specifically AGO1 protein, to unwind the strands (Arribas-Hernández et al., 2016; Iki, 2017). Degeneration of a single strand is achieved by sRNA degrading nuclease in exosome and the other strand is put into miRISC containing AGO (Sun et al., 2012). However, the above model has been revised by several experimental evidence in recent reports (Bologna et al., 2018; Zhang et al., 2020). Bologna et al. (2018) have shown that upon maturation and methylation inside the nucleus, mature miRNAs are loaded into AGO1, and then the AGO1:miRNA complex is exported to the cytosol via CRM1 (EXPORTINH1)/NES export pathway (Bologna et al., 2018). According to Zhang et al. (2020), the MIR gene transcription, pri and pre-miRNA processing, and nuclear export of mature-miRNA are promoted by TREX-2 complex which interacts and colocalizes with RNA pol II to promote the MIR genes transcription and also interacts with SE to promote the processing in the nucleoplasm. At the nuclear envelope THP1, a component of TREX-2 interacts and colocalizes with the nucleoporin protein NUP1, to export the miRNA: AGO1 complex into the cytosol. In our recent review the complex mechanism of miRNA processing has been discussed in detail (Jodder, 2021). The mature miRNA with the miRISC then approaches the target in a sequence-dependent manner, highly homologous strands are cleaved and the strands sharing less complementarity with the miRNAs are silenced (Sun et al., 2012). siRNAs are 50 phosphorylated dsRNA molecules of 21e25 nucleotide length with overhangs of nearly two nucleotides at 30 end formed after cleavage by dicer enzyme from a long dsRNA and target homologous sequence for gene silencing (Bernstein et al., 2001; Elbashir et al., 2001; Fire et al., 1998; Hamilton & Baulcombe, 1999; Tuschl, 2001). After getting loaded into the siRISC, the sense strand of siRNA having the same sequence as the target gets degenerated. The antisense siRNA strand gets assimilated into siRISC with AGO protein which further carries out the process of mRNA targeting and silencing, this complex could be activated multiple times for gene repression. In Arabidopsis, ten different types of AGO proteins (Henderson et al., 2006) and four different types of Dicerlike enzymes have been reported which are required for small RNA biogenesis (Henderson et al., 2006; Vaucheret, 2008). Regulation of morphogenesis and development in food crops: role of small RNA 217 RNA interference (RNAi) also known as post-transcriptional gene silencing (PTGS) is a biological phenomenon in which small doublestranded RNAs are involved in the suppression of target genes’ expression in a sequence-specific manner. The introduction of RNAi has been proved to be a useful strategy to introduce desired characteristics in plants by regulating major genes essential in physiological and morphological activities (Bej & Basak, 2014; Djami-Tchatchou & Dubery, 2015; SananMishra et al., 2009). miRNA sequences are highly evolutionarily conserved in nature (Cuperus et al., 2011; Sun et al., 2012). Numerous MIR genes and corresponding miRNAs have been identified and collected in the database named miRbase. For example, nearly 592 miRNA gene sequences were identified in rice, 116 in wheat, 321 in maize, 69 in barley, 413 in apple, etc. and all of them have been found to be involved in regulating development, growth, biotic and abiotic stresses (http://www.mirbase.org; Li et al., 2012; Zhang et al., 2009). Here in this chapter, the role of small RNA in regulating the morphogenesis and development of food crops has been discussed in detail. 2. Modes of sRNA transport to cell sRNA molecules proficient in obstructing gene expression are highly flexible due to their heterogeneity and thus can regulate nearly every gene in an organism (Djami-Tchatchou & Dubery, 2015; Lelandais-Brière et al., 2010; Pantaleo et al., 2010; Sanan-Mishra et al., 2009; Sun, 2012). sRNA molecules mainly follow two distinct modes of transport. Shortand long-range movements of sRNA molecules are cell to cell and systematic movements respectively (Melnyk et al., 2011). The movement to local cells by plasmodesmata is symplastic (Lough & Lucas, 2006). Systemic movement of sRNAs takes place by bulk flow, which requires the involvement of vascular tissues in which the leaf acts as a source and immature plant organs as a sink (Buhtz et al., 2008; Yoo et al., 2004). Phloem sap and grafted plants have been seen to have RNA silencing signals containing sRNA molecules (Brosnan et al., 2007; Dunoyer et al., 2010; Molnar et al., 2010; Palauqui et al., 1997; Schwach et al., 2005), Rapeseed and Pumpkin were shown to have sRNA molecule in their phloem sap, which were fluoresce labeled to show their mode of transport which was found to be through the phloem (Buhtz et al., 2008) and pumpkin (Yoo et al., 2004). 218 Plant Small RNA in Food Crops 3. Role of small RNAs in development, morphogenesis, and yield of the plant From seed germination to plant maturation the whole life cycle of the plant has mainly two phases, the vegetative phase, and the reproductive phase. Vegetative growth starts from the apical meristem and gives rise to aboveground vegetative parts like all the leaves and branches etc. After the completion of vegetative growth, the apical meristem undergoes some alteration and changes to the inflorescence meristem and thus the plants enter into their reproductive phase. Initially, the inflorescence meristem gives rise to small leaves and then floral meristems. From the floral meristems, the flowers start to develop, and eventually, fruits and seeds are produced. Behind all these growth and developmental phases, lots of genetic reprogramming events are involved. Alteration of phase-specific gene expressions is crucial for the success in each and every developmental event. A literature survey depicts that almost all the developmental stages and transitions are regulated by many miRNAs. For example, miR319 regulates TEOSINTE BRANCHED1 CYCLOIDEA AND PCF FAMILY (TCP2) mRNA, this transcript cleavage controls leaf morphogenesis (Palatnik et al., 2003). The development of compound leaf in tomatoes is also regulated by the action of miR319on LANCEOLATE (Ori et al., 2007). Overexpression of miR156 at appropriate levels in switchgrass can greatly enhance biomass yield by increasing the tiller number. miR156 can target SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes that act downstream to the gene family that controls the transition from vegetative to reproductive phase change. Thus, miR156 mediated downregulation of SPL genes resulted in a longer vegetative period and also induce the increase in tiller number resulting in an increase in biomass in rice, Arabidopsis, and maize (Chuck et al., 2007; Schwab et al., 2005; Xie et al., 2006). These kinds of miRNA-mediated regulations have been utilized in RNAi technology to improve basic architecture in plants including height, inflorescence, flowering, and growth (Kamthan et al., 2015; Wilson & Doudna, 2013). Similarly, Rice has been manipulated to produce grains with better yield and quality (Jiao et al., 2010; Miura et al., 2010; Springer, 2010; Wang et al., 2012). During the non-fertile period, rice plants with a knockout of OsDWARF4 showed higher adaptation leading to better yield (Feldmann, 2006). Down-regulation of enzyme GA20-oxidase (OsGA20ox2) resulted in rice plants with higher seed production, bigger panicle length, and increased weight of the grains from the Regulation of morphogenesis and development in food crops: role of small RNA 219 QX1 variety of rice (Qiao et al., 2007). A higher expression of OsSPL14 (Squamosa promoter binding protein-like 14) and OsSPL16 (inducer of cell proliferation) gene by miR156 resulted in higher grain production in rice plants (Guo et al., 2013; Jiao et al., 2010; Miura et al., 2010). In maize miR156, miR166, miR167, and miR169 were the five dominating MIR genes that were identified to be responsible for development and growth. Their target genes were supposed to be squamosa promoter-binding protein, auxin response factor, HD-ZIP TF, CCAATbinding factor, and HAP-2-like proteins respectively (Mica et al., 2006). The development of endosperm in maize is regulated by 95 conserved MIR genes belonging to 20 families such as miR156a, miR160a, miR165e, miR164a, miR167d, miR168, miR169a, miR393a, etc. (Gu et al., 2013). Genes for early morphological and physiological changes were set to be regulated by miR528a, miR167a, and miR160b which are auxin response regulators (Gu et al., 2013). Nearly 72 genes leading to 117 transcripts were targeted by 62 miRNAs such as miR159a, miR164a, miR171c, miR398a, miR408a, miR528a, etc. to play role in early development (Li et al., 2015). In Arabidopsis development of stomata has been found to be regulated by various miRNA (Kutter et al., 2007). Some of the miRNAs and their target genes regulating different developmental functions in food crops are enlisted in Table 8.1. 3.1 miRNAs are involved in vegetative to floral shift After the completion of vegetative growth, the leaf meristem undergoes some changes and transforms into a floral meristem. From this floral meristem development of flowers initiate. This phase-changing event and the eventual floral organ developments are regulated by different small RNAs. miR164 targets and maintains the accumulation of CUPSHAPEPCOTYLEDON (CUC) transcripts, CUC1 and CUC2 which in turn regulates leaf morphogenesis and flower development (Baker et al., 2005; Laufs et al., 2004; Mallory et al., 2004; Nikovics et al., 2006; Sieber et al., 2007). Another gene miR172 targets mRNA of Glossy 15 in maize (Lauter et al., 2005), and APETALA2 (AP2) gene (floral organ identity gene) in Arabidopsis leads to the transition from the vegetative phase to the flowering phase specifying all four floral whorls, stamen, pistil, sepals and petals (Aukerman & Sakai, 2003; Chen, 2004; Jofuku et al., 1994). Overexpression of osmiR393 showed higher tillering and early flowering (Jiao et al., 2010). Table 8.1 Role of some miRNA and their target genes in the development and morphogenesis of food crops. Name of miRNA Target genes Food crops Function References miR156 SPL Zea mays Floral development Oryza sativa Plant architecture, floral development Solanum lycopersicum Ovary and fruit development Grapes Berry development and ripening Senescence Liu et al. (2014), Chuck et al. (2007), Hong & Jackson (2015) Jiao et al. (2010), Wang et al. (2015), Xie et al. (2006), Hong & Jackson (2015) Silva et al. (2014), Zhang et al. (2011), Hong & Jackson (2015) Cui et al. (2018) miR159 MYB Zea mays miR172 AP2 miR319 miR164 LANCEOLATE (TCP homolog) NAC Glycine max Phaseolus vulgaris Solanum lycopersicum Zea mays miR396 GRF Zea mays miR393 TIR/AFB Oryza sativa TIR homolog Cucumber miR397a LAC Pear fruit miR529a SPL Oryza sativa Nodule formation Nodule formation Leaf development Lateral root development Grain development Flowering and tillering Fruit/seed set development and leaf morphogenesis Stone cell development Plant height, tiller number, panicle architecture and grain size Wu et al. (2016) Yan et al. (2013) Nova-Franco et al. (2015) Ori et al. (2007) Li et al. (2012) Zhang et al. (2015) Xia et al. (2012) Xu et al. (2017) Xue et al. (2019) Yan et al. (2021) Regulation of morphogenesis and development in food crops: role of small RNA 221 3.2 Small RNA in fruit development and quality improvement Tomato is widely cultivated worldwide as a fruit crop with antioxidants, minerals, and vitamins (Rajam et al., 2006), apple is economical fruit with high levels of phenols and flavonoids which reduces the risk of many chronic diseases (Boyer & Liu, 2004). Spoilage of crops and fruits after harvesting due to ripening leads to huge monetary loss. RNAi could be used to delay fruit ripening. Climacteric fruits like tomatoes start ripening after the accumulation of ethylene. miR1917, a negative regulator of ethylene response, was found to play an essential role during tomato fruit ripening (Moxon et al., 2008). Apart from phase transition, miR172 is also involved in fruit maturation, it targets AP2, which suppresses fruit ripening in tomato fruit, and mutant AP2 (SlAP2a) leads to overly ripped uneven tomato fruit (Karlova et al., 2011). miR172-AP2 follows different pathways for fruit ripening in different types of fruits. Parthenocarpy is a process in which seedless fruits develop directly from the ovary without fertilization. Seedless tomato fruits were produced after the upregulation of miR172 (J.-L. Yao et al., 2016). A decrease in fruit size was observed due to the overproduction of miR172, resulting in the silencing of the AP2 gene in apples (Yao et al., 2015). As miR172 is also involved in floral organ development, trans-engineering of miR172 for the development of fruits can also negatively or positively affect floral development. RNAi has been widely utilized in tomato plants in order to make them transgenic to produce enhanced levels of flavonoids and carotenoids. DET1 gene is a photomorphogenesis regulatory gene downregulation that results in tomato fruit with increased levels of carotenoids and flavonoids (Davuluri et al., 2005). RNAi has been utilized to increase zeaxanthin, violaxanthin, b-carotene, and lutein accumulation in rapeseed (Brassica napus) by repressing the activity of the lycopene epsilon cyclase (ε-CYC) gene (Yu et al., 2008). Increased seed size and panicle branching were observed when OsmiR397 was over-expressed with repression of L-ascorbate oxidase (OsLAC) (Zhang et al., 2013). 4. Small RNA regulating growth and development related hormone signaling Auxin and cytokinin are the major hormones that regulate the growth and development of plants. AUXIN RESPONSE FACTORS (ARFs) are transcription factors involved in auxin signaling pathways (Zouine et al., 2014). 222 Plant Small RNA in Food Crops There are some miRNAs that can target these ARF factors and thereby control the auxin signaling which in turn regulates various developmental pathways. miR160/167 are known to target different ARFs during auxin signaling. miR160 can target ARF10, ARF16, and ARF17 during root cap formation in Arabidopsis (Wang et al., 2005), it is also involved in the regulation of floral identity, leaf, and fruit development in tomatoes (Jones-Rhoades & Bartel, 2004). miR160 resistant ARF10/16/17 results in abnormal root tissue (Liu et al., 2007; Mallory et al., 2005; Wang et al., 2005). miR167 regulates ARF6, and ARF8 to develop ovule and anther in tomatoes, whereas the resistant varieties show abnormal growth in these floral organs (Wu et al., 2006). Auxin perception is also regulated by miRNA. miR393 can target the SCFTIR-1 complex protein, Auxin receptors transport inhibitor response1 (TIR-1), and auxin F-box protein 2 (AFB2) during seed germination and seedling development in rice (Chen et al., 2015; Guo et al., 2016; Windels et al., 2014; Wójcik & Gaj, 2016). NAC transcription factors are the target of miR164 (Fang et al., 2014; Feng et al., 2014). During lateral root development, this miR164-mediated regulation of NAC plays a crucial role (Guo et al., 2005). Lateral root development is also regulated by a network of ARFs, miR390, and TAS3-derived trans-acting short interfering RNAs (tasiRNAs) (Marin et al., 2010). Development in potatoes is also regulated by the action of miR390 on the stCDPK1 gene (Santin et al., 2017). In the model plant Arabidopsis, miR847 has been found to regulate the auxin repressor gene IAA28 mRNA during the proliferation of cells and lateral organ development (Wang & Guo, 2015). Cytokinins promote cell division in roots and shoots and induce the growth of buds. There are several small RNAs that can regulate the cytokinin pathway. Two siRNAs id4 and id65 can target an orthologue of cytokinin synthase, IPT3 in A. thaliana, were identified from the RNA library of Vitis vinifera (Carra et al., 2009). miR171h has been found to target the cytokinin-responsive GRAS family transcription factor, Nodulation Signaling Pathway (NSP2), involved in early nodulation in Medicago trancatula (Ariel et al., 2012). 5. Small RNA in disease resistance and stress tolerance in plants Apart from having major roles in development and morphogenesis, miRNAs play the crucial task of eliminating biotic stresses from crops. Pathogenic diseases are a major cause of crop damage, tons of crops are Regulation of morphogenesis and development in food crops: role of small RNA 223 spoiled leading to major economic losses. miRNA works in mitigating fungal, viral, and bacterial stresses in plants by regulating the expression of plant genes that are involved in disease resistance (Chen, 2009; Khraiwesh et al., 2012; Ruiz-Ferrer & Voinnet, 2009; Sunkar et al., 2012; Zhu & Helliwell, 2011). Five miRNAs, miR156, miR160, miR166, miR167, and miR169 have been characterized in maize which are involved in the growth, development, and stress tolerance (Mica et al., 2006). Fatty acids produced during infection play a vital role in stress resistance. Blocking of the genes involved in fatty acid synthesis during infection to increase stress tolerance in crop plants by RNAi is an emerging solution. Blast, a fungal disease in rice due to the attack of Magnaporthe grisea and bacterial disease, leaf blight due to Xanthomonas oryzae can be countered by the silencing of the OsSSI2 gene (Jiang et al., 2009). Omega-3 fatty acid genes OsFAD7 and OsFAD8 can also be silenced to increase the resistance of M. grisea in rice (Yara et al., 2007). Another example is the enhanced resistance of soybean from Sclerotinia sclerotiorum after targeting of lignin producing genes (Peltier et al., 2009) and wheat powdery mildew caused by fungus Blumeria graminis f. sp. tritici (Bgt) can also be limited by miRNA technology. 24 different miRNAs are also identified working on the same purpose (Xin et al., 2010). 6. Conclusion and future prospects Plants are the major source of nourishment, therapeutics, and shade for humans and animals, but as their demand is getting increased their yield due to man-made activities, pathogenic diseases and other abiotic stresses are getting decreased. There is a need for the development of methods to combat these challenges to increase yield, nutrition availability, and quality of crops. In mitigation of biotic stress due to bacteria, viruses, and fungus, in development, growth flowering, hormone signaling, and plant immunity small RNAs play a crucial role (Balmer & Mauch-Mani, 2013; DjamiTchatchou & Dubery, 2015; Li et al., 2010; Sun et al., 2012). RNAi has been proved to be an effective technology to produce improved agronomic traits in food crops. RNAi is being utilized by silencing and regulating the activities of major genes by targeting transcripts through miRNA/siRNA methods. Production of transgenic plants by editing and regulation of sRNA is emerging in recent times by upregulation or downregulation of miRNA 224 Plant Small RNA in Food Crops genes. These small non-coding RNAs are proving themselves to be the best tool to produce crops with resistant genes to avoid the pathogenic attack, improved agricultural value, and improved yield. Post-transcriptional gene regulation by use of miRNA has become the topic of research to manipulate genetic data of any crop plant (Kamthan et al., 2015). Constitutive overexpression and suppression of miRNA targets could become a novel strategy for quality improvement for plants (Yang et al., 2013). This seemingly practical in-vitro technology can give a tough time to researchers in the field. Though highly successful the genes getting targeted by miRNAs not only have multiple functions but also their over-expression, down-regulation, and knock-out can affect the growth of the plant leading to pleiotropic changes (Kamthan et al., 2015). miRNAs are evolutionarily highly preserved and this can be used to avoid these changes by manufacturing artificial miRNAs with transcript specify that can be synthesized to circumvent non-target gene binding, thereby having the least effect on the morphological and physiological role of major genes in plants (Schwab et al., 2010; Zhang et al., 2011). Genes homologous to the RNAi construct can also be silenced, which can lead to the silencing of genes responsible for growth, stress tolerance, and morphogenesis. Even though this strategy seems to be tough, proper knowledge, studies, and genuinely synthesized RNAi constructs implemented with the correct plan of action can reduce the possibility of unintentional damage due to similarity in the gene sequence. The use of small RNAs is important in the manipulation of food crops which could be a master plan in the agronomic world for a better future. References Ariel, F., Brault-Hernandez, M., Laffont, C., Huault, E., Brault, M., Plet, J., Moison, M., Blanchet, S., Ichanté, J. L., Chabaud, M., Carrere, S., Crespi, M., Chan, R. L., & Frugier, F. (2012). Two direct targets of cytokinin signaling regulate symbiotic nodulation in Medicago truncatula. The Plant Cell Online, 24(9), 3838e3852. https:// doi.org/10.1105/tpc.112.103267 Arribas-Hernández, L., Marchais, A., Poulsen, C., Haase, B., Hauptmann, J., Benes, V., Meister, G., & Brodersen, P. (2016). The slicer activity of ARGONAUTE1 is required specifically for the phasing, not production, of trans-acting short interfering RNAs in Arabidopsis. The Plant Cell Online, 28(7), 1563e1580. https://doi.org/10.1105/ tpc.16.00121 Aukerman, M. J., & Sakai, H. (2003). Regulation of flowering time and floral organ identity by a microRNA and its APETALA2-like target genes. The Plant Cell Online, 15(11), 2730e2741. https://doi.org/10.1105/tpc.016238 Regulation of morphogenesis and development in food crops: role of small RNA 225 Baker, C. C., Sieber, P., Wellmer, F., & Meyerowitz, E. M. (2005). The early extra petals1 mutant uncovers a role for microRNA miR164c in regulating petal number in Arabidopsis. Current Biology, 15(4), 303e315. https://doi.org/10.1016/j.cub.2005.02.017 Balmer, D., & Mauch-Mani, B. (2013). Small yet mighty e microRNAs in plant-microbe interactions. MicroRNA, 2(1), 73e80. https://doi.org/10.2174/2211536611302010008 Bartel, D. P. (2004). MicroRNAs: genomics, biogenesis, mechanism, and function. Cell, 116(2), 281e297. https://doi.org/10.1016/S0092-8674(04)00045-5 Bej, S., & Basak, J. (2014). MicroRNAs: the potential biomarkers in plant stress response. American Journal of Plant Sciences, 05(05), 748e759. https://doi.org/10.4236/ ajps.2014.55089 Bernstein, E., Caudy, A. A., Hammond, S. M., & Hannon, G. J. (2001). Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature, 409(6818), 363e366. https://doi.org/10.1038/35053110 Bologna, N. G., Iselin, R., Abriata, L. A., Sarazin, A., Pumplin, N., Jay, F., Grentzinger, T., Dal Peraro, M., & Voinnet, O. (2018). Nucleo-cytosolic shuttling of ARGONAUTE1 prompts a revised model of the plant microRNA pathway. Molecular Cell, 69(4). https:// doi.org/10.1016/j.molcel.2018.01.007, 709-719.e5. Boyer, J., & Liu, R. H. (2004). Apple phytochemicals and their health benefits. Nutrition Journal, 3, 1e45. https://doi.org/10.1186/1475-2891-3-1 Brosnan, C. A., Mitter, N., Christie, M., Smith, N. A., Waterhouse, P. M., & Carroll, B. J. (2007). Nuclear gene silencing directs reception of long-distance mRNA silencing in Arabidopsis. Proceedings of the National Academy of Sciences, 104(37), 14741e14746. https://doi.org/10.1073/pnas.0706701104 Buhtz, A., Springer, F., Chappell, L., Baulcombe, D. C., & Kehr, J. (2008). Identification and characterization of small RNAs from the phloem of Brassica napus. The Plant Journal, 53(5), 739e749. https://doi.org/10.1111/j.1365-313X.2007.03368.x Carra, A., Mica, E., Gambino, G., Pindo, M., Moser, C., Pè, M. E., & Schubert, A. (2009). Cloning and characterization of small non-coding RNAs from grape. The Plant Journal, 59(5), 750e763. https://doi.org/10.1111/j.1365-313X.2009.03906.x Chen, X. (2004). A microRNA as a translational repressor of APETALA2 in Arabidopsis flower development. Science, 303(5666), 2022e2025. https://doi.org/10.1126/ science.1088060 Chen, X. (2009). Small RNAs and their roles in plant development. Annual Review of Cell and Developmental Biology, 25, 21e44. https://doi.org/10.1146/ annurev.cellbio.042308.113417 Chen, Z., Hu, L., Han, N., Hu, J., Yang, Y., Xiang, T., Zhang, X., & Wang, L. (2015). Overexpression of a miR393-resistant form of transport inhibitor response protein 1 (mTIR1) enhances salt tolerance by increased osmoregulation and Naþ exclusion in Arabidopsis thaliana. Plant and Cell Physiology, 56(1), 73e83. https://doi.org/10.1093/ pcp/pcu149 Chen, C., Zeng, Z., Liu, Z., & Xia, R. (2018). Small RNAs, emerging regulators critical for the development of horticultural traits. Horticulture Research, 5(1). https://doi.org/ 10.1038/s41438-018-0072-8 Chuck, G., Meeley, R., Irish, E., Sakai, H., & Hake, S. (2007). Overexpression of the maize corngrass1 microRNA prevents flowering, improves digestibility, and increases starch content of switch grass. Nature Genetics, 39(12), 1517e1521. https://doi.org/10.1038/ ng.2007.20 Cui, M., Wang, C., Zhang, W., et al. (2018). Characterization of Vv-miR156: Vv-SPL pairs involved in the modulation of grape berry development and ripening. Molecular Genetics and Genomics, 293, 1333e1354. https://doi.org/10.1007/s00438-018-1462-1 226 Plant Small RNA in Food Crops Cuperus, J. T., Fahlgren, N., & Carrington, J. C. (2011). Evolution and functional diversification of MIRNA genes. The Plant Cell Online, 23(2), 431e442. https://doi.org/ 10.1105/tpc.110.082784 Davuluri, G. R., Van Tuinen, A., Fraser, P. D., Manfredonia, A., Newman, R., Burgess, D., Brummell, D. A., King, S. R., Palys, J., Uhlig, J., Bramley, P. M., Pennings, H. M. J., & Bowler, C. (2005). Fruit-specific RNAi-mediated suppression of DET1 enhances carotenoid and flavonoid content in tomatoes. Nature Biotechnology, 23(7), 890e895. https://doi.org/10.1038/nbt1108 Djami-Tchatchou, A. T., & Dubery, I. A. (2015). Lipopolysaccharide perception leads to dynamic alterations in the microtranscriptomes of Arabidopsis thaliana cells and leaf tissues. BMC Plant Biology, 15(1). https://doi.org/10.1186/s12870-015-0465-x Dunoyer, P., Schott, G., Himber, C., Meyer, D., Takeda, A., Carrington, J. C., & Voinnet, O. (2010). Small RNA duplexes function as mobile silencing signals between plant cells. Science, 328(5980), 912e916. https://doi.org/10.1126/science.1185880 Elbashir, S. M., Lendeckel, W., & Tuschl, T. (2001). RNA interference is mediated by 21and 22-nucleotide RNAs. Genes & Development, 15(2), 188e200. https://doi.org/ 10.1101/gad.862301 Fang, Y., Xie, K., & Xiong, L. (2014). Conserved miR164-targeted NAC genes negatively regulate drought resistance in rice. Journal of Experimental Botany, 65(8), 2119e2135. https://doi.org/10.1093/jxb/eru072 Feldmann, K. A. (2006). Steroid regulation improves crop yield. Nature Biotechnology, 24(1), 46e47. https://doi.org/10.1038/nbt0106-46 Feng, H., Duan, X., Zhang, Q., Li, X., Wang, B., Huang, L., Wang, X., & Kang, Z. (2014). The target gene of tae-miR164, a novel NAC transcription factor from the NAM subfamily, negatively regulates resistance of wheat to stripe rust. Molecular Plant Pathology, 15(3), 284e296. https://doi.org/10.1111/mpp.12089 Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E., & Mello, C. C. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, 391(6669), 806e811. https://doi.org/10.1038/35888 Gu, Y., Liu, Y., Zhang, J., Liu, H., Hu, Y., Du, H., Li, Y., Chen, J., Wei, B., & Huang, Y. (2013). Identification and characterization of microRNAs in the developing maize endosperm. Genomics, 102(5e6), 472e478. https://doi.org/10.1016/j.ygeno.2013. 08.007 Guo, F., Han, N., Xie, Y., Fang, K., Yang, Y., Zhu, M., Wang, J., & Bian, H. (2016). The miR393a/target module regulates seed germination and seedling establishment under submergence in rice (Oryza sativa L.). Plant, Cell and Environment, 39(10), 2288e2302. https://doi.org/10.1111/pce.12781 Guo, H. S., Xie, Q., Fei, J. F., & Chua, N. H. (2005). MicroRNA directs mRNA cleavage of the transcription factor NAC1 to downregulate auxin signals for arabidopsis lateral root development. The Plant Cell Online, 17(5), 1376e1386. https://doi.org/10.1105/ tpc.105.030841 Guo, S., Xu, Y., Liu, H., Mao, Z., Zhang, C., Ma, Y., Zhang, Q., Meng, Z., & Chong, K. (2013). The interaction between OsMADS57 and OsTB1 modulates rice tillering via DWARF14. Nature Communications, 4. https://doi.org/10.1038/ncomms2542 Hamilton, A. J., & Baulcombe, D. C. (1999). A novel species of small anti-sense RNA in post transcriptional gene silencing. Science, 286(5441), 950e952. https://doi.org/ 10.1126/science.286.5441.950 Henderson, I. R., Zhang, X., Lu, C., Johnson, L., Meyers, B. C., Green, P. J., & Jacobsen, S. E. (2006). Dissecting Arabidopsis thaliana DICER function in small RNA processing, gene silencing and DNA methylation patterning. Nature Genetics, 38(6), 721e725. https://doi.org/10.1038/ng1804 Regulation of morphogenesis and development in food crops: role of small RNA 227 Hong, Y., & Jackson, S. (2015). Floral induction and flower formationethe role and potential applications of miRNAs. Plant Biotechnology Journal, 13, 282e292. https:// doi.org/10.1111/pbi.12340 Iki, T. (2017). Messages on small RNA duplexes in plants. Journal of Plant Research, 130(1), 7e16. https://doi.org/10.1007/s10265-016-0876-2 Jiang, C. J., Shimono, M., Maeda, S., Inoue, H., Mori, M., Hasegawa, M., Sugano, S., & Takatsuji, H. (2009). Suppression of the rice fatty-acid desaturase gene OsSSI2 enhances resistance to blast and leaf blight diseases in rice. Molecular Plant-Microbe Interactions, 22(7), 820e829. https://doi.org/10.1094/MPMI-22-7-0820 Jiao, Y., Wang, Y., Xue, D., Wang, J., Yan, M., Liu, G., Dong, G., Zeng, D., Lu, Z., Zhu, X., Qian, Q., & Li, J. (2010). Regulation of OsSPL14 by OsmiR156 defines ideal plant architecture in rice. Nature Genetics, 42(6), 541e544. https://doi.org/10.1038/ ng.591 Jodder, J. (2021). Regulation of pri-MIRNA processing: mechanistic insights into the miRNA homeostasis in plant. Plant Cell Reports, 40(5), 783e798. https://doi.org/ 10.1007/s00299-020-02660-7 Jofuku, K. D., den Boer, B. G., Van Montagu, M., & Okamuro, J. K. (1994). Control of Arabidopsis flower and seed development by the homeotic gene APETALA2. The Plant Cell Online, 6(9), 1211e1225. https://doi.org/10.1105/tpc.6.9.1211 Jones-Rhoades, M. W., & Bartel, D. P. (2004). Computational identification of plant microRNAs and their targets, including a stress-induced miRNA. Molecular Cell, 14(6), 787e799. https://doi.org/10.1016/j.molcel.2004.05.027 Jones-Rhoades, M. W., Bartel, D. P., & Bartel, B. (2006). MicroRNAs and their regulatory roles in plants. Annual Review of Plant Biology, 57, 19e53. https://doi.org/10.1146/ annurev.arplant.57.032905.105218 Kamthan, A., Chaudhuri, A., Kamthan, M., & Datta, A. (2015). Small RNAs in plants: recent development and application for crop improvement. Frontiers of Plant Science, 6. https://doi.org/10.3389/fpls.2015.00208 Karlova, R., Rosin, F. M., Busscher-Lange, J., Parapunova, V., Do, P. T., Fernie, A. R., Fraser, P. D., Baxter, C., Angenent, G. C., & de Maagd, R. A. (2011). Transcriptome and metabolite profiling show that APETALA2a is a major regulator of tomato fruit ripening. The Plant Cell Online, 23(3), 923e941. https://doi.org/10.1105/ tpc.110.081273 Khraiwesh, B., Zhu, J. K., & Zhu, J. (2012). Role of miRNAs and siRNAs in biotic and abiotic stress responses of plants. Biochimica et Biophysica ActadGene Regulatory Mechanisms, 1819(2), 137e148. https://doi.org/10.1016/j.bbagrm.2011.05.001 Kutter, C., Schöb, H., Stadler, M., Meins, F., & Si-Ammour, A. (2007). MicroRNAmediated regulation of stomatal development in Arabidopsis. The Plant Cell Online, 19(8), 2417e2429. https://doi.org/10.1105/tpc.107.050377 Laufs, P., Peaucelle, A., Morin, H., & Traas, J. (2004). microRNA regulation of the CUC genes is required for boundary size control in Arabidopsis meristems. Development, 131(17), 4311e4322. https://doi.org/10.1242/dev.01320 Lauter, N., Kampani, A., Carlson, S., Goebel, M., & Moose, S. P. (2005). microRNA172 down-regulates glossy15 to promote vegetative phase change in maize. Proceedings of the National Academy of Sciences of the United States of America, 102(26), 9412e9417. https:// doi.org/10.1073/pnas.0503927102 Lelandais-Brière, C., Sorin, C., Declerck, M., Benslimane, A., Crespi, M., & Hartmann, C. (2010). Small RNA diversity in plants and its impact in development. Current Genomics, 11(1), 14e23. https://doi.org/10.2174/138920210790217918 Li, J., Guo, G., Guo, W., Guo, G., Tong, D., Ni, Z., Sun, Q., & Yao, Y. (2012). miRNA164-directed cleavage of ZmNAC1 confers lateral root development in maize (Zea mays L.). BMC Plant Biology, 12. https://doi.org/10.1186/1471-2229-12-220 228 Plant Small RNA in Food Crops Liu, P. P., Montgomery, T. A., Fahlgren, N., Kasschau, K. D., Nonogaki, H., & Carrington, J. C. (2007). Repression of AUXIN RESPONSE FACTOR10 by microRNA160 is critical for seed germination and postgermination stages. The Plant Journal, 52(1), 133e146. https://doi.org/10.1111/j.1365-313X.2007.03218.x Li, F., Wang, W., Zhao, N., Xiao, B., Cao, P., Wu, X., Ye, C., Shen, E., Qiu, J., Zhu, Q. H., Xie, J., Zhou, X., & Fan, L. (2015). Regulation of nicotine biosynthesis by an endogenous target mimicry of microRNA in tobacco. Plant Physiology, 169(2), 1062e1071. https://doi.org/10.1104/pp.15.00649 Li, Y., Zhang, Q. Q., Zhang, J., Wu, L., Qi, Y., & Zhou, J. M. (2010). Identification of microRNAs involved in pathogen-associated molecular pattern-triggered plant innate immunity. Plant Physiology, 152(4), 2222e2231. https://doi.org/10.1104/ pp.109.151803 Liu, H., Qin, C., Chen, Z., Zuo, T., Yang, X., Zhou, H., Xu, M., Cao, S., Shen, Y., Lin, H., He, X., Zhang, Y., Li, L., Ding, H., Lübberstedt, T., Zhang, Z., & Pan, G. (2014). Identification of miRNAs and their target genes in developing maize ears by combined small RNA and degradome sequencing. BMC Genomics, 15(1), 1e18. https://doi.org/10.1186/1471-2164-15-25 Llave, C., Kasschau, K. D., Rector, M. A., & Carrington, J. C. (2002). Endogenous and silencing-associated small RNAs in plants. The Plant Cell Online, 14(7), 1605e1619. https://doi.org/10.1105/tpc.003210 Lough, T. J., & Lucas, W. J. (2006). Integrative plantbiology: role of phloem long- distance macromolecular trafficking. Annual Review of Plant Biology, 57, 203e232. https:// doi.org/10.1146/annurev.arplant.56.032604.144145 Mallory, A. C., Bartel, D. P., & Bartel, B. (2005). MicroRNA-directed regulation of Arabidopsis AUXIN RESPONSE FACTOR17 is essential for proper development and modulates expression of early auxin response genes. The Plant Cell Online, 17(5), 1360e1375. https://doi.org/10.1105/tpc.105.031716 Mallory, A. C., Dugas, D. V., Bartel, D. P., & Bartel, B. (2004). microRNA regulation of NAC-domain targets is required for proper formation and separation of adjacent embryonic, vegetative, and floral organs. Current Biology, 14(12), 1035e1046. https:// doi.org/10.1016/j.cub.2004.06.022 Marin, E., Jouannet, V., Herz, A., Lokerse, A. S., Weijers, D., Vaucheret, H., Nussaume, L., Crespi, M. D., & Maizel, A. (2010). miR390, Arabidopsis TAS3 tasiRNAs, and their AUXIN RESPONSE FACTOR targets define an autoregulatory network quantitatively regulating lateral root growth. The Plant Cell Online, 22(4), 1104e1117. https:// doi.org/10.1105/tpc.109.072553 Melnyk, C. W., Molnar, A., & Baulcombe, D. C. (2011). Intercellular and systemic movement of RNA silencing. EMBO Journal, 30(17), 3553e3563. https://doi.org/ 10.1038/emboj.2011.274 Mica, E., Gianfranceschi, L., & Pè, M. E. (2006). Characterization of five microRNA families in maize. Journal of Experimental Botany, 57(11), 2601e2612. https://doi.org/ 10.1093/jxb/erl013 Miura, K., Ikeda, M., Matsubara, A., Song, X. J., Ito, M., Asano, K., Matsuoka, M., Kitano, H., & Ashikari, M. (2010). OsSPL14 promotes panicle branching and higher grain productivity in rice. Nature Genetics, 42(6), 545e549. https://doi.org/10.1038/ ng.592 Molnar, A., Melnyk, C. W., Bassett, A., Hardcastle, T. J., Dunn, R., & Baulcombe, D. C. (2010). Small silencing RNAs in plants are mobile and direct epigenetic modification in recipient cells. Science, 328(5980), 872e875. https://doi.org/10.1126/science.1187959 Moxon, S., Jing, R., Szittya, G., Schwach, F., Rusholme Pilcher, R. L., Moulton, V., & Dalmay, T. (2008). Deep sequencing of tomato short RNAs identifies microRNAs Regulation of morphogenesis and development in food crops: role of small RNA 229 targeting genes involved in fruit ripening. Genome Research, 18(10), 1602e1609. https://doi.org/10.1101/gr.080127.108 Nikovics, K., Blein, T., Peaucelle, A., Ishida, T., Morin, H., Aida, M., & Laufs, P. (2006). The balance between the MIR164A and CUC2 genes controls leaf margin serration in Arabidopsis. The Plant Cell Online, 18(11), 2929e2945. https://doi.org/10.1105/ tpc.106.045617 Nova-Franco, B., Íñiguez, L. P., Valdés-López, O., Alvarado-Affantranger, X., Leija, A., Fuentes, S. I., et al. (2015). The micro-RNA172c-APETALA2-1 node as a key regulator of the common bean-Rhizobium etli nitrogen fixation symbiosis. Plant Physiology, 168, 273e291. https://doi.org/10.1104/pp.114.255547 Ori, N., Cohen, A. R., Etzioni, A., Brand, A., Yanai, O., Shleizer, S., Menda, N., Amsellem, Z., Efroni, I., Pekker, I., Alvarez, J. P., Blum, E., Zamir, D., & Eshed, Y. (2007). Regulation of LANCEOLATE by miR319 is required for compound-leaf development in tomato. Nature Genetics, 39(6), 787e791. https://doi.org/10.1038/ ng2036 Palatnik, J. F., Allen, E., Wu, X., Schommer, C., Schwab, R., Carrington, J. C., & Weigel, D. (2003). Control of leaf morphogenesis by microRNAs. Nature, 425(6955), 257e263. https://doi.org/10.1038/nature01958 Palauqui, J. C., Elmayan, T., Pollien, J. M., & Vaucheret, H. (1997). Systemic acquired silencing: transgene-specific post-transcriptional silencing is transmitted by grafting from silenced stocks to non-silenced scions. EMBO Journal, 16(15), 4738e4745. https:// doi.org/10.1093/emboj/16.15.4738 Pantaleo, V., Szittya, G., Moxon, S., Miozzi, L., Moulton, V., Dalmay, T., & Burgyan, J. (2010). Identification of grapevine microRNAs and their targets using high-throughput sequencing and degradome analysis. The Plant Journal, 62(6). https://doi.org/10.1111/ j.0960-7412.2010.04208.x Papp, I., Mette, M. F., Aufsatz, W., Daxinger, L., Schauer, S. E., Ray, A., Van Der Winden, J., Matzke, M., & Matzke, A. J. M. (2003). Evidence for nuclear processing of plant micro RNA and short interfering RNA precursors. Plant Physiology, 132(3), 1382e1390. https://doi.org/10.1104/pp.103.021980 Park, W., Li, J., Song, R., Messing, J., & Chen, X. (2002). CARPEL FACTORY, a Dicer homolog, and HEN1, a novel protein, act in microRNA metabolism in Arabidopsis thaliana. Current Biology, 12(17), 1484e1495. https://doi.org/10.1016/S0960-9822(02) 01017-5 Park, M. Y., Wu, G., Gonzalez-Sulser, A., Vaucheret, H., & Poethig, R. S. (2005). Nuclear processing and export of microRNAs in Arabidopsis. Proceedings of the National Academy of Sciences, 102(10), 3691e3696. https://doi.org/10.1073/pnas.0405570102 Peláez, P., Trejo, M. S., Iñiguez, L. P., Estrada-Navarrete, G., Covarrubias, A. A., Reyes, J. L., & Sanchez, F. (2012). Identification and characterization of microRNAs in Phaseolus vulgaris by high-throughput sequencing. BMC Genomics, 13(1). https:// doi.org/10.1186/1471-2164-13-83 Peltier, A. J., Hatfield, R. D., & Grau, C. R. (2009). Soybean stem lignin concentration relates to resistance to Sclerotinia sclerotiorum. Plant Disease, 93(2), 149e154. https:// doi.org/10.1094/PDIS-93-2-0149 Qiao, F., Yang, Q., Wang, C.-L., Fan, Y.-L., Wu, X.-F., & Zhao, K.-J. (2007). Modification of plant height via RNAi suppression of OsGA20ox2 gene in rice. Euphytica, 158(1e2), 35e45. https://doi.org/10.1007/s10681-007-9422-6 Rajam, M. V., Madhulatha, P., Pandey, R., Hazarika, P. J., & Razdan, M. K. (2006). Applications of genetic engineering in tomato. In Genetic improvement of Solanaceous crops Volume 2: Tomato (pp. 285e311). CRC Press. https://www.taylorfrancis.com/books/ e/9781439844069. 230 Plant Small RNA in Food Crops Reinhart, B. J., Weinstein, E. G., Rhoades, M. W., Bartel, B., & Bartel, D. P. (2002). MicroRNAs in plants. Genes & Development, 16(13), 1616e1626. https://doi.org/ 10.1101/gad.1004402 Ruiz-Ferrer, V., & Voinnet, O. (2009). Roles of plant small RNAs in biotic stress responses. Annual Review of Plant Biology, 60, 485e510. https://doi.org/10.1146/ annurev.arplant.043008.092111 Sanan-Mishra, N., Kumar, V., Sopory, S. K., & Mukherjee, S. K. (2009). Cloning and validation of novel miRNA from basmati rice indicates crosstalk between abiotic and biotic stresses. Molecular Genetics and Genomics, 282(5), 463e474. https://doi.org/ 10.1007/s00438-009-0478-y Santin, F., Bhogale, S., Fantino, E., Grandellis, C., Banerjee, A. K., & Ulloa, R. M. (2017). Solanum tuberosum StCDPK1 is regulated by miR390 at the posttranscriptional level and phosphorylates the auxin efflux carrier StPIN4 in vitro, a potential downstream target in potato development. Physiologia Plantarum, 159(2), 244e261. https://doi.org/ 10.1111/ppl.12517 Schwab, R., Ossowski, S., Warthmann, N., & Weigel, D. (2010). Directed gene silencing with artificial microRNAs. Methods in Molecular Biology, 592, 71e88. https://doi.org/ 10.1007/978-1-60327-005-2_6 Schwab, R., Palatnik, J. F., Riester, M., Schommer, C., Schmid, M., & Weigel, D. (2005). Specific effects of microRNAs on the plant transcriptome. Developmental Cell, 8(4), 517e527. https://doi.org/10.1016/j.devcel.2005.01.018 Schwach, F., Vaistij, F. E., Jones, L., & Baulcombe, D. C. (2005). An RNA dependent RNA-polymerase prevents meristem invasion by Potatovirus X and is required for the activity but not the production of asystemic silencing signal. Plant Physiology, 138(4), 1842e1852. https://doi.org/10.1104/pp.105.063537 Sieber, P., Wellmer, F., Gheyselinck, J., Riechmann, J. L., & Meyerowitz, E. M. (2007). Redundancy and specialization among plant microRNAs: role of the MIR164 family in developmental robustness. Development, 134(6), 1051e1060. https://doi.org/10.1242/ dev.02817 Silva, G. F. F. E, Silva, E. M., da Silva Azevedo, M., Guivin, M. A. C., Ramirio, D. A., Figueiredo, C. R., Carrer, H., Peres, L. E. P., & Nogueira, F. T. S. (2014). micro RNA 156-targeted SPL/SBP box transcription factors regulate tomato ovary and fruit development. The Plant Journal, 78(4), 604e618. Springer, N. (2010). Shaping a better rice plant. Nature Genetics, 42(6), 475e476. https:// doi.org/10.1038/ng0610-475 Sun, G. (2012). MicroRNAs and their diverse functions in plants. Plant Molecular Biology, 80(1), 17e36. https://doi.org/10.1007/s11103-011-9817-6 Sunkar, R., Li, Y. F., & Jagadeeswaran, G. (2012). Functions of microRNAs in plant stress responses. Trends in Plant Science, 17(4), 196e203. https://doi.org/10.1016/ j.tplants.2012.01.010 Sun, G., Stewart, C. N., Xiao, P., & Zhang, B. (2012). MicroRNA expression analysis in the cellulosic biofuel crop Switchgrass (Panicum virgatum) under abiotic stress. PLoS One, 7(3). https://doi.org/10.1371/journal.pone.0032017 Tuschl, T. (2001). RNA interference and small interfering RNAs. ChemBioChem, 2, 239. https://doi.org/10.1002/1439-7633(20010401)2:4<239::AID-CBIC239> 3.0.CO Vaucheret, H. (2008). Plant ARGONAUTES. Trends in Plant Science, 13(7), 350e358. https://doi.org/10.1016/j.tplants.2008.04.007 Wang, J. J., & Guo, H. S. (2015). Cleavage of INDOLE-3-ACETIC ACID INDUCIBLE28 mRNA by microRNA847 upregulates auxin signaling to modulate cell proliferation and lateral organ growth in Arabidopsis. The Plant Cell Online, 27(3), 574e590. https://doi.org/10.1105/tpc.15.00101 Regulation of morphogenesis and development in food crops: role of small RNA 231 Wang, L., Sun, S., Jin, J, Fu, D., Yang, X., Weng, X., Xu, C., Li, X., Xiao, J., & Zhang, Q. (2015). Coordinated regulation of vegetative and reproductive branching in rice. Proceedings of the National Academy of Sciences, 112(50), 15504e15509. Wang, J. W., Wang, L. J., Mao, Y. B., Cai, W. J., Xue, H. W., & Chen, X. Y. (2005). Control of root cap formation by microRNA-targeted auxin response factors in Arabidopsis. The Plant Cell Online, 17(8), 2204e2216. https://doi.org/10.1105/ tpc.105.033076 Wang, S., Wu, K., Yuan, Q., Liu, X., Liu, Z., Lin, X., Zeng, R., Zhu, H., Dong, G., Qian, Q., Zhang, G., & Fu, X. (2012). Control of grain size, shape and quality by OsSPL16 in rice. Nature Genetics, 44(8), 950e954. https://doi.org/10.1038/ng.2327 Wilson, R. C., & Doudna, J. A. (2013). Molecular mechanisms of RNA interference. Annual Review of Biophysics, 42(1), 217e239. https://doi.org/10.1146/annurevbiophys-083012-130404 Windels, D., Bielewicz, D., Ebneter, M., Jarmolowski, A., Szweykowska-Kulinska, Z., & Vazquez, F. (2014). miR393 is required for production of proper auxin signalling outputs. PLoS One, 9(4), e95972. Wójcik, A. M., & Gaj, M. D. (2016). miR393 contributes to the embryogenic transition induced in vitro in Arabidopsis via the modification of the tissue sensitivity to auxin treatment. Planta, 244(1), 231e243. https://doi.org/10.1007/s00425-016-2505-7 Wu, X., Ding, D., Shi, C., Xue, Y., Zhang, Z., Tang, G, et al. (2016). MicroRNAdependent gene regulatory networks in maize leaf senescence. BMC Plant Biology, 16(73). https://doi.org/10.1186/s12870-016-0755-y Wu, M. F., Tian, Q., & Reed, J. W. (2006). Arabidopsis microRNA167 controls patterns of ARF6 and ARF8 expression, and regulates both female and male reproduction. Development, 133(21), 4211e4218. https://doi.org/10.1242/dev.02602 Xia, K., Wang, R., Ou, X., Fang, Z., Tian, C., Duan, J., Wang, Y., & Zhang, M. (2012). OsTIR1 and OsAFB2 downregulation via OsmiR393 overexpression leads to more tillers, early flowering and less tolerance to salt and drought in rice. PloS One, 7(1), e30039. https://doi.org/10.1371/journal.pone.0030039 Xie, K., Wu, C., & Xiong, L. (2006). Genomic organization, differential expression, and interaction of Squamosa promoter-binding-like transcription factors and microRNA156 in rice. Plant Physiology, 142(1), 280e293. https://doi.org/10.1104/pp.106.084475 Xin, M., Wang, Y., Yao, Y., Xie, C., Peng, H., Ni, Z., & Sun, Q. (2010). Diverse set of microRNAs are responsive to powdery mildew infection and heat stress in wheat (Triticumaestivum L.). BMC Plant Biology, 10. https://doi.org/10.1186/1471-2229-10123 Xu, J., Li, J., Cui, L., Zhang, T., Wu, Z., Zhu, P. Y., Meng, Y. J., Zhang, K. J., Yu, X. Q., Lou, Q. F., & Chen, J. F. (2017). New insights into the roles of cucumber TIR1 homologs and miR393 in regulating fruit/seed set development and leaf morphogenesis. BMC Plant Biology, 17(1), 1e14. https://doi.org/10.1186/s12870-017-1075-6 Xue, C., Yao, J. L., Qin, M. F., Zhang, M. Y., Allan, A. C., Wang, D. F., & Wu, J. (2019). PbrmiR397a regulates lignification during stone cell development in pear fruit. Plant Biotechnology Journal, 17(1), 103e117. https://doi.org/10.1111/pbi.12950 Yan, Z., Hossain, M. S., Wang, J., Valdez-Lopez, O., Liang, Y., Libault, M., et al. (2013). miR172 regulates soybean nodulation. Molecular Plant-Microbe Interactions, 2, 1371e1377. https://doi.org/10.1094/mpmi-04-13-0111-r Yan, Y., Wei, M., Li, Y., Tao, H., Wu, H., Chen, Z., Li, C., & Xu, J. H. (2021). MiR529a controls plant height, tiller number, panicle architecture and grain size by regulating SPL target genes in rice (Oryza sativa L.). Plant Science, 302(110728), p.110728. https:// doi.org/10.1016/j.plantsci.2020.110728 Yang, C., Li, D., Mao, D., Liu, X., Ji, C., Li, X., Zhao, X., Cheng, Z., Chen, C., & Zhu, L. (2013). Overexpression of microRNA319 impacts leaf morphogenesis and leads to 232 Plant Small RNA in Food Crops enhanced cold tolerance in rice (Oryza sativa L.). Plant, Cell and Environment, 36(12), 2207e2218. https://doi.org/10.1111/pce.12130 Yao, J.-L., Tomes, S., Xu, J., & Gleave, A. P. (2016). How microRNA172 affects fruit growth in different species is dependent on fruit type. Plant Signaling & Behavior, 11(4), e1156833. https://doi.org/10.1080/15592324.2016.1156833 Yao, J. L., Xu, J., Cornille, A., Tomes, S., Karunairetnam, S., Luo, Z., Bassett, H., Whitworth, C., Rees-George, J., Ranatunga, C., Snirc, A., Crowhurst, R., De Silva, N., Warren, B., Deng, C., Kumar, S., Chagné, D., Bus, V. G. M., Volz, R. K., & Gleave, A. P. (2015). A microRNA allele that emerged prior to apple domestication may underlie fruit size evolution. The Plant Journal, 84(2), 417e427. https://doi.org/ 10.1111/tpj.13021 Yara, A., Yaeno, T., Hasegawa, M., Seto, H., Montillet, J. L., Kusumi, K., Seo, S., & Iba, K. (2007). Disease resistance against Magnaporthe grisea is enhanced intransgenic rice with suppression of o-3fattyacid desaturases. Plant and Cell Physiology, 48(9), 1263e1274. https://doi.org/10.1093/pcp/pcm107 Yoo, B. C., Kragler, F., Varkonyi-Gasic, E., Haywood, V., Archer-Evans, S., Lee, Y. M., Lough, T. J., & Lucas, W. J. (2004). A systemic smallRNA signalling system in plants. The Plant Cell Online, 16(8), 1979e2000. https://doi.org/10.1105/tpc.104.023614 Yu, B., Lydiate, D. J., Young, L. W., Schäfer, U. A., & Hannoufa, A. (2008). Enhancing the carotenoid content of Brassicanapus seeds by downregulating lycopene epsilon cyclase. Transgenic Research, 17(4), 573e585. https://doi.org/10.1007/s11248-007-9131-x Zhang, L., Chia, J.-M., Kumari, S., Stein, J. C., Liu, Z., Narechania, A., Maher, C. A., Guill, K., McMullen, M. D., Ware, D., & Ecker, J. R. (2009). A genome-wide characterization of microRNA genes in maize. PLoS Genetics, 5(11), e1000716. https://doi.org/10.1371/journal.pgen.1000716 Zhang, K., Shi, X., Zhao, X., Ding, D., Tang, J., & Niu, J. (2015). Investigation of miR396 and growth-regulating factor regulatory network in maize grain filling. Acta Physiologiae Plantarum, 37(28). https://doi.org/10.1007/s11738-014-1767-6 Zhang, B., You, C., Zhang, Y., Zeng, L., Hu, J., Zhao, M., & Chen, X. (2020). Linking key steps of microRNA biogenesis by TREX-2 and the nuclear pore complex in Arabidopsis. Nature Plants, 6(8), 957e969. https://doi.org/10.1038/s41477-020-0726-z Zhang, Y. C., Yu, Y., Wang, C. Y., Li, Z. Y., Liu, Q., Xu, J., Liao, J. Y., Wang, X. J., Qu, L. H., Chen, F., Xin, P., Yan, C., Chu, J., Li, H. Q., & Chen, Y. Q. (2013). Overexpression of microRNA OsmiR397 improves rice yield by increasing grain size and promoting panicle branching. Nature Biotechnology, 31(9), 848e852. https:// doi.org/10.1038/nbt.2646 Zhang, X., Zou, Z., Zhang, J., Zhang, Y., Han, Q., Hu, T., Xu, X., Liu, H., Li, H., & Ye, Z. (2011). Over-expression of sly-miR156a in tomato results in multiple vegetative and reproductive trait alterations and partial phenocopy of the sft mutant. FEBS Letters, 585(2), 435e439. https://doi.org/10.1016/j.febslet.2010.12.036 Zhu, Q. H., & Helliwell, C. A. (2011). Regulation of flowering time and floral patterning by miR172. Journal of Experimental Botany, 62(2), 487e495. https://doi.org/10.1093/ jxb/erq295 Zouine, M., Fu, Y., Chateigner-Boutin, A.-L., Mila, I., Frasse, P., Wang, H., Audran, C., Roustan, J.-P., Bouzayen, M., & Maas, S. (2014). Characterization of the tomato ARF gene family uncovers a multi-levels post-transcriptional regulation including alternative splicing. PLoS One, 9(1), e84203. https://doi.org/10.1371/journal.pone.0084203 CHAPTER 9 Small RNA e regulator of biotic stress and pathogenesis in food crops Ilamathi Raja and Jebasingh Tennyson Department of Plant Sciences, School of Biological Sciences, Madurai Kamaraj University, Madurai, Tamil Nadu, India 1. Introduction Food crops are grown worldwide for human consumption and play critical roles for human wellbeing. Food crops include wide range of crops such as rice, maize, wheat, barley etc., and oil crops like soybeans, oilseed, etc., cereals, legumes, vegetables, fruits and nuts. Development and productivity of plants are seriously affected by different kinds of abiotic stresses like cold, drought, etc., and biotic stress like fungus, bacteria, virus, viroids, oomycetes, nematodes, and other parasitic plants (Chauhan et al., 2017; Huang et al., 2016). Plants have combined defense mechanisms which include physiological, biochemical, and regulatory mechanisms (Kuo & Falk, 2020). One such important defense mechanism is to regulate their gene expression and signal transduction pathways (Ali et al., 2020). It is a very complicated process involving a series of gene expression which are regulated at posttranscriptional level thereby altering the production of corresponding proteins and the accumulation of metabolites (Khraiwesh et al., 2012). Small RNAs modifications are also crucial to regulate their function and abundance. In case of plants, modifications are occurred majorly at the 3ʹ end and confer the stability to sRNAs which prevent them from degradation (Borges & Martienssen, 2015). These small RNA modifications are found to be responsible for small RNA diversity in Arabidopsis thaliana due to its differential expression in various cell types or under certain growth conditions. Apart from contributing to plant immunity, sRNAs play an important role in generating disease resistant plants. These sRNAs are dynamic and capable of moving within and between organisms. sRNA’s and their targets are found to be conserved between various organisms. Plant Small RNA in Food Crops ISBN 978-0-323-91722-3 https://doi.org/10.1016/B978-0-323-91722-3.00010-5 © 2023 Elsevier Inc. All rights reserved. 233 234 Plant Small RNA in Food Crops 2. Plant immunity and small RNAs in plant defense Plants have developed the mechanism to resist the pathogen by either activating or suppressing its important genes (Jones & Dangl, 2006). Plants are capable of recognize the pathogens with their molecular patterns upon pathogen attack (Huang et al., 2016; Zvereva & Pooggin, 2012) by its pattern-recognition receptors (PRRs) localized in the plasma membrane. PRRs are mainly trans-membrane receptor-like kinases (RLKs) or receptor-like proteins (RLPs) (Cao et al., 2014; Huang et al., 2016). Molecular patterns in pathogen are called as pathogens associated molecular patterns (PAMPs) and in bacteria it includes peptidoglycan, muramyl dipeptide, lipopolysaccharides, triacyl lipopeptides, lipoteichoic acid, flagellin. In the case of viruses, it includes envelope glycoproteins, CpG DNA, dsDNA, ssRNA, dsRNA and 50 -triphosphate RNA, GPI-mucin for protozoa; and b-glycan, chitin, mannan, zymosan, phospholipomannan and xylanase for fungi (Turgut-Kara et al., 2020). These PAMPs are recognized by PRR activates the PAMP triggered immunity (PTI) subsequently induces production of reactive oxygen species (ROS), increased synthesis of salicylic acid, callose deposition, and expression of pathogenesis related (PR) genes (Hou et al., 2019; Song et al., 2014; Sun et al., 2013). PTI is the first line of induced defense in plants. Pathogens produce effectors molecules which can be proteins or small peptides and oligosaccharides. These pathogen effector molecules (also called avirulence (Avr) proteins) are recognized by resistance (R) gene-coded plant cell receptors (PRR). PRR contains nucleotide-binding site (NBS) and leucine-rich repeats (LRRs). If a pathogen successfully invade with effectors and suppress the PTI, it results in another response called effector-triggered susceptibility (ETS) which ultimately results in immune response known as effector-triggered immunity (ETI) (Cui et al., 2015; Zhu et al., 2019). Subsequent to ETI, special type of response called hypersensitive response is produced at the site of infection which is very similar to programmed cell death (PCD) to limit the further growth of the pathogens (Gupta et al., 2018; Hou et al., 2019). Due to the evolution of the effector protein of pathogen and evolution of its counter attack R protein in plants an array of new effectors proteins and NBS-LRR proteins are produced now and then to maintain the plant immunity (Bigeard et al., 2015; Liu et al., 2014). Simplified overview of plant defense mechanism is represented in Fig. 9.1. The role of small non-coding RNAs (sRNAs) in stress resistant crops are extensively studied over a decade as they are a key player as genetic and Small RNA e regulator of biotic stress and pathogenesis in food crops 235 Figure 9.1 Overview of plant defense mechanism. epigenetic regulators across a plethora of plant species, ranging from the DNA modifications to the modulating the abundance of coding or noncoding RNAs. sRNA are small regulatory RNAs, 20e30 nucleotides in length. In plants, according to their biogenesis and functions, sRNAs are mainly classified into microRNA (miRNA) primarily engaging in posttranscriptional regulation and short interfering RNA (siRNA) playing pivotal role in transcriptional regulation. 3. MicroRNA of plants MicroRNAs are a class of endogenous small non-coding RNAs present in eukaryotes with 20e24 nucleotides in length. It does not code for proteins instead it regulate their target gene expression post-transcriptionally. miRNA act as a key regulator in plants by regulating their targeted genes through either cleavage or by repressing the transcribed mRNA (Ali et al., 2020). Plant miRNAs influence their growth and development and also regulates the physiological and biochemical process during the biotic and abiotic stresses (Djami-Tchatchou et al., 2017). 3.1 Biogenesis of miRNA miRNAs are widely conserved and are the most important functional sRNAs in plants. Biogenesis of miRNA is a convoluted process involving an array of proteins. Initially, MIR (miRNA gene) is coded form the DNA by RNA polymerase II and generates a primary transcript (pri-miRNA) 236 Plant Small RNA in Food Crops with 50 cap and 30 poly-A tail with a self complementary stem loop structure of 100e120 nucleotides long. Precursor miRNA (pre-miRNA) is formed by the sequential slicing of pri-miRNAs in the nucleus by the RNase III endo ribonuclease DICER-LIKE1 (DCL1) protein along with other proteins like a nuclear cap-binding complex (CBC), dsRNA-binding protein HYPONASTIC LEAVES (HYL1), and a C2H2-type zinc finger (SE) G-patch structural protein TGH to form the precursor miRNA (premiRNA) (Gao et al., 2021; Axtell & Meyers, 2018; Brant & Budak, 2018). First cleavage occurs at the base of the stem yielding an approximately 70 bp pre-miRNA which is a miRNA/miRNA* (guide strand/passenger strand) duplex. Pre-miRNA also has 30 overhangs with 2 or 3 nucleotides. 20e24 nucleotides apart from first cleavage site, second cleavage occurs thus releasing the miRNA/miRNA* duplex from the stem. Small RNA degrading nuclease (SDN) class of endonucleases protects the duplex from degradation by 20 -O-methylation at the 30 terminal residues by HUA ENHANCER1 (HEN1) and they may be transferred from nucleus to cytoplasm with the aid of HST (HASTY, the plant homolog of exportin-5/ Exp5) and other unknown factors (Park et al., 2005). The movement of miRNA duplex from nucleus to cytoplasm occurs at this stage or later is still unclear. Still few recent researches suggest that movement of miRNA in to cytoplasm might occur at post incorporation into RISC complex (Bologna et al., 2018). The simplified miRNA biogenesis in plants is shown in Fig. 9.2. The guide strand from mature miRNA is then incorporated into an Argonaute 1 (AGO1) protein is a part of RNA-induced silencing complex (RISC) (Axtell et al., 2011; Voinnet, 2009). RISC complex guides mature miRNA with almost perfect complementarity to target mRNA there by regulate its expression either by cleaving the transcript or by inhibiting the translation. The maturation process of miRNAs can be summarized in to transcription, post-transcriptional processing and stabilization of miRNA after processing (Gao et al., 2021). 3.2 Regulatory role of miRNA in plants Plant sRNAs play very important role in regulation of wide range of biological processes and metabolic pathways in response to the developmental changes, various abiotic stress and pathogen/pest attacks. miRNA are proven to regulate the plant growth and development, organogenesis, auxin signaling and play an important role in combating the biotic and Small RNA e regulator of biotic stress and pathogenesis in food crops AGO1 DCL1 SE HEN1 DRB1 HYL1 RISC 237 Argonaute Dicer-like 1 serrate Hua enhancer1 Double strand RNA binding protein1 Hyponastic leaves1 RNA induced silencing complex Figure 9.2 miRNA biogenesis in plants. abiotic stress conditions (Zhang et al., 2017). miRNAs are grouped into miRNA families which are found to be conserved in terms of sequence and functions across various plant species whereas few miRNAs are completely diverged across species (Dezulian et al., 2005; Fahlgren et al., 2010; Secic & Kogel, 2021). Different families of miRNA are responding differently to biotic stresses. In case of positive regulation, pathogen induced miRNA increases disease resistance thereby over-expression of that miRNA in plants might be an effective strategy to overcome the pathogen infection. However in case of negative regulation, the suppression of expression of mRNA with miRNA would be a perfect strategy to improve plant stress tolerance (Ali et al., 2020). Few miRNAs majorly regulating the biotic stress of food crop plants are discussed below. 3.2.1 miR393 miR393 was the first miRNA identified to be upregulated during biotic stress condition in 15 plant species (Navarro et al., 2006; Windels & Vazquez, 2011). miR393 targets the auxin signaling F-box auxin coreceptors 238 Plant Small RNA in Food Crops and transport inhibitor response 1 (TIR1) which are proven to interfere in auxin signaling by suppressing its production which in turn induces salicylic acid (SA) production thus increasing plant’s resistance to biotrophic microbes (Naseem et al., 2015; Secic & Kogel, 2021). 3.2.2 miR160 and miR167 miR160 and miR167 function similar to miR393 upon infection targets the genes involved in auxin signaling such as ARF6, ARF8, ARF10, ARF16 and ARF17. miR160 and miR167 upon bacterial infection in Arabidopsis, up-regulated which differentially targeting ARF genes. In rice, miR160 induction enhanced disease resistant upon infection with Magnaporthe oryzae fungus (Li et al., 2014) whereas in loblolly pine (Pinus taeda), miR160 was down-regulated upon infection with Cronartium quercuum (pine-oak rust) fungus (Lu et al., 2007). Infection of pathogenic Verticillium dahliae fungus in eggplant, down regulated the expression of miR167 (Yang et al., 2013). Hence, miRNA160 and miR167 were found to respond differentially with respect to the pathogens and the plant infected by the particular pathogens. 3.2.3 miR164 miR164 is shown to target the members of the NAC domain-encoding transcription factor genes in Arabidopsis and other plants. NAC domain includes ATAF1/2- Arabidopsis transcription activation factor, NAM-no apical meristem, and CUCe cup-shaped cotyledon domains (Guo et al., 2005; Sieber et al., 2007). This mRNA was up-regulated in cotton and rice against the infection of V. dahliae and M. oryzae, respectively (Hu et al., 2020). 3.2.4 miR168 This miRNA plays major role in maintaining AGO1 homeostasis. AGO1 is a crucial protein involved in the biogenesis of miRNA and siRNA and act as a key player in plant-pathogen interactions (Secic & Kogel, 2021). 3.2.5 miR169 miR169 targets Heme activator protein2 (HAP2), a transcription factor involved in hormone homeostasis and biotic stress regulation. In Arabidopsis, the considerable number of loci encoding this miRNA was identified and its target genes are involved in development, stress-response and defense signaling processes (Zhang et al., 2017; Secic & Kogel, 2021). Small RNA e regulator of biotic stress and pathogenesis in food crops 239 3.2.6 miR398 miR398 regulate the reactive oxygen species (ROS) production which is a first process in hypersensitive response against any infection (Lamb & Dixon, 1997; Secic & Kogel, 2021) miRNAs and their target genes involved in biotic stress conditions in model plant, Arabidopsis are listed below (Table 9.1). 3.3 miRNA’s involved in biotic stress regulation of important food crops 3.3.1 Rice (Oryza sativa) Rice is the staple cereal food consumed by half of world’s population. Wang et al. (2004) initially identified 20 new miRNAs to regulate various metabolic processes in rice. Sunkar et al. (2005) then identified another 14 novel miRNAs. Till date, a total of more than 600 microRNA sequences of rice are found in miRbase (Kozomara et al., 2019). miR393 targets OST1R1 and OSAFB2, auxin receptor gene homologs in multiple stress conditions. Rice yields are greatly affected by the fungus M. oryzae. An increased resistance was observed against rice blast disease with the expression of Osa-miR7695 in rice plants (Sanchez-Sanuy et al., 2019). Another newly identified miRNA from rice miR9664 upon down regulation/knocked out plants showed increased resistance against M. oryzae (Li et al., 2021). Many number of the miRNAs showed interaction with fungal elicitor reflected the complexity of miRNA regulation (Baldrich et al., 2015). Alteration of this microRNA can lead to fine tuning of gene expression to resist this fungal pathogen. Nonstructural protein 3 (NS3) encoded by Rice stripe virus (RSV) interacts with OsDRB1, to enhance viral infection and pathogenesis in rice (Zheng et al., 2017). OsDRB1 is an important component of the rice miRNA biogenesis and processing.. 3.3.2 Maize (Zea mays) Exserohilum turcicum fungus causes a prominent disease Northern leaf blight in maize. Wu et al. (2014) have identified four miRNAs such as miR811, miR829, miR845, and miR408 regulated differently in maize plant infected with E. turcicum. Kaur et al. (2020) has identified 30 putative miRNA candidates in maize contributed prominent role in plant defense. 3.3.3 Wheat Wheat is another important economic and staple food crop worldwide. miR169 and miR171 are known to be involved in powdery mildew 240 Table 9.1 List of miRNAs and their target genes identified to regulate biotic stress in Arabidopsis. Target gene Regulation Source miR156/157 SQUAMOSA promoter binding protein-like (SBP or SPL) family of transcription factors Pentatricopeptide repeat (PPR) MYB transcription factor TCP transcription factor GAMyb-like1/2 ACC synthase Auxin response factor (ARF) Pentatricopeptide repeat (PPR) Dicer-like (DCL) MYB-like DNA-binding protein S-adenosylmethioninedependent methyltransferase (SAMT) NAC domain transcription factor NO APICAL MERISTEM family protein HD-ZIPIII transcription factor Auxin response factor Argonaute (AGO) Up (bacteria) Down (fungus) Fahlgren et al. (2007), Chauhan et al. (2017) miR158 miR159 miR160 miR161 miR162 miR163 miR164 miR165/166 miR167 miR168 Up Up Up Not known Not known Not known Down (bacteria) Up (fungus) Differential Up Up Plant Small RNA in Food Crops miRNA miR169 miR170/171 miR173 miR390/miR391 miR393 miR394 miR395 miR396 miR397 miR398 miR399 miR400 miR402 Not known Not known Not known Down Up Not known Not known Down Not known Differential Up Not known Not known Not known 241 miR403 Differential Small RNA e regulator of biotic stress and pathogenesis in food crops miR172 HAP2 transcription factor CCAAT-binding transcription factor Scarecrow-like transcription factor (SCL) Apetala2-like transcription factor (AP2) TAS1, TAS2 TAS3 Transport inhibitor response 1 (TIR1)/Auxin F-box (AFB) bHLH transcription factor F-box ATP-sulfurylase (APS) Sulfate transporter (AST) Growth regulating factor (GRF) Laccase Cytochrome-c oxidase, superoxide dismutase Copper superoxide dismutase E2 ubiquiting-conjugating protein Pentatricopeptide repeat (PPR) HhH-GPD super family base excision DNA repair protein Argonaute (AGO) Continued Target gene Regulation miR408 miR447 Laccase plantacyanin-like 2-Phosphoglycerate kinaserelated CC-NBS-LRR NBS-LRR DNA (cytosine-5-)-methyltransferase F-box Galactosyltransferase SET-domain Cation/hydrogen exchanger MADS-box transcription factor Remorin, zinc finger, homeobox gene family, frataxin SPX (SYG1/Pho81/XPR1) domain/Zinc finger (C3HC4type) Jacalin lectin Kinase Jacalin lectin Cation/hydrogen exchanger, zinc transporter Laccase MYB transcription factor F-box Differential Not known miR472 miR482 miR773 miR774 miR775 miR778 miR780.1/miR780.2 miR824 miR825 miR827 miR842 miR844 miR846 miR856 miR857 miR858 miR859 Not known Down Down Not Not Not Not Not Up known known known known known Not known Not Not Not Not known known known known Not known Not known Not known Source Plant Small RNA in Food Crops miRNA 242 Table 9.1 List of miRNAs and their target genes identified to regulate biotic stress in Arabidopsis.dcont'd Small RNA e regulator of biotic stress and pathogenesis in food crops 243 infection in wheat. Leaf rust in wheat is caused by Puccinia triticina. Kumar et al. (2014) identified 22 miRNAs expressed differentially in defense related functions in the leaf rust susceptible wheat cultivars. Han et al. (2013) identified that miRNAs- miR1436, miR1439, miR5067 and miR5205 are involved in disease resistance in wheat plants. 3.3.4 Barley Barley is another important crop grown for human consumption and livestock feeds. Hunt et al. (2019) have shown that few predicted miRNAs such as miR166/165, miR398, and miR528 are shown to be differentially expressed in barley upon Blumeria graminis f. sp. Hordei (Bgh) infection. Other 73 miRNAs expressed differentially in barley upon Barley yellow dwarf virus infection (Jarosova et al., 2020) (Table 9.2). 3.3.5 Soyabean Upon Soybean mosaic virus (SMV) infection, few miRNAs such as miR168a, miR403a, miR162b and miR1515a were up regulated, regulates the expression of miRNA biogenesis genes like AGO1, AGO2, DCL1 and DCL2. Similarly, miR1507a, miR1507c and miR482a were expected to regulate NBS-LRR family disease resistance genes in Soyabean (Bao et al., 2018). Many conserved miRNAs of soyabean such as miR159, miR171, miR398, miR399, and miR408 and legume specific miRNAs like miR1512, miR2119, and miR9750 were found to be important candidates against the soyabean cyst nematodes infection (Tian et al., 2017). 3.4 Mechanism of action of miRNA in biotic stress regulation Plant miRNA uses two different mechanisms to regulate the targets gene expression by binding to mRNA causes either transcript cleavage or repression of translation (Rogers & Chen, 2013). In transcript cleavage, cleavage of the target mRNA occurs preferentially at 5’ monophosphate under the guidance of miRNA. PIWI domain of AGO proteins (AGO1, AGO2, AGO4, AGO 7, AGO 10) play a significant role in this cleavage mechanism by forming RNAseH fold with endonuclease activity (Choudhary et al., 2021). The cleaved 50 and 30 ends are further degraded by the exonuclease. Inhibition of translation by miRNA is a less frequent method comparatively. To cope up with the stress conditions, miRNA play a significant role in targeting the transcription factors to decrease the growth and development of plants thereby enhancing the expression of target genes responsible for stress tolerance. Differential expression of miRNA could be miRNAs Role in biotic stress References Rice (Oryza sativa) miR160, miR166, miR398, miR7695, miR159, miR162 miR164,miR169, miR167,miR396, miR319, miR9664 miR164, miR396, miR530, miR1846, miR1858 and miR2097 Positive regulators of M. oryzae Negative regulators of M. oryzae Sanchez-Sanuy et al. (2019) Li et al. (2014) Differential expression due to southern rice blackstreaked dwarf virus (SRBSDV) infection Differential expression with rice stripe virus infection Xu et al. (2014) Differential expression in response to Exserohilum turcicum Up-regulation with response to powdery mildew infection Down regulation due to powdery mildew infection Differential expression observed on leaf rust infection Differential expression upon infection with Blumeria graminis f. sp. hordei (Bgh) fungus, Wu et al. (2014) Maize (Zea Mays) Wheat (Triticum aestivum) Barley (Hordeum vulgare) miR168, miR159, miR172, and miR miR811, miR408 miR156, miR396, miR 535, miR166, miR167, miR528 444 miR829, miR845, miR393, miR444, and miR827 miR156, miR159, miR164, miR171, and miR396 miR169 and miR1122 miR156, miR159, miR160, miR164, hvu-miR165/hvumiR166, miR169, miR171, and miR396. Guo et al. (2012) Xin et al. (2010) Kumar et al. (2014) Hunt et al. (2019) Plant Small RNA in Food Crops Food crops 244 Table 9.2 miRNAs regulate the biotic stress in important food crops. Soyabean (Glycine max) Cowpea (Vigna unguiculata) Sorghum (Sorghum bicolor) miR159, miR171, miR398, miR399, miR408, miR1512, miR2119, miR9750 miR171, miR396, miR2111, miR156 miR2118, miR482, miR160, miR169, miR396 miR397, miR166, miR396, miR894, miR164, miR171, miR398, and miR408 miR160a miR398b and miR773 Tomato (Solanum lycopersicum) Potato (Solanum tuberosum) Bao et al. (2018) Differential expression with an infection of cowpea severe mosaic virus (CPSMV) Differential expression in response to bacterial wilt (BW) disease Martins et al. (2020) Positive regulator of anthracnose disease Negative regulator of anthracnose disease Differential expression in response to Root-knot nematodes Differential expression due to Bemisia tabaci and Tomato chlorosis virus infection Fu et al. (2020) Potato virus A Li et al. (2017) Tian et al. (2017) Zhao et al. (2015) Kaur et al. (2017) Yue et al. (2021) 245 miR393, miR482, miR1446 and miR156, miR164, miR319 and miR1446 Sly-miR159, sly-miR9471, and sly-miR162, slymiR6022 sly-miR171, miR166, miR164 miR156, miR160, miR164, miR172, miR390, miR408, miR482, miR 530, miR6027 Infection of soyabean mosaic virus causes upregulation expression Differential expression up on soyabean cyst nematode infection Small RNA e regulator of biotic stress and pathogenesis in food crops Peanut (Arachis hypogea) miR168a, miR403a, miR162b and miR1515a 246 Plant Small RNA in Food Crops observed in different parts of the plant during the stress and non-stress conditions (Kantar et al., 2011). 4. Small interfering RNA Small interfering RNA (siRNA)/short interfering RNA/silencing RNA is generally 20e27bp in length. It is a non-coding double-stranded RNA similar to miRNA in function, and operates by the RNA interference (RNAi) pathway. RNAi based on siRNA is an important tool widely used in reverse genetics application. It has been applied to mask the expression of genes in plants to achieve disease and pest resistance, enhance nutritional values, removes toxic compounds that alter plant architecture and flowering time and to improve commercial traits of fruits and flowers, and allergens, and to develop economically important industrial products (Guo et al., 2016; Kamthan et al., 2015) 4.1 siRNA to decide the fate of mRNA? There are various regulatory steps involved to guide the fate of mRNAs after transcription, either translate into a protein which is the primary fate of mRNAs or to eventually get degraded. These mRNA either get eliminated by decay or enter into RNA interference (RNAi) pathway. Generally mRNAs are degraded by the exonucleases from the ends whereas in RNAi, the RNA forms a double-stranded (dsRNA) conformation. This dsRNA acts as a substrate for DICER-family proteins processing them into small interfering RNAs (siRNAs) usually of 21e22 bp in length. 4.2 RNAi in plants sRNAs involves in regulating the plant genes via either by translational repression of mRNAs or by target it for degradation post-transcriptionaly is collectively called as RNAi (RNA interference) (Pattanayak et al., 2013). RNAi uses reverse genetics approach to silence a gene at posttranscriptional level. RNAi is a natural defense mechanism of plant immune system against biotic stress. Single stranded RNAs- miRNA and siRNA are key players in RNAi in plant. The siRNAs are incorporated into an ARGONAUTE (AGO)-family protein, which is responsible for the specificity of siRNA to target the particular mRNA by complementarity base pairing. After the incorporation of AGO protein with siRNA, it associates with other proteins to form the RNA-induced silencing complex (RISC) (Pratt & MacRae, 2009). The Small RNA e regulator of biotic stress and pathogenesis in food crops 247 RISC complex is capable of either repressing the mRNA at translation level or to cleave the target mRNA that acts as template for another round of dsRNA formation thus initiating the 2nd cycle of RNAi to generate the secondary siRNAs. The mechanism by which specific mRNAs are selected undergo RNAi is determined by the internal cleavage by endonuclease in RISC complex. RISC complex guides the miRNA or siRNA to undergo cleavage and the sliced mRNA into double stranded RNA by RNA dependent RNA polymerase or into degradation by the exonucleases. This step determines the fate of mRNA either its degradation or it enter into the feed forward cycle of RNAi (Hung & Slotkin, 2021). 4.3 Types of siRNAs Based on the diverse biogenesis pathways, siRNAs of plants are further classified into secondary phased siRNAs (phasiRNAs) also known as trans acting siRNAs (ta-siRNAs), natural antisense transcripts-derived siRNAs (nat-siRNAs), heterochromatic siRNAs (hc-siRNAs), and long siRNAs (lsiRNAs) (Huang et al., 2019; Katiyar-Agarwal & Jin, 2010) (Fig. 9.3). The first reported phasiRNA was tasiRNA derived from long noncoding RNA loci close to TAS genes tasiRNA generation is triggered from non-coding TAS transcripts by miRNA in a phased pattern by headto-tail arrangement from a specific nucleotide (Allen et al., 2005; Chen et al., 2010; Fei et al., 2013; Yoshikawa et al., 2005). Figure 9.3 Biogenesis of various types of siRNAs in plants. 248 Plant Small RNA in Food Crops nat-siRNAs also called as cis-natural antisense transcript siRNAs (cisNATS) are generated from the hybrid fraction of sense and antisense transcripts of genomic DNA. There are evidences that nat-siRNAs play an important regulatory role in stress conditions but their exact function in stress is yet to be investigated (Borsani et al., 2005, Katiyar-Agarwal & Jin, 2010). The proteins involved in production of nat-siRNAs are DCL1 and/or DCL2, HYL1, and HEN1 and also partially dependent on RDR6, SGS3, and Pol IV (Katiyar-Agarwal et al., 2006; Katiyar-Agarwal & Jin, 2010). hc-siRNAs or ra-siRNAs are usually 24 nt in length which are basically derived from retrotransposons, centromeric repeat elements, heterochromatin regions and extrachromosomal elements like viruses and viriods (Chan et al., 2005; Rosa et al., 2018; Sanan-Mishra et al., 2021). Their biogenesis hc-siRNAs is dependent on the DCL3-RDR2-Pol IV pathway (Katiyar-Agarwal & Jin, 2010). hc-siRNAs or ra-siRNAs regulates histone modification and/or DNA methylation at the target sites (Katiyar-Agarwal & Jin, 2010). Another class of lsiRNAs which are of 30e40 nt in length (KatiyarAgarwal, 2007). lsiRNAs biogenesis is majorly dependent on proteins like DCL1, AGO7, HEN1, HST and HYL1 and minorly dependent on RDR6 and Pol IV (Katiyar-Agarwal et al., 2007; Katiyar-Agarwal & Jin, 2010). miRNAs and tasiRNAs together regulates various biological processes in plants, of which their regulation in auxin production is widely studied (Marin et al., 2010). Unlike miRNAs, most of the siRNAs are produced from dsRNAs. siRNA formation is aided by number of different DCL proteins which process by cleaving dsRNAs into various classes of siRNA, varying in size of 21e24 nucleotides in length. The similarity observed between miRNAs and siRNAs in loading of siRNAs into RISCs which is targeted for post-transcriptional regulation later (Martinez de Alba et al., 2015). The primary proteins that are involved in tasiRNA biogenesis include RDR6, DCL4, SGS3, AGO1, AGO7, and DOUBLE-STRANDED RNA BINDING FACTOR4 (Adenot et al., 2006; Fukudome et al., 2011; Peragine et al., 2004; Vazquez et al., 2004; Xie et al., 2005). 4.4 Regulatory roles of siRNA in plants There are plethoras of mechanism is available in plants to fight against viral pathogens and production of viral derived siRNA is one of the mechanisms. In tobacco, siRNAs produced from intron hairpin RNA (ihpRNA) Small RNA e regulator of biotic stress and pathogenesis in food crops 249 induced resistance against the coat protein (CP) of Tomato yellow leaf curl virus (TYLCV) (Zrachya et al., 2007). siRNAs as short hairpin RNAs (shRNAs) in plant showed resistance against Potato virus X (PVX) and Potato virus Y (PVY) (Tabassum et al., 2016). siRNAs is also provide protection for plants against the bacterial infections. nat-siRNAATGB2 with a size of 22 nucleotides siRNA was effective against Pseudomonas syringae (Ps) in Arabidopsis (Katiyar-Agarwal et al., 2006). siRNAATGB2 derived from the overlapped sequence of Rab2-like small GTP-binding protein gene and a penta-tricopeptiderepeat protein-like gene (PPRL). Avirulence gene (avrRpt2) of P. syringae (Ps) induces nat-siRNAATGB2 in Arabidopsis whereas other genes do not have any impact on nat-siRNAATGB2 induction (Ali et al., 2020) (Table 9.3). 5. Importance of small RNAs in biotic stress regulation Major problem accounting for major loss in productivity and its quality is by attack of pathogens like bacteria, fungi, oomycetes and viruses and also by insect pests. Hence to induce the biotic stress responses in plants it is very important to develop innovative tools to protect crops from the pathogens and pests (Bebber & Gurr, 2015). Several RNAi strategies have been employed in crop plants against various biotic stresses to improve the defense mechanism against virus, bacteria, fungi, nematodes, and insects. 5.1 Viruses and viroids Several studies proved that small RNAs are a major player in plants to protect it from viral infection (Bao et al., 2018; Bester et al., 2017; Kundu et al., 2017). The viral RNAs silencing takes place by small RNAs, derived from virus-derived dsRNAs through siRNA pathways (Balmer & MauchMani, 2013). Plant infecting viruses replicate in the host by transferring their genetic material which is mainly RNA in most viruses. Hence RNAi is used as a natural defense mechanism in plant to combat viral infection. Other plant pathogens like fungi, oomycetes, nematodes and parasitic plants unlike viruses do not produce the RNA in the cytoplasm of host cell during the infection. Instead, pathogens have endogenous RNAi activity within their cell. Thus rather than targeting the pathogen RNAs, if RNA inducers from plants are used to target the pathogen RNA it might be possible to induce RNAi effects inside the pathogen. This can be an effective tool for 250 siRNA Pathogen/Nematode Plant species Role of siRNA References Nat-siRNAATGB2 Pseudomonas syringae Arabidopsis thaliana Katiyar-Agarwal et al. (2006) AtlsiRNAs Pseudomonas syringae Arabidopsis thaliana Supress PPR (pentatricopeptide repeats) protein-like gene (PPRL, At4g35850) Silences AtRAP gene siRNA6, siRNA9, siRNA29, siRNA32, siRNA41, siRNA46, siRNA50, siRNA52, siRNA54 rasiRNAs, tasiRNAs Heterodera schachtii Arabidopsis thaliana Suppress genes causing cyst nematode parasitism Meloidogyne javanica Arabidopsis thaliana Responsible for gall formation through ARFs Katiyar-Agarwal et al. (2007) Hewezi et al. (2008) Cabrera et al. (2015) Plant Small RNA in Food Crops Table 9.3 Various siRNAs of plant against various pathogens. Small RNA e regulator of biotic stress and pathogenesis in food crops 251 disease control in the crop plants (Kuo & Falk, 2020). Virus-induced gene silencing (VIGS) is a RNA-mediated PTGS mechanism to protect plants against pathogens (Beclin et al., 2002; Ding et al., 2010; Huang et al., 2016) (Fig. 9.4). VIGS is an effective functional genomics tool to knock out target genes expression in few plants. 5.2 Bacteria In plants, bacterial diseases spread very fast hence it is very difficult to suppress its infection. In Arabidopsis, two genes namely iaaM (tryptophan monooxygenase) and ipt (Isopentenyltransferase) which are involved in crown gall tumor formation by Agrobacterium tumefaciens were controlled significantly by RNAi mediated silencing of genes (Dunoyer et al., 2006). Fatty acids and their derivatives are reported to be important signaling molecule in regulating plant’s resistance against bacterial disease (Jiang et al., 2009). As mentioned earlier, miR393 provides resistance against P. syringae is the best example of sRNA regulation of bacterial stress condition. Major insight into miRNA function was attained after the discovery of various sRNA families target genes of plant NBS-LRR in Solanaceae (Li et al., 2012) and Legumes (Zhai et al., 2011). MiR482/2118 family of miRNAs targeted NBS-LRR mRNAs encoding bacterial disease resistance proteins Figure 9.4 Plants defend against virus attack through RNAi mechanism by silencing viral DNA/RNA genome involving microRNAs (miRNAs) and virus-derived small interfering RNAs (vsiRNAs) (Huang et al., 2016). RDR-RNA dependent RNA polymerase. 252 Plant Small RNA in Food Crops in various members of Solanaceae family mainly in tomato (Shivaprasad et al., 2012). The sRNAs responding to the pathogens are either up regulated or down regulated upon bacterial infection. In a particular case, where sRNA acts as positive regulator, strategy can be developed for disease resistance by over-expression of the particular sRNA in the transgenic plant. In the case where sRNA act as negative regulator, over-expression of target genes might result in plant stress tolerance (Franco-Zorrilla et al., 2007). 5.3 Fungi In rice, RNAi-mediated suppression of a particular gene OsSSI2 resulted in enhanced resistance against fungus Magnaporthe grisea to suppress rice blast disease (Jiang et al., 2009). Similarly, in soyabean, increased resistance to phytopathogen Sclerotinia sclerotiorum was achieved by targeting of genes involved in lignin production through RNAi based approach (Peltier et al., 2009). In another study on wheat crop biotic stress regulation, 24 miRNAs were involved in resistance response to fungus B. graminis f. sp. tritici (Bgt) which causes wheat-powdery mildew (Xin et al., 2010). The fungus S. sclerotiorum is known to infect over 600 plant species, produces at least 374 distinct highly abundant sRNAs targeting various genes which were down-regulated during the infection. Entry of microbes activates the plant immunity which in turn up regulates/down regulates the miRNA and siRNA expression to regulate the synthesis of plant hormones like auxin, ABA, salicylic acid and jasmonic acid to combat the infection (Fig. 9.5). 5.3.1 Insects Insects do not infect plant cells directly, but they act as a vector to inject certain pathogens such as bacteria, fungi and viruses. Many miRNA were found to be present in both plant and insects in common. One such example is miR393 identified in Arabidopsis also present in many vectors. sRNA are known to regulate the production of phytochemicals toxic to insects the mechanism of which is unclear yet. 6. Circular RNA and its biogenesis Circular RNA (circRNA) is a non-coding RNA with circular nature with a size of 100bp-4Kbp without 50 e30 polarities and poly-A tails and resistant to RNase R. It is generated from precursor mRNA during its processing by Small RNA e regulator of biotic stress and pathogenesis in food crops 253 Figure 9.5 Biotic stress regulation of sRNAs. ETI, effector triggered immunity; ETS, effector triggered susceptibility; PTI, Pathogen triggered immunity; PRR, Pattern recognition receptors, nucleotide-binding site (NBS) and leucine-rich repeats (LRRs). back splicing (Jeck et al., 2013). Back-splicing occurs either by base pairing between splice donor of a downstream exon with upstream splice acceptor of a mRNA (Ivanov et al., 2015), or by dimerization of RNA binding proteins (RBPs) on specific motifs in the flanking region of introns (Fig. 9.6A) (Conn et al., 2015). CircRNAs are also formed from the lariat precursor during exon-skipping or from intron lariats which is escape from debranching (Fig. 9.6B) (Kelly et al., 2015). CircRNAs could derive from exonic, intronic, and intergenic regions of a genome (Chen, 2016). Though circRNA found both in animal and plants, plant circRNAs are slightly differ from animal circRNAs. In animal circRNAs, reverse complementary elements are found in the flanking regions of introns of circRNAs (Jeck & Sharpless, 2014) and few circRNAs regulate the expression of target genes by acting as miRNA sponges. Plants circRNAs contain comparatively fewer repetitive and reverse complementary sequences in the flanking introns (Lu et al., 2015; Ye et al., 2015) and do not act like miRNA sponges (Hansen et al., 2013; Memczak et al., 2013; Westholm et al., 2014). circRNAs are widely distributed and abundantly found in plant species. It has been reported in various plants like O. sativa (Lu et al., 2015; Ye et al., 2017), Arabidopsis thaliana (Chen et al., 2017; Pan et al., 2018), barley 254 Plant Small RNA in Food Crops Figure 9.6 Role of cis sequence and trans factor in circRNA biogenesis (Guria et al., 2020). The non-coding intronic region in pre-mRNA harbors the highly conserved sequence in the 50 and 30 , which is essential for splicing by spliceosomal machinery (I). In addition to the conserved sequence, flanking introns consist of a repeat sequence or RBP site, which help to bring the 50 and 30 ends of intervening exon closer together due to either the base pairing (IIa, IIc) or by the binding of RBP (IIb, IId). Due to this proximity, circRNAs are generated by exon skipping or direct backsplicing (IIIa, IVa). Exon skipping produces linear RNA first, followed by the circularization of an intervening exon along with the formation of a lariat containing flanking introns (IIIb). Subsequent splicing yields a circular exonic RNA (IVb). In contrast, in direct backsplicing, exonic circRNA is generated first (IIIc, IVc) and is then followed by an exoneintron lariat (IIId). The latter is processed further to convert it into linear RNA (IVd). The pictorial representation is not to scale. (Darbani et al., 2016), maize (Chen et al., 2018b; Tang et al., 2018), tomato (Tan et al., 2017), wheat (Wang et al., 2017), soybean (Chen et al., 2018a; Zhao et al., 2017), kiwifruit (Wang, Yifei, et al. 2017), sea buckthorn fruit (Zhang et al., 2017), tea (Tong et al., 2018) and cotton (Xiang et al., 2018; Zhao et al., 2017). Small RNA e regulator of biotic stress and pathogenesis in food crops 255 6.1 Regulatory roles of circRNA in plants The formation of circRNAs with the response to pathogen infection was reported first time in Arabidopsis leaves (Sun et al., 2016). 584 circRNAs was expressed differently in kiwifruit with the response to P. syringae pathogen (Wang, Yifei, et al. 2017) (Table 9.4). 2098 circRNAs from susceptible Valor potato cultivars (1404) and disease tolerant BP1 potato cultivar (1337) were found to expressed with response to Pectobacterium carotovorum infection and from which half (931, 38%) of them were intergenic circRNAs. The expression analysis between Valor and BP1 potato cultivars detected 429 circRNAs significantly regulate the mRNAs and sponging the miRNAs (Zhou et al., 2018). Gene Ontology (GO) enrichment analysis between the parental genes and miRNAs targeted mRNAs identified the differentially expressed (DE) circRNAs were involved in defense response (GO:0006952), ADP binding (GO:0043531), phosphorylation (GO:0016310), cell wall (GO:0005199) and kinase activity (GO:0016301). Expression and weighted gene co-expression network analysis (WGCNA) revealed that few circRNAs are produced as plant defense response against P. carotovorum infection. In another study, 183 circRNAs were found to be expressed in tomato plants with tomato yellow leaf curl virus (TYLCV) infection and 114 (62%) circRNAs from 183 were derived from exons (Wang et al., 2018). The knock down of parent gene Solyc07g043420.2.1 in tomato plant abolishes the formation of Slcirc107 Table 9.4 Differential expression of circRNAs in response to pathogen infection. Plant Pathogen Kiwifruit Pseudomonas syringae Tomato yellow leaf curl virus Maize Iranian mosaic virus Verticillium dahlia Tomato Maize Cotton Tomato Pectobacterium carotovorum Number of differentially expressed circRNAs 584 115 160 280 429 References Wang, Yifei, et al. (2017) Wang et al. (2018) Ghorbani et al. (2018) Xiang et al. (2018) Zhou et al. (2018) 256 Plant Small RNA in Food Crops which significantly reduce the accumulation of TYLCV. 1443 circRNAs were identified in maize Iranian mosaic virus (MIMV) infected maize plants (Ghorbani et al., 2018). 155 and 5 circRNAs were up regulated and down regulated, respectively due to MIMV infection in maize. Xiang et al. (2018) observed the formation of 686 circRNAs in cotton plant with response to Verticillium dahlia infection which causes Verticillium wilt disease. The study of circRNAs showed that the numbers of circRNAs in CSSL-1 and CSSL4 was not different, and there were many common circRNAs were found in V. dahlia between two chromosome segment substitution lines, CSSL-1 (a highly resistant line) and CSSL-4 (a susceptible line) with response to wilt response. However, the difference in the expression of circRNAs in CSSL4 was much greater than that in CSSL-1 with response to V. dahlia infection. Source genes of these circRNAs include 17 differentially expressed circRNAs from 13 non-redundant source genes were related to a defense response, and 11 from the 13 source genes were correspond to nuclear binding site (NBS) family genes. GO analysis showed maximum number of circRNAs derived from the NBS gene family plays a prominent role in detecting the effectors of pathogens. circRNA formed from the source genes NBS regulates the expression of NBS family genes play an important role in regulating the resistance of Verticillium wilt resistance (Xiang et al., 2018). 7. Small RNA movement between organisms to regulate plant-pathogen interactions sRNA can greatly influence plantepathogen interactions by its movement within and between organisms. In 1997, short distance cell-to-cell movement of sRNA was observed first time in plants (Palauqui et al., 1997; Voinnet & Baulcombe, 1997). Intra and interspecies long-distance mobility has now been observed over a decade, which includes both plants to pathogen transportation and from pathogen to plant (Knip et al., 2014). Cross-kingdom RNAi is the phenomenal mechanism in which gene silencing was induced with sRNAs between unrelated species like between plant host and its interacting microorganism. The modulation in transcription between Rhizobia and soybean is another cross-kingdom RNAi revealed that tRFs (tRNA derived fragments) formed from the bacteria can silence the specific plant genes (Ren et al., 2019). Further experimental evidences in this aspect on tRFs can prove that they can play a role in coping with biotic stress. tRFs could act on a cannonical pathway of Small RNA e regulator of biotic stress and pathogenesis in food crops 257 microRNA will explore its role in biotic stress in food crops (Alves & Nogueira, 2021). rRFs have been identified in various studies in plants and it might also regulate the expression of host genes similar to tRF but their definite roles in biotic stress are yet to be revealed (Guan & Grigoriev, 2021). Many sRNAs were found to be present in phloem sap and plants might use symplastic movement through plasmodesmata to perform cell to cell movement (Zhang et al., 2009) thus allowing its movement to meristamatic points and sink organs from photosynthetic tissues but the exact mechanism of movement is still unidentified (Dunoyer et al., 2013; Lewsey et al., 2016). This sRNA movement within plasmodesmata is found to be one of the initial host responses to the diseases. sRNA mobility is also observed during virus induced gene silencing (VIGS) (Dunoyer & Voinnet, 2005). Another example is observed for a fungal pathogen, Botrytis cinerea which releases its sRNAs into host plant cells to suppress the AGO proteins of host and targets genes responsible for defense mechanisms (Weiberg et al., 2013). Pelaez et al. (2017) found that several T-DNA encoded genes are mainly constituted of phasiRNA-producing loci. This may result in post transational gene silencing induced dsRNA-derived sRNAs. They also suggested that sRNAs generated from bacteria’s T-DNA might target its plant genes to induce RNA silencing. Similar process could have happened in T-DNA genes of the Ri plasmid of Agrobacterium rhizogenes in hairy roots formation in common bean (Pelaez et al., 2017). Hence, movement of sRNAs from Agrobacterium to plant may play a crucial role in natural plant transformation mechanism. Plants also have similar mechanisms to fight against pathogens known as host induced gene silencing (HIGS) in which plant sRNAs invade the pathogen to suppress the disease causing target genes. Different mechanisms are adapted in different organisms and two commonly proposed methods are transfer of sRNAs occurred through vesicular transportation in the case of bacteria and through haustoria (tube from hypha) in the case of fungi (Brant & Budak, 2018; Hua et al., 2018; Nowara et al., 2010; Zhang et al., 2012). In insect pests, the mode of sRNA transfer is occurred through feeding tubes while feeding (Fig. 9.7). 8. RNA based technologies for plant disease control 8.1 Host induced gene silencing (HIGS) In many commercial crops, artificial RNAi signals were induced to suppress the target genes of causative mRNAs in parasitic nematodes, bacterial, 258 Plant Small RNA in Food Crops Figure 9.7 Various modes of small RNA mobility, between plant cells and bacteria (vesicular transportation), plant cells and fungi/parasitic plants (haustoria), and plant cells and insect herbivores (feeding tube) (Brant & Budak, 2018). fungal and oomycete pathogens, thus can generate pest- and pathogenresistant crops (Katoch et al., 2013; Koch & Kogel., 2014). HIGS increase the resistance in plants against wide range of pathogen and insect pests which can be an effective strategy for food crop protection. Barley (Hordeum vulgare) and wheat (Triticum spp.) plants with the expression of artificial siRNAs showed resistance to fungal pathogens B. graminis and Fusarium graminearum, respectively (Koch et al., 2013; Nowara et al., 2010). There are several other examples showing that artificial siRNAs can be used as a tool in agriculture for crop protection. This is possible because of the movement of sRNAs between different organisms (Fig. 9.8). 8.2 Artificial manipulation of microRNA (AmiRNAs) Artificial microRNAs (amiRNAs) are an excellent tool to silence the endogenous specific genes. From a miRNA precursor, amiRNAs are formed by exchanging the miRNA/miRNA (*) sequence to target the specific gene (Sablok et al., 2011). AmiRNAs have fewer bio-safety risks compared to other technologies. Thus amiRNA is could be an effective tool in conferring disease resistance against pathogens in several food crops. Small RNA e regulator of biotic stress and pathogenesis in food crops 259 Figure 9.8 A flowchart on various RNA based technologies used to develop disease resistant food crops. 8.3 CRISPR/Cas9 CRISPR-Cas9 (Clustered regularly interspaced short palindromic repeatsassociated 9) is capable of generating double-strand break (DSB) in the genome, which is a significant tool currently used for genome editing across wide range of food crops. Hong et al. (2021) has used this CRISPR/Cas9 system to knock out two miRNAs- miR482b and miR482c responsible for Late blight disease in tomato. Transgenic lines with knock out of these miRNAs showed increased resistance to Light blight disease compared to normal lines (Hong et al., 2021). Hence, this gene editing tool is a very promising RNA based tool to develop resistant crops against various biotic stresses. 8.4 Hairpin RNA Hairpin RNA is an artificial RNA mediated gene silencing method used to knock-down a target gene which is often combined with RNAi technology. This is yet another powerful tool to develop disease resistant crops. One such example of using Hairpin RNA construct to confer disease tolerance was performed in tomato. A fungal pathogen Fusarium oxysporum f. sp causes Fusarium wilt disease in wide range of food crops. According to the report by Singh et al. (2020) using RNAi technology they have silenced the ornithine decarboxylase (ODC) gene of the fungal pathogen which is a key player in pathogenesis. In order to silence ODC gene of fungal 260 Plant Small RNA in Food Crops pathogen, the fragment of gene was cloned in the hairpin RNA construct showed siRNA formation in transgenic tomato lines exhibited disease resistance to wilt disease (Singh et al., 2020). 9. Conclusion This book chapter summarizes the biogenesis and the regulatory functions of various small RNAs (miRNAs, siRNAs and circRNAs) present in crop plants. The details of various plants small RNAs involved in biotic stress discussed in this chapter are provided in Table 9.5. To cope up with the demand of food crops due to increasing population, exploring the role of sRNAs in crop plants can help in enhancing the plant immunity against wide range of pathogens and pests thereby increasing its productivity and yield. sRNAs of plants are also essential for Table 9.5 Comprehensive table on various plant small RNAs involved in biotic stress. CircRNAs are excluded in the table. Small RNAs Types miRNAs miR156/157, miR158, miR159, miR160, miR161, miR162, miR163 miR164, miR165/166, miR167, miR168, miR169, miR170/171, miR172,miR173, miR390/miR391, miR393, miR394, miR395 miR396, miR397, miR398, miR399, miR400, miR402, miR403, miR408, miR447, miR472, miR482, miR773, miR774, miR775, miR778, miR780.1/miR780.2, miR824, miR825, miR827, miR842, miR844, miR846, miR856, miR857, miR858, miR859 Nat-siRNAATGB2, AtlsiRNAs, siRNA6, siRNA9, siRNA29, siRNA32, siRNA41, siRNA46, siRNA50, siRNA52, siRNA54 rasiRNAs, tasiRNAs siRNAs Small RNA e regulator of biotic stress and pathogenesis in food crops 261 plant growth and development apart from its regulatory role in defense mechanism. Hence it is a very important role of sRNAs to equilibrize the expression of plants hormones and R genes to balance the plant growth and defense mechanisms. As mentioned above, RNA based technologies such as AmiRNAs mediated gene silencing, HIGS, CRISPR/Cas9 and hairpin RNAs are being widely studied to combat the responses of crop plants against biotic stress. But still lot of researches needs to be carried out to overcome the challenges in applying these small RNA technologies in plants. Different transgenic approaches are being carried out to develop disease resistant crops but bio-safety regulations against usage of engineered agricultural products needs to be addressed. Hence it is very important to understand the small RNA regulatory mechanisms to design strategies that can result in desired traits in crops with minimal side effects for human consumption. References Adenot, X., Elmayan, T., Lauressergues, D., Boutet, S., Bouché, N., Gasciolli, V., & Vaucheret, H. (2006). DRB4-dependent TAS3 trans-acting siRNAs control leaf morphology through AGO7. Current Biology, 16(9), 927e932. Ali, M., Javaid, A., Naqvi, S. H., Batcho, A., Kayani, W. K., Lal, A., … Nwogwugwu, J. O. (2020). Biotic stress triggered small RNA and RNAi defense response in plants. Molecular Biology Reports, 47(7), 5511e5522. Allen, E., Xie, Z., Gustafson, A. M., & Carrington, J. C. (2005). microRNA-directed phasing during trans-acting siRNA biogenesis in plants. Cell, 121(2), 207e221. Alves, C. S., & Nogueira, F. T. (2021). Plant small RNA world growing bigger: tRNAderived fragments, longstanding players in regulatory processes. Frontiers in Molecular Biosciences, 8, 529. Axtell, M. J., & Meyers, B. C. (2018). Revisiting criteria for plant microRNA annotation in the era of big data. The Plant Cell, 30(2), 272e284. Axtell, M. J., Westholm, J. O., & Lai, E. C. (2011). Vive la différence: Biogenesis and evolution of microRNAs in plants and animals. Genome Biology, 12(4), 1e13. Baldrich, P., Campo, S., Wu, M. T., Liu, T. T., Hsing, Y. I. C., & Segundo, B. S. (2015). MicroRNA-mediated regulation of gene expression in the response of rice plants to fungal elicitors. RNA Biology, 12(8), 847e863. Balmer, D., & Mauch-Mani, B. (2013). Small yet mightyemicroRNAs in plant-microbe interactions. MicroRNA, 2(1), 73e80. Bao, D., Ganbaatar, O., Cui, X., Yu, R., Bao, W., Falk, B. W., & Wuriyanghan, H. (2018). Down-regulation of genes coding for core RNAi components and disease resistance proteins via corresponding microRNAs might be correlated with successful Soybean mosaic virus infection in soybean. Molecular Plant Pathology, 19(4), 948e960. Bebber, D. P., & Gurr, S. J. (2015). Crop-destroying fungal and oomycete pathogens challenge food security. Fungal Genetics and Biology, 74, 62e64. Beclin, C., Boutet, S., Waterhouse, P., & Vaucheret, H. (2002). A branched pathway for transgene-induced RNA silencing in plants. Current Biology, 12(8), 684e688. 262 Plant Small RNA in Food Crops Bester, R., Burger, J. T., & Maree, H. J. (2017). Differential expression of miRNAs and associated gene targets in grapevine leafroll-associated virus 3-infected plants. Archives of Virology, 162(4), 987e996. Bigeard, J., Colcombet, J., & Hirt, H. (2015). Signaling mechanisms in pattern-triggered immunity (PTI). Molecular Plant, 8(4), 521e539. Bologna, N. G., Iselin, R., Abriata, L. A., Sarazin, A., Pumplin, N., Jay, F., … Voinnet, O. (2018). Nucleo-cytosolic shuttling of ARGONAUTE1 prompts a revised model of the plant microRNA pathway. Molecular Cell, 69(4), 709e719. Borges, F., & Martienssen, R. A. (2015). The expanding world of small RNAs in plants. Nature Reviews Molecular Cell Biology, 16(12), 727e741. Borsani, O., Zhu, J., Verslues, P. E., Sunkar, R., & Zhu, J. K. (2005). Endogenous siRNAs derived from a pair of natural cis-antisense transcripts regulate salt tolerance in Arabidopsis. Cell, 123(7), 1279e1291. Brant, E. J., & Budak, H. (2018). Plant small non-coding RNAs and their roles in biotic stresses. Frontiers in Plant Science, 9, 1038. Cabrera, J, et al. (2015). Differentially expressed small RNAs in Arabidopsis galls formed by Meloidogyne javanica. Wiley Online Library. Cao, Y., Liang, Y., Tanaka, K., Nguyen, C. T., Jedrzejczak, R. P., Joachimiak, A., & Stacey, G. (2014). The kinase LYK5 is a major chitin receptor in Arabidopsis and forms a chitin-induced complex with related kinase CERK1. eLife, 3, e03766. Chan, S. W. L., Henderson, I. R., & Jacobsen, S. E. (2005). Gardening the genome: DNA methylation in Arabidopsis thaliana. Nature Reviews Genetics, 6(5), 351e360. Chauhan, S., Yogindran, S., & Rajam, M. V. (2017). Role of miRNAs in biotic stress reactions in plants. Indian Journal of Plant Physiology, 22(4), 514e529. Chen, L. L. (2016). The biogenesis and emerging roles of circular RNAs. Nature Reviews Molecular Cell Biology, 17(4), 205e211. Chen, H. M., Chen, L. T., Patel, K., Li, Y. H., Baulcombe, D. C., & Wu, S. H. (2010). 22-Nucleotide RNAs trigger secondary siRNA biogenesis in plants. Proceedings of the National Academy of Sciences, 107(34), 15269e15274. Chen, G., Cui, J., Wang, L., Zhu, Y., Lu, Z., & Jin, B. (2017). Genome-wide identification of circular RNAs in Arabidopsis thaliana. Frontiers in Plant Science, 8, 1678. Chen, L., Ding, X., Zhang, H., He, T., Li, Y., Wang, T., … Gai, J. (2018a). Comparative analysis of circular RNAs between soybean cytoplasmic male-sterile line NJCMS1A and its maintainer NJCMS1B by high-throughput sequencing. BMC Genomics, 19(1), 1e14. Chen, L., Zhang, P., Fan, Y., Lu, Q., Li, Q., Yan, J., … Li, L. (2018b). Circular RNAs mediated by transposons are associated with transcriptomic and phenotypic variation in maize. New Phytologist, 217(3), 1292e1306. Choudhary, A, et al. (2021). Circular RNA as an additional payer in the conflicts between the host and the virus. Frontiers in Immunology, 12, 602006. Conn, S. J., Pillman, K. A., Toubia, J., Conn, V. M., Salmanidis, M., Phillips, C. A., … Goodall, G. J. (2015). The RNA binding protein quaking regulates formation of circRNAs. Cell, 160(6), 1125e1134. Cui, H., Tsuda, K., & Parker, J. E. (2015). Effector-triggered immunity: From pathogen perception to robust defense. Annual Review of Plant Biology, 66, 487e511. Darbani, B., Noeparvar, S., & Borg, S. (2016). Identification of circular RNAs from the parental genes involved in multiple aspects of cellular metabolism in barley. Frontiers in Plant Science, 7, 776. Dezulian, T., Palatnik, J. F., Huson, D., & Weigel, D. (2005). Conservation and divergence of microRNA families in plants. Genome Biology, 6(11), 1e25. Small RNA e regulator of biotic stress and pathogenesis in food crops 263 Ding, X. S., Ballard, K., Nelson, R. S., Antoun, H., Avis, T., Brisson, L., … Trepanier, M. (2010). Improving virus induced gene silencing (VIGS) in rice through Agrobacterium infiltration. Biology of Molecular Plant-Microbe Interactions, 7. Djami-Tchatchou, A. T., Sanan-Mishra, N., Ntushelo, K., & Dubery, I. A. (2017). Functional roles of microRNAs in agronomically important plants-potential as targets for crop improvement and protection. Frontiers in Plant Science, 8, 378. Dunoyer, P., Himber, C., & Voinnet, O. (2006). Induction, suppression and requirement of RNA silencing pathways in virulent Agrobacterium tumefaciens infections. Nature Genetics, 38(2), 258e263. Dunoyer, P., Melnyk, C., Molnar, A., & Slotkin, R. K. (2013). Plant mobile small RNAs. Cold Spring Harbor Perspectives in Biology, 5(7), a017897. Dunoyer, P., & Voinnet, O.. (2005). The complex interplay between plant viruses and host RNA-silencing pathways. Current Opinion in Plant Biology, 8(4), 415e423. Fahlgren, N., Howell, M. D., Kasschau, K. D., Chapman, E. J., Sullivan, C. M., Cumbie, J. S., … Carrington, J. C. (2007). High-throughput sequencing of Arabidopsis microRNAs: evidence for frequent birth and death of MIRNA genes. PLoS One, 2(2), e219. Fahlgren, N., Jogdeo, S., Kasschau, K. D., Sullivan, C. M., Chapman, E. J., Laubinger, S., … Carrington, J. C. (2010). MicroRNA gene evolution in Arabidopsis lyrata and Arabidopsis thaliana. The Plant Cell, 22(4), 1074e1089. Fei, Q., Xia, R., & Meyers, B. C. (2013). Phased, secondary, small interfering RNAs in posttranscriptional regulatory networks. The Plant Cell, 25(7), 2400e2415. Franco-Zorrilla, J. M., Valli, A., Todesco, M., Mateos, I., Puga, M. I., Rubio-Somoza, I., … Paz-Ares, J. (2007). Target mimicry provides a new mechanism for regulation of microRNA activity. Nature Genetics, 39(8), 1033e1037. Fu, F., Girma, G., & Mengiste, T. (2020). Global mRNA and microRNA expression dynamics in response to anthracnose infection in sorghum. BMC Genomics, 21(1), 1e16. Fukudome, A., Kanaya, A., Egami, M., Nakazawa, Y., Hiraguri, A., Moriyama, H., & Fukuhara, T. (2011). Specific requirement of DRB4, a dsRNA-binding protein, for the in vitro dsRNA-cleaving activity of Arabidopsis Dicer-like 4. RNA, 17(4), 750e760. Gao, Z., Nie, J., & Wang, H. (2021). MicroRNA biogenesis in plant. Plant Growth Regulation, 93(1), 1e12. Ghorbani, A., Izadpanah, K., Peters, J. R., Dietzgen, R. G., & Mitter, N. (2018). Detection and profiling of circular RNAs in uninfected and maize Iranian mosaic virus-infected maize. Plant Sciences, 274, 402e409. Guan, L., & Grigoriev, A. (2021). Computational meta-analysis of ribosomal RNA fragments: Potential targets and interaction mechanisms. Nucleic Acids Research, 49(7), 4085e4103. Guo, Q., Liu, Q., A Smith, N., Liang, G., & Wang, M. B. (2016). RNA silencing in plants: Mechanisms, technologies and applications in horticultural crops. Current Genomics, 17(6), 476e489. Guo, W., Wu, G., Yan, F., Lu, Y., Zheng, H., Lin, L., … Chen, J. (2012). Identification of novel Oryza sativa miRNAs in deep sequencing-based small RNA libraries of rice infected with Rice stripe virus. PLoS One, 7, e46443. Guo, H. S., Xie, Q., Fei, J. F., & Chua, N. H. (2005). MicroRNA directs mRNA cleavage of the transcription factor NAC1 to downregulate auxin signals for Arabidopsis lateral root development. The Plant Cell, 17(5), 1376e1386. Gupta, O. P., Pandey, V., Meena, N. L., Karkute, S. G., Banerjee, S., & Dahuja, A. (2018). Small noncoding RNA-based regulation of plant immunity. In Molecular aspects of plantpathogen interaction (pp. 203e217). Singapore: Springer. Guria, A, Sharma, P, Natesan, S, & Pandi, G (2020). Circular RNAsdThe road less traveled. Frontiers in Molecular Biosciences, 6, 146. 264 Plant Small RNA in Food Crops Han, J., Kong, M. L., Xie, H., Sun, Q. P., Nan, Z. J., Zhang, Q. Z., & Pan, J. B. (2013). Identification of miRNAs and their targets in wheat (Triticum aestivum L.) by EST analysis. Genetics and Molecular Research, 12(3793), 805. Hansen, T. B., Jensen, T. I., Clausen, B. H., Bramsen, J. B., Finsen, B., Damgaard, C. K., & Klems, J. (2013). Natural RNA circles function as efficient microRNA sponges. Nature, 495, 384e388. Hewezi, T., Howe, P., Maier, T. R., Baum, T. J., et al. (2008). Arabidopsis small RNAs and their targets during cyst nematode parasitism. Molecular Plant-Microbe Interactions, 21(12), 1622e1634. Hong, Y., Meng, J., He, X., Zhang, Y., Liu, Y., Zhang, C., … Luan, Y. (2021). Editing miR482b and miR482c simultaneously by CRISPR/Cas9 enhanced tomato resistance to Phytophthora infestans. Phytopathology, 111(6), 1008e1016. Hou, S., Liu, Z., Shen, H., & Wu, D. (2019). Damage-associated molecular patterntriggered immunity in plants. Frontiers in Plant Science, 10, 646. Huang, C. Y., Wang, H., Hu, P., Hamby, R., & Jin, H. (2019). Small RNAsebig players in plant-microbe interactions. Cell Host & Microbe, 26(2), 173e182. Huang, J., Yang, M., & Zhang, X. (2016). The function of small RNAs in plant biotic stress response. Journal of Integrative Plant Biology, 58(4), 312e327. Hua, C., Zhao, J. H., & Guo, H. S. (2018). Trans-kingdom RNA silencing in plantefungal pathogen interactions. Molecular Plant, 11(2), 235e244. Hu, G., Hao, M., Wang, L., Liu, J., Zhang, Z., Tang, Y., … Wu, J. (2020). The cotton miR477-CBP60A module participates in plant defense against Verticillium dahlia. Molecular Plant-Microbe Interactions, 33(4), 624e636. Hung, Y. H., & Slotkin, R. K. (2021). The initiation of RNA interference (RNAi) in plants. Current Opinion in Plant Biology, 61, 102014. Hunt, M., Banerjee, S., Surana, P., Liu, M., Fuerst, G., Mathioni, S., … Wise, R. P. (2019). Small RNA discovery in the interaction between barley and the powdery mildew pathogen. BMC Genomics, 20(1), 1e21. Ivanov, A., Memczak, S., Wyler, E., Torti, F., Porath, H. T., Orejuela, M. R., Piechotta, M., Levanon, E. Y., Landthaler, M., Dieterich, C., & Rajewsky, N. (2015). Analysis of intron sequences reveals hallmarks of circular RNA biogenesis in animals. Cell Reports, 10(2), 170e177. Jarosova, J., Singh, K., Chrpova, J., & Kundu, J. K. (2020). Analysis of small RNAs of barley genotypes associated with resistance to Barley Yellow Dwarf Virus. Plants, 9(1), 60. Jeck, W. R., Jessica, A. S., Kai, W., Michael, K. S., Christin, E. B., Jinze, L., William, F. M., & Norman, E. S. (2013). Circular RNAs are abundant, conserved, and associated with ALU repeats. RNA, 19(2), 141e157. Jeck, W. R., & Sharpless, N. E. (2014). Detecting and characterizing circular RNAs. Natural Biotechnology, 32, 453e461. Jiang, C. J., Shimono, M., Maeda, S., Inoue, H., Mori, M., Hasegawa, M., … Takatsuji, H. (2009). Suppression of the rice fatty-acid desaturase gene OsSSI2 enhances resistance to blast and leaf blight diseases in rice. Molecular Plant-Microbe Interactions, 22(7), 820e829. Jones, J. D., & Dangl, J. L. (2006). The plant immune system. Nature, 444(7117), 323e329. Kamthan, A., Chaudhuri, A., Kamthan, M., & Datta, A. (2015). Small RNAs in plants: Recent development and application for crop improvement. Frontiers in Plant Science, 6, 208. Kantar, M., et al. (2011). miRNA expression patterns of Triticum dicoccoides in response to shock drought stress. Planta, 233(3), 471e484. Katiyar-Agarwal, S., Gao, S., Vivian-Smith, A., & Jin, H. (2007). A novel class of bacteriainduced small RNAs in Arabidopsis. Genes & Development, 21(23), 3123e3134. Katiyar-Agarwal, S., & Jin, H. (2010). Role of small RNAs in host-microbe interactions. Annual Review of Phytopathology, 48, 225e246. Small RNA e regulator of biotic stress and pathogenesis in food crops 265 Katiyar-Agarwal, S., Morgan, R., Dahlbeck, D., Borsani, O., Villegas, A., Zhu, J. K., … Jin, H. (2006). A pathogen-inducible endogenous siRNA in plant immunity. Proceedings of the National Academy of Sciences, 103(47), 18002e18007. Katoch, R., Sethi, A., Thakur, N., & Murdock, L. L. (2013). RNAi for insect control: Current perspective and future challenges. Applied Biochemistry and Biotechnology, 171(4), 847e873. Kaur, K., Duhan, N., Singh, J., Kaur, G., & Vikal, Y. (2020). Computational identification of maize miRNA and their gene targets involved in biotic and abiotic stresses. Journal of Biosciences, 45(1), 1e17. Kaur, P., Shukla, N., Joshi, G., VijayaKumar, C., Jagannath, A., Agarwal, M., … Kumar, A. (2017). Genome-wide identification and characterization of miRNAome from tomato (Solanum lycopersicum) roots and root-knot nematode (Meloidogyne incognita) during susceptible interaction. PLoS One, 12(4), e0175178. Kelly, S., Greenman, C., Cook, P. R., & Papantonis, A. (2015). Exon skipping is correlated with exon circularization. Journal of Molecular Biology, 427, 2414e2417. Khraiwesh, B., Zhu, J. K., & Zhu, J. (2012). Role of miRNAs and siRNAs in biotic and abiotic stress responses of plants. Biochimica et Biophysica Acta (BBA)-Gene Regulatory Mechanisms, 1819(2), 137e148. Knip, M., Constantin, M. E., & Thordal-Christensen, H. (2014). Trans-kingdom cross-talk: Small RNAs on the move. PLoS Genetics, 10(9), e1004602. Koch, A., & Kogel, K. H. (2014). New wind in the sails: Improving the agronomic value of crop plants through RNAi-mediated gene silencing. Plant Biotechnology Journal, 12(7), 821e831. Koch, A., Kumar, N., Weber, L., Keller, H., Imani, J., & Kogel, K. H. (2013). Hostinduced gene silencing of cytochrome P450 lanosterol C14a-demethylaseeencoding genes confers strong resistance to Fusarium species. Proceedings of the National Academy of Sciences, 110(48), 19324e19329. Kozomara, A., Birgaoanu, M., & Griffiths-Jones, S. (2019). miRBase: from microRNA sequences to function. Nucleic Acids Research, 47(D1), D155eD162. Kumar, D., Singh, D., Kanodia, P., Prabhu, K. V., Kumar, M., & Mukhopadhyay, K. (2014). Discovery of novel leaf rust responsive microRNAs in wheat and prediction of their target genes. Journal of Nucleic Acids, 2014, 570176. Kundu, A., Paul, S., Dey, A., & Pal, A. (2017). High throughput sequencing reveals modulation of microRNAs in Vigna mungo upon Mungbean Yellow Mosaic India Virus inoculation highlighting stress regulation. Plant Science, 257, 96e105. Kuo, Y. W., & Falk, B. W. (2020). RNA interference approaches for plant disease control. Biotechniques, 69(6), 469e477. Lamb, C., & Dixon, R. A. (1997). The oxidative burst in plant disease resistance. Annual Review of Plant Biology, 48(1), 251e275. Lewsey, M. G., Hardcastle, T. J., Melnyk, C. W., Molnar, A., Valli, A., Urich, M. A., … Ecker, J. R. (2016). Mobile small RNAs regulate genome-wide DNA methylation. Proceedings of the National Academy of Sciences, 113(6), E801eE810. Li, R., Gao, S., Hernandez, A. G., Wechter, W. P., Fei, Z., & Ling, K. S. (2012). Deep sequencing of small RNAs in tomato for virus and viroid identification and strain differentiation. PLoS One, 7(5), e37127. Li, Y., Hu, X., Chen, J., Wang, W., Xiong, X., & He, C. (2017). Integrated mRNA and microRNA transcriptome analysis reveals miRNA regulation in response to PVA in potato. Scientific Reports, 7(1), 1e16. Li, Y., Lu, Y. G., Shi, Y., Wu, L., Xu, Y. J., Huang, F., … Wang, W. M. (2014). Multiple rice microRNAs are involved in immunity against the blast fungus Magnaporthe oryzae. Plant Physiology, 164(2), 1077e1092. 266 Plant Small RNA in Food Crops Liu, T., Song, T., Zhang, X., Yuan, H., Su, L., Li, W., … Dou, D. (2014). Unconventionally secreted effectors of two filamentous pathogens target plant salicylate biosynthesis. Nature Communications, 5(1), 1e10. Li, J., Zhang, H., Yang, R., Zeng, Q., Han, G., Du, Y., … Luo, Q. (2021). Identification of miRNAs contributing to the broad-spectrum and durable blast resistance in the Yunnan local rice germplasm. Frontiers in Plant Science, 12, 749919. Lu, T., Cui, L., Zhou, Y., Zhu, C., Fan, D., Gong, H., Zhao, Q., Zhou, C., Zhao, Y., Lu, D., Luo, J., Wang, Y., Tian, Q., Feng, Q., Huang, T., & Hanet, B. (2015). Transcriptome wide investigation of circular RNAs in rice. RNA, 21, 2076e2087. Lu, S., Sun, Y. H., Amerson, H., & Chiang, V. L. (2007). MicroRNAs in loblolly pine (Pinus taeda L.) and their association with fusiform rust gall development. The Plant Journal, 51(6), 1077e1098. Marin, E., Jouannet, V., Herz, A., Lokerse, A. S., Weijers, D., Vaucheret, H., … Maizel, A. (2010). miR390, Arabidopsis TAS3 tasiRNAs, and their AUXIN RESPONSE FACTOR targets define an autoregulatory network quantitatively regulating lateral root growth. The Plant Cell, 22(4), 1104e1117. Martinez de Alba, A. E., Moreno, A. B., Gabriel, M., Mallory, A. C., Christ, A., Bounon, R., … Maizel, A. (2015). In plants, decapping prevents RDR6-dependent production of small interfering RNAs from endogenous mRNAs. Nucleic Acids Research, 43(5), 2902e2913. Martins, T. F., Souza, P. F., Alves, M. S., Silva, F. D. A., Arantes, M. R., Vasconcelos, I. M., & Oliveira, J. T. (2020). Identification, characterization, and expression analysis of cowpea (Vigna unguiculata [L.] Walp.) miRNAs in response to cowpea severe mosaic virus (CPSMV) challenge. Plant Cell Reports, 39(8), 1061e1078. Memczak, S., Jens, M., Elefsinioti, A., Torti, F., Krueger, J., Rybak, A., Maier, L., Mackowiak, S. D., Gregersen, L. H., Munschauer, M., Loewer, A., Ziebold, U., Landthaler, M., Kocks, C., Noble, F.le, & Rajewsky, N. (2013). Circular RNAs are a large class of animal RNAs with regulatory potency. Nature, 495, 333e338. Naseem, M., Kaltdorf, M., & Dandekar, T. (2015). The nexus between growth and defence signalling: Auxin and cytokinin modulate plant immune response pathways. Journal of Experimental Botany, 66(16), 4885e4896. Navarro, L., Dunoyer, P., Jay, F., Arnold, B., Dharmasiri, N., Estelle, M., … Jones, J. D. (2006). A plant miRNA contributes to antibacterial resistance by repressing auxin signaling. Science, 312(5772), 436e439. Nowara, D., Gay, A., Lacomme, C., Shaw, J., Ridout, C., Douchkov, D., … Schweizer, P. (2010). HIGS: Host-induced gene silencing in the obligate biotrophic fungal pathogen Blumeria graminis. The Plant Cell, 22(9), 3130e3141. Palauqui, J. C., Elmayan, T., Pollien, J. M., & Vaucheret, H. (1997). Systemic acquired silencing: Transgene-specific post-transcriptional silencing is transmitted by grafting from silenced stocks to non-silenced scions. The EMBO Journal, 16(15), 4738e4745. Pan, T., Sun, X., Liu, Y., Li, H., Deng, G., Lin, H., & Wang, S. (2018). Heat stress alters genome-wide profiles of circular RNAs in Arabidopsis. Plant Molecular Biology, 96(3), 217e229. Park, M. Y., Wu, G., Gonzalez-Sulser, A., Vaucheret, H., & Poethig, R. S. (2005). Nuclear processing and export of microRNAs in Arabidopsis. Proceedings of the National Academy of Sciences, 102(10), 3691e3696. Pattanayak, D., Solanke, A. U., & Kumar, P. A. (2013). Plant RNA interference pathways: Diversity in function, similarity in action. Plant Molecular Biology Reporter, 31(3), 493e506. Pelaez, P., Hernandez-Lopez, A., Estrada-Navarrete, G., & Sanchez, F. (2017). Small RNAs derived from the T-DNA of Agrobacterium rhizogenes in hairy roots of Phaseolus vulgaris. Frontiers in Plant Science, 8, 96. Small RNA e regulator of biotic stress and pathogenesis in food crops 267 Peltier, A. J., Hatfield, R. D., & Grau, C. R. (2009). Soybean stem lignin concentration relates to resistance to Sclerotinia sclerotiorum. Plant Disease, 93(2), 149e154. Peragine, A., Yoshikawa, M., Wu, G., Albrecht, H. L., & Poethig, R. S. (2004). SGS3 and SGS2/SDE1/RDR6 are required for juvenile development and the production of trans-acting siRNAs in Arabidopsis. Genes & Development, 18(19), 2368e2379. Pratt, A. J., & MacRae, I. J. (2009). The RNA-induced silencing complex: A versatile genesilencing machine. Journal of Biological Chemistry, 284(27), 17897e17901. Ren, B., Wang, X., Duan, J., & Ma, J. (2019). Rhizobial tRNA-derived small RNAs are signal molecules regulating plant nodulation. Science, 365(6456), 919e922. Rogers,K, & Chen, X. (2013). Biogenesis, turnover, and mode of action of plant microRNAs. The Plant Cell, 25(7), 2383e2399. Rosa, C., Kuo, Y. W., Wuriyanghan, H., & Falk, B. W. (2018). RNA interference mechanisms and applications in plant pathology. Annual Review of Phytopathology, 56, 581e610. Sablok, G., Perez-Quintero, A. L., Hassan, M., Tatarinova, T. V., & Lopez, C. (2011). Artificial microRNAs (amiRNAs) engineeringeOn how microRNA-based silencing methods have affected current plant silencing research. Biochemical and Biophysical Research Communications, 406(3), 315e319. Sanan-Mishra, N., Jailani, A. A. K., Mandal, B., & Mukherjee, S. K. (2021). Secondary siRNAs in plants: Biosynthesis, various functions, and applications in virology. Frontiers in Plant Science, 12(610283). Sanchez-Sanuy, F., Peris-Peris, C., Tomiyama, S., Okada, K., Hsing, Y. I., San Segundo, B., & Campo, S. (2019). Osa-miR7695 enhances transcriptional priming in defense responses against the rice blast fungus. BMC Plant Biology, 19(1), 1e16. Secic, E., & Kogel, K. H. (2021). Requirements for fungal uptake of dsRNA and gene silencing in RNAi-based crop protection strategies. Current Opinion in Biotechnology, 70, 136e142. Shivaprasad, P. V., Dunn, R. M., Santos, B. A., Bassett, A., & Baulcombe, D. C. (2012). Extraordinary transgressive phenotypes of hybrid tomato are influenced by epigenetics and small silencing RNAs. The EMBO Journal, 31(2), 257e266. Sieber, P., Wellmer, F., Gheyselinck, J., Riechmann, J. L., & Meyerowitz, E. M. (2007). Redundancy and specialization among plant microRNAs: Role of the MIR164 family in developmental robustness. Agronomy, 7(48), 1051e1060. Singh, N., Mukherjee, S. K., & Rajam, M. V. (2020). Silencing of the ornithine decarboxylase gene of Fusarium oxysporum f. sp. lycopersici by host-induced RNAi confers resistance to Fusarium wilt in tomato. Plant Molecular Biology Reporter, 38(3), 419e429. Song, S., Huang, H., Gao, H., Wang, J., Wu, D., Liu, X., … Xie, D. (2014). Interaction between MYC2 and ETHYLENE INSENSITIVE3 modulates antagonism between jasmonate and ethylene signaling in Arabidopsis. The Plant Cell, 26(1), 263e279. Sunkar, R., Girke, T., Jain, P. K., & Zhu, J. K. (2005). Cloning and characterization of microRNAs from rice. The Plant Cell, 17(5), 1397e1411. Sun, X. Y., Wang, L., Ding, J. C., Wang, Y. R., Wang, J. S., Zhang, X. Y., … Zhao, H. (2016). Integrative analysis of Arabidopsis thaliana transcriptomics reveals intuitive splicing mechanism for circular RNA. FEBS Letters, 590, 3510e3516. Sun, S., Wang, H., Yu, H., Zhong, C., Zhang, X., Peng, J., & Wang, X. (2013). GASA14 regulates leaf expansion and abiotic stress resistance by modulating reactive oxygen species accumulation. Journal of Experimental Botany, 64(6), 1637e1647. Tabassum, B., Nasir, I. A., Khan, A., Aslam, U., Tariq, M., Shahid, N., & Husnain, T. (2016). Short hairpin RNA engineering: In planta gene silencing of potato virus Y. Crop Protection, 86, 1e8. Tang, B., Hao, Z., Zhu, Y., Zhang, H., & Li, G. (2018). Genome-wide identification and functional analysis of circRNAs in Zea mays. PLoS One, 13, e0202375. 268 Plant Small RNA in Food Crops Tan, J., Zhou, Z., Niu, Y., Sun, X., & Deng, Z. (2017). Identification and functional characterization of tomato CircRNAs derived from genes involved in fruit pigment accumulation. Scientific Reports, 7(8594). Tian, B., Wang, S., Todd, T. C., Johnson, C. D., Tang, G., & Trick, H. N. (2017). Genome-wide identification of soybean microRNA responsive to soybean cyst nematodes infection by deep sequencing. BMC Genomics, 18(1), 1e13. Tong, W., Yu, J., Hou, Y., Li, F., Zhou, Q., Wei, C., & Bennetzen, J. L. (2018). Circular RNA architecture and differentiation during leaf bud to young leaf development in tea (Camellia sinensis). Planta, 248, 1417e1429. Turgut-Kara, N., Arikan, B., & Celik, H. (2020). Epigenetic memory and priming in plants. Genetica, 148(2), 47e54. Vazquez, F., Vaucheret, H., Rajagopalan, R., Lepers, C., Gasciolli, V., Mallory, A. C., … Crété, P. (2004). Endogenous trans-acting siRNAs regulate the accumulation of Arabidopsis mRNAs. Molecular Cell, 16(1), 69e79. Voinnet, O. (2009). Origin, biogenesis, and activity of plant microRNAs. Cell, 136(4), 669e687. Voinnet, O., & Baulcombe, D. C. (1997). Systemic signalling in gene silencing. Nature, 389(6651), 553. Wang, Y., Ming, Y., Shimei, W., Fujun, Q., Huijie, Z., & Biao, S. (2017). Identification of circular RNAs and their targets in leaves of Triticum aestivum L. under dehydration stress. Frontiers in Plant Science, 7(202), 2024. Wang, Z., Yifei, L., Dawei, L., Li, L., Qiong, Z., Shuaibin, & W., & Hongwen, H. (2017). Identification of circular RNAs in kiwifruit and their species-specific response to bacterial canker pathogen invasion. Frontiers in Plant Science, 8, 413. Wang, J., Yuwen, Y., Lamei, J., Xitie, L., Tingli, L., Tianzi, C., Yinghua, J., Wengui, Y., & Baolong, Z. (2018). Re-analysis of long non-coding RNAs and prediction of circRNAs reveal their novel roles in susceptible tomato following TYLCV infection. BMC Plant Biology, 18(104). Wang, J. F., Zhou, H., Chen, Y. Q., Luo, Q. J., & Qu, L. H. (2004). Identification of 20 microRNAs from Oryza sativa. Nucleic Acids Research, 32(5), 1688e1695. Weiberg, A., Wang, M., Lin, F. M., Zhao, H., Zhang, Z., Kaloshian, I., … Jin, H. (2013). Fungal small RNAs suppress plant immunity by hijacking host RNA interference pathways. Science, 342(6154), 118e123. Westholm, J. O., Miura, P., Olson, S., Shenker, S., Joseph, B., Sanfilippo, P., Celniker, S. E., Brenton, R. G., & Eric, C. L. (2014). Genome-wide analysis of drosophila circular RNAs reveals their structural and sequence properties and agedependent neural accumulation. Cell Reports, 9, 1966e1980. Windels, D., & Vazquez, F. (2011). miR393: integrator of environmental cues in auxin signaling? Plant Signaling & Behavior, 6(11), 1672e1675. Wu, F., Shu, J., & Jin, W. (2014). Identification and validation of miRNAs associated with the resistance of maize (Zea mays L.) to Exserohilum turcicum. PLoS One, 9(1), e87251. Xiang, L., Chaowei, C., Jieru, C., Lu, W., Chaofeng, W., Yuzhen, S., Jingzhi, L., Lin, He, Yushan, D., Xiao, Z., Youlu, Y., & Cai, Y. (2018). Identification of circularRNAs and their targets in Gossypium under Verticillium wilt stress based on RNA-seq. PeerJ, 6, e4500. Xie, Z., Allen, E., Wilken, A., & Carrington, J. C. (2005). DICER-LIKE 4 functions in trans-acting small interfering RNA biogenesis and vegetative phase change in Arabidopsis thaliana. Proceedings of the National Academy of Sciences, 102(36), 12984e12989. Xin, M., Wang, Y., Yao, Y., Xie, C., Peng, H., Ni, Z., & Sun, Q. (2010). Diverse set of microRNAs are responsive to powdery mildew infection and heat stress in wheat (Triticum aestivum L.). BMC Plant Biology, 10(1), 1e11. Small RNA e regulator of biotic stress and pathogenesis in food crops 269 Xu, D., Mou, G., Wang, K., & Zhou, G. (2014). MicroRNAs responding to southern rice black-streaked dwarf virus infection and their target genes associated with symptom development in rice. Virus Research, 190, 60e68. Yang, L., Jue, D., Li, W., Zhang, R., Chen, M., & Yang, Q. (2013). Identification of MiRNA from eggplant (Solanum melongena L.) by small RNA deep sequencing and their response to Verticillium dahliae infection. PLoS One, 8(8), e72840. Ye, C. Y., Chen, L., Liu, C., Zhu, Q. H., & Fan, L. (2015). Widespread noncoding circular RNAs in plants. New Phytologist, 208, 88e95. Ye, C. Y., Zhang, X., Chu, Q., Liu, C., Yu, Y., Jiang, W., Zhu, Q.-H., Fan, L., & Guo, L. (2017). Full-length sequence assembly reveals circular RNAs with diverse non-GT/AG splicing signals in rice. RNA Biology, 14, 1055e1063. Yoshikawa, M., Peragine, A., Park, M. Y., & Poethig, R. S. (2005). A pathway for the biogenesis of trans-acting siRNAs in Arabidopsis. Genes & Development, 19(18), 2164e2175. Yue, H., Huang, L. P., Ding-Yi-Hui Lu, Z. H., Zhang, Z. Z., Zhang, D. Y., Zheng, L. M., … Liu, Y. (2021). Integrated analysis of microRNA and mRNA transcriptome reveals the molecular mechanism of Solanum lycopersicum response to bemisia tabaci and tomato chlorosis virus. Frontiers in Microbiology, 12, 693574. Zhai, J., Jeong, D. H., De Paoli, E., Park, S., Rosen, B. D., Li, Y., … Meyers, B. C. (2011). MicroRNAs as master regulators of the plant NB-LRR defense gene family via the production of phased, trans-acting siRNAs. Genes & Development, 25(23), 2540e2553. Zhang, G., Duan, A., Zhang, J., & He, C. (2017). Genome-wide analysis of long noncoding RNAs at the mature stage of sea buckthorn (Hippophae rhamnoides Linn) fruit. Gene, 596, 130e136. Zhang, S., Sun, L., & Kragler, F. (2009). The phloem-delivered RNA pool contains small noncoding RNAs and interferes with translation. Plant Physiology, 150(1), 378e387. Zhang, Y., Wiggins, B. E., Lawrence, C., Petrick, J., Ivashuta, S., & Heck, G. (2012). Analysis of plant-derived miRNAs in animal small RNA datasets. BMC Genomics, 13(1), 1e8. Zhao, W., Cheng, Y., Zhang, C., You, Q., Shen, X., Guo, W., & Jiao, Y. (2017). Genome-wide identification and characterization of circular RNAs by high throughput sequencing in soybean. Scientific Reports, 7(1), 5636. Zhao, C., Xia, H., Cao, T., Yang, Y., Zhao, S., Hou, L., … Wang, X. (2015). Small RNA and degradome deep sequencing reveals peanut microRNA roles in response to pathogen infection. Plant Molecular Biology Reporter, 33(4), 1013e1029. Zheng, L., Zhang, C., Shi, C., Yang, Z., Wang, Y., Zhou, T., … Li, Y. (2017). Rice stripe virus NS3 protein regulates primary miRNA processing through association with the miRNA biogenesis factor OsDRB1 and facilitates virus infection in rice. PLoS Pathogens, 13(10), e1006662. Zhou, R., Yongxing, Z., Jiao, Z., Zhengwu, F., Shuping, W., Junliang, Y., Zhaohui, C., & Dongfang, M. (2018). Transcriptome-wide identification and characterization of potato circular RNAs in response to Pectobacterium carotovorum subspecies brasiliense infection. International Journal Of Molecular Sciences, 19, 1e12. Zhu, C., Liu, T., Chang, Y. N., & Duan, C. G. (2019). Small RNA functions as a trafficking effector in plant immunity. International Journal of Molecular Sciences, 20(11), 2816. Zrachya, A., Glick, E., Levy, Y., Arazi, T., Citovsky, V., & Gafni, Y. (2007). Suppressor of RNA silencing encoded by Tomato yellow leaf curl virus-Israel. Virology, 358(1), 159e165. Zvereva, A. S., & Pooggin, M. M. (2012). Silencing and innate immunity in plant defense against viral and non-viral pathogens. Viruses, 4(11), 2578e2597. This page intentionally left blank CHAPTER 10 Small RNA networking: hostmicrobe interaction in food crops Uzma Afreen, Manish Kumar and Kunal Mukhopadhyay Department of Bioengineering and Biotechnology, Birla Institute of Technology, Ranchi, Jharkhand, India 1. Introduction Over the past few years research community has centered its efforts to unravel the world of small RNA (sRNA) molecules that are not translated into proteins yet play regulatory functions during host-microbe interactions (Morin et al., 2008). Small RNAs are short length molecules of 20e24 nt, are non-coding RNAs that regulate post-transcriptional gene expression (Morin et al., 2008) during plant immunity, health, and response toward abiotic and biotic stress (Chuck et al., 2009; Moldovan et al., 2010; Su et al., 2017). Most plant small RNAs have a prominent role in defense response and in microbes, some of them are evident in modulating pathogenesis. They follow the common strategy to communicate during host-microbe interactions i.e through Extracellular vesicles (EVs). These EVs are lipid bilayer cargos that are secreted from the donor and are up taken by the recipient. EVs are of two types first is plants EVs by encompassing and delivering small RNAs to microbes to silence their virulence and secondly, microbial EVs carry secreted proteins and regulatory molecules (sRNAs) to deliver to host plants to silence defense genes and exacerbate infection demonstrating a mechanism of Cross-kingdom RNAi (RNA interference) (Cai et al., 2019). This sRNA encapsulated EVs based movement accelerates Cross-kingdom RNAi and proved to be an environment-friendly innovative tool for plant protection. For example, Botrytis cinerea exports small RNAs (BcesRNAs) to plants to suppress defense-related genes and cause gray mold disease (Wang et al., 2016). Plant Small RNA in Food Crops ISBN 978-0-323-91722-3 https://doi.org/10.1016/B978-0-323-91722-3.00017-8 © 2023 Elsevier Inc. All rights reserved. 271 272 Plant Small RNA in Food Crops 2. Differentially expressed stress responsive small RNA Some RNAs arise de novo at different time points of plant-microbe interactions. Their expression either makes the plant resistant or susceptible towards pathogen, hence facilitating or inhibiting the microbe association with plants. Dutta et al., in 2017 has identified leaf rust responsive differentially expressed novel ta-siRNA in wheat. Comparative expression study of ta-siRNA has revealed that sRNAs express themselves at different time points of disease progression. High-throughput sequencing techniques are used to analyze small RNA libraries. Small RNAs are isolated from the uninoculated and inoculated plants with their pathogenic or beneficial microbes at different infection time points. Computational predictions with the help of bioinformatics tools we further identify the novel and conserved small RNAs (Kumar et al., 2017) (Fig. 10.1). 3. Pathogenic interaction-triggers plant immunity Plants constantly encounter by microbes. Imparting pathogenicity to plant, microbes evades plant interior after entering through natural openings (stomata and hydathodes), or gain access via wound or by penetrating the leaf or root surface directly (Chisholm et al., 2006). Then the plant-microbe Fig. 10.1 Computational pipeline for identifying stress responsive novel plant miRNA using various bioinformatics softwares. Abbreviations: B2G, Blast2GO (GOGene Ontology) tool; miRBase, microRNA database; miRCat, miRNA categorization tool; psRNATarget, plant small RNA target analysis server; Rfam, RNA family database. (CLC genomics workbench: https://www.qiagenbioinformatics.com.) Small RNA networking: host-microbe interaction in food crops 273 interactions evolve cascades of reactions in plants. The prefatory interaction between host and microbe elicits PAMP-triggered immunity (PTI) in plants (Chisholm et al., 2006; Dodds & Rathjen, 2010). PAMP (PathogenAssociated Molecular Pattern) are highly conserved microbial elicitors present outside the host cell, such as bacterial flagellin or fungal chitin (Dodds & Rathjen, 2010). All microbes do not lie under pathogenic member classes so the conserved patterns as well as referred as microbeassociated molecular patterns (MAMPs) (Boller & Felix, 2009). Once the microbe cross plant cell wall as an obstacle PAMPs/MAMPs confront extracellular surface receptors called pattern recognition receptors (PRRs) (Boller & Felix, 2009). Stimulation of PRRs initiates an active defense response which leads MAP kinase signaling cascades (Asai et al., 2002) resulting in the expression of pathogen-responsive genes, production of free radicals (ROS), and deposition of callose polymer for the cell wall reinforcement at sites of penetration, all of which helps in prevention of microbial colonization (Nurnberger et al., 2004). Generally, PAMPs/ MAMPs-PRRs constitute the first defense active response that are also referred as basal immunity in plants. Recognition of PAMPs by PRRs is best understood in case of the Arabidopsis thaliana receptor kinase FlAGEllIn SEnSInG 2 (FlS2), which binds bacterial flagellin directly and then activates signaling complex. For their survivability in host cells, some pathogenic microbes acquire the ability to suppress PAMP triggered immunity and deliver virulence molecules known as effectors against which plants activate a second defense known as Effector-triggered immunity (ETI) (Chisholm et al., 2006). Gram-negative bacteria adapt a type III secretion system (TTSS) through which they transfer their effector genes directly into the host thus spoofing PTI (Chisholm et al., 2006). The intended or indirect interaction of effector proteins and the product of plant Resistance (R) genes are considered as gene-for-gene theory (Martin et al., 2003; Nimchuk et al., 2003). The gene-for-gene hypothesis was proposed (Flor et al., 1942) is a simple explanation given after the study on the Inheritance of pathogenicity in the rust fungus in Melampsora lini (Flor et al., 1971). R genes upon becoming functional, activate defense reactions like a hypersensitive response (HR), leading to apoptosis and local necrosis (Martin et al., 2003; Nimchuk et al., 2003). Most functional R genes are categorized under LRRs (Leucine-Rich Repeats) family, for example, LRR-RK: leucine-rich repeat receptor kinase RLK: receptor-like kinase (Shiu & Bleecker, 2003). 274 Plant Small RNA in Food Crops 4. MiRNAs In 1993 the first microRNA of 22 nucleotides (nt) in length was discovered in Caenorhabditis elegans called lin-4. Lin-4 is essential for controlling the normal timing of postembryonic developmental events during early larval stage in C. elegans (Ambros, 1989; Ambros & Horvitz, 1984, 1987). Lin-4 transcripts contain sequences complementary at 30 untranslated region (UTR) of LIN-14 mRNA therefore negatively regulate the expression level of LIN-14 protein (Lee et al., 1993). Later in 2002, plant miRNA been extensively studied in Arabidopsis by several groups (Llave et al., 2002; Park et al., 2002; Reinhart et al., 2002). MiRNA are endogenous, noncoding, single stranded tiny RNA about 20e24 nt in length, acts as regulators for gene expression at post transcriptional level in all eukaryotes (Reinhart et al., 2002). They are partially or fully complementary to mRNA sequences of target genes resulting in cleavage induced degradation and thereby regulating gene expression at post transcriptional level or rarely by translational repression in plants (Bartel & Bartel, 2003; Chapman & Carrington, 2007). The characteristic of miRNA is its predicted stem loop hairpin secondary structure of precursor sequence. The minimum free energy value should be less than 30 kcal/mol (Bonnet et al., 2004). Most miRNAs are conserved among several plant species but with the development of advanced bioinformatics tools and application of next-generation deep sequencing technology numerous miRNAs have been identified which are induced when plants encounter stress and so they are referred as differentially expressed novel miRNAs. Identification of the novel miRNAs has energetically increased in the past few years as they are potentially involved in the regulation of expression of defense responsive genes (Wei et al. 2009, 2015; Zhang et al., 2015). In 2002, an online database and primary public repository of miRNA sequences and annotation were established known as miRBase (http:// microrna.sanger.ac.uk/) (Griffiths-Jones et al., 2007). The current release (v22) of this database collected from 271 organisms which contain 38 589 hairpin precursor sequences and 48 860 mature miRNAs (Kozomara et al., 2019). MiRNAs play a regulatory role in almost all metabolic and biological processes and have an evident role in biotic stress response (Sun et al., 2012). Interestingly, most of the miRNAs act in overlapping networks rather than working independently they play coordinating roles in regulating the target gene expression (Mallory et al., 2006). Now, in this section, we will discuss the responses aided by miRNA in plant-microbe Small RNA networking: host-microbe interaction in food crops 275 interaction when infected by different types of pathogens like bacteria, virus and fungi. 4.1 Plant-bacteria pathogenic interaction According to bacterial pathologists in an association with the Journal of Molecular Plant Pathology most pathogenic plant-bacteria are Pseudomonas syringae pathovars, Ralstonia solanacearum, Agrobacterium tumefaciens, Xanthomonas sp., Erwinia amylovora, Xylella fastidiosa, Dickeya, Pectobacterium carotovorum that rates economic importance worldwide as they have a very broad range of affected crops as a host (Mansfield et al., 2012). Plants infected with these pathogens show symptoms like spots on leaves or fruits, blights, mosaics and necrosis of tissue on leaves, stems or tree trunks (Vidaver & Lambrecht, 2004). The first plant miRNA discovered during Arabidopsis e P. syringae interaction is miR393. Bacterial flagellin of 22 amino acid peptide (flg 22) derived PAMP induces the expression of miR393 in Arabidopsis negatively regulating the transcript of F-box auxin receptors TIR1 (transport inhibitor response 1), AFB2 (auxin signaling F-box protein 2), and AFB3 restricts the growth of P. syringae. MiR393 was the first plant miRNA identified that has key role in the defense and increase antibacterial resistance efficiency of plants (Navarro et al., 2006). Isoforms of miR167 (miR167a, miR167b, miR167c, miR167d) has definite roles in plant developmental process and during abiotic stresses (Arora et al., 2019; Liu et al. 2012, 2014) but its differential regulation during biotic stresses has also been depicted in various studies (Kumar et al., 2017). MiR167a was found as stress-responsive miRNA in tomato during bacterial infection. It mediates the downregulation of auxin signaling genes (ARF 6 and ARF 8) during biotic stresses in tomato (Jodder et al., 2017; Liu et al., 2021). Next-generation sequencing technique was used to construct two small RNA libraries from Manihot esculenta tissue infected and uninfected with Xanthomonas axonopodis pv. Manihotis (Xam). From these libraries 56 conserved and 12 novel differentially expressed miRNAs identified in the M. esculenta (Cassava)-Xam interaction. Some conserved miRNAs showed high expression during bacterial infection regulating auxin response factors (ARFs) while some miRNAs get repressed, mediates defense through regulating leucine-rich repeats (LRRs) disease resistance proteins (PérezQuintero et al., 2012). Mainly bacterial responsive miRNAs are known to modulate hormone signaling, for example miR160, miR167, miR 390 276 Plant Small RNA in Food Crops and miR393 involved in auxin signaling, miR159 and miR 319 involved in ABA and jasmonic acid signaling respectively (Table 10.1). 4.2 Plant-fungus pathogenic interaction Based on economic importance fungal pathologists with association with the Journal of Molecular Plant Pathology ranked top 10 fungal pathogens. The list includes (1) Magnaporthe oryzae; (2) B. cinerea; (3) Puccinia spp.; (4) Fusarium graminearum; (5) Fusarium oxysporum; (6) Blumeria graminis; (7) Mycosphaerella graminicola; (8) Colletotrichum spp.; (9) Ustilago maydis; (10) M. lini. These diseases are threat to food security as these not only affect grain yield but also reduces grain quality causing great economic losses (Dean et al., 2012). A large group of disease-causing phytopathogenic fungi fulfills their nutritional requirements by attacking crop plants. Recent studies have shown the involvement of miRNAs conferring resistance against fungal attack by regulating several R (Resistance) genes (Kulshrestha et al., 2020; Zhang et al., 2016). Lu et al., in 2007 tested whether miRNA are involved in biotic stress response in loblolly pine (Pinus taeda L.) against rust fungus Cronartium quercuum f. sp. Fusiforme. This endemic rust fungus cause development of spindle-shaped galls (cankers) on branches or stems of pine. This fungus cause greater destruction to timber quality and quantity produced per unit area. From the infection site 26 miRNAs were identified in pine tree having defense-regulated targets such as NB-LRR receptors, ubiquitin ligases, laccases and peroxidases. This research concreates the fact that small RNA network play a part in host-fungal interaction. Verticillium longisporum, a pathogenic fungi that cause vascular disease in oilseed rape (Brassica napus). In this plant-fungal interaction, V. longisporum regulates plant gene expression to establish their infection through miRNAs. In total 893 differentially expressed endogenous miRNAs were identified, representing 360 conserved and 533 novel miRNAs (Shen et al., 2014). Recent studies on cotton-Verticillium dahlia interaction identify a novel defense strategy of host plants by exporting specific upregulated miRNAs to induce cross-kingdom gene silencing in the pathogenic fungi and confer resistance against wilt disease (Zhang et al., 2016). Bread wheat is a widely grown crop worldwide, feeding 30% of the world population (http://www.faostat.fao.org) been tremendously affected by rust diseases caused by different species of the fungal pathogen Puccinia. For sustainable production of wheat molecular approaches were studied using sRNA Table 10.1 Defense role of miRNAs against bacterial pathogens in various crops. Target gene Crops affected Pathogen Reference Osa-miR396f Osa-miR166m_R-1 Osa-miR171b Osa-miR156a Osa-miR535e5p Osa-miR159a_1R-3 Rice (Oryza sativa L.) Dickeya zeae Li et al. (2019) miR396a-5p Growth-regulating factor Leucine zipper family protein GRAS family transcription factor SBP transcription factor Squamosa promoter-like 11 Myb domain protein 33 RING/U-box superfamily protein Leucine-rich repeat protein kinase Myb domain protein 65 MAP kinase 20 Pre-mRNA processing splicing factor Growth regulating factors Tomato (Solanum pimpinellifolium. L) Phytophthora infestans miR393b MEMB12 Benthi (Nicotiana benthamiana) Pseudomonas syringae mdm-miR169a, mdm-miR160e, mdmmiR167b-g, and mdm-miR168a,b NF-YA (Fragaria vesca nuclear transcription factor Y subunit A) Apple (Malus domestica) Erwinia amylovora Chen et al. (2015) Zhang et al. (2011) Kaja et al. (2015) Osa-miR5072 _L-4 Small RNA networking: host-microbe interaction in food crops miRNA 277 278 Plant Small RNA in Food Crops libraries of susceptible and resistant wheat varieties infected and noninfected with leaf rust pathogen and 497 conserved and 559 novel miRNAs were identified that were related to enhancing plant immunity and providing resistance to wheat plants (Kumar et al., 2017) (Table 10.2). 4.3 Plant-virus pathogenic interaction From various studies, it is becoming evident that miRNA are playing role in antiviral defense in host plants. In Molecular Plant Pathology Journal top 10 viruses are listed according to their economically and scientifically importance (1) Tobacco mosaic virus, (2) Tomato spotted wilt virus, (3) Tomato yellow leaf curl virus, (4) Cucumber mosaic virus, (5) Potato virus Y, (6) Cauliflower mosaic virus, (7) African cassava mosaic virus, (8) Plum pox virus, (9) Brome mosaic virus and (10) P. virus X (Scholthof et al., 2011). Brassica rapa is a source of seed oil and is considered as one of the most economically important crops. Specifically, infection with Turnip mosaic virus (TuMVs) upregulation of two novel miRNAs -miR 158 and bra-miR 1885 occurs in B. rapa. These two miRNAs cleave the mRNA of the Toll/interleukin-1, nucleotide-binding site leucine-rich repeat (TIR-NBS-LRR) disease-resistant gene (He et al., 2008). The biological significance of miRNA being documented in this research indicates elevated expression of miR159ab is associated with induction of severe symptoms of viral disease (Du et al., 2014). Pepper plants are highly susceptible to Pepper mild mottle virus (PMMoVs). The study of virus-derived small-interfering RNAs (vsiRNAs) in infected pepper plants showed the majority is 21e22 nucleotide long and mainly targets physiological pathways like the stress response, cell regulation, and metabolism process (Jiao et al., 2022). In a study of Brassicaceae family plants and cauliflower mosaic virus (CaMVs) interaction, 15 loci of vsiRNAs were identified which are regulating the same targets in all three plants of Brassicaceae (turnip, oilseed rape and A. thaliana). The targets are involved in photosynthesis and stress response (Leonetti et al., 2021) (Table 10.3). 5. MicroRNA like RNA Study of Lee et al. (2010), revealed the existence of other sRNAs, one of them being miRNA-like RNAs (milRNAs) found in the filamentous fungus Neurospora crassa (Lee et al., 2010). Through next-generation sequencing, it was confirmed that these sRNAs also exist in the plant pathogenic fungus Sclerotinia sclerotiorum. Small_RNA library analysis of miRNA Target gene Crops affected Pathogen Reference miR166 Cotton (Gossypium hirsutum) Verticillium dahlia Zhang et al. (2016) miR397b Ca2þ-dependent cysteine protease (Clp-1) isotrichodermin C-15 hydroxylase (HiC-15), Lignin biosynthesis TaCLP1. A type of a plantacyanin protein WRKY transcription factor Botryosphaeria dothidea Puccinia striiformis f. sp. Tritici Alternaria alternata sp. Mali Yu et al. (2020) miR399b and miR 9664 Md-miR156ab Chinese Crab apple (Malus hupehensis) Wheat (Triticum aestivum) miR159 Apple (Malus domestica) Ramachandran et al. (2020) Zhang et al. (2017) Small RNA networking: host-microbe interaction in food crops Table 10.2 Defensive role of miRNAs against fungal pathogens in various crops. 279 280 miRNA Target gene miR482 Nucleotide-binding site (NBS) and leucine-rich repeat (LRR) miR171a, miR167b, and miR159a miRNA168 miRNA3623 miRNA319 miRNA395 miRNA396 P1/HCPro Nta-miR 6019 and ntamiR 6020 TIR-NBS-LLR protein repress the translation of Ago 1 mRNA nucleobase-ascorbate transporter (NAT) family and sulfate adenylyltransferase 3 Arabidopsis growth-regulating factors and negatively regulates cell division and cell cycle Toll and Interleukin-1 receptor-NBLRR immune receptor Crops affected Tomato (Solanum lycopersicum) Benthi (Nicotiana benthamiana) Grapevine (Vitis vinifera) Tobacco (Nicotiana tabacum) Pathogen Reference Turnip crinkle virus, Cucumber mosaic virus, and Tobacco rattle virus Plum pox virus Shivaprasad et al. (2012) Grapevine vein clearing virus Tobacco mosaic virus SimónMateo et al. (2006) Singh et al. (2012) Li et al. (2012) Plant Small RNA in Food Crops Table 10.3 Defense role of miRNAs against viral pathogens in various crops. Small RNA networking: host-microbe interaction in food crops 281 S. sclerotiorum uncovered two milRNAs and 42 milRNA candidates (Zhou et al., 2012). Verticillium dahliae is a plant pathogenic fungus affecting many crop plants with wilt diseases. Novel milRNA named as VdmilR1 was identified in V. dahliae spores which were collected from cotton plants. It targets VdHy1 gene which is responsible for fungal virulence. Thus this fungal sRNA plays role in biotic stress response in cotton plants (Jin et al., 2019). MilRNAs are exogenous small RNAs being produced during hostmicrobe interaction by the microbes. Identification of milRNA was mostly of fungal origin and their loci could be mapped on fungal genomes (Mueth et al., 2015). During the interaction of Puccinia triticina with wheat plants, two mil-RNAs were identified by using next-generation sequencing technology. These P. triticina mil-RNAs (pt-mil-RNAs) targets many transcripts of the host having functions like ROS related, disease resistance, metabolic processes, transporter, apoptotic inhibitor, and transcription factors. Pt-mil-RNAs play a regulatory role in biotrophic growth and fungal infection (Dubey et al., 2019). In another study, three milRNAs were identified in P. triticina which causes leaf rust infection in wheat plants. These Puccinia derived milRNA mediate RNAi in host plants. Precursor sequences of these milRNAs ePTmilR1, PTmilR2, and PTmilR3 were predicted and with the help of bioinformatics tools their precursor molecules were found to fold they were folded into stem-loop like secondary similar to structure miRNAs (Dutta et al., 2019) (Fig. 10.2). 6. Si-RNA Short interfering RNAs (siRNAs) are small noncoding RNAs of 21e24 nucleotide, arising from double-stranded RNA (dsRNA) precursor. The mechanism behind siRNA is RNA interference which is responsible for diversified natural functions such as maintaining genome integrity, DNA methylation, antiviral defense and regulating stress-responsive gene expression. Dicer gene family DCL2 and DCL3 process the production of viral siRNA and transposon siRNA (Baulcombe, 2004). Sometimes from 282 Plant Small RNA in Food Crops Fig. 10.2 Secondary structure of PTmilR1 precursor sequence (Dutta et al., 2019). the action of miRNA targeting mRNA generate secondary siRNA by the activity of RNA-dependent RNA polymerase (RDR) (de Felippes, 2019). Small RNA library sequencing provided hundreds of endogenous siRNAs in rice plants. Most of them are 21 nucleotides in length, they are differently accumulated in different developmental stages and are tissue specific. These siRNAs genes target genes whose functional annotation suggests that they are involved in preventing transposon mobility, therefore maintaining genome structural integrity (Sunkar et al., 2005). However, the biological roles of endogenous siRNA during microbial interaction in crop plants are not well understood. We have discussed some of its functions during interaction in this section. 6.1 Plant-bacterial pathogenic interaction The first plant endogenous siRNA, nat-siRNAATGB2 generated from the overlapping region of two genes, Rab2-like small GTP-binding protein gene (ATGB2) and a PPR (pentatricopeptide repeats) protein-like gene (PPRL) are NAT pairs of gene. This study demonstrated that siRNAs are involved in antibacterial defense and play a role in regulating R gene mediated ETI. Distinctive biogenesis pathway produces nat-siRNAATGB2 that requires DCL1, HYL1, HEN1, RDR6, SGS3, and RNA polymerase Iva. This nat-siRNA is of 22 nucleotides in length specifically induced in plants upon infection with effector protein avrRpt2 carried by the bacterial pathogen P. syringae. Expression of nat-siRNAATGB2 elicitate plant Small RNA networking: host-microbe interaction in food crops 283 immunity by silencing of antisense PPRL gene which leads to specific disease resistance. As pathogens regulate plant machinery to spread their infection and growth, plants rapidly counter defense mechanisms in order to increase plant survivability, growth and development. One of the endogenous small RNA, AtlsiRNA-1 produced from SRRLK/AtRAP NAT pair of genes induced particularly by the bacterium P. syringae. It is a long siRNA (lsiRNA) of 30e40 nucleotides in length. AtlsiRNA-1 negatively regulates the expression of antisense mRNA of AtRAP by triggering silencing of the gene. AtRAP gene encodes a RNA-binding protein containing a putative RNA-binding RAP domain (RNA complexes). AtRAP is a negative regulator of plant immunity, hence AtlsiRNA-1 mediated cleavage leads to enhanced plant resistance (Katiyar-Agarwal et al., 2007). 6.2 Plant-viral pathogenic interaction High-throughput sequencing of small RNA of wild type and transgenic anti-Rice stripe virus (RSVs) rice confirmed that differentially induced specific siRNA is a major player in providing resistance against the virus rather than specifically induced miRNA in rice plants (Guo et al., 2015). Insertional mutation in DCL2 gene of Arabidopsis helps in identifying its critical functions in the biogenesis of viral siRNA. The loss of viral siRNA activity in dcl2 mutant plants increases the susceptibility of plants against turnip crinkle virus. Hence, siRNA play an important role in development, maintaining chromatin structure and defense (Xie et al., 2004). In Arabidopsis, a 21 nucleotide siRNA known as vasiRNAs (virusactivated siRNAs) has been discovered which is characteristically different from other classified siRNA. The biogenesis pathway of vasiRNA requires DCL4 and RDR1, and loss of XRN4/EIN5. In vivo it binds with Argonaute 2 which is essentially needed for silencing its target mRNA suppressor protein 2b of Cucumber mosaic virus (CMV). Induction of endogenous vasiRNA on infection with CMV encourages antiviral defenses in Arabidopsis (Cao et al., 2014; Lu et al., 2007). 7. Plant-microbe beneficial interactions Plants with arbuscular mycorrhizal fungi, nitrogen-fixing rhizobia, and fungal and bacterial endophytes are enrolled in beneficial interactions with microbes. These interactions increase the nutrient uptake ability, improve nitrogenase activity and reduce oxidative stress by regulating stress 284 Plant Small RNA in Food Crops hormones of plants, thereby increasing plant yield, growth and resistance towards biotic and abiotic stresses of economically important crops (Kafle et al., 2018). A symbiotic association of soil-borne microorganisms that fixes inert atmospheric nitrogen into ammonia with the help of solar energy. These microorganisms are also known as Nitrogen efixers are of different varieties like species of Rhizobium for legumes, blue-green algae or cyanobacteria and Azolla for wet land rice and Azotobacter/Azospirillum for several crops (Lindström & Mousavi, 2020; Shridhar, 2012). The symbiosis relation of Rhizobia and legume develop a new specialize organ on roots and sometimes on stem known as nodules (Johnston et al., 2007). To unravel the molecular mechanisms and to elucidate this symbiotic mutual interaction, the regulators involved at different stages of nodule development were discovered. Two strategies have been followed for the identification of specific miRNA expressed during the interaction e First, homology search of known miRNA in other plant species to identify conserved miRNA or siRNA. Second, through high throughput sequencing technique analysis of mock and rhizobia inoculated plant to discover differentially expressed non-coding RNAs (Subramanian et al., 2008). Intensive research on soybean plants having symbiotic interaction with beneficial bacteria provided an insight of miRNA involvement at different development stages of nodules (in mtr- Medicago truncatula; gma, Glycine max) formation in roots are (a) Nitrogen deprived conditions for leguminous plants-mtr-miR396 and mtr-miR166 expressed on roots (b) Recognition and attachment of rhizobium in roots of legume plantsgma-miR168, gma-miR172, gma-miR159, gma-miR393, gmamiR160 and gma-miR169 are expressed (c) Growing of infection thread and bacteroid development-mtr-miR169, mtr-miR107, mtr-miR162, mtr-miR398 and mtr-miR166 are found to play roles in this stage of development (d) Fixation of nitrogen in mature nodulesdgma-miR167 gma-miR172 gma-miR396 gma-miR399 gma-miR1507, gma-miR1508 gmamiR1509 and gma-miR1510 are identified (Katiyar-Agarwal & Jin, 2010; Lelandais-Brière et al., 2009; Subramanian et al., 2008; Wang et al., 2009) In response of the symbiotic association of Bradyrhizobium japonicum with soybean roots identified miR169 sRNA. This miRNA targets CCAAT-binding transcription factor MtHAP2-1 transcripts of M. truncatula Small RNA networking: host-microbe interaction in food crops 285 (Combier et al., 2006), which is significant for nodule development. This result shows strong support to miRNA regulating nodule development (Subramanian et al., 2008). Endogenous sRNAs are either up or downregulated in this mutualistic relationship which regulates different stages in the development of nitrogen-fixing nodules (Katiyar-Agarwal & Jin, 2010). In a study, roots of model legume M. truncatula during the symbiotic interaction with Sinorhizobium meliloti result in selective employment of mRNA in early phase of nodule development. This differential translation of mRNA is achieved by miRNA-miR169 and miR172 regulating gene expression of targets NF-YA/HAP2 and AP2 transcription factors (Reynoso et al., 2013). There was another strong evidence suggesting that miRNA not only play role during nodule organogenesis but also in bacterial infections and in nodule function. Two sets of specific miRNAs miR171c and miR 397 identified from Lotus japonicas leguminous plant. MiR171c targets Nodulation Signaling Pathway 2 transcription factor and upregulated only in nodules respectively (De luis et al., 2012). Azospirillum brasilense and Herbaspirillum seropedicae belongs to a group of diazotrophic bacteria and are nitrogen fixers in economically important maize crop. Newly expressed small RNA through small RNA deep sequencing analysis revealed 15 novel miRNAs and 25 conserved miRNA and siRNA in maize plants. SiRNA was only induced when plants are inoculated by bacteria species. The two conserved sRNA, miR398 and miR 408 were highly up-regulated in maize plants. These results depict that small RNA plays an important role in the host plant for the association with rhizobia (Thiebaut et al., 2014). The establishment of mutualistic symbiosis requires a tolerant relationship between host and microbe. Intolerant interaction viral growth doesn’t show a notable effect on plant survivability, growth and reproduction. Tolerating the invasion and proliferation of beneficial microbes require controlled regulation of host resistance genes which are regulated by small RNAs regulators (Kri
znik et al., 2020). 8. Small RNA in crop improvement Small non-coding RNAs (miRNA & siRNA) are major regulators in plant-microbe interactions. They are differentially expressed under stress conditions and regulate their target gene expression in sequence complementary manner (Dutta et al., 2020). Small RNA by post-transcriptional gene silencing (PTGS) phenomenon silence their target gene’s activity is 286 Plant Small RNA in Food Crops a natural mechanism known as RNA interference (RNAi) (Baulcombe, 2004). Nowadays, RNAi is used as an emerging tool to alter the plant genomes for attaining crop yield and quality, increase shelf life of fruits and vegetables and resistance toward stresses for producing transgenic crops (Kamthan et al., 2015; Kerschen et al., 2004). Cross kingdom RNAi is a well-studied mechanism of pathogens to suppress host immunity. It was seen in B. cinerea (pathogenic fungus) sRNAs binds with Arabidopsis Ago 1 making them to work for itself. This strategy of fungus helps them to deliver their sRNA into hosts to increase their pathogenicity (Weiberg et al., 2013). The inherent capability of RNAi in the form of RNA-based biocontrol products will provide an alternative of eco-friendly and chemical-free pesticides for the management of crop yield losses caused by epidemics (Taning et al., 2020). This research potentially opened the gate to generate artificial small RNA mediated RNAi gene silencing in crop improvement (Table 10.4). 8.1 Small RNA sprays Genetic engineering requires fine-tuning of the genome and a highly skilled plant molecular biologist. Small RNA or dsRNA uptake by plants targeting pathogen virulent genes makes it easier for managing disease in agronomically important crop plants. Like spraying of dsRNA/sRNA on vegetables, fruit and flowers prevent gray mold fungal disease caused by B. cinerea (Wang et al., 2016). Mitter et al. (2017) in her work introduced an alternative to transgenic RNA interference (RNAi). She found out an efficient way to make plant virus-resistant by using pathogen-specific dsRNA. The stable binding of naked dsRNA on designed, layered double hydroxide (LDH) clay nanosheets applied by spraying over plant leaves. Plants achieve resistance for at least 20 days on one spray (Mitter et al., 2017). This type of gene silencing on the host by exogenous small RNA or dsRNA (double-stranded RNA) are referred as Spray-induced gene silencing (SIGS) (Wang et al., 2017). Werner et al. (2020) used SIGS-based strategies on designed dsRNAs targeting ARGONAUTE and DICER genes of F. graminearum (Fg) protected barley leaves from fungal infection. A recent study also showed the effectiveness of SIGS technology toward plant-fungal disease control is largely dependent on pathogen’s RNA uptake efficiency. In this study, topical application of dsRNA protects rice (Oryza sativa) from fungal diseasedrice sheath blight caused by Rhizoctonia solani. It also illustrates the external application of dsRNA that targets vesicle trafficking pathway genes i. e vacuolar protein sorting 51 (VPS51), dynactin (DCTN1) and suppressor of actin (SAC1) inhibited the infection of B. cinerea, S. sclerotiorum and Aspergillus niger (Qiao et al., 2021). Small RNA networking: host-microbe interaction in food crops 287 Table 10.4 List of artificial small RNAs in transgenic plants. miRNA Target gene amiR 171 2b amiR-P69159 and amiRHC-Pro159 amiR-AV1-1 P69 and HC-Pro AC1amiRNA1 and AC1amiRNA2 atasiRNA Crops affected Pathogen Reference Nicotiana tabacum (Tobacco) Arabidopsis thaliana Cucumber mosaic virus Turnip yellow mosaic virus Tomato leaf curl New Delhi virus Qu et al. (2007) Niu et al. (2006) Van Vu et al. (2013) Sharma et al. (2020) AV1 and AV2 AC1 Solanum lycopersicum (Tomato) AC2/ AC1 and AC4/ AC1 Nicotiana tabacum (tobacco) and Solanum lycopersicum (tomato) Solanum lycopersicum (Tomato) Singh et al. (2015) 9. Conclusion Exploring the world of sRNA drives most researchers to enhance their knowledge in this field. Stress-responsive sRNAs regulate biotic and abiotic stresses and their tendency to get absorbed in leaves makes them to be used in therapeutics during epidemics. These non-coding RNAs regulate gene expression without altering the plant genome and coevolves with plantmicrobe interaction. Single sRNA targets multiple resistant gene expression and the same miRNA gets activated for different diseases, therefore in near future this integrated knowledge of sRNAs network in host-microbe interactions might help to tackle different types of diseases at the same time, other than modeling transgenic crops. References Ambros, V. (1989). A hierarchy of regulatory genes controls a larva-to-adult developmental switch in C. elegans. Cell, 57(1), 49e57. Ambros, V., & Horvitz, H. R. (1984). Heterochronic mutants of the nematode Caenorhabditis elegans. Science, 226(4673), 409e416. 288 Plant Small RNA in Food Crops Ambros, V., & Horvitz, H. R. (1987). The lin-14 locus of Caenorhabditis elegans controls the time of expression of specific postembryonic developmental events. Genes & Development, 1(4), 398e414. Arora, S., Pandey, D. K., & Chaudhary, B. (2019). Target-mimicry-based diminution of miRNA167 reinforced flowering-time phenotypes in tobacco via spatial-transcriptional biases of flowering-associated miRNAs. Gene, 682, 67e80. Asai, T., Tena, G., Plotnikova, J., Willmann, M. R., Chiu, W. L., Gomez-Gomez, L., Boller, T., Ausubel, F. M., & Sheen, J. (2002). MAP kinase signalling cascade in Arabidopsis innate immunity. Nature, 415(6875), 977e983. Bartel, B., & Bartel, D. P. (2003). MicroRNAs: At the root of plant development? Plant Physiology, 132(2), 709e717. Baulcombe, D. (2004). RNA silencing in plants. Nature, 431(7006), 356e363. Boller, T., & Felix, G. (2009). A renaissance of elicitors: Perception of microbe-associated molecular patterns and danger signals by pattern-recognition receptors. Annual Review of Plant Biology, 60, 379e406. Bonnet, E., Wuyts, J., Rouzé, P., & Van de Peer, Y. (2004). Detection of 91 potential conserved plant microRNAs in Arabidopsis thaliana and Oryza sativa identifies important target genes. Proceedings of the National Academy of Sciences, 101(31), 11511e11516. Cai, Q., He, B., Weiberg, A., Buck, A. H., & Jin, H. (2019). Small RNAs and extracellular vesicles: New mechanisms of cross-species communication and innovative tools for disease control. PLoS Pathogens, 15(12), e1008090. Cao, M., Du, P., Wang, X., Yu, Y. Q., Qiu, Y. H., Li, W., Gal-On, A., Zhou, C., Li, Y., Ding, S. W., et al. (2014). Virus infection triggers widespread silencing of host genes by a distinct class of endogenous siRNAs in Arabidopsis. Proceedings of the National Academy of Sciences, 111(40), 14613e14618. Chapman, E. J., & Carrington, J. C. (2007). Specialization and evolution of endogenous small RNA pathways. Nature Reviews Genetics, 8(11), 884e896. Chen, L., Luan, Y., & Zhai, J. (2015). Sp-miR396a-5p acts as a stress-responsive genes regulator by conferring tolerance to abiotic stresses and susceptibility to Phytophthora nicotianae infection in transgenic tobacco. Plant Cell Reports, 34(12), 2013e2025. Chisholm, S. T., Coaker, G., Day, B., & Staskawicz, B. J. (2006). Host-microbe interactions: Shaping the evolution of the plant immune response. Cell, 124(4), 803e814. Chuck, G., Candela, H., & Hake, S. (2009). Big impacts by small RNAs in plant development. Current Opinion in Plant Biology, 12(1), 81e86. Combier, J. P., Frugier, F., de Billy, F., Boualem, A., El-Yahyaoui, F., Moreau, S., Vernié, T., Ott, T., Gamas, P., Crespi, M., & Niebel, A. (2006). MtHAP2-1 is a key transcriptional regulator of symbiotic nodule development regulated by microRNA169 in Medicago truncatula. Genes & Development, 20(22), 3084e3088. De Luis, A., Markmann, K., Cognat, V., Holt, D. B., Charpentier, M., Parniske, M., Stougaard, J., & Voinnet, O. (2012). Two microRNAs linked to nodule infection and nitrogen-fixing ability in the legume Lotus japonicus. Plant Physiology, 160(4), 2137e2154. Dean, R., Van Kan, J. A., Pretorius, Z. A., Hammond-Kosack, K. E., Di Pietro, A., Spanu, P. D., Rudd, J. J., Dickman, M., Kahmann, R., Ellis, J., & Foster, G. D. (2012). The Top 10 fungal pathogens in molecular plant pathology. Molecular Plant Pathology, 13(4), 414e430. Dodds, P. N., & Rathjen, J. P. (2010). Plant immunity: Towards an integrated view of plant-pathogen interactions. Nature Reviews Genetics, 11(8), 539e548. Dubey, H., Kiran, K., Jaswal, R., Jain, P., Kayastha, A. M., Bhardwaj, S. C., Mondal, T. K., & Sharma, T. R. (2019). Discovery and profiling of small RNAs from Puccinia triticina by deep sequencing and identification of their potential targets in wheat. Functional & Integrative Genomics, 19(3), 391e407. Small RNA networking: host-microbe interaction in food crops 289 Du, Z., Chen, A., Chen, W., Westwood, J. H., Baulcombe, D. C., & Carr, J. P. (2014). Using a viral vector to reveal the role of microRNA159 in disease symptom induction by a severe strain of Cucumber mosaic virus. Plant Physiology, 164(3), 1378e1388. Dutta, S., Afreen, U., Kumar, M., & Mukhopadhyay, K. (2020). Small RNA in tolerating various biotic stresses. In Plant small RNA (pp. 155e176). Academic Press. Dutta, S., Jha, S. K., Prabhu, K. V., Kumar, M., & Mukhopadhyay, K. (2019). Leaf rust (Puccinia triticina) mediated RNAi in wheat (Triticum aestivum L.) prompting host susceptibility. Functional & Integrative Genomics, 19(3), 437e452. Dutta, S., Kumar, D., Jha, S., Prabhu, K. V., Kumar, M., & Mukhopadhyay, K. (2017). Identification and molecular characterization of a trans-acting small interfering RNA producing locus regulating leaf rust responsive gene expression in wheat (Triticum aestivum L.). Planta, 246(5), 939e957. de Felippes, F. F. (2019). Gene regulation mediated by microRNA-triggered secondary small RNAs in plants. Plants, 8(5), 112. Flor, H. H. (1942). Inheritance of pathogenicity in Melampsora lini. Phytopathology, 32, 653e669. Flor, H. H. (1971). Current status of the gene-for-gene concept. Annual Review of Phytopathology, 9(1), 275e296. Griffiths-Jones, S., Saini, H. K., Van Dongen, S., & Enright, A. J. (2007). miRBase: tools for microRNA genomics. Nucleic Acids Research, 36(Suppl. 1), D154eD158. Guo, C., Li, L., Wang, X., & Liang, C. (2015). Alterations in siRNA and miRNA expression profiles detected by deep sequencing of transgenic rice with siRNAmediated viral resistance. PLoS One, 10(1), e0116175. He, X. F., Fang, Y. Y., Feng, L., & Guo, H. S. (2008). Characterization of conserved and novel microRNAs and their targets, including a TuMV-induced TIReNBSeLRR class R gene-derived novel miRNA in Brassica. FEBS Letters, 582(16), 2445e2452. Jiao, Y., Zhao, X., Hao, K., Gao, X., Xing, D., Wang, Z., An, M., Xia, Z., & Wu, Y. (2022). Characterization of small interfering RNAs derived from pepper mild mottle virus in infected pepper plants by high-throughput sequencing. Virus Research, 307, 198607. Jin, Y., Zhao, J. H., Zhao, P., Zhang, T., Wang, S., & Guo, H. S. (2019). A fungal milRNA mediates epigenetic repression of a virulence gene in Verticillium dahliae. Philosophical Transactions of the Royal Society B, 374(1767), 20180309. Jodder, J., Basak, S., Das, R., & Kundu, P. (2017). Coherent regulation of miR167a biogenesis and expression of auxin signaling pathway genes during bacterial stress in tomato. Physiological and Molecular Plant Pathology, 100, 97e105. Johnston, A. W., Todd, J. D., Curson, A. R., Lei, S., Nikolaidou-Katsaridou, N., Gelfand, M. S., & Rodionov, D. A. (2007). Living without Fur: The subtlety and complexity of iron-responsive gene regulation in the symbiotic bacterium Rhizobium and other a-proteobacteria. Biometals, 20(3), 501e511. Kafle, A., Garcia, K., Peta, V., Yakha, J., Soupir, A., & Bücking, H. (2018). Beneficial plant microbe interactions and their effect on nutrient uptake, yield, and stress resistance of soybeans. In Soybean-biomass, yield and productivity. IntechOpen. Kaja, E., Szczesniak, M. W., Jensen, P. J., Axtell, M. J., McNellis, T., & Makałowska, I. (2015). Identification of apple miRNAs and their potential role in fire blight resistance. Tree Genetics & Genomes, 11(1), 812. Kamthan, A., Chaudhuri, A., Kamthan, M., & Datta, A. (2015). Small RNAs in plants: Recent development and application for crop improvement. Frontiers of Plant Science, 6, 208. Katiyar-Agarwal, S., Gao, S., Vivian-Smith, A., & Jin, H. (2007). A novel class of bacteriainduced small RNAs in Arabidopsis. Genes & Development, 21(23), 3123e3134. 290 Plant Small RNA in Food Crops Katiyar-Agarwal, S., & Jin, H. (2010). Role of small RNAs in host-microbe interactions. Annual Review of Phytopathology, 48, 225e246. Kerschen, A., Napoli, C. A., Jorgensen, R. A., & Müller, A. E. (2004). Effectiveness of RNA interference in transgenic plants. FEBS Letters, 566(1e3), 223e228. Kozomara, A., Birgaoanu, M., & Griffiths-Jones, S. (2019). miRBase: from microRNA sequences to function. Nucleic Acids Research, 47(D1), D155eD162. Kri
znik, M., Baebler,
S., & Gruden, K. (2020). Roles of small RNAs in the establishment of tolerant interaction between plants and viruses. Current Opinion in Virology, 42, 25e31. Kulshrestha, C., Pathak, H., Kumar, D., Dave, S., & Sudan, J. (2020). Elucidating micro RNAs role in different plant-pathogen interactions. Molecular Biology Reports, 47, 1e9. Kumar, D., Dutta, S., Singh, D., Prabhu, K. V., Kumar, M., & Mukhopadhyay, K. (2017). Uncovering leaf rust responsive miRNAs in wheat (Triticum aestivum L.) using highthroughput sequencing and prediction of their targets through degradome analysis. Planta, 245(1), 161e182. Lee, R. C., Feinbaum, R. L., & Ambros, V. (1993). The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell, 75(5), 843e854. Lee, H. C., Li, L., Gu, W., Xue, Z., Crosthwaite, S. K., Pertsemlidis, A., Lewis, Z. A., Freitag, M., Selker, E. U., Mello, C. C., & Liu, Y. (2010). Diverse pathways generate microRNA-like RNAs and Dicer-independent small interfering RNAs in fungi. Molecular Cell, 38(6), 803e814. Lelandais-Brière, C., Naya, L., Sallet, E., Calenge, F., Frugier, F., Hartmann, C., Gouzy, J., & Crespi, M. (2009). Genome-wide Medicago truncatula small RNA analysis revealed novel microRNAs and isoforms differentially regulated in roots and nodules. The Plant Cell Online, 21(9), 2780e2796. Leonetti, P., Ghasemzadeh, A., Consiglio, A., Gursinsky, T., Behrens, S. E., & Pantaleo, V. (2021). Endogenous activated small interfering RNAs in virus-infected Brassicaceae crops show a common host gene-silencing pattern affecting photosynthesis and stress response. New Phytologist, 229(3), 1650e1664. Li, W., Jia, Y., Liu, F., Wang, F., Fan, F., Wang, J., Zhu, J., Xu, Y., Zhong, W., & Yang, J. (2019). Integration analysis of small RNA and degradome sequencing reveals microRNAs responsive to Dickeya zeae in resistant rice. International Journal of Molecular Sciences, 20(1), 222. Lindström, K., & Mousavi, S. A. (2020). Effectiveness of nitrogen fixation in rhizobia. Microbial Biotechnology, 13(5), 1314e1335. Li, F., Pignatta, D., Bendix, C., Brunkard, J. O., Cohn, M. M., Tung, J., Sun, H., Kumar, P., & Baker, B. (2012). MicroRNA regulation of plant innate immune receptors. Proceedings of the National Academy of Sciences, 109(5), 1790e1795. Liu, X., Huang, S., & Xie, H. (2021). Advances in the regulation of plant development and stress response by miR167. Frontiers in Bioscience, 26(9), 655e665. Liu, H., Jia, S., Shen, D., Liu, J., Li, J., Zhao, H., Han, S., & Wang, Y. (2012). Four AUXIN RESPONSE FACTOR genes downregulated by microRNA167 are associated with growth and development in Oryza sativa. Functional Plant Biology, 39(9), 736e744. Liu, N., Wu, S., Van Houten, J., Wang, Y., Ding, B., Fei, Z., Clarke, T. H., Reed, J. W., & Van Der Knaap, E. (2014). Down-regulation of AUXIN RESPONSE FACTORS 6 and 8 by microRNA 167 leads to floral development defects and female sterility in tomato. Journal of Experimental Botany, 65(9), 2507e2520. Llave, C., Xie, Z., Kasschau, K. D., & Carrington, J. C. (2002). Cleavage of Scarecrow-like mRNA targets directed by a class of Arabidopsis miRNA. Science, 297(5589), 2053e2056. Lu, S, Sun, Y. H., Amerson, H., Chiang, V. L., et al. (2007). MicroRNAs in loblolly pine (Pinus taeda L.) and their association with fusiform rust gall development. The Plant Journal, 51(6), 1077e1098. Small RNA networking: host-microbe interaction in food crops 291 Mallory, A. C., & Vaucheret, H. (2006). Erratum: Functions of microRNAs and related small RNAs in plants. Nature Genetics, 38(7), 850. Mansfield, J., Genin, S., Magori, S., Citovsky, V., Sriariyanum, M., Ronald, P., Dow, M. A. X., Verdier, V., Beer, S. V., Machado, M. A., & Toth, I. A. N. (2012). Top 10 plant pathogenic bacteria in molecular plant pathology. Molecular Plant Pathology, 13(6), 614e629. Martin, G. B., Bogdanove, A. J., & Sessa, G. (2003). Understanding the functions of plant disease resistance proteins. Annual Review of Plant Biology, 54(1), 23e61. Mitter, N., Worrall, E. A., Robinson, K. E., Li, P., Jain, R. G., Taochy, C., Fletcher, S. J., Carroll, B. J., Lu, G. M., & Xu, Z. P. (2017). Clay nanosheets for topical delivery of RNAi for sustained protection against plant viruses. Nature Plants, 3(2), 1e10. Moldovan, D., Spriggs, A., Yang, J., Pogson, B. J., Dennis, E. S., & Wilson, I. W. (2010). Hypoxia-responsive microRNAs and trans-acting small interfering RNAs in Arabidopsis. Journal of Experimental Botany, 61(1), 165e177. Morin, R. D., Aksay, G., Dolgosheina, E., Ebhardt, H. A., Magrini, V., Mardis, E. R., Sahinalp, S. C., & Unrau, P. J. (2008). Comparative analysis of the small RNA transcriptomes of Pinus contorta and Oryza sativa. Genome Research, 18(4), 571e584. Mueth, N. A., Ramachandran, S. R., & Hulbert, S. H. (2015). Small RNAs from the wheat stripe rust fungus (Puccinia striiformis f. sp. tritici). BMC Genomics, 16(1), 1e16. Navarro, L., Dunoyer, P., Jay, F., Arnold, B., Dharmasiri, N., Estelle, M., Voinnet, O., & Jones, J. D. (2006). A plant miRNA contributes to antibacterial resistance by repressing auxin signaling. Science, 312(5772), 436e439. Nimchuk, Z., Eulgem, T., Holt, B. F., III, & Dangl, J. L. (2003). Recognition and response in the plant immune system. Annual Review of Genetics, 37(1), 579e609. Niu, Q. W., Lin, S. S., Reyes, J. L., Chen, K. C., Wu, H. W., Yeh, S. D., & Chua, N. H. (2006). Expression of artificial microRNAs in transgenic Arabidopsis thaliana confers virus resistance. Nature Biotechnology, 24(11), 1420e1428. Nürnberger, T., Brunner, F., Kemmerling, B., & Piater, L. (2004). Innate immunity in plants and animals: Striking similarities and obvious differences. Immunological Reviews, 198(1), 249e266. Park, W., Li, J., Song, R., Messing, J., & Chen, X. (2002). CARPEL FACTORY, a Dicer homolog, and HEN1, a novel protein, act in microRNA metabolism in Arabidopsis thaliana. Current Biology, 12(17), 1484e1495. Pérez-Quintero, Á. L., Quintero, A., Urrego, O., Vanegas, P., & López, C. (2012). Bioinformatic identification of cassava miRNAs differentially expressed in response to infection by Xanthomonas axonopodis pv. manihotis. BMC Plant Biology, 12(1), 1e11. Qiao, L., Lan, C., Capriotti, L., Ah-Fong, A., Sanchez, J. N., Hamby, R., … Jin, H. (2021). Spray-induced gene silencing for disease control is dependent on the efficiency of pathogen RNA uptake. BioRxiv, 19, 1756e1768. Qu, J., Ye, J., & Fang, R. (2007). Artificial microRNA-mediated virus resistance in plants. Journal of Virology, 81(12), 6690e6699. Ramachandran, S. R., Mueth, N. A., Zheng, P., & Hulbert, S. H. (2020). Analysis of miRNAs in two wheat cultivars infected with Puccinia striiformis f. sp. tritici. Frontiers of Plant Science, 10, 1574. Reinhart, B. J., Weinstein, E. G., Rhoades, M. W., Bartel, B., & Bartel, D. P. (2002). MicroRNAs in plants. Genes & Development, 16(13), 1616e1626. Reynoso, M. A., Blanco, F. A., Bailey-Serres, J., Crespi, M., & Zanetti, M. E. (2013). Selective recruitment of m RNA s and mi RNA s to polyribosomes in response to rhizobia infection in Medicago truncatula. The Plant Journal, 73(2), 289e301. Scholthof, K. B. G., Adkins, S., Czosnek, H., Palukaitis, P., Jacquot, E., Hohn, T., Hohn, B., Saunders, K., Candresse, T., Ahlquist, P., & Hemenway, C. (2011). Top 10 plant viruses in molecular plant pathology. Molecular Plant Pathology, 12(9), 938e954. 292 Plant Small RNA in Food Crops Sharma, N., & Prasad, M. (2020). Silencing AC1 of Tomato leaf curl virus using artificial microRNA confers resistance to leaf curl disease in transgenic tomato. Plant Cell Reports, 39(11), 1565e1579. Shen, D., Suhrkamp, I., Wang, Y., Liu, S., Menkhaus, J., Verreet, J. A., Fan, L., & Cai, D. (2014). Identification and characterization of micro RNA s in oilseed rape (Brassica napus) responsive to infection with the pathogenic fungus Verticillium longisporum using Brassica AA (Brassica rapa) and CC (Brassica oleracea) as reference genomes. New Phytologist, 204(3), 577e594. Shiu, S. H., & Bleecker, A. B. (2003). Expansion of the receptor-like kinase/Pelle gene family and receptor-like proteins in Arabidopsis. Plant Physiology, 132(2), 530e543. Shivaprasad, P. V., Chen, H. M., Patel, K., Bond, D. M., Santos, B. A., & Baulcombe, D. C. (2012). A microRNA superfamily regulates nucleotide binding siteeleucine-rich repeats and other mRNAs. The Plant Cell Online, 24(3), 859e874. Shridhar, B. S. (2012). Nitrogen fixing microorganisms. International Journal of Microbiology Research, 3(1), 46e52. Simón-Mateo, C., & García, J. A. (2006). MicroRNA-guided processing impairs Plum pox virus replication, but the virus readily evolves to escape this silencing mechanism. Journal of Virology, 80(5), 2429e2436. Singh, K., Talla, A., & Qiu, W. (2012). Small RNA profiling of virus-infected grapevines: Evidences for virus infection-associated and variety-specific miRNAs. Functional & Integrative Genomics, 12(4), 659e669. Singh, A., Taneja, J., Dasgupta, I., & Mukherjee, S. K. (2015). Development of plants resistant to tomato geminiviruses using artificial trans-acting small interfering RNA. Molecular Plant Pathology, 16(7), 724e734. Subramanian, S., Fu, Y., Sunkar, R., Barbazuk, W. B., Zhu, J. K., & Yu, O. (2008). Novel and nodulation-regulated microRNAs in soybean roots. BMC Genomics, 9(1), 1e14. Sun, G. (2012). MicroRNAs and their diverse functions in plants. Plant Molecular Biology, 80(1), 17e36. Sunkar, R., Girke, T., & Zhu, J. K. (2005). Identification and characterization of endogenous small interfering RNAs from rice. Nucleic Acids Research, 33(14), 4443e4454. Su, Y., Zhang, Y., Huang, N., Liu, F., Su, W., Xu, L., Ahmad, W., Wu, Q., Guo, J., & Que, Y. (2017). Small RNA sequencing reveals a role for sugarcane miRNAs and their targets in response to Sporisorium scitamineum infection. BMC Genomics, 18(1), 1e19. Taning, C. N., Arpaia, S., Christiaens, O., Dietz-Pfeilstetter, A., Jones, H., Mezzetti, B., … Smagghe, G. (2020). RNA-based biocontrol compounds: Current status and perspectives to reach the market. Pest Management Science, 76(3), 841e845. Thiebaut, F., Rojas, C. A., Grativol, C., Motta, M. R., Vieira, T., Regulski, M., Martienssen, R. A., Farinelli, L., Hemerly, A. S., & Ferreira, P. C. (2014). Genomewide identification of microRNA and siRNA responsive to endophytic beneficial diazotrophic bacteria in maize. BMC Genomics, 15(1), 1e18. Van Vu, T., Choudhury, N. R., & Mukherjee, S. K. (2013). Transgenic tomato plants expressing artificial microRNAs for silencing the pre-coat and coat proteins of a begomovirus, Tomato leaf curl New Delhi virus, show tolerance to virus infection. Virus Research, 172(1e2), 35e45. Vidaver, A. K., & Lambrecht, P. A. (2004). Bacteria as plant pathogens. The Plant Health Instructor, 10. Wang, Y., Li, P., Cao, X., Wang, X., Zhang, A., & Li, X. (2009). Identification and expression analysis of miRNAs from nitrogen-fixing soybean nodules. Biochemical and Biophysical Research Communications, 378(4), 799e803. Wang, M., Thomas, N., & Jin, H. (2017). Cross-kingdom RNA trafficking and environmental RNAi for powerful innovative pre-and post-harvest plant protection. Current Opinion in Plant Biology, 38, 133e141. Small RNA networking: host-microbe interaction in food crops 293 Wang, M., Weiberg, A., Lin, F. M., Thomma, B. P., Huang, H. D., & Jin, H. (2016). Bidirectional cross-kingdom RNAi and fungal uptake of external RNAs confer plant protection. Nature Plants, 2(10), 1e10. Weiberg, A., Wang, M., Lin, F. M., Zhao, H., Zhang, Z., Kaloshian, I., Huang, H. D., & Jin, H. (2013). Fungal small RNAs suppress plant immunity by hijacking host RNA interference pathways. Science, 342(6154), 118e123. Wei, R., Qiu, D., Wilson, I. W., Zhao, H., Lu, S., Miao, J., Feng, S., Bai, L., Wu, Q., Tu, D., & Ma, X. (2015). Identification of novel and conserved microRNAs in Panax notoginseng roots by high-throughput sequencing. BMC Genomics, 16(1), 1e10. Wei, L., Zhang, D., Xiang, F., & Zhang, Z. (2009). Differentially expressed miRNAs potentially involved in the regulation of defense mechanism to drought stress in maize seedlings. International Journal of Plant Sciences, 170(8), 979e989. Werner, B. T., Gaffar, F. Y., Schuemann, J., Biedenkopf, D., & Koch, A. M. (2020). RNAspray-mediated silencing of Fusarium graminearum AGO and DCL genes improve barley disease resistance. Frontiers of Plant Science, 11, 476. Xie, Z., Johansen, L. K., Gustafson, A. M., Kasschau, K. D., Lellis, A. D., Zilberman, D., Jacobsen, S. E., Carrington, J. C., & Weigel, D. (2004). Genetic and functional diversification of small RNA pathways in plants. PLoS Biology, 2(5), e104. Yu, X., Gong, H., Cao, L., Hou, Y., & Qu, S. (2020). MicroRNA397b negatively regulates resistance of Malus hupehensis to Botryosphaeria dothidea by modulating MhLAC7 involved in lignin biosynthesis. Plant Science, 292, 110390. Zhang, Q., Li, Y., Zhang, Y., Wu, C., Wang, S., Hao, L., Wang, S., & Li, T. (2017). MdmiR156ab and Md-miR 395 target WRKY transcription factors to influence apple resistance to leaf spot disease. Frontiers of Plant Science, 8, 526. Zhang, S., Shi, W., Chen, Y., Xu, Z., Zhu, J., Zhang, T., Huang, W., Ni, R., Lu, C., & Zhang, X. (2015). Overexpression of SYF2 correlates with enhanced cell growth and poor prognosis in human hepatocellular carcinoma. Molecular and Cellular Biochemistry, 410(1), 1e9. Zhang, X., Zhao, H., Gao, S., Wang, W. C., Katiyar-Agarwal, S., Huang, H. D., Raikhel, N., & Jin, H. (2011). Arabidopsis Argonaute 2 regulates innate immunity via miRNA393*-mediated silencing of a Golgi-localized SNARE gene, MEMB12. Molecular Cell, 42(3), 356e366. Zhang, T., Zhao, Y. L., Zhao, J. H., Wang, S., Jin, Y., Chen, Z. Q., Fang, Y. Y., Hua, C. L., Ding, S. W., & Guo, H. S. (2016). Cotton plants export microRNAs to inhibit virulence gene expression in a fungal pathogen. Nature Plants, 2(10), 1e6. Zhou, J., Fu, Y., Xie, J., Li, B., Jiang, D., Li, G., & Cheng, J. (2012). Identification of microRNA-like RNAs in a plant pathogenic fungus Sclerotinia sclerotiorum by highthroughput sequencing. Molecular Genetics and Genomics, 287(4), 275e282. This page intentionally left blank CHAPTER 11 Small RNAs involved in salt stress tolerance of food crops Zahra-Sadat Shobbara, Nazanin Amirbakhtiarb, Raheleh Mirdar Mansuria, Fatemeh Lonia, Alireza Akbaria and Mahboube Sasaninezhada a Department of Systems Biology, Agricultural Biotechnology Research Institute of Iran (ABRII), Agricultural Research Education and Extension Organization (AREEO), Karaj, Iran; bGenetic Research Department, Seed and Plant Improvement Institute, Agricultural Research, Education and Extension Organization, Karaj, Iran 1. Salt stress is a main abiotic stress restricting growth and production of crops Salt stress is one of the most important constraints for crop production in the world (Rasool et al., 2013, pp. 1e24). It is reported that over 80 million hectares of agricultural land (40% of entire irrigated land) are influenced by salinization in the world, and it is expected that saline soils will increase up to more than 50% because of irrigation and climate change by 2050 (Parmar et al., 2020). Salinity stress has adverse effects on plants, such as osmotic stress, ion toxicity, nutrient limitations and oxidative stress, which lead to restrictions on plant growth and development (Shrivastava & Kumar, 2015). Plants have developed numerous tactics to deal with salinity stress. These strategies comprise a series of signal transduction pathways engaged in different actions ranging from salinity sensing to the expression of salt-responsive genes, which regulate processes comprising ion transport, osmotic homeostasis and detoxification (Zhao et al., 2021). Because of the disruptive effect of abiotic stress on normal cellular functions, a rapid and inclusive reprogramming at the molecular level is needed to respond to these disruptions (Megha et al., 2018). This reprogramming is the outcome of the expression regulation of the stress responsive genes at transcriptional, post-transcriptional and translational levels (Amirbakhtiar et al., 2021; Lu et al., 2011; Zhao et al., 2021). In recent years, researchers have recognized a large number of noncoding RNAs (ncRNAs) involved in salt stress response, including long non-coding RNAs (lncRNAs) and small RNAs (sRNAs) (Huang et al., Plant Small RNA in Food Crops ISBN 978-0-323-91722-3 https://doi.org/10.1016/B978-0-323-91722-3.00003-8 © 2023 Elsevier Inc. All rights reserved. 295 296 Plant Small RNA in Food Crops 2019; Song et al., 2019; Yuan et al., 2019). MicroRNAs (miRNAs), which are abundant, endogenous, small non-coding RNA molecules act as the key players in gene regulation networks by transcript degradation or translational repression (Bartel, 2004). MicroRNAs are considered as the main regulators of gene expression in eukaryotes, since each miRNA can control expression of many genes, and together they organize a miRNAgene(s) network. There are several such miRNA-gene regulatory networks in plants related to one or more physiological processes. In plants, miRNA adjusts the developmental processes and responses to biotic and abiotic stresses using an endogenous gene silencing mechanism (Singh et al., 2018). Engagement of miRNAs in salt stress response and tolerance has been reported in various plants (Liu et al., 2021; Sun et al., 2015; Xie et al., 2017; Yaish et al., 2015; Yang et al., 2020; Zhang et al., 2020). Microarray and small RNA-sequencing are the two most widely used methods for identification of salt-responsive miRNAs in plants (Carnavale Bottino et al., 2013; Ding et al., 2009; Eren et al., 2015; Kohli et al., 2014; Tian et al., 2014). Plants can deal with osmotic stress and stabilize dehydrated enzymes and membranes by accumulating compatible solutes such as sucrose, trehalose, raffinose and fructans under salt stress (Van den Ende & Peshev, 2013, pp. 285e307; Yang & Guo, 2018). Some salt-responsive miRNAs target genes engaged in synthetizing compatible solutes (Han et al., 2018). Ion toxicity is the other adverse effect of salt stress which is the result of disrupted ion homeostasis and ion imbalance in plants in response to salt stress (Guo et al., 2020). Consequently, the reconstruction of ion homeostasis is of great importance for improving salt tolerance. Some genes involved in ion homeostasis like potassium transporter KUP3p (targeted by miR414) and potassium channel akt2-like (targeted by pda-miR535) are regulated by miRNAs under salt stress (Sun et al., 2015; J. Xie et al., 2015). Moreover, high salinity induces oxidative stress due to generation of reactive oxygen species (ROSs) (Isayenkov & Maathuis, 2019). Involvement of miRNAs in targeting genes dealing with oxidative stress caused by salinity has been reported in various studies. Targeting copper/zinc superoxide dismutase by miR398A in flax and NADH dehydrogenase 1 and cytochrome P450 by novel miRNA-52 and miR1131, respectively, in bread wheat, indicates the role of miRNAs in regulating genes associated with ROS homeostasis (Han et al., 2018; He et al., 2021; Yu et al., 2016). Reversible epigenetic modifications help plants to adjust their transcriptome in a way to survive stress. Some epigenetic modification-related genes such as genes encoding Small RNAs involved in salt stress tolerance of food crops 297 histone variants, histone acetyltransferase and chromatin structure remodeling factors are targeted by miRNAs under salinity stress (Han et al., 2018). So, the reported salt-responsive small RNAs in food crops (Table 11.1), their regulatory networks and their possible role in salt tolerance improvement are discussed in this chapter. 2. Salt-responsive small RNAs are identified in different food crops 2.1 Rice (Oryza sativa) Rice is categorized as the most salt sensitive crop in both seedling and reproductive stages (Mirdar Mansuri et al., 2020). Consequently, salt stress is one of the main reasons of rice losses in the world (Parmar et al., 2020). This is despite the fact that rice is a crucial crop that provides a main source of calories for many people (Xing & Zhang, 2010). Hence, the rising saline cultivable land is in conflict with the demand for rice yield in future, which must drive up from the current production of 500 million tons to 800 million tons (Virk et al., 2004) to supply the demand of 9.6 billion people by 2050 (http://www.fao.org). Many studies have been performed to understand the molecular mechanisms of salt stress response in rice as a well-studied model organism (Quan et al., 2018; Walia et al., 2005; Zhang et al., 2018), but the findings of these studies have chiefly focused on identification of salt stress responsive protein-coding genes. Although numerous genes coding regulator and effector proteins have been identified, such as genes encoding proteins involved in ion homeostasis, oxidative stress and transcriptional regulation, rice breeding programs for the achievement of salt tolerant varieties have not been successful until now. Over the last years, several studies have shown that sRNAs such as microRNAs (miRNAs) act as important players responding to salt stress in rice (Ai et al., 2021; Barrera-Figueroa et al., 2012; Huang et al., 2019). These achievements revealed that sRNAs as crucial epigenetic factors regulate expression of genes responding to salt stress (Kumar et al., 2018; Lotfi et al., 2017). To determine salt stress-responsive miRNAs, the expression profiles of miRNAs were subsequently compared in several rice cultivars under normal and salt stress conditions (Barrera-Figueroa et al., 2012; Huang et al., 2019; Jeong et al., 2011; Sunkar et al., 2008; Tripathi et al., 2018). In this context, several novel and conserved salt-responsive miRNAs have been found in rice (Table 11.1). They reported the major part of salt-responsive miRNAs were down-regulated in rice Table 11.1 Reported salt-responsive small RNAs in food crops. Conserved/ known small RNA# Novel small RNA# Differentially expressed small RNA under salinity # Approach Threshold for responsive miRNAs Plant References Oryza sativa (Rice) BarreraFigueroa et al. (2012) 149 miRNAs 67 miRNAs 10 miRNAs (9 downregulated, 1 up-regulated) Deep sequencing of small RNAs |Log2(FC)| 1 Oryza sativa (Rice) Huang et al. (2019) 545 miRNAs 648 miRNAs Sunkar et al. (2008) 242 miRNAs 23 miRNAs Oryza sativa (Rice) Tripathi et al. (2018) 357 miRNAs 87 miRNAs Oryza sativa (Rice) Parmar et al. (2020) 25 miRNAs in root, 39 miRNAs in leaf 236 miRNAs in root, 318 miRNAs in leaf Deep sequencing of small RNAs Deep sequencing of small RNAs Deep sequencing of small RNAs Deep sequencing of small RNAs |Log2(FC)| 2 Oryza sativa (Rice) 156 miRNAs (52 downregulated, 104 up-regulated) __ 29 miRNAs (18 downregulated, 11 up-regulated) __ Tissue Fluorescence tissues (rachis, branches, spikelets) Root Salt stress treatment 400 mM NaCl/24 h 150 mM NaCl/12 h __ Seedling 150 mM NaCl/24 h |Log2(FC)| 2 Leaf 200 mM NaCl __ Root, leaf 256 mM/9 h Triticum aestivum (Bread wheat) Eren et al. (2015) __ __ Triticum aestivum (Bread wheat) He et al. (2021) 108 miRNAs 579 miRNAs Triticum aestivum (Bread wheat) Han et al. (2018) Not reported Not reported Triticum aestivum (Bread wheat) Pandey et al. (2014) 47 miRNAs 49 miRNAs 18 miRNAs in salt sensitive cultivar (7 downregulated, 11 up-regulated), 29 miRNAs in salt tolerant cultivar (12 downregulated, 17 up-regulated) 210 miRNAs (151 downregulated, 59 up-regulated) 195 miRNAs (98 downregulated, 97 up-regulated) 15 miRNAs (12 downregulated, 2 up-regulated, 1 up-regulated in low salt concentration and downregulated in high salt concentration) miRNAmicroarray analysis |Log2(FC)| > 1.45, P  0.05 Root 200 mM NaCl/48 h Deep sequencing of small RNAs Deep sequencing of small RNAs Deep sequencing of small RNAs |Log2(FC)|  1, FDR 0 Root 200 mM NaCl/24 h |Log2(FC)|  1, FDR  0.001 Root 200 mM NaCl/24 h __ Shoot, root, leaf, spikelet, seedling 150, 250 mM NaCl/3, 6, 12, 24 h Continued Table 11.1 Reported salt-responsive small RNAs in food crops.dcont'd Conserved/ known small RNA# Novel small RNA# Differentially expressed small RNA under salinity # Plant References Triticum aestivum (Bread wheat) Agharbaoui et al. (2015) __ __ 55 miRNAs Zea mays (Maize) Fu et al. (2017) Ding et al. (2009) 200 miRNAs in leaf, 150 miRNAs in root, 76 miRNAs in leaf and root __ 8 miRNAs (4 downregulated, 4 up-regulated) Zea mays (Maize) 1040 miRNAs (278 miRNAs in root, 314 miRNAs in leaf) 98 miRNAs Hordeum vulgare (Barley) Kuang et al. (2019) 243 miRNAs 124 miRNAs Hordeum vulgare (Barley) Deng et al. (2015) 142 miRNAs 10 miRNAs 7 miRNAs (4 downregulated, 3 up-regulated) 160 miRNAs (106 downregulated, 54 up-regulated) 55 miRNAs (30 upregulated and 25 downregulated) Approach Threshold for responsive miRNAs Tissue Salt stress treatment |Log2(FC)| > 2, FDR  0.05 Aerial part |Log2(FC)|  1, P  0.05 Leaf, root miRNAmicroarray analysis P  0.01 Root 200 mM NaCl/0.5, 5, 24 h Deep sequencing of small RNAs Deep sequencing of small RNAs |Log2(FC)|  0.5 Leaf, root 100 mM/ 7 days |Log2(FC)|  1, P < 0.00001 Seedling 100 mmol/L NaCl/3, 27 h Deep sequencing of small RNAs Deep sequencing of small RNAs 200 mM NaCl/1, 3, 5, 7, 12, 15 days 250 mM NaCl/12 h Hordeum vulgare (Barley) Lv et al. (2012) 126 miRNAs 133 miRNAs Pennisetum glaucum (Pearl millet) Shinde et al. (2020) 81 miRNAs 14 miRNAs Setaria viridis (Green foxtail) Pegler et al. (2020) 217 miRNAs 118 miRNA Eleusine coracana (Finger millet) Jagadeesh Selvam et al. (2015) 48 miRNAs 35 miRNAs Vicia faba (Broad bean) Alzahrani et al. (2019) 527 miRNAs in Hassawi-3, 693 miRNAs in ILB4347 35 miRNAs Glycine max (Soybean) Sun et al. (2016) 71 miRNAs 22 miRNAs 6 miRNAs (1 up-regulated, 5 downregulated) 38 miRNAs (21 downregulated, 17 up-regulated) 33 miRNAs (12 downregulated, 21 up-regulated) 28 miRNAs (17 downregulated, 11 up-regulated) 527 miRNAs (284 upregulated and 243 downregulated) in Hassawi-3, 693 miRNAs (298 up-regulated, 395 downregulated) in ILB4347 6 miRNAs (2 up-regulated, 4 downregulated) Deep sequencing of small RNAs Deep sequencing of small RNAs Deep sequencing of small RNAs Deep sequencing of small RNAs Deep sequencing of small RNAs Deep sequencing of small RNAs __ Seedling, root, leaf 200 mM NaCl/ 15 days |Log2(FC)| > 2, P  0.01 Leaf 250 mM NaCl/ 18 days P  0.05 Root, shoot 150 mM NaCl/7 days __ Leaf 300 mM NaCl/ 21 days |Log2(FC)|  1, FDR  0.001 Seedling 150 mM NaCl |Log2(FC)| > 0.1, FDR < 0.01 Root 75 mM NaCl Continued Table 11.1 Reported salt-responsive small RNAs in food crops.dcont'd Novel small RNA# Differentially expressed small RNA under salinity # 122 miRNAs 59 miRNAs Not reported Ma et al. (2021) 128 miRNAs Not reported Glycine max (Soybean) Dong et al. (2013) 220 miRNAs in the NSN, 194 miRNAs in the SSN 116 novel miRNAs in the NSN, 94 novel miRNAs in the SSN Glycine max (Soybean) Wang et al. (2020) 253 miRNAs 38 miRNAs 29 miRNAs (16 downregulated, 13 up-regulated) 47 and 46 miRNAs respectively revealed increased or decreased expression in the SSN compared with that in the NSN 31 miRNAs (29 upregulated) Plant References Conserved/ known small RNA# Cicer arietinum (Chickpea) Kohli et al. (2014) Medicago sativa (Alfalfa) Threshold for responsive miRNAs Tissue Deep sequencing of small RNAs Deep sequencing of small RNAs Deep sequencing of small RNAs __ Root 150 mM NaCl/12 h |Log2(FC)|  1, P < 0.05) Leaf 250 mM/ 72 h __ Root 125 mM NaCl/6 h Deep sequencing of small RNAs |Log2(FC)| > 1, P < 0.0001 Root 80 mmol/L NaCl Approach Salt stress treatment Raphanus sativus (Radish) Sun et al. (2015) 136 miRNAs 68 miRNAs Brassica oleracea (Broccoli) Tian et al. (2014) 97 miRNAs 326 miRNAs Ipomoea batatas (Sweet potato) Yang et al. (2020) 475 miRNAs 175 miRNAs 49 known miRNAs (18 downregulated, 31 up-regulated), 22 novel miRNAs (12 downregulated, 10 up-regulated) 42 known miRNAs, 39 novel miRNAs 83 known miRNAs (61 downregulated, 22 up regulated) in leaf, 19 known miRNAs (7 downregulated, 12 up-regulated) in root, 44 novel miRNAs (15 downregulated, 29 up-regulated) in leaf, 3 novel miRNAs (2 down- Deep sequencing of small RNAs |Log2(FC)| > 0.5, P < 0.05 Root 200 mM NaCl/3, 6, 12, 24, 48, 96 h Deep sequencing of small RNAs Deep sequencing of small RNAs |Log2(FC)| >2 or  1.2 Flower 80 mM NaCl/ 15 day |Log2(FC)| > 1, P < 0.05 Root, leaf 150 mM NaCl/2 days Continued Table 11.1 Reported salt-responsive small RNAs in food crops.dcont'd Plant References Conserved/ known small RNA# Novel small RNA# Differentially expressed small RNA under salinity # Approach Threshold for responsive miRNAs Tissue Salt stress treatment regulated, 1 up-regulated) in root Cucurbita moschata, C. maxima (Pumpkin) Xie et al. (2015a) 58 miRNAs 33 miRNAs 12 known miRNAs (2 downregulated, 10 up regulated) in C. maxima, 10 novel miRNAs (4 downregulated, 6 up regulated) in C. maxima, 13 known miRNAs (0 downregulated, 13 up regulated) in C. moschata, 7 novel miRNAs (1 downregulated, 6 up regulated) in C. moschata Deep sequencing of small RNAs |Log2(FC)| 1 Root 100 mM NaCl/4 h Linum usitatissimum (Flax) Yu et al. (2016) 120 miRNAs 212 miRNAs Musa acuminata (Banana) Lee et al. (2015) 181 miRNAs 56 miRNAs Citrus junos (Yuzu) Xie et al. (2017) Not reported Not reported 101 known miRNAs (14 up-regulated, 87 dawnregulated), 56 novel miRNAs (16 upregulated, 40 dawnregulated) 43 known miRNAs, 16 novel miRNAs 41 known miRNAs (40 downregulated, 1 up-regulated), 21 novel miRNAs (18 downregulated, 3 up-regulated) Deep sequencing of small RNAs |Log2(FC)| > 1, FDR < 0.001 Seedling 500 mM NaCl/18 h Deep sequencing of small RNAs Deep sequencing of small RNAs |Log2(FC)| > 1, FDR < 0.05 |Log2(FC)|  1, Q < 0.01 Root 100 mM, 300 mM NaCl/48 h Root 300 mM NaCl/24 h Continued Table 11.1 Reported salt-responsive small RNAs in food crops.dcont'd Plant References Conserved/ known small RNA# Phoenix dactylifera (Date palm) Yaish et al. (2015) 89 miRNA variants 180 miRNAs Sesamum indicum (Sesame) Zhang et al. (2020) 351 miRNAs 91 miRNAs Novel small RNA# Differentially expressed small RNA under salinity # 30 known miRNAs (2 downregulated, 28 up regulated) in leaf, 27 novel miRNAs (17 downregulated, 10 up-regulated) in leaf, 9 known miRNAs (6 downregulated, 3 up regulated) in root, 16 novel miRNAs (0 downregulated, 16 up regulated) in root 93 known miRNAs, 23 novel miRNAs Threshold for responsive miRNAs Tissue Salt stress treatment Deep sequencing of small RNAs FDR < 0.05 Seedling, leaf, root 300 mM NaCl/72 h Deep sequencing of small RNAs P < 0.05 Seedling, shoot 150 mM NaCl/2, 6, 12, 24 h Approach Small RNAs involved in salt stress tolerance of food crops 307 (Barrera-Figueroa et al., 2012; Huang et al., 2019; Tripathi et al., 2018). Whereas it is also reported that most stress-regulated miRNAs were upregulated by drought or cold stress in rice (Barrera-Figueroa et al., 2012). Among the identified miRNAs in rice fluorescence tissues, some miRNA families such as miR159, miR160, miR319, miR394, miR528, and miR530 were also previously reported to be regulated by salt stress in Arabidopsis thaliana and Populus euphratica (Barrera-Figueroa et al., 2012; B. Li et al., 2011; Sunkar & Zhu, 2004). While miR1866, as a salt-regulated miRNA, was identified by Barrera-Figueroa et al., in rice fluorescence tissues for the first time (Barrera-Figueroa et al., 2012). Some of these identified miRNAs were shown to be regulated by diverse abiotic stresses. For example, miR394 was up-regulated by cold stress, while this miRNA was down-regulated by salt stress (Barrera-Figueroa et al., 2012). F-Box proteins were identified as the predicted target for miR394 (BarreraFigueroa et al., 2012). F-Box proteins are engaged in various developmental processes e.g., self-incompatibility, photomorphogenesis, circadian clock regulation and floral meristem and floral organ recognition (Jain et al., 2007). Further, comparative miRNA profiling of the contrasting rice genotypes revealed some specific miRNAs involved in salt stress (Huang et al., 2019; Parmar et al., 2020). It is reported that osa-miR169b, osa-miR396d, osa-miR171b, osa-miR1425-3p, osa-miR164a and osa-miR166e-3p were exclusively up-regulated in JCQ’s root (as a salt tolerant rice genotype) versus susceptible genotype (IR26) (Huang et al., 2019). It is remarkable that mRNAs encoding nuclear transcription factors (TFs) were predicted as targets for some of them, e.g., osa-miR164e and osa-miR164d (Huang et al., 2019). Also, comparative miRNA analysis between the root and shoot of Pokkali (rice salt tolerant) and Badami (rice salt-sensitive) revealed that miR167f was only down-regulated in the root of Pokkali, while it was up-regulated in both shoot and root of Badami (Parmar et al., 2020). In contrast, miR159a was down-regulated in both shoot and root in the salttolerant genotype (Pokkali), however it was down-regulated merely in Badami’s root (as a salt-sensitive genotype) (Parmar et al., 2020). The Myb transcription factor engaged in diverse biological processes, such as defense and stress responses, was predicted as a target for miR159a (Mondal et al., 2015, 2018). This result suggested that Pokkali is more tolerant than Badami, because of overexpression of the Myb transcription factors, which caused elevated salinity tolerance in several plants (Ganesan et al., 2012; He et al., 2012). 308 Plant Small RNA in Food Crops Furthermore, deep sequencing of small RNAs revealed 23 new candidate miRNAs responding to salt stress in rice; six of them, including osa-miR1436, belong to the osa-miR444 family (Sunkar et al., 2008). The osa-miR444 family was identified as conserved miRNA in several other monocots (Sunkar et al., 2008). It is reported that osa-miR444 regulates rice root growth (Jiao et al., 2020). Five homologous MADS-box transcription repressors were targeted by miR444 (Jiao et al., 2020). miR444 positively regulated brassinosteroid (BR) biosynthesis through its MADS-box targets (Jiao et al., 2020). It has been reported that a key BR biosynthetic gene, transcription of BRdeficient dwarf 1 (OsBRD1), was directly repressed by it (Jiao et al., 2020). Moreover, the signaling cascade of miR444-OsBRD1 was induced by NH4þ in rice root (Jiao et al., 2020). Overexpression of OsMADS57, as a target of miR444, showed hyposensitivity to NH4þ in root elongation, which was related to BR content reduction (Jiao et al., 2020). Intriguingly, some miRNAs were identified which are involved in ion homeostasis, such as miR827 (Jeong et al., 2011). It is reported that miR827 was strongly induced by potassium starvation under salt stress (Jeong et al., 2011). Also, miR827 is induced by cold, H2O2, and phosphate starvation (Jeong et al., 2011; T. Li, et al., 2011). These findings support that miR827 plays an important role in oxidative stress as common to all the mentioned stress conditions (Jeong et al., 2011). Genes encoding the syringaldazine peroxidase (SPX) domain were predicted as the target of miR827 (Jeong et al., 2011). However, the role of SPX domain proteins has not been identified under potassium starvation and salt stress conditions (Jeong et al., 2011). Further, overexpression of several miRNAs e.g., miR1861h, miR164b, and osa-miR820 showed enhancement of tolerance to salinity in rice (Ai et al., 2021; Jiang et al., 2019; Sharma et al., 2021). It has been clearly affirmed that miRNAs act as great actors in salt stress responses in rice. Some miRNAs such as osa-miR408, osa-miR414 and osa-miR164e, as well as their targeted genes, are only expressed in initial response to salt stress (Macovei & Tuteja, 2012), whereas others are expressed in specific tissues. These findings suggest that miRNA expression is controlled at transcriptional level (Sunkar et al., 2007). MiRNAs connect to regulatory networks, which control diverse physiological processes (Sunkar et al., 2007). Salt tolerance is a complex process in rice and controlled by multicomponent signaling pathways; while the involvement of miRNAs increases these complexities. Thus, identification and understanding of a near complete set of miRNA-target interactions will provide opportunities for researchers to improve rice tolerance to salinity. Small RNAs involved in salt stress tolerance of food crops 309 2.2 Wheat (Triticum aestivum) Wheat, as one of the most important cereals, is used by at least 40% of the population of the world (Han et al., 2018). Around 17% of the entire cultivated area is under cultivation of wheat in the world. Global warming, industry derived pollution, absence of water resources, and nonstandard irrigation has resulted in enhanced salinization of soil in the important wheat-producing zones of the world that is a serious danger for wheat production worldwide (Nadeem et al., 2020). Developing salt tolerant cultivars is the best way to deal with this problem. Wheat is relatively sensitive to salt stress. Breeding crops for salt tolerance could be quickened by clarifying the molecular foundation of the plant response to salt stress (Han et al., 2018). Eren et al. (2015) investigated the miRNA profiles of roots in two wheat cultivars of Seri-82 (salt tolerant) and Bezostaja (salt sensitive) using a miRNA-microarray analysis. All known plant miRNAs were used as probes (11862). They discovered 44 salt-responsive miRNAs in the studied cultivars. Transporters and transcription factors including bHLH135-like, AP2/ERBP and MADS-box were recognized as the target genes of salinity-responsive miRNAs. The results obtained indicated that three miRNAs of hvu-miR5049a, ppt-miR1074, and osa-miR444b.2 (out of 44 differentially regulated miRNAs) had significantly higher expression in Bezostaja in comparison with Seri-82 (Eren et al., 2015). NGS technologies have greatly fastened the discovery of abiotic stress responsive miRNAs in crop plants (Barrera-Figueroa et al., 2012). Sequencing of small RNA libraries provided from different tissues and under various abiotic stresses resulted in discovering 47 known miRNAs and 49 novel miRNAs in wheat cv “PBW343” (Pandey et al., 2014). Investigating the expression profile of some of the identified miRNAs under salt stress using the qPCR method showed that conserved miRNAs of miR156, miR160, miR164, miR166, miR167a, miR171a, miR396d and miR5139 and 9 novel miRNAs responded to salt stress. In addition, RLM-RACE method identified target genes of miR156, miR160 and miR164 as follows: SPL-like, ARF10 and NAC1, respectively (Pandey et al., 2014). Han et al., (2018) used small RNA sequencing to find salinity and alkalinity responsive miRNAs in the roots of a bread wheat cultivar (JN177) and two breeder’s lines of SR3 and SR4. SR3 and SR4 derived from an asymmetric somatic hybrid formed between JN177 and tall wheatgrass 310 Plant Small RNA in Food Crops (Thinopyrum ponticum), with SR3 being more salt tolerant and SR4 being more alkaline tolerant compared to JN177 (Liu & Xia, 2014; Meng et al., 2017; Xia et al., 2003). In JN177, 97 and 98 miRNAs showed upregulation and down-regulation, respectively, in response to salt stress. Comparing the salt stressed SR3 library (SR3S) with the salt stressed JN177 library (JNS) (SR3S/JNS) showed that 81 and 69 miRNAs were significantly up-regulated and down-regulated, respectively. Target prediction together with degradome sequencing were utilized to find miRNA targets. GO and KEGG analysis were used to define function of the target genes. GO analysis revealed that the target genes of salt-responsive miRNAs were engaged in two classes of molecular function and 8 classes of biological process, with “Binding” and “Regulation of biological process” being the most plentiful terms in molecular function and biological process categorizations, respectively. KEGG analysis indicated that salinity-responsive genes were enriched in “one carbon pool by folate”, “phenylalanine metabolism”, “sphingolipid metabolism”, “glycerophospholipid metabolism” and “glycerolipid metabolism” pathways. Eventually, auxin mediated regulation and epigenetic modifications pathways were identified as significant pathways for both salinity and alkalinity tolerance, whereas jasmonate signaling and carbohydrate metabolism were recognized as crucial pathways for salt tolerance and proton transport was identified as an important pathway for tolerance to alkalinity. In addition, the virusinduced gene silencing (VIGS) method was used to transiently overexpress two stress responsive miRNAs (tae-miR1120c and tae-miR9664) in wheat seedlings. Plants over-expressing tae-miR1120c were more tolerant to salt stress and less tolerant to alkalinity stress in comparison with plants having only an empty vector. Plants over-expressing tae-miR9664 showed an enhanced tolerance to both stresses (Han et al., 2018). He et al. (2021) discovered 8 salt tolerance-related miRNAs and their target genes in a salt tolerant bread wheat variety, QM6, using highthroughput sequencing. Tae-miR319 and tae-miR9666b-3p played role in wheat salinity tolerance through regulation of MYB transcription factor. Tae-miR1131 enhanced salt tolerance in wheat by regulating CYP450 and increasing antioxidant stress capacity of wheat. Furthermore, tae-miR1131 together with Novel_72 miRNA control POT (an important nutrient absorption transporter) (Newstead, 2017) expression and improves wheat nutrient absorption under salt stress, leading to salt tolerance. They also indicated that tae-miR9774 and tae-miR9668-5p down-regulated under salt stress whereas their target gene from the NB-ARC family up-regulated Small RNAs involved in salt stress tolerance of food crops 311 in response to salinity, which led to salt tolerance. In addition, they speculated that tae-miR1122a, which showed significant down-regulation under salinity conditions, regulated the lipid balance of wheat under salt stress via adjusting GDSL-like lipase He et al. (2021). Salt-responsive microRNAs can also be identified by applying bioinformatics tools. In this method, conserved miRNAs identified in other plants can be used as queries to search for expressed sequenced tags to identify corresponding orthologues (Singh et al., 2018). Pandey et al. (2013) used a computational approach which was a combination of bioinformatics software and perl script to discover abiotic stress induced miRNAs in wheat. They blasted all known plant miRNAs against abiotic-stressed EST libraries of wheat. They succeeded in finding 5 new and 4 known abiotic stress-responsive miRNAs in wheat. Results obtained indicated that the new miRNA of Ta-miR855 and the known miRNA of Ta-miR1133 were salt-responsive. In addition, they used UEA sRNA workbench to identify the putative target sites of identified miRNAs. Myb transcription factor and FCA -like protein were identified as potential target genes of Ta-miR855 and Ta-miR1133, respectively (Pandey et al., 2013). Agharbaoui et al. (2015) identified salt, cold, and toxic aluminum responsive miRNAs in wheat using a comprehensive miRNAome analysis including both generating and sequencing small RNA libraries and bioinformatic prediction. They succeeded in finding a sum of 165 abiotic stress responsive miRNAs, among which 55 were recognized as salt-responsive miRNAs in aerial parts of the winter wheat cultivar Clair. ApMir_20602, apMir_14769, apMir_54471, apMir_22246 (tae-miR160) and apMir_20968 (miR395-21) were identified as salt-responsive miRNAs. The results indicated that apMir_20602 was commonly regulated under cold, salt and Al and interacted with glutathione peroxidase. They suggested that apMir_20602 intermediates crosstalk between response to abiotic stresses via adjusting metabolism of glutathione (Agharbaoui et al., 2015). Furthermore, expression analysis by real-time PCR and northern blot analysis were utilized to identify salt-responsive miRNAs in wheat. Lu et al. (2011) studied the expression patterns of 32 wheat miRNAs available in the microRNA database under non-stress and salt stress conditions. Semiquantitative RT-PCR and quantitative real qRT-PCR results indicated that Ta-miR159a, Ta-miR160, Ta-miR167, Ta-miR174, Ta-miR399, Ta-miR408, Ta-miR11124 and Ta-miR1133 responded to salt stress, with an up-regulation pattern in response to salinity. They identified 2 to 7 putative target genes for the salt-inducible miRNAs (except for 312 Plant Small RNA in Food Crops Ta-miR399) by blasting against the NCBI GenBank database. They indicated that the putative target genes were engaged in different biological processes including signaling perception (e.g. RLK4 kinase interacting with Ta-miR174), protein phosphorylation (e.g. phosphoinositide-specific phospholipase C having interaction with Ta-miR159a), primary metabolism (e.g. putative pyruvate decarboxylase interacting with Ta-miR159a), phytohormone response (e.g. auxin response factor 16 interacting with TaMIR160), transcriptional regulation (e.g. putative auxin response transcription factor 6 having interaction with Ta-miR167), defensive response (e.g. putative salt-inducible protein interacting with Ta-miR1133), nucleic acid metabolism (e.g. endonuclease having interaction with Ta-miR1124), RNA editing (e.g. pentatricopeptide repeat (PPR)-containing protein interacting with Ta-miR1133), and apoptosis (e.g. NB-ARC domain protein having interaction with Ta-miR1133) and cell division (e.g. plastid division regulator MinD interacting with Ta-miR408) (Lu et al., 2011). In another study, the expression of thirty one known miRNAs were evaluated under wounding, cold and salt stresses in the wheat genotype Suwon11 using RT-qPCR and northern blot analysis. The results indicated that 8 miRNAs were responsive to high-salinity treatment based on RT-qPCR results, of which only 3 miRNAs of miRNA159, miRNA393 and miRNA171 were found as salinity responsive by northern blot analysis. They also indicated that miR159, miR393 and miR398 responded to multiple stresses. They identified MYB3 (TC368630) as a potential target of miR159 and evaluated its expression in response to cold, wounding and salinity stresses using qRT-PCR. MYB3 displayed different expression patterns in three stresses (Wang et al., 2014). Gupta et al. (2014) showed that conserved miRNAs of miR172 and miR393 up-regulated under salt stress and conserved miRNAs of miR159, miR164, miR168, miR397 and miR1029 down-regulated in response to salt stress in bread wheat, C-306 genotype by qRT-PCR. They also indicated that a novel conserved miRNA, miR855, down-regulated after 1 h exposing to salinity stress while miR855 displayed severe up-regulation at later time points (12 and 24 h). miR855 which is expressed in wheat sheath in response to salinity stress (Gupta et al., 2014), targets MYB TF that is engaged in regulating leaf development (Sun, 2012). Downregulation of miR855 after 1 h of salt exposure might be associated with activating MYB based signaling to manage salt stress, while up-regulation at later time points could be because of repressing a MYB based tolerance mechanism in the studied genotype (Gupta et al., 2014). Small RNAs involved in salt stress tolerance of food crops 313 2.3 Maize (Zea mays) High-throughput sequencing and miRNA-microarray have been employed to detect the salt stress responsive small RNAs and their target genes in maize varieties treated with different concentrations (125e200 mM) of NaCl and various time courses (0 h to 15 days) (Ding et al., 2009; Fu et al., 2017; Luan et al., 2014, Luan et al., 2015). Highthroughput sequencing led to the identification of 1040 miRNAs in Z. mays including 726 and 762 known miRNAs from roots and leaves, respectively (Fu et al., 2017). Moreover, 200 novel miRNAs were identified, including mir-29 and mir-36, which were classified as novel members of the miR167 and miR164 families, respectively. Mir-330, mir-250, mir-205 and mir-17 are some novel miRNAs, which down-regulated in the leaves or roots of maize under salinity stress; then, their target genes for including casein kinase II (CK2a), GPX, IF-1, P5CS, and some other significant genes were also revealed among the up-regulated genes. CK2a subunits have also been reported to be up-regulated by high salinity and affect diverse developmental and stress-responsive pathways in plants, such as suggested targets for miR330 including photomorphogenesis, circadian rhythms, flowering time, lateral root development, cell cycle and cell division, cell expansion, auxin signaling, seed storage and salt responses (Fu et al., 2017). Glutathione peroxidase (GPx), which are a group of antioxidant enzymes, was proposed as a target for mir-250. GPxs protect plants against oxidative stress, salt stress, and membrane damage (Matamoros et al., 2015). Further, proline is one of the most accumulated osmolytes in salinity and water deficit conditions in plants. Pyrroline-5-carboxylate synthetase (P5CS), which is a regulatory enzyme playing a crucial role in proline biosynthesis, has been suggested as a target for miR205 (Silva-Ortega et al., 2008). Translation initiation factors (IFs) play various important roles in plants and IF1 was predicted as the target for miR17, regulating gene expression under salt stress (Fu et al., 2017). The conserved miR169 miRNA family is the largest miRNA family in maize; however, only a few members have been annotated with specific functions (Luan et al., 2015). It is interesting to note that a set of the similar miRNA family were differentially expressed in response to salt and drought stress. For example, drought stress significantly repressed the expression of most zma-miR169 genes, whereas, zma-miR169 genes were up-regulated under salt stress conditions. Moreover, the expression level of zma-miR169s were not correlated with ZmNF-YA in maize leaves in response to salt stress in 314 Plant Small RNA in Food Crops contrast to those discovered previously in maize roots. Zma-miR169 expression down-regulated under salt stress in maize roots (Luan et al., 2014). Furthermore, miRNA-microarray analysis of small RNAs revealed 18 new putative miRNAs responding to salt stress in maize roots (Ding et al., 2009). It has been predicted that Myb, NAC1 and homeodomainleucine zipper protein (HD-ZIP) transcription factors would be the targets of zma-miR159a/b, zma-miR164a/b/c/d and zma-miR166l/m, respectively (Kohli et al., 2014; Shan et al., 2020). It is reported that miR164, miR156 and miR167 were down-regulated in root under salinity (Ding et al., 2009). Additionally, reduction of miR167 and miR164 lead to up-regulation of ARF8 and NAC1 improving the auxin response and consequently enhance shoot and leaf development. 2.4 Barley (Hordeum vulgare) To define miRNAs in barley plants, several efforts have also been done in recent years. Some of these studies utilized small RNA sequencing and high-throughput sequencing to define miRNA and mRNA expression profiling in root and leaf of barley in treated plants with different concentrations (100e200 mM) of NaCl with various time courses (3 h to 15 days) (Deng et al., 2015; Kuang et al., 2019; Lv et al., 2012). Deep sequencing and computational analysis led to characterization of several salt-responsive miRNAs from shoots in two barley cultivars of Golden Promise (GP) and XZ16 (Kuang et al., 2019). MYB33, which was targeted by miR159a in shoots, significantly induced in response to salt stress in both the barley genotypes. MiR159a might modulate osmotic balance and ROS scavenging through regulating MYB33. Intriguingly, NFYA5 is regulated by miR169i. NFYA5 was significantly up-regulated in shoots of XZ16 under salt stress. Nuclear transcription factor Y (NF-Y), which is an important CCAAT-binding transcription factor, has an essential role in growth, development and abiotic stress response (Kuang et al., 2019). Furthermore, differential expression of miR156d, miR164a, miR169i, miR172b, miR319a, miR393a and a novel miRNA called PC-miR124 were revealed by comparative miRNA analysis in GP and XZ16 genotypes. It has been suggested that these differentially expressed miRNAs could represent the diverse salt tolerance characterization of the two genotypes (Kuang et al., 2019). Deng et al. (2015) reported that most stress-regulated miRNAs were down-regulated by salt stress in barley. MYB transcription Small RNAs involved in salt stress tolerance of food crops 315 factor, F-box and Nodulation signaling pathway 2 proteins have been proposed as targets for miR159, miR164, miR167, miR168, miR171, miR172 and miR393 that seems to be involved in salt stress signal transduction (Deng et al., 2015). In addition, three miRNAs, including miR319, miR393 and miR396, were targeting genes involved in plant morphological modulation such as leaf and root development. Moreover, 126 conserved miRNAs from 58 families and 133 novel miRNAs belonging to 50 families responding to salt stress in barley were discovered by high-throughput sequencing of small RNAs. Lv et al. (2012), also reported the expression of MiR156d upon drought and salt stress in barley to be induced in leaves, while miR396d and miR399b, up-regulated only in drought stressed leaves (Lv et al., 2012). 2.5 Green foxtail (Setaria viridis) Several salt-responsive miRNAs have been identified from shoot and root in S. viridis (L.) Beauv accessions, A10 and ME034V, using small RNA sequencing (Pegler et al., 2020). Differential expressions of miR169, miR395, miR396, miR397, miR398 and miR408 under salinity conditions were experimentally validated using RT-qPCR. RT-qPCR was further applied to profile the molecular response of the miR160 and miR167 regulatory modules to salt stress. This analysis revealed accessionand tissue-specific responses for the miR160 and miR167 regulatory modules in A10 and ME034V shoot and root tissues exposed to salinity. The findings reported here form the first crucial step in the identification of the miRNA regulatory modules to target for molecular manipulation to determine if such modification provides S. viridis with higher salt tolerance. In shoots, 5 and 3 miRNAs were up and down-regulated in A10, and 6and 2 miRNAs were up- and down-regulated in ME034V under salt stress, respectively. In roots, totally 4 and 5 miRNAs were up- and downregulated in A10, While 6 and 2 were up- and down-regulated in ME034V under salinity conditions, respectively. 2.6 Finger millet (Eleusine coracana) Several salinity responsive miRNAs were identified from shoot in two contrasting finger millet cultivars of CO 12 (susceptible) and Trichy 1 (tolerant) using small RNA sequencing (Jagadeesh Selvam et al., 2015). Data analysis led to detection of many conserved miRNAs exhibiting significant salinity responsive differential expression in finger millet. In the 316 Plant Small RNA in Food Crops tolerant Trichy 1, some miRNAs, including miR160, miR162, miR169, miR894 and miR2914 showed more than 10 fold-up-regulation during salinity compared to the susceptible CO 12. One of the miRNAs, namely miR159, was found to be 25 fold less abundant in Trichy 1 during salinity stress, which was predicted to target key salinity tolerance related genes such as MYB transcription factor(s) and calmodulin-related calcium sensor protein. In shoots, 6 up- and 7 down-regulated miRNAs in CO 12 (susceptible) and 5 up- and 10 down-regulated miRNAs in Trichy 1 (tolerant) were detected under stress. 2.7 Pearl millet (Pennisetum glaucum) A pearl millet salinity tolerant genotype, ICMB 01222, has been used for investigation of salinity stress-related miRNA profiles. In total; small RNA sequencing detected 81 conserved and 14 novel miRNAs in pearl millet to be differentially expressed (Shinde et al., 2020). An important role in response to salinity stress has been proposed for MiR164 and its target genes (NAC92 transcription factor genes) in pearl millet. MADS-box transcription factors were identified as the predicted target for miR444c. Most of the MADS-box family members are involved in developmental processes such as flowering time, short vegetative phase and fruit formation (Parenicová et al., 2003). Whereas, previous studies have shown that a greater number of miRNAs that were down-regulated in response to salinity stress, miR156 and miR160 exhibited differential expression patterns and they were the most significantly up-regulated family. Both miR166 and miR159 families have played important roles in abiotic stress responses (Abdelrahman et al., 2018; Li et al., 2017a). 2.8 Chickpea (Cicer arietinum) Legumes are the third largest family of flowering plants, and grain legumes are fundamental section of the human plant-based foods (Chand Jha et al., 2021). Legumes are a significant source of proteins, oil, fiber, and are loaded with twice the amount of micronutrients including iron, zinc, selenium, magnesium, manganese, copper, and nickel compared to cereals, therefore legume crops serve as a necessary component for sustaining global food safety (Graham & Vance, 2003; Ohri et al., 2021). Recently, the salt stress responsive expression profiles of nine miRNAs were studied in C. arietinum. The mature miRNAs of miR160, miR166, miR169, miR171, miR172 and miR396 were selected from a prior study Small RNAs involved in salt stress tolerance of food crops 317 in Medicago (Reynoso et al., 2013). Based on the achieved results, all nine miRNAs (miR159, miR160, miR166, miR167, miR169, miR171, miR172, miR393 and miR396) were differentially expressed under salinity conditions compared to the control (Jatan et al., 2019). The expression of miR160 was significantly reduced (-2.2 fold) in salt treatment. The transcript levels of miR169 and miR396 were also decreased (-2.0 fold) at 1 h of salinity stress compared to the control while their expression gradually up-regulated up to 72 h. Prior studies were also shown that miR160 is down-regulated under abiotic stresses in Populus tomentosa under salt stress (Ren et al., 2013). It is reported that miR160 targets three auxins responsive factor (ARF) genes (ARF10, ARF16, and ARF17) (Wang et al., 2005). ARFs play a crucial role in physiological and morphological mechanisms mediated through auxins leading to stress adaptation (Tombuloglu, 2019). Down-regulation of miR169 was also reported in soybean (Ramesh et al., 2019), and Medicago (Mantri et al., 2013) under salt stress. In Arabidopsis and wheat, down-regulation of miR169 under drought stress leads to the up-regulation of NF-YA, which regulates the expression of several drought-responsive genes, leading to higher tolerance (Luan et al., 2014; Wang et al., 2011). GmNFYA3 is recognized as the target gene for miR169 in soybean. The expression of GmNFYA3 was up-regulated by ABA and abiotic stresses, including PEG, NaCl, and cold (Ni et al., 2013). The gene regulation by miRNAs under salt stresses were studied in chickpea using high-throughput deep sequencing (Kohli et al., 2014). In addition to 122 conserved miRNAs from 25 various families, 59 novel miRNAs were identified. Four legume-specific miRNAs (miR2111, miR2118, miR5232 and miR5213) were recognized in the chickpea libraries, which were formerly reported in Medicago (Jagadeeswaran et al., 2009). Furthermore, several conserved salt-responsive miRNAs were identified, which among them, miR156, miR396 and miR319 were induced by salt stress. MiR390 targeted protein kinases and the CZF1 TF, which is associated with intracellular signal transduction. MiR396 was upregulated under salt stress in the chickpea while it was also salt-responsive in rice. Furthermore, salt and alkali stress tolerances were decreased in transgenic lines over-expressing osa-mir396c compared to wild type plants (Gao et al., 2010). Moreover, the achieved results indicated that serine/threonine protein kinases and MAPK protein kinases are the target genes of miR172, miR319, miR171, miR390 and miR396, which are involved in signaling pathways. 318 Plant Small RNA in Food Crops 2.9 Soybean (Glycine max) A general analysis of the small RNA transcriptome of soybean root tips was performed under normal and salt stress conditions (Sun et al., 2016), which led to identification of 71 miRNA candidates, containing known and novel variants of 59 miRNA families (Sun et al., 2016). Also, 66 salt-responsive miRNAs were found in soybean root meristem, of which 22 were identified as novel miRNAs. Furthermore, auxin-responsive cis-elements were discovered in the promoters of lots of differentially expressed miRNAs. This means that auxin is possibly involved in regulating these miRNAs and auxin signaling exerts a crucial role in adjusting miRNAome plasticity and root development in soybean. Based on functional analysis, the salinityresponsive miRNA of miR399 has a fundamental role in adjusting developmental plasticity of soybean root (Sun et al., 2016). In addition, expression of gma-miR1512b was reduced significantly in response to salinity stress in soybean root tips, indicating the involvement of this miRNA in the development of soybean plastic root under salinity conditions. 2.10 Faba bean (Vicia faba) Two contrasting genotypes of faba bean were subjected to sRNA sequencing to identify salt-responsive miRNAs. The results indicated that 527 miRNAs in the salt-sensitive genotype and 693 miRNAs in the salttolerant genotype responded to salt stress. Comparing the salt responsive miRNAs in the investigated genotypes showed that miR166, miR171, miR398, miR396, and miR1432 were recognized as salt-responsive in both faba bean genotypes. miR171 targets MYB TFs and is involved in adjusting osmotic balance under salinity treatment (Alzahrani et al., 2019). miR396 was identified as another salt responsive miRNA in both genotypes. Based on a previous study, growth factor-like TF and its putative heat-shock protein were recognized as putative targets of miR396 (Kantar et al., 2011). 2.11 Cowpea (Vigna unguiculata) The role of miRNAs responding to salinity stress were studied cowpea (V. unguiculata) and 18 conserved miRNAs (e.g., miR160, miR156/157, miR159, miR169, miR172, miR408) were identified in the root tissues and 15 corresponding target genes were recognized as TFs (e.g., ARF, SBP, AP2, TCP) (Paul et al., 2011). Small RNAs involved in salt stress tolerance of food crops 319 2.12 Banana (Musa acuminata) Using sRNA and RNA sequencing, 59 orthologous and Musa-specific miRNAs and their corresponding targets were found in banana roots to be salt stress responsive. Comparison analysis of the expression patterns between the miRNAs and their target genes showed that the expression levels of the major part of responsive miRNAs were declined under salinity condition, while expression levels of their targets were increased. Stress signaling, stress defense, transport, cellular homeostasis and metabolism were identified as biological processes in which the target genes were engaged. Some predicted targets of salt responsive miRNAs were transcription factors such as AP2 transcription factor (interacted with miR172) and GRAS transcription factor (interacted with miR166 and miR171). In addition, transcripts encoding the heat shock protein (interacted with macmiR827), the salt responsive protein (interacted with miR159), the osmotic stress-activated protein (interacted with miR397), glutamate synthase (interacted with miR169), tropine dehydrogenase (interacted with miR156), chloride channel (interacted with miR37) and oligopeptide transporter 7-like (interacted with miR66) were also recognized as targets of differentially expressed miRNAs (Lee et al., 2015). 2.13 Date palm (Phoenix dactylifera) Utilizing sRNA sequencing in date palm resulted in finding 54 and 25 miRNAs that responded to salinity in leaves and roots, respectively. Around 70% and 76% of the responsive miRNAs were up-regulated in leaves and roots, respectively. Among the predicted targets, genes coding for potassium channel AKT2-like proteins, vacuolar protein sorting-associated protein, calcium-dependent proteins and mitogen-activated proteins, known as salt tolerance involved genes, were observed. Furthermore, the KEGG tool was employed for functional annotation of the predicted target genes. Some important pathways involved in salt tolerance like purine metabolism, glycolysis/gluconeogenesis, fructose, mannose metabolism, starch and sucrose metabolism were identified to be significantly enriched based on KEGG analysis (Yaish et al., 2015). 2.14 Yuzu (Citrus junos) sRNA-seq and RNA-seq approaches were utilized to define miRNA and mRNA expression profiling in the roots of Citrus junos Siebold cv. ‘Ziyang’ under dehydration and salt treatments. sRNA-seq analysis showed that 41 320 Plant Small RNA in Food Crops known miRNAs and 21 novel miRNAs responded to salt stress, the majority of which were down-regulated. The results indicated that 21 responsive miRNAs were found both under salt and dehydration treatments. Performing KEGG enrichment analysis showed that the target genes were engaged in the pathways relating to plant hormone signal transduction, oxidative phosphorylation, ascorbate and aldarate metabolism, flavonoid biosynthesis and phenylalanine metabolism under salinity and dehydration conditions while tryptophan metabolism, propanoate metabolism and fatty acid metabolism were uniquely found as dominant pathways responding to salt stress. In fact, molecular players responding to drought and salt stresses were partially overlapping. It was proposed that the roots of citrus regulates transcription factors, which then integrate carbohydrate metabolism, polyamine pathway, ROS system and hormone signaling pathway into a complicated network to deal with salt and dehydration stress (Xie et al., 2017). 2.15 Sesame (Sesamum indicum) A mixed analysis of sRNA and degradome sequencing was utilized to clarify the complicated mechanisms engaged in salinity condition response through identifying microRNAs and their targets in two opposing genotypes of sesame. Totally, 116 miRNAs and 210 target genes were recognized to be salt responsive. Salinity stress led to decrease in the expression of majority of miRNAs. Furthermore, network analysis of DE-microRNAs and their target mRNAs indicated that some key models involved some miRNA family members and several stress-associated genes coding for ATP sulfurylase 1, growth-regulating factors, NAC, homeobox-leucine zipper proteins, low affinity sulfate transporter 3 and TCP 4. Furthermore, several miRNAs from miR156, miR160 and miR319 families participated in signaling pathways mediated by brassinosteroid, auxin and gibberellin through adjusting their target genes, respectively. Expression profiles of miRNAs responding to salt stress are genotype-specific in sesame and though many salinity-responsive miRNAs are conserved in plants, some miRNAs showed diverse regulatory patterns in different species (Zhang et al., 2020). 2.16 Radish (Raphanus sativus) Totally, 49 known miRNAs and 22 new candidate miRNAs were recognized as salt responsive miRNAs in roots of R. sativus inbred line Small RNAs involved in salt stress tolerance of food crops 321 “NAU-YH” using sRNA sequencing. Target prediction for DE-miRNAs indicated that many of the target genes coded for stress-associated transcription factors like SPB-like proteins (SPLs), MYBs, auxin response factors (ARFs), APETALA2 (AP2), NACs, nuclear transcription factor Y (NF-YA and NF-YB) and bZIP. MiR169/NF-YA module, broadly regulated under drought and salinity stress, conditions entire plant growth via adjusting carbohydrate metabolism and cell elongation (Stephenson et al., 2007; Zhao et al., 2009). Conserved module of miR160/ARF exerts a main role in response of plant to different abiotic stresses via adjusting plant growth and development under stress situation. miR164/NAC, miR156/SPL, miR172/AP2 and miR159/MYB modules were also recognized as salt responsive modules in radish. Besides main TFs, several target genes coding for important enzymes or functional proteins like APX (ascorbate peroxidase), CSD (superoxide dismutase), APS (ATP sulfurylase), LACs (laccase), UBC (ubiquitin-conjugating enzyme), Ca2þ-mediated signal-associated proteins (CAM7, CIPK21 and CDPK9), were also found to implement key roles in response to salt stress in radish (Sun et al., 2015). 2.17 Sweet potato (Ipomoea batatas) sRNA sequencing and bioinformatic analysis in sweet potato resulted in recognition of 127 and 22 miRNAs responsive to salinity in roots and leaves. Using degradome sequencing to find target genes of DE-miRNAs indicated that the main part of the target genes were transcription factors such as NACs, SPLs, AP2, TCP and NF-Y and some conserved modules in abiotic stress response like miR156/SPL, miR172/AP2 and miR319/TCP were also engaged in response of sweet potato to salinity stress (Yang et al., 2020). The squamosa promoter binding protein-like (SPL) exerts a main regulatory role in plant tolerance to abiotic stresses (Hou et al., 2018; Ning et al., 2017). AP2 TFs play a key role in regulating transcription of biological processes engaged in growth and development and response to abiotic stresses (Aukerman & Sakai, 2003; Gutterson & Reuber, 2004). TCP is a plant-specific TF which has a main role in plant growth and development and response to biotic/abiotic stresses (Martín-Trillo & Cubas, 2010; Nicolas & Cubas, 2016). In addition, Performing KEGG analysis for the target genes showed that some metabolic pathways in which the target genes participated were environmental adaptation, lipid metabolism and nucleotide metabolism (Yang et al., 2020). 322 Plant Small RNA in Food Crops 2.18 Broccoli (Brassica oleracea) Utilizing sRNA sequencing in Broccoli resulted in discovering 42 putative conserved and 39 novel miRNAs that responded to salt stress. The study revealed that the expression of miR393 and miR855 as conserved miRNAs and miR3 and miR34 as new candidate miRNAs were highly declined under salinity condition. Among the up-regulated miRNAs, conserved miR838-5p and miR396a and novel miR37 were the most highly up regulated under salt stress. Performing KEGG analysis for the target genes of salt responsive miRNAs indicated that they were engaged in various cellular processes such as cell cycle, plant hormone signal transduction, sulfur metabolism and calcium reabsorption. Totally, KO and GO analysis of the target genes indicated that many of themiRNAs target genes were associated with salt stress (Tian et al., 2014). 2.19 Pumpkin (Cucurbita) Small RNA sequencing was used to define salt responsive miRNAs in two germplasm of Cucurbita, N12 (Cucurbita maxima) and N15 (Cucurbita moschata), with various patterns of sodium accumulation. While Naþ was collectedin the shoots in N12, it was collected in the roots in N15. As a result, 20 and 22 DE-miRNAs were recognized in N15 and N12, respectively. Target prediction using PsRNA target revealed that the target genes of conserved salt responsive miRNAs were transcription factors and genes engaged in metabolism and plant response to environmental stimulus. Transcription factors and salinity-responsive proteins such as dehydrationinduced protein, cation/Hþ antiporter 18, and CBL-interacting serine/ threonine-protein kinase were identified as targets of novel responsive miRNAs. In addition, the results indicated that the salt overly sensitive (SOS) pathway together with ethylene may play key roles in N12 response to salinity, while the NAC TF was of great importance for salt stress response in N15 (J. Xie et al., 2015). 2.20 Indian mustard (Brassica juncea) NGS method together with computational approaches were used for identification of abiotic stress responsive miRNAs in B. juncea and resulted in finding 34 conserved and 112 novel abiotic stress responsive microRNAs. Among the stress responsive miRNAs, six conserved and 46 new candidate miRNAs exclusively responded to salt stress. Target prediction for responsive miRNAs using open source software of “plant small RNA Small RNAs involved in salt stress tolerance of food crops 323 target” showed that novel miRNA of BjuN21 (up-regulated under salt stress) targets glycine decarboxylase (GDC) P-protein (Bhardwaj et al., 2014) which is involved in betaine accumulation, so regulating glycine decarboxylase by BjuN21 probably justifies glycine betaine levels. Glycine betaine acts as an osmoprotectant (Bhuiyan et al., 2007). b-Glucosidase 16 like gene was determined as a potential target of novel miRNA of BjuN35 (repressed below salinity stress) (Bhardwaj et al., 2014). In b-glucosidase gene family, some members are engaged in modulating cellular ABA levels in Arabidopsis. It has also been reported that ABA is inactivated by glucosebinding. In fact, glucose is eliminated from ABA by b-glucosidase in response to dehydration condition, which results in filling the cellular ABA pool (Lee et al., 2006). So, BjuN35 possibly controls active levels of ABA in Indian mustardvia regulating levels of b-glucosidase transcripts. (Bhardwaj et al., 2014). 3. Common small RNAs involved in salt stress response of different food crops and their target genes As reviewed in the previous section, plenty of salt-responsive miRNAs and their target genes have been identified in different food crops (Table 11.1), due to availability of high-throughput techniques such as transcriptome, small RNA and degradome sequencing and advanced computational analysis. Various salt stress treatments have been applied including different concentrations (80e600 mM) of NaCl and treatment time/duration (3 h to 15 days), and different plant tissues (e.g. root, leaf, stem, and flower) were used for the miRNAs detection. Both up-regulation and down-regulation of miRNAs have been observed in response to salt stress. Many common salt-responsive miRNAs have been reported from different plants (Fig. 11.1). In many cases, miRNAs and their functions appear to be conserved in response to salinity. However, some miRNAs or various members of a miRNA family seem to play diverse roles in salt tolerance of different plants. Moreover, members of a miRNA family are differentially regulated in various plant tissues (e.g. roots and shoots) and developmental stages or in response to diverse stresses (such as salinity and drought). It is interesting that up- or down-regulated expression profiles of some miRNAs were different among plant species under salt stress, suggesting the same miRNA may have diverse function in various plant species under 324 Plant Small RNA in Food Crops miR396 miR156 miR319 miR169 miR159 miR166 miR168 miR393 miR394 miR399 miR482 miR477 miR157 miR858 miR444 miR530 miR162 miR827 miR6173 miR535 miR528 miR189 miR165 miR414 miR160 miR167 miR164 miR172 miR390 miR171 miR408 miR397 miR395 miR398 1 0.5 0 −0.5 −1 Linum.usitatissimum Ipomoea.batatas Musa.acuminata Triticum.aestivum Pennisetum.glaucum Oryza.glaberrima Hordeum.vulgare Zea.mays Vigna.Unguiculata Eleucine.coracana Solanum.linnaeanum Medicago.sativa Glycinemax Citrus.junos Oryza.Sativa Cicer.arietinum Sesamum.indicum_ST Sesamum.indicum_SS Cucurbita.moschata Cucurbita.maxima Raphanus.sativus Brassica.oleracea Setaria.viridis Phoenix.dactylifera Figure 11.1 Common salt-responsive small RNAs among different food crops. Red: Up-regulated, Blue: Down-regulated, Yellow: Not reported. salinity. For instance, miR396 was down-regulated in rice, cotton and barley (Deng et al., 2015; Ding et al., 2009; Lv et al., 2012; F. Xie et al., 2015), while it was up-regulated in Arabidopsis (Liu et al., 2008). Similarly, the expression of miR167 was reduced in maize and barley (Deng et al., 2015; Ding et al., 2009), but induced in Arabidopsis in response to salinity conditions (Liu et al., 2008). Small RNAs involved in salt stress tolerance of food crops 325 Many salt-responsive miRNAs target stress associated transcription factors, and the regulated TFs adjust the expression of stress related genes (Sun et al., 2015; J. Xie et al., 2015). The regulatory module of miR172/ IDS1 is a good example of how a miRNA controls the expression of stress associated genes via regulating a transcription factor. MiR172 is a positive regulator of salt tolerance in rice and wheat. IDS1, an AP2/ERF TF, is directly suppressed by miR172. The miR172-regulated IDS1 directly represses the expression of a set of ROS-scavenging genes, which shows the involvement of this module in redox homeostasis under salt stress. Furthermore, some conserved modules, such as miR164/NAC, miR156/ SPL, miR172/AP2, miR159/MYB and miR159/ARF, in which miRNAs target stress related TFs, are involved in salt stress response of various plants (Pandey et al., 2014; Sun et al., 2015; Wang et al., 2014; Yang et al., 2020). Apart from transcription factors, some genes engaged in Caþ2 mediated signaling under salt stress are also regulated by miRNAs. Caþ2 mediated signaling proteins such as CIPK21, CAM7 and CDPK9 have been identified as targets of miR414, rsa-mir3 and rsa-mir5, respectively (Sun et al., 2015; J. Xie et al., 2015). In addition, some miRNAs target genes encoding stress associated functional proteins. Osmotic stress is the first consequence of salt stress on plants (Munns, 2005; Rahnama et al., 2010). Phytohormone signaling is a crucial regulator for root growth under salt stress. The plant hormone auxin influences several aspects of plant development and the identity of many miRNA targets suggests roles of miRNAs in auxin signaling. MiR393a is complementary to F-box proteins (AFBs) and transport inhibitor response protein (TIR1) and regulates auxin signaling through auxin response factors (ARFs) as auxin receptors. miR393 has been reported to be regulated by salt stress which targets OsTIR1 and OsAFB2 in rice (Bai et al., 2017), HvAFB2 and HvTIR1 in barley (Kuang et al., 2019), four F-box genes in A. thaliana (Abdelrahman et al., 2018; Jones-Rhoades & Bartel, 2004; Parry et al., 2009) and pearl millet (Shinde et al., 2020). Whereas, miR162a was down-regulated by salinity stress, and can target ARF transcription factor genes, which are positive regulators of auxin signaling. Additionally, miR162 is conserved and involved in multiple abiotic stress responses in plants. For example, in cotton (Salih et al., 2018), Maize (Ding et al., 2009), and rice miR162 was down-regulated by drought stress while this miRNA was up-regulated by drought stress in Arabidopsis (Tian et al., 2015). Conserved miR169 is one of the largest miRNA families playing significant roles in response to abiotic stresses in plants. MiR169 is 326 Plant Small RNA in Food Crops up-regulated under high soil salinity (Zhao et al., 2009), drought (Li et al., 2008; Zhang et al., 2011) and low temperature (Lee et al., 2010; Zhou et al., 2008). Mir169 regulates the expression of NF-YA transcription factor encoding genes through transcript cleavage. NF-Y encodes a CCAATbinding TF, which regulates the expression levels of several genes. The function of miR169 in transcription regulation and signal transduction of NF-Y genes is conserved in barley (Pacak et al., 2016), rice (Li et al., 2017b) and A. thaliana (Rao et al., 2020). 4. Salt tolerance improvement of food crops through manipulation of small RNAs Even though a great number of salt-responsive miRNAs have been discovered, only a limited number of them have been functionally characterized through approaches such as overexpression or repression of the miRNAs or their targets (Table 11.2 and Fig. 11.2). Plant miR164 family members are conserved regulating targets from NAC transcription factors (Guo et al., 2005; Kim et al., 2009; Mallory et al., 2004). The accumulation of NAC1 resulting from the downregulation of miR164 might increase the auxin response, and thus improve root development. It has been revealed that the miR164: NAC module play a critical role in abiotic stress tolerance (Guo et al., 2005). Overexpression of miR164b-resistant OsNAC2 mutant gene, improved salt and drought tolerance of rice through an ABA-mediated pathway, and some stress responsive genes such as OsLEA3 were up-regulated in the mutant plants (Mondal et al., 2018). Plants overexpressing mOsNAC2 had strong root systems and high yields (Mondal et al., 2018). Higher expression of miR164 in Arabidopsis plants led to reduced NAC1 mRNA levels via the cleavage of its mRNA, while NAC1 is involved in auxin signal transduction for lateral root emergence (Kim et al., 2009). In another study, transgenic Arabidopsis plants over-expressing PeNAC070 (the target gene of miR164) exhibits promoted lateral root development (Lu et al., 2017). In barley roots, miR164a, which has been proposed to play a role in salt tolerance, targets NAC079, which is involved in auxin signaling and improves root growth (Kuang et al., 2019). It is reported that zma-miR164 was down-regulated in root under salinity, leading to up-regulation of its target genes (Ding et al., 2009). Significant salt stress phenotypes were observed in Arabidopsis plants overexpressing zma-miR164a, while PCAM-35S: zma-MIM164a (target Table 11.2 Salt tolerance related small RNAs along their target genes in food crops. Target gene Oryza sativa (Rice) osa-miR164b OsNAC2 Oryza sativa (Rice) osa-miR820 OsDRM2 Oryza sativa (Rice) osa-miR1861h The putative targets (120) appeared to be involved in a wide range of biological processes, and most of them were classified as retrotransposons, transcription factors, methyltransferase, and functional proteins. Salt tolerance related phenotype Improved salt tolerance in rice via ABA-mediated pathways Transgenic plants exhibited enhanced vigor, w25%e30% increase in the number of spikelets per panicle and increased grain filling, under normal and salt stress conditions. Overexpression of miR1861h increases tolerance to salt stress in rice (Oryza sativa L.) Approach References Overexpression of miR164b-resistant OsNAC2 mutant gene in rice Overexpression of osa-miR820 in rice Jiang et al. (2019) Overexpression of miR1861h in rice Sharma et al. (2021) Ai et al. (2021) 327 miRNA name Small RNAs involved in salt stress tolerance of food crops Plant specious Continued Table 11.2 Salt tolerance related small RNAs along their target genes in food crops.dcont'd Target gene Oryza sativa (Rice) osa-miR396c Members of miR396 often target growthregulating factor (GRF) genes encoding putative transcription factors that regulate plant growth. Oryza sativa (Rice) osa-miR171c Oryza sativa (Rice) osa-miR393 HAM (hairy meristem) genes, which encode members of the GRAS (GAI-RGASCR) transcription factor family OsTIR1 and OsAFB2 Salt tolerance related phenotype Over-expression of osa-MIR396c decreases salt and alkali stress tolerance. osaMIR396c likely functions as a negative regulator to target GRF and other regulatory proteins and mediates plant saltalkali stress responses. Overexpressing osa-miR171c decreases salt stress tolerance in rice. OsmiR393 overexpression leads to more tillers, early flowering and less tolerance to salt and drought in rice Approach References Over-expression of osa-MIR396c in rice Gao et al. (2010) Over-expression of miR171c in rice Yang et al. (2017) Over-expression of osa-miR393 in rice Xia et al. (2012) Plant Small RNA in Food Crops miRNA name 328 Plant specious osa-miR1848 OsCYP51G3 Oryza sativa (Rice) osa-miR393 Oryza sativa (Rice) osa-miR528 Eight putative target including transport inhibitor response proteins, oxidoreductase, the phytosulfokine receptor precursor, GRF-interacting factor (GIF) AsAAO and COPPER ION BINDING PROTEIN1 Triticum aestivum (Bread wheat) tae-miR1120c Traes_2BS_5C 64FC44A.2 Osa-miR1848-ox and OsCYP51G3RNAi plants were more sensitive to salt stress. Transgenic plants were more sensitive to salt and alkali treatment compared to wildtype plants. Enhances tolerance to salinity stress and nitrogen starvation in creeping bentgrass Improved salt tolerance and increased fresh weight of seedlings Over-expression of osa-miR1848 in rice Xia et al. (2015) Over-expression of osa-miR393 in rice Gao et al. (2011) Over-expression of osa-miR528 in creeping bentgrass Yuan et al. (2015) Transient overexpression of tae-miR1120c using VIGS method in bread wheat Han et al. (2018) 329 Continued Small RNAs involved in salt stress tolerance of food crops Oryza sativa (Rice) 330 Table 11.2 Salt tolerance related small RNAs along their target genes in food crops.dcont'd miRNA name Target gene Triticum aestivum (Bread wheat) tae-miR9664 Traes_2BS_50 45C640C.2 Improved salt and alkalinity tolerance and increased fresh weight of seedlings Triticum aestivum (Bread wheat) TaemiR408 TaCP, TaMP, TaBCP, TaKRP, TaABP and TaFP Triticum aestivum (Bread wheat) TaMIR172-1B TaIDS1 Enhanced salt tolerance, improved photosynthesis function, phenotype and biomass and elevated osmolytes under salt stress Increased salt tolerance, enhanced fresh weight, improved ROS scavenging system and less sensitivity to toxic ROS Approach References Transient overexpression of tae-miR9664 using VIGS method in bread wheat Overexpression of TaemiR408 in tobacco Han et al. (2018) Overexpression of TaMIR172-1B in bread wheat Liu et al. (2021) Bai et al. (2018) Plant Small RNA in Food Crops Salt tolerance related phenotype Plant specious Tae-miR408 TaCLP1 Zea maize (Maize) zma-miR164a Glycine max (Soybean) gma-miR172a GRMZM2G114 850 (the NAC transcription factor) and GRMZM 2G008819 (electron carrier). SSAC (salt suppressed AP2 domaincontaining) Glycine max (Soybean) Gma-miR172c APETALA2 Glycine max (Soybean) MiR172c NNC1 Significantly increased cell growth under high salinity and Cu2þ stresses zma-MIM164a (target mimicry) transgenic Arabidopsis plants showed improved salt tolerance Enhanced tolerance to salt stress through cleaving and degrading the SSAC1 Enhanced tolerance to salinity stress Overexpression and knockdown of miR172c activity resulted in substantially enhanced and decrease root sensitivity to salt stress, respectively Overexpressing TaCLP1 in yeast Feng et al. (2013) Overexpress zmaMIR164a, target mimic zmaMIM164a in Arabidopsis Shan et al. (2020) Overexpressiongma-miR172a in soybean Pan et al. (2016) Overexpression of gma-miR172c in Arabidopsis Overexpress miR172c and knockdown miR172c in soybean Li et al. (2016) Sahito et al. (2017) 331 Continued Small RNAs involved in salt stress tolerance of food crops Triticum aestivum (Bread wheat) 332 Table 11.2 Salt tolerance related small RNAs along their target genes in food crops.dcont'd miRNA name Target gene Medicago sativa (Alfalfa) miR156OE SPL family Malus domestica (Apple) miR156a MdSPL13 Solanum pimpinellifolium (Tomato) Sp-miR396a-5p GRF1, GRF3, GRF7, GRF8 Salt tolerance related phenotype Enhanced tolerance to salinity stress, enhanced shoot biomass and number of branches Overexpressing MiR156a weakened salt tolerance in apple, whereas MdSPL13 strengthened Enhanced tolerance to salt, drought and cold stresses Approach References OverexpressionmiR156 in alfalfa Arshad et al. (2017) Overexpression of MiR156a and SPL13 in apple Ma et al. (2021) Overexpressiing Sp-miR396a-5p in tobacco Chen et al. (2015b) Plant Small RNA in Food Crops Plant specious Small RNAs involved in salt stress tolerance of food crops 333 Figure 11.2 Functionally characterized small RNAs of food crops involved in salt tolerance and their molecular mechanism. mimicry; blocking the cleavage by miR164a leading to accumulation of the target genes) transgenic plants showed improved salt tolerances (Shan et al., 2020). It has been reported that the NAC1 transcription factor was predicted as the target of zma-miR164a/b/c/d (Kohli et al., 2014; Shan et al., 2020). MiR393 members are belonged to a conserved miRNA family in plants. Osa-MIR393 is salt-responsive, and miR393 overexpression in rice and Arabidopsis decreased the plant salt tolerance (Gao et al., 2011). In Arabidopsis, induction of miR393 led to the reduction of transport inhibitor response 1 (TIR1) and Auxin signaling F-box 2 (AFB2) (Iglesias et al., 2014). Transgenic Arabidopsis plants overexpressing miR393resistant TIR1 revealed improved salt tolerance (Z. Chen et al., 2015). It is suggested that miR393 regulates redox-related components as well as lateral root initiation, emergence and elongation under salt stress conditions (Iglesias et al., 2014). The miR396 family members are ancient miRNAs that usually regulate transcription factors from the Growth-Regulating Factor (GRF) family as targets (Debernardi et al., 2012). Under salinity, osa-MIR396c is significantly down-regulated in Oryza sativa. The salinity tolerance was decreased in the transgenic rice and Arabidopsis lines over-expressing osa-MIR396c. Gene ontology analysis results showed that the genes encoding GRF transcription factors were significantly enriched (Gao et al., 2010). Moreover, the predicted targets were modulated by various stress or hormonal treatments including abscisic acid (ABA), transzeatin and gibberellin (Gao et al., 2010). MiR528, as a conserved monocot-specific small RNA, plays a mediating role responding to multiple stresses. Overexpression of rice miR528 in 334 Plant Small RNA in Food Crops creeping bentgrass showed enhanced salt tolerance through increased capacity of ROS scavenging, water retention, cell membrane integrity, chlorophyll content, capacity for preserving potassium homeostasis. Also, two targets of miR528, including ASCORBIC ACID OXIDASE (AAO) and COPER ION BINDING PROTEIN1 (CBP1) were identified as responding to salt stress and nitrogen starvation. Based on the prior reports, AAO and CBP1 were significantly down-regulated in miR528overexpressing transgenic plants, which are involved in several functions such as cell signaling, photosynthesis, and various reactions of oxidases and reductases (Yuan et al., 2015). TaemiR408 is a wheat (T. aestivum) member of a highly conserved miRNA family in plants, which is involved in abiotic stress responses (Bai et al., 2018; Ma et al., 2015). Up-regulation of TaemiR408 under salt stress and Pi starvation conditions and its down-regulation by the recovery treatments have been reported. Transgenic tobacco plants with TaemiR408 overexpression showed improved salinity tolerance. They exhibited enhanced biomass, photosynthesis and osmolytes under salt stress compared to the wild type. Conclusively, TaemiR408 is involved in adaptation of plants to salt stress via regulating ABA signaling pathway, photosynthesis and osmolyte biosynthesis under salt stress (Bai et al., 2018). It was demonstrated that six target genes of TaemiR408 were involved in mediating plant responses to salt stress, encoding proteins engaged in biochemical metabolism, microtubule organization, and signal transduction. TaemiR408 mediates mRNA cleavage of the target genes confirmed by 50 and 30 -RACE analyses (Bai et al., 2018). MiR172 is a positive regulator of salinity tolerance in both monocots (e.g. rice and wheat) and dicots (e.g. Arabidopsis and soybean) (Li et al., 2016; Liu et al., 2021; Pan et al., 2016). Despite this conserved biological role in various crops, and the fact that the target genes of miR172 in rice/ wheat (IDS1) and soybean (SSAC1) both encode TF genes containing AP2/ERF domain, their downstream signaling pathways appear to be diverged (Liu et al., 2021). In rice and wheat, the miR172/IDS1 regulatory module maintains ROS homeostasis during salt stress via regulating a group of ROS-scavenging genes (Liu et al., 2021). IDS1 (Indeterminate Spikelet1), which is an AP2-type TF gene, functions as a negative regulator of salinity tolerance (Cheng et al., 2018). IDS1 was identified as one of the downstream target genes that is cleaved and suppressed by miR172. It is proved that the miR172-regulated IDS1 directly represses the expression of a cluster of ROS-scavenging genes which creates a decisive link between the Small RNAs involved in salt stress tolerance of food crops 335 miR172/IDS1 module and the redox detoxification system during salinity tolerance (Liu et al., 2021). In soybean, miR172a enhances salt tolerance mostly via cleaving the target mRNA of a salt suppressed AP2 domain-containing gene (SSAC1) to dismiss its protein suppression of THI1 (thiamine biosynthesis) gene, which codes for a salt tolerance positive regulator. Some well-known stress related genes, including RD22 and NCED3, are regulated by miR172a, which may also participate in salt tolerance. Moreover, miR172a may play a role as a root to shoot long distance signal (Pan et al., 2016). The expression of gma-miR172c was up-regulated by salt stress. Transgenic Arabidopsis plants overexpressing gma-mir172c revealed enhanced stress tolerance with altered levels of stress/ABA-responsive genes and physiological indicators (Li et al., 2016). Overexpression and knockdown of miR172c in soybean led to a significant increase and decrease of salt stress root sensitivity, respectively. Furthermore, it was shown that NNC1 is the target gene of miR172c and acts as a negative regulator of root response to salt stress in soybean. Knockdown of NNC1 increased the salt tolerance of roots. Moreover, soybean miR172c may also be involved in various biological processes including hormonal crosstalk (e.g. ABA, auxin and cytokinin), lateral root development, ROS and ion homeostasis (Sahito et al., 2017). MiR156 is a conserved microRNA family in plants, which regulate SPL (SQUAMOSA PROMOTER BINDING PROTEIN-LIKE) genes. SPLs are involved in plants’ abiotic stress tolerance, and there is increasing evidence that the conserved module of miR156/SPL plays an important role in adjusting stress responses and plant growth (Cui et al., 2014; Wang et al., 2019; Wang & Wang, 2015). Overexpression of miR156 in Alfalfa enhanced tolerance to salinity stress. Upregulation of miR156 leads to a rise in the uptake of favorable ions and a reduction in toxic ions contributing to salinity tolerance. The biomass, number of branches and time to complete growth stages were increased in the transgenic plants, while plant height was decreased under control and salinity stress conditions. Under salinity stress, miR156 down-regulated SPL transcription factor family genes, changed expression levels of other significant transcription factors, and downstream salt stress responsive genes (Arshad et al., 2017). The role of miR156 was revealed in modulating commercially main features of alfalfa (Medicago sativa) under salinity stress (Arshad et al., 2017). There are some reports indicating different expression profiles of the miR156/SPL module in response to salt stress in woody plants and 336 Plant Small RNA in Food Crops herbaceous plants, which might be related to the difference in their life cycle (Gentile et al., 2015; Khraiwesh et al., 2012; Ma et al., 2021). The miR156/SPL module is also associated with salt stress response in apple. In order to clarify the mechanism by which this module controls salt stress response in apple, genetic transformation technology was used. While overexpression of MIR156a in apple resulted in decreased salt tolerance, overexpression of its target, MdSPL13, led to enhanced salt tolerance. Furthermore, a yeast one-hybrid assay revealed that MdSPL13 binds directly to the promoter of MdWRKY100 and positively regulates its expression. Generating MdWRKY100 overexpressing lines and evaluating them under salt stress showed that they had more RWC and chlorophyll content and less H2O2 and malondialdehyde (MDA) levels compared to MdWRKY100RNAi lines and wild type, indicating improved salt tolerance (Ma et al., 2021). WRKY TFs are known to be involved in plant salt tolerance through ABA biosynthesis, ROS-scavenging and ionic homeostasis (Chen et al., 2012; Ding et al., 2015). MiR396 is one of the plants conserved small RNA families targeting GRFs (growth-regulating factors). In tomato, Sp-miR396a-5p, which targets the GFR transcription factor, is significantly induced by salt and drought stress. Real-time quantitative analysis revealed that SpGRF1 and SpGRF3, as target genes of Sp-miR396a-5p, down-regulated under salt and drought stress. In order to clarify the role of tomato miR396a-5p in response to salinity treatment, transgenic tobacco plants overexpressing Sp-miR396a-5p were generated and investigated under salt stress. The results indicated that overexpression of Sp-miR396a-5p in tobacco increased salt tolerance. Transgenic plants showed higher relative water content (RWC), higher root weight and higher proline content compared to wild types under salt stress. On the other hand, catalase and peroxidase enzymes had higher activity in transgenic plants which resulted in less accumulation of ROS (Chen et al., 2015b; L. Chen et al., 2015). References Abdelrahman, M., Jogaiah, S., Burritt, D. J., & Tran, L. S. P. (2018). Legume genetic resources and transcriptome dynamics under abiotic stress conditions. Plant, Cell and Environment, 41(9), 1972e1983. Agharbaoui, Z., Leclercq, M., Remita, M. A., Badawi, M. A., Lord, E., Houde, M., Danyluk, J., Diallo, A. B., & Sarhan, F. (2015). An integrative approach to identify hexaploid wheat miRNAome associated with development and tolerance to abiotic stress. BMC Genomics, 16(1), 339. Small RNAs involved in salt stress tolerance of food crops 337 Ai, B., Chen, Y., Zhao, M., Ding, G., Xie, J., & Zhang, F. (2021). Overexpression of miR1861h increases tolerance to salt stress in rice (Oryza sativa L.). Genetic Resources and Crop Evolution, 68(1), 87e92. Alzahrani, S. M., Alaraidh, I. A., Khan, M. A., Migdadi, H. M., Alghamdi, S. S., & Alsahli, A. A. (2019). Identification and characterization of salt-responsive microRNAs in Vicia faba by high-throughput sequencing. Genes, 10(4), 303. Amirbakhtiar, N., Ismaili, A., Ghaffari, M.-R., Mirdar Mansuri, R., Sanjari, S., & Shobbar, Z.-S. (2021). Transcriptome analysis of bread wheat leaves in response to salt stress. PLoS One, 16(7), e0254189. Arshad, M., Gruber, M. Y., Wall, K., & Hannoufa, A. (2017). An insight into microRNA156 role in salinity stress responses of alfalfa. Frontiers of Plant Science, 8, 356. Aukerman, M. J., & Sakai, H. (2003). Regulation of flowering time and floral organ identity by a microRNA and its APETALA2-like target genes. The Plant Cell, 15(11), 2730e2741. Bai, B., Bian, H., Zeng, Z., Hou, N., Shi, B., Wang, J., Zhu, M., & Han, N. (2017). miR393-mediated auxin signaling regulation is involved in root elongation inhibition in response to toxic aluminum stress in barley. Plant and Cell Physiology, 58(3), 426e439. Bai, Q., Wang, X., Chen, X., Shi, G., Liu, Z., Guo, C., & Xiao, K. (2018). Wheat miRNA TaemiR408 acts as an essential mediator in plant tolerance to Pi deprivation and salt stress via modulating stress-associated physiological processes. Frontiers in Plant Science, 9, 499. Barrera-Figueroa, B. E., Gao, L., Wu, Z., Zhou, X., Zhu, J., Jin, H., Liu, R., & Zhu, J.-K. (2012). High throughput sequencing reveals novel and abiotic stress-regulated microRNAs in the inflorescences of rice. BMC Plant Biology, 12(1), 1e11. Bartel, D. P. (2004). MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell, 116(2), 281e297. Bhardwaj, A. R., Joshi, G., Pandey, R., Kukreja, B., Goel, S., Jagannath, A., Kumar, A., Katiyar-Agarwal, S., & Agarwal, M. (2014). A genome-wide perspective of miRNAome in response to high temperature, salinity and drought stresses in Brassica juncea (Czern) L. PLoS One, 9(3), e92456. Bhuiyan, N. H., Hamada, A., Yamada, N., Rai, V., Hibino, T., & Takabe, T. (2007). Regulation of betaine synthesis by precursor supply and choline monooxygenase expression in Amaranthus tricolor. Journal of Experimental Botany, 58(15e16), 4203e4212. Carnavale Bottino, M., Rosario, S., Grativol, C., Thiebaut, F., Rojas, C. A., Farrineli, L., Hemerly, A. S., & Ferreira, P. C. G. (2013). High-throughput sequencing of small RNA transcriptome reveals salt stress regulated microRNAs in sugarcane. PLoS One, 8(3), e59423. Chand Jha, U., Nayyar, H., Mantri, N., & Siddique, K. H. (2021). Non-coding RNAs in legumes: Their emerging roles in regulating biotic/abiotic stress responses and plant growth and development. Cells, 10(7), 1674. Chen, L., Song, Y., Li, S., Zhang, L., Zou, C., & Yu, D. (2012). The role of WRKY transcription factors in plant abiotic stresses. Biochimica et Biophysica Acta (BBA)-Gene Regulatory Mechanisms, 1819(2), 120e128. Chen, Z., Hu, L., Han, N., Hu, J., Yang, Y., Xiang, T., Zhang, X., & Wang, L. (2015a). Overexpression of a miR393-resistant form of transport inhibitor response protein 1 (mTIR1) enhances salt tolerance by increased osmoregulation and Naþ exclusion in Arabidopsis thaliana. Plant and Cell Physiology, 56(1), 73e83. Chen, L., Luan, Y., & Zhai, J. (2015b). Sp-miR396a-5p acts as a stress-responsive genes regulator by conferring tolerance to abiotic stresses and susceptibility to Phytophthora nicotianae infection in transgenic tobacco. Plant Cell Reports, 34(12), 2013e2025. 338 Plant Small RNA in Food Crops Cheng, X., Zhang, S., Tao, W., Zhang, X., Liu, J., Sun, J., Zhang, H., Pu, L., Huang, R., & Chen, T. (2018). INDETERMINATE SPIKELET1 recruits histone deacetylase and a transcriptional repression complex to regulate rice salt tolerance. Plant Physiology, 178(2), 824e837. Cui, L. G., Shan, J. X., Shi, M., Gao, J. P., & Lin, H. X. (2014). The miR156-SPL 9-DFR pathway coordinates the relationship between development and abiotic stress tolerance in plants. The Plant Journal, 80(6), 1108e1117. Debernardi, J. M., Rodriguez, R. E., Mecchia, M. A., & Palatnik, J. F. (2012). Functional specialization of the plant miR396 regulatory network through distinct microRNAtarget interactions. PLoS Genetics, 8(1), e1002419. Deng, P., Wang, L., Cui, L., Feng, K., Liu, F., Du, X., Tong, W., Nie, X., Ji, W., & Weining, S. (2015). Global identification of microRNAs and their targets in barley under salinity stress. PLoS One, 10(9), e0137990. Ding, D., Zhang, L., Wang, H., Liu, Z., Zhang, Z., & Zheng, Y. (2009). Differential expression of miRNAs in response to salt stress in maize roots. Annals of Botany, 103(1), 29e38. Ding, Z. J., Yan, J. Y., Li, C. X., Li, G. X., Wu, Y. R., & Zheng, S. J. (2015). Transcription factor WRKY 46 modulates the development of Arabidopsis lateral roots in osmotic/ salt stress conditions via regulation of ABA signaling and auxin homeostasis. The Plant Journal, 84(1), 56e69. Dong, Z., Shi, L., Wang, Y., Chen, L., Cai, Z., Wang, Y., Jin, J., & Li, X. (2013). Identification and dynamic regulation of microRNAs involved in salt stress responses in functional soybean nodules by high-throughput sequencing. International Journal of Molecular Sciences, 14(2), 2717e2738. Eren, H., Pekmezci, M., Okay, S., Turktas, M., Inal, B., Ilhan, E., Atak, M., Erayman, M., & Unver, T. (2015). Hexaploid wheat (Triticum aestivum) root miRNome analysis in response to salt stress. Annals of Applied Biology, 167(2), 208e216. Feng, H., Zhang, Q., Wang, Q., Wang, X., Liu, J., Li, M., Huang, L., & Kang, Z. (2013). Target of tae-miR408, a chemocyanin-like protein gene (TaCLP1), plays positive roles in wheat response to high-salinity, heavy cupric stress and stripe rust. Plant Molecular Biology, 83(4), 433e443. Fu, R., Zhang, M., Zhao, Y., He, X., Ding, C., Wang, S., Feng, Y., Song, X., Li, P., & Wang, B. (2017). Identification of salt tolerance-related microRNAs and their targets in maize (Zea mays L.) using high-throughput sequencing and degradome analysis. Frontiers in Plant Science, 8, 864. Ganesan, G., Sankararamasubramanian, H., Harikrishnan, M., Ashwin, G., & Parida, A. (2012). A MYB transcription factor from the grey mangrove is induced by stress and confers NaCl tolerance in tobacco. Journal of Experimental Botany, 63(12), 4549e4561. Gao, P., Bai, X., Yang, L., Lv, D., Li, Y., Cai, H., Ji, W., Guo, D., & Zhu, Y. (2010). Overexpression of osa-MIR396c decreases salt and alkali stress tolerance. Planta, 231(5), 991e1001. Gao, P., Bai, X., Yang, L., Lv, D., Pan, X., Li, Y., Cai, H., Ji, W., Chen, Q., & Zhu, Y. (2011). osa-MIR393: a salinity-and alkaline stress-related microRNA gene. Molecular Biology Reports, 38(1), 237e242. Gentile, A., Dias, L. I., Mattos, R. S., Ferreira, T. H., & Menossi, M. (2015). MicroRNAs and drought responses in sugarcane. Frontiers in Plant Science, 6, 58. Graham, P. H., & Vance, C. P. (2003). Legumes: Importance and constraints to greater use. Plant Physiology, 131(3), 872e877. Guo, H., Huang, Z., Li, M., & Hou, Z. (2020). Growth, ionic homeostasis, and physiological responses of cotton under different salt and alkali stresses. Scientific Reports, 10(1), 1e20. Small RNAs involved in salt stress tolerance of food crops 339 Guo, H.-S., Xie, Q., Fei, J.-F., & Chua, N.-H. (2005). MicroRNA directs mRNA cleavage of the transcription factor NAC1 to downregulate auxin signals for Arabidopsis lateral root development. The Plant Cell, 17(5), 1376e1386. Gupta, O. P., Meena, N. L., Sharma, I., & Sharma, P. (2014). Differential regulation of microRNAs in response to osmotic, salt and cold stresses in wheat. Molecular Biology Reports, 41(7), 4623e4629. Gutterson, N., & Reuber, T. L. (2004). Regulation of disease resistance pathways by AP2/ ERF transcription factors. Current Opinion in Plant Biology, 7(4), 465e471. Han, H., Wang, Q., Wei, L., Liang, Y., Dai, J., Xia, G., & Liu, S. (2018). Small RNA and degradome sequencing used to elucidate the basis of tolerance to salinity and alkalinity in wheat. BMC Plant Biology, 18(1), 1e17. He, Y., Li, W., Lv, J., Jia, Y., Wang, M., & Xia, G. (2012). Ectopic expression of a wheat MYB transcription factor gene, TaMYB73, improves salinity stress tolerance in Arabidopsis thaliana. Journal of Experimental Botany, 63(3), 1511e1522. He, X., Han, Z., Yin, H., Chen, F., Dong, Y., Zhang, L., Lu, X., Zeng, J., Ma, W., & Mu, P. (2021). High-throughput sequencing-based identification of miRNAs and their target mRNAs in wheat variety Qing Mai 6 under salt stress condition. Frontiers in Genetics, 1467. Hou, H., Jia, H., Yan, Q., & Wang, X. (2018). Overexpression of a SBP-box gene (VpSBP16) from Chinese wild Vitis species in Arabidopsis improves salinity and drought stress tolerance. International Journal of Molecular Sciences, 19(4), 940. Huang, X., Feng, J., Wang, R., Zhang, H., & Huang, J. (2019). Comparative analysis of microRNAs and their targets in the roots of two cultivars with contrasting salt tolerance in rice (Oryza sativa L.). Plant Growth Regulation, 87(1), 139e148. Iglesias, M. J., Terrile, M. C., Windels, D., Lombardo, M. C., Bartoli, C. G., Vazquez, F., Estelle, M., & Casalongué, C. A. (2014). MiR393 regulation of auxin signaling and redox-related components during acclimation to salinity in Arabidopsis. PLoS One, 9(9), e107678. Isayenkov, S. V., & Maathuis, F. J. (2019). Plant salinity stress: Many unanswered questions remain. Frontiers in Plant Science, 10, 80. Jagadeesh Selvam, H. R. N., Senthil, N., Manikanda Boopathi, N., & Raveendran, M. (2015). Computational identification of salinity responsive microRNAs in contrasting genotypes of Finger millet (Eleusinecoracana L.). Research Journal of Biotechnology, 10, 9. Jagadeeswaran, G., Zheng, Y., Li, Y. F., Shukla, L. I., Matts, J., Hoyt, P., Macmil, S. L., Wiley, G. B., Roe, B. A., & Zhang, W. (2009). Cloning and characterization of small RNAs from Medicago truncatula reveals four novel legume-specific microRNA families. New Phytologist, 184(1), 85e98. Jain, M., Nijhawan, A., Arora, R., Agarwal, P., Ray, S., Sharma, P., Kapoor, S., Tyagi, A. K., & Khurana, J. P. (2007). F-box proteins in rice. Genome-wide analysis, classification, temporal and spatial gene expression during panicle and seed development, and regulation by light and abiotic stress. Plant Physiology, 143(4), 1467e1483. Jatan, R., Chauhan, P. S., & Lata, C. (2019). Pseudomonas putida modulates the expression of miRNAs and their target genes in response to drought and salt stresses in chickpea (Cicer arietinum L.). Genomics, 111(4), 509e519. Jeong, D.-H., Park, S., Zhai, J., Gurazada, S. G. R., De Paoli, E., Meyers, B. C., & Green, P. J. (2011). Massive analysis of rice small RNAs: Mechanistic implications of regulated microRNAs and variants for differential target RNA cleavage. The Plant Cell, 23(12), 4185e4207. Jiang, D., Zhou, L., Chen, W., Ye, N., Xia, J., & Zhuang, C. (2019). Overexpression of a microRNA-targeted NAC transcription factor improves drought and salt tolerance in rice via ABA-mediated pathways. Rice, 12(1), 1e11. 340 Plant Small RNA in Food Crops Jiao, X., Wang, H., Yan, J., Kong, X., Liu, Y., Chu, J., Chen, X., Fang, R., & Yan, Y. (2020). Promotion of BR biosynthesis by miR444 is required for ammonium-triggered inhibition of root growth. Plant Physiology, 182(3), 1454e1466. Jones-Rhoades, M. W., & Bartel, D. P. (2004). Computational identification of plant microRNAs and their targets, including a stress-induced miRNA. Molecular Cell, 14(6), 787e799. Kantar, M., Lucas, S. J., & Budak, H. (2011). miRNA expression patterns of Triticum dicoccoides in response to shock drought stress. Planta, 233(3), 471e484. Khraiwesh, B., Zhu, J.-K., & Zhu, J. (2012). Role of miRNAs and siRNAs in biotic and abiotic stress responses of plants. Biochimica et Biophysica Acta (BBA)-Gene Regulatory Mechanisms, 1819(2), 137e148. Kim, J. H., Woo, H. R., Kim, J., Lim, P. O., Lee, I. C., Choi, S. H., Hwang, D., & Nam, H. G. (2009). Trifurcate feed-forward regulation of age-dependent cell death involving miR164 in Arabidopsis. Science, 323(5917), 1053e1057. Kohli, D., Joshi, G., Deokar, A. A., Bhardwaj, A. R., Agarwal, M., Katiyar-Agarwal, S., Srinivasan, R., & Jain, P. K. (2014). Identification and characterization of wilt and salt stress-responsive microRNAs in chickpea through high-throughput sequencing. PLoS One, 9(10), e108851. Kuang, L., Shen, Q., Wu, L., Yu, J., Fu, L., Wu, D., & Zhang, G. (2019). Identification of microRNAs responding to salt stress in barley by high-throughput sequencing and degradome analysis. Environmental and Experimental Botany, 160, 59e70. Kumar, V., Khare, T., Shriram, V., & Wani, S. H. (2018). Plant small RNAs: The essential epigenetic regulators of gene expression for salt-stress responses and tolerance. Plant Cell Reports, 37(1), 61e75. Lee, K. H., Piao, H. L., Kim, H.-Y., Choi, S. M., Jiang, F., Hartung, W., Hwang, I., Kwak, J. M., Lee, I.-J., & Hwang, I. (2006). Activation of glucosidase via stress-induced polymerization rapidly increases active pools of abscisic acid. Cell, 126(6), 1109e1120. Lee, H., Yoo, S. J., Lee, J. H., Kim, W., Yoo, S. K., Fitzgerald, H., Carrington, J. C., & Ahn, J. H. (2010). Genetic framework for flowering-time regulation by ambient temperature-responsive miRNAs in Arabidopsis. Nucleic Acids Research, 38(9), 3081e3093. Lee, W. S., Gudimella, R., Wong, G. R., Tammi, M. T., Khalid, N., & Harikrishna, J. A. (2015). Transcripts and microRNAs responding to salt stress in Musa acuminata Colla (AAA Group) cv. Berangan roots. PLoS One, 10(5), e0127526. Li, W.-X., Oono, Y., Zhu, J., He, X.-J., Wu, J.-M., Iida, K., Lu, X.-Y., Cui, X., Jin, H., & Zhu, J.-K. (2008). The Arabidopsis NFYA5 transcription factor is regulated transcriptionally and posttranscriptionally to promote drought resistance. The Plant Cell, 20(8), 2238e2251. Li, T., Li, H., Zhang, Y.-X., & Liu, J.-Y. (2011). Identification and analysis of seven H2O2responsive miRNAs and 32 new miRNAs in the seedlings of rice (Oryza sativa L. ssp. indica). Nucleic Acids Research, 39(7), 2821e2833. Li, B., Qin, Y., Duan, H., Yin, W., & Xia, X. (2011). Genome-wide characterization of new and drought stress responsive microRNAs in Populus euphratica. Journal of Experimental Botany, 62(11), 3765e3779. Li, X., Xie, X., Li, J., Cui, Y., Hou, Y., Zhai, L., Wang, X., Fu, Y., Liu, R., & Bian, S. (2017a). Conservation and diversification of the miR166 family in soybean and potential roles of newly identified miR166s. BMC plant biology, 17, 1e18. Li, Y., Zhao, S.-L., Li, J.-L., Hu, X.-H., Wang, H., Cao, X.-L., Xu, Y.-J., Zhao, Z.-X., Xiao, Z.-Y., & Yang, N. (2017b). Osa-miR169 negatively regulates rice immunity against the blast fungus Magnaporthe oryzae. Frontiers in Plant Science, 8, 2. Liu, J., Chen, T., & Wang, H. (2021). The miR172/IDS1 signaling module confers salt tolerance through maintaining ROS homeostasis in cereal crops. New Phytologist, 1. Small RNAs involved in salt stress tolerance of food crops 341 Liu, H. H., Tian, X., Li, Y.-J., Wu, C.-A., & Zheng, C. C. (2008). Microarray-based analysis of stress-regulated microRNAs in Arabidopsis thaliana. RNA, 14, 836e843. Liu, S., & Xia, G. (2014). The place of asymmetric somatic hybridization in wheat breeding. Plant Cell Reports, 33(4), 595e603. Li, W., Wang, T., Zhang, Y., & Li, Y. (2016). Overexpression of soybean miR172c confers tolerance to water deficit and salt stress, but increases ABA sensitivity in transgenic Arabidopsis thaliana. Journal of Experimental Botany, 67(1), 175e194. Lu, X., Dun, H., Lian, C., Zhang, X., Yin, W., & Xia, X. (2017). The role of peu-miR164 and its target PeNAC genes in response to abiotic stress in Populus euphratica. Plant Physiology and Biochemistry, 115, 418e438. Luan, M., Xu, M., Lu, Y., Zhang, Q., Zhang, L., Zhang, C., Fan, Y., Lang, Z., & Wang, L. (2014). Family-wide survey of miR169s and NF-YAs and their expression profiles response to abiotic stress in maize roots. PLoS One, 9(3), e91369. Lotfi, A., Pervaiz, T., Jiu, S., Faghihi, F., Jahanbakhshian, Z., Khorzoghi, E. G., & Fang, J. (2017). Role of microRNAs and their target genes in salinity response in plant s. Plant Growth Regulation, 82(3), 377e390. Lu, W., Li, J., Liu, F., Gu, J., Guo, C., Xu, L., Zhang, H., & Xiao, K. (2011). Expression pattern of wheat miRNAs under salinity stress and prediction of salt-inducible miRNAs targets. Frontiers of Agriculture in China, 5(4), 413e422. Luan, M., Xu, M., Lu, Y., Zhang, L., Fan, Y., & Wang, L. (2015). Expression of zmamiR169 miRNAs and their target ZmNF-YA genes in response to abiotic stress in maize leaves. Gene, 555(2), 178e185. Lv, S., Nie, X., Wang, L., Du, X., Biradar, S. S., Jia, X., & Weining, S. (2012). Identification and characterization of microRNAs from barley (Hordeum vulgare L.) by highthroughput sequencing. International Journal of Molecular Sciences, 13(3), 2973e2984. Ma, Y., Xue, H., Zhang, F., Jiang, Q., Yang, S., Yue, P., Wang, F., Zhang, Y., Li, L., & He, P. (2021). The miR156/SPL module regulates apple salt stress tolerance by activating MdWRKY100 expression. Plant Biotechnology Journal, 19(2), 311e323. Ma, C., Burd, S., & Lers, A. (2015). miR408 is involved in abiotic stress responses in Arabidopsis. The Plant Journal, 84(1), 169e187. Macovei, A., & Tuteja, N. (2012). microRNAs targeting DEAD-box helicases are involved in salinity stress response in rice (Oryza sativa L.). BMC Plant Biology, 12(1), 1e12. Mallory, A. C., Dugas, D. V., Bartel, D. P., & Bartel, B. (2004). MicroRNA regulation of NAC-domain targets is required for proper formation and separation of adjacent embryonic, vegetative, and floral organs. Current Biology, 14(12), 1035e1046. Mantri, N., Basker, N., Ford, R., Pang, E., & Pardeshi, V. (2013). The role of microribonucleic acids in legumes with a focus on abiotic stress response. The Plant Genome, 6(3) (plantgenome2013.2005.0013). Martín-Trillo, M., & Cubas, P. (2010). TCP genes: A family snapshot ten years later. Trends in Plant Science, 15(1), 31e39. Matamoros, M. A., Saiz, A., Peñuelas, M., Bustos-Sanmamed, P., Mulet, J. M., Barja, M. V., Rouhier, N., Moore, M., James, E. K., & Dietz, K.-J. (2015). Function of glutathione peroxidases in legume root nodules. Journal of Experimental Botany, 66(10), 2979e2990. Megha, S., Basu, U., & Kav, N. N. (2018). Regulation of low temperature stress in plants by microRNAs. Plant, Cell and Environment, 41(1), 1e15. Meng, C., Quan, T.-Y., Li, Z.-Y., Cui, K.-L., Yan, L., Liang, Y., Dai, J.-L., Xia, G.-M., & Liu, S.-W. (2017). Transcriptome profiling reveals the genetic basis of alkalinity tolerance in wheat. BMC Genomics, 18(1), 1e14. Mirdar Mansuri, R., Shobbar, Z.-S., Babaeian Jelodar, N., Ghaffari, M., Mohammadi, S. M., & Daryani, P. (2020). Salt tolerance involved candidate genes in rice: An integrative meta-analysis approach. BMC Plant Biology, 20(1), 1e14. 342 Plant Small RNA in Food Crops Mondal, T. K., Ganie, S. A., & Debnath, A. B. (2015). Identification of novel and conserved miRNAs from extreme halophyte, Oryza coarctata, a wild relative of rice. PLoS One, 10(10), e0140675. Mondal, T. K., Panda, A. K., Rawal, H. C., & Sharma, T. R. (2018). Discovery of microRNA-target modules of African rice (Oryza glaberrima) under salinity stress. Scientific Reports, 8(1), 1e11. Munns, R. (2005). Genes and salt tolerance: Bringing them together. New Phytologist, 167(3), 645e663. Nadeem, M., Ali, M., Kubra, G., Fareed, A., Hasan, H., Khursheed, A., Gul, A., Amir, R., Fatima, N., & Khan, S. U. (2020). Role of osmoprotectants in salinity tolerance in wheat. In Climate change and food security with emphasis on wheat (pp. 93e106). Elsevier. Newstead, S. (2017). Recent advances in understanding proton coupled peptide transport via the POT family. Current Opinion in Structural Biology, 45, 17e24. Nicolas, M., & Cubas, P. (2016). TCP factors: New kids on the signaling block. Current Opinion in Plant Biology, 33, 33e41. Ni, Z., Hu, Z., Jiang, Q., & Zhang, H. (2013). GmNFYA3, a target gene of miR169, is a positive regulator of plant tolerance to drought stress. Plant Molecular Biology, 82(1e2), 113e129. Ning, K., Chen, S., Huang, H., Jiang, J., Yuan, H., & Li, H. (2017). Molecular characterization and expression analysis of the SPL gene family with BpSPL9 transgenic lines found to confer tolerance to abiotic stress in Betula platyphylla Suk. Plant Cell, Tissue and Organ Culture, 130(3), 469e481. Ohri, P., Bhardwaj, R., Kaur, R., Jasrotia, S., Parihar, R. D., & Sharma, N. (2021). MicroRNAs and abiotic stress tolerance in legumes. In Abiotic stress and legumes (pp. 303e336). Elsevier. Pan, W.-J., Tao, J. J., Cheng, T., Bian, X. H., Wei, W., Zhang, W. K., & Zhang, J. S. (2016). Soybean miR172a improves salt tolerance and can function as a long-distance signal. Molecular Plant, 9(9), 1337e1340. Pandey, B., Gupta, O. P., Pandey, D. M., Sharma, I., & Sharma, P. (2013). Identification of new stress-induced microRNA and their targets in wheat using computational approach. Plant Signaling & Behavior, 8(5), e23932. Pandey, R., Joshi, G., Bhardwaj, A. R., Agarwal, M., & Katiyar-Agarwal, S. (2014). A comprehensive genome-wide study on tissue-specific and abiotic stress-specific miRNAs in Triticum aestivum. PLoS One, 9(4), e95800. Pacak, A. M., Kruszka, K., Swida-Barteczka, A., Nuc, P., Karlowski, W., Jarmołowski, A., & Szweykowska-Kuli nska, Z. (2016). Developmental changes in barley microRNA expression profiles coupled with miRNA targets analysis. Acta Biochimica Polonica, 63(4), 799e809. Parenicová, L., de Folter, S., Kieffer, M., Horner, D. S., Favalli, C., Busscher, J., Cook, H. E., Ingram, R. M., Kater, M. M., & Davies, B. (2003). Molecular and phylogenetic analyses of the complete MADS-box transcription factor family in Arabidopsis: New openings to the MADS world. The Plant Cell Online, 15(7), 1538e1551. Parmar, S., Gharat, S. A., Tagirasa, R., Chandra, T., Behera, L., Dash, S. K., & Shaw, B. P. (2020). Identification and expression analysis of miRNAs and elucidation of their role in salt tolerance in rice varieties susceptible and tolerant to salinity. PLoS One, 15(4), e0230958. Parry, G., Calderon-Villalobos, L., Prigge, M., Peret, B., Dharmasiri, S., Itoh, H., Lechner, E., Gray, W., Bennett, M., & Estelle, M. (2009). Complex regulation of the TIR1/AFB family of auxin receptors. Proceedings of the National Academy of Sciences, 106(52), 22540e22545. Small RNAs involved in salt stress tolerance of food crops 343 Paul, S., Kundu, A., & Pal, A. (2011). Identification and validation of conserved microRNAs along with their differential expression in roots of Vigna unguiculata grown under salt stress. Plant Cell, Tissue and Organ Culture, 105(2), 233e242. Pegler, J. L., Nguyen, D. Q., Grof, C. P., & Eamens, A. L. (2020). Profiling of the salt stress responsive microRNA landscape of C4 genetic model species Setaria viridis (L.) Beauv. Agronomy, 10(6), 837. Quan, R., Wang, J., Hui, J., Bai, H., Lyu, X., Zhu, Y., Zhang, H., Zhang, Z., Li, S., & Huang, R. (2018). Improvement of salt tolerance using wild rice genes. Frontiers in Plant Science, 8, 2269. Rahnama, A., James, R. A., Poustini, K., & Munns, R. (2010). Stomatal conductance as a screen for osmotic stress tolerance in durum wheat growing in saline soil. Functional Plant Biology, 37(3), 255e263. Ramesh, S., Govindasamy, V., Rajesh, M., Sabana, A., & Praveen, S. (2019). Stressresponsive miRNAome of Glycine max (L.) Merrill: Molecular insights and way forward. Planta, 249(5), 1267e1284. Rao, S., Balyan, S., Jha, S., & Mathur, S. (2020). Novel insights into expansion and functional diversification of MIR169 family in tomato. Planta, 251(2), 1e17. Rasool, S., Hameed, A., Azooz, M., Siddiqi, T., & Ahmad, P. (2013). Salt stress: Causes, types and responses of plants. Ecophysiology and responses of plants under salt stress (pp. 1e24). Springer. Ren, Y., Chen, L., Zhang, Y., Kang, X., Zhang, Z., & Wang, Y. (2013). Identification and characterization of salt-responsive microRNAs in Populus tomentosa by high-throughput sequencing. Biochimie, 95(4), 743e750. Reynoso, M. A., Blanco, F. A., Bailey-Serres, J., Crespi, M., & Zanetti, M. E. (2013). Selective recruitment of m RNA s and mi RNA s to polyribosomes in response to rhizobia infection in Medicago truncatula. The Plant Journal, 73(2), 289e301. Sahito, Z. A., Wang, L., Sun, Z., Yan, Q., Zhang, X., Jiang, Q., Ullah, I., Tong, Y., & Li, X. (2017). The miR172c-NNC1 module modulates root plastic development in response to salt in soybean. BMC Plant Biology, 17(1), 1e12. Salih, H., Gong, W., Mkulama, M., & Du, X. (2018). Genome-wide characterization, identification, and expression analysis of the WD40 protein family in cotton. Genome, 61(7), 539e547. Shan, T., Fu, R., Xie, Y., Chen, Q., Wang, Y., Li, Z., Song, X., Li, P., & Wang, B. (2020). Regulatory mechanism of maize (Zea mays L.) miR164 in salt stress response. Russian Journal of Genetics, 56(7), 835e842. Sharma, N., Kumar, S., & Sanan-Mishra, N. (2021). Osa-miR820 regulatory node primes rice plants to tolerate salt stress in an agronomically advantageous manner. bioRxiv. Shinde, H., Dudhate, A., Anand, L., Tsugama, D., Gupta, S. K., Liu, S., & Takano, T. (2020). Small RNA sequencing reveals the role of pearl millet miRNAs and their targets in salinity stress responses. South African Journal of Botany, 132, 395e402. Shrivastava, P., & Kumar, R. (2015). Soil salinity: A serious environmental issue and plant growth promoting bacteria as one of the tools for its alleviation. Saudi Journal of Biological Sciences, 22(2), 123e131. Silva-Ortega, C. O., Ochoa-Alfaro, A. E., Reyes-Agüero, J. A., Aguado-Santacruz, G. A., & Jiménez-Bremont, J. F. (2008). Salt stress increases the expression of p5cs gene and induces proline accumulation in cactus pear. Plant Physiology and Biochemistry, 46(1), 82e92. Singh, A. K., Singh, R., Kumar, R., Chaurasia, S., & Yadav, S. (2018). Genomics technologies for improving salt tolerance in wheat. In Engineering practices for management of soil salinity (pp. 251e304). Apple Academic Press. Song, X., Li, Y., Cao, X., & Qi, Y. (2019). MicroRNAs and their regulatory roles in planteenvironment interactions. Annual Review of Plant Biology, 70, 489e525. 344 Plant Small RNA in Food Crops Stephenson, T. J., McIntyre, C. L., Collet, C., & Xue, G.-P. (2007). Genome-wide identification and expression analysis of the NF-Y family of transcription factors in Triticum aestivum. Plant Molecular Biology, 65(1e2), 77e92. Sun, G. (2012). MicroRNAs and their diverse functions in plants. Plant Mol Biol, 80(1), 17e36. https://doi.org/10.1007/s11103-011-9817-6. Epub 2011 Aug 27. PMID: 21874378. Sun, X., Xu, L., Wang, Y., Yu, R., Zhu, X., Luo, X., Gong, Y., Wang, R., Limera, C., & Zhang, K. (2015). Identification of novel and salt-responsive miRNAs to explore miRNA-mediated regulatory network of salt stress response in radish (Raphanus sativus L.). BMC Genomics, 16(1), 1e16. Sun, Z., Wang, F. Mou, Tian, Y., Chen, L., Zhang, S., Jiang, Q., & Li, X. (2016). Genomewide small RNA analysis of soybean reveals auxin-responsive microRNAs that are differentially expressed in response to salt stress in root apex. Frontiers of Plant Science, 6, 1273. Sunkar, R., Chinnusamy, V., Zhu, J., & Zhu, J.-K. (2007). Small RNAs as big players in plant abiotic stress responses and nutrient deprivation. Trends in Plant Science, 12(7), 301e309. Sunkar, R., Zhou, X., Zheng, Y., Zhang, W., & Zhu, J.-K. (2008). Identification of novel and candidate miRNAs in rice by high throughput sequencing. BMC Plant Biology, 8(1), 1e17. Sunkar, R., & Zhu, J.-K. (2004). Novel and stress-regulated microRNAs and other small RNAs from Arabidopsis. The Plant Cell Online, 16(8), 2001e2019. Tian, Y., Tian, Y., Luo, X., Zhou, T., Huang, Z., Liu, Y., Qiu, Y., Hou, B., Sun, D., & Deng, H. (2014). Identification and characterization of microRNAs related to salt stress in broccoli, using high-throughput sequencing and bioinformatics analysis. BMC Plant Biology, 14(1), 1e13. Tian, C., Zuo, Z., & Qiu, J.-L. (2015). Identification and characterization of ABAresponsive microRNAs in rice. Journal of Genetics and Genomics, 42(7), 393e402. Tombuloglu, H. (2019). Genome-wide analysis of the auxin response factors (ARF) gene family in barley (Hordeum vulgare L.). Journal of Plant Biochemistry and Biotechnology, 28(1), 14e24. Tripathi, A., Chacon, O., Lata Singla-Pareek, S., K Sopory, S., & Sanan-Mishra, N. (2018). Mapping the microRNA expression profiles in glyoxalase overexpressing salinity tolerant rice. Current Genomics, 19(1), 21e35. Van den Ende, W., & Peshev, D. (2013). Sugars as antioxidants in plants. Crop improvement under adverse conditions. Springer. Virk, P., Khush, G., & Peng, S. (2004). Breeding to enhance yield potential of rice at IRRI: the ideotype approach. International Rice Research Notes. Wang, B., Sun, N. Song, Wei, J.-p., Wang, X.-j., Feng, H., Yin, Z.-y., & Kang, Z.-s. (2014). MicroRNAs involving in cold, wounding and salt stresses in Triticum aestivum L. Plant Physiology and Biochemistry, 80, 90e96. Wang, T., Chen, L., Zhao, M., Tian, Q., & Zhang, W.-H. (2011). Identification of drought-responsive microRNAs in Medicago truncatula by genome-wide highthroughput sequencing. BMC Genomics, 12(1), 1e11. Walia, H., Wilson, P. Condamine, Liu, X., Ismail, A. M., Zeng, L., Wanamaker, S. I., Mandal, J., Xu, J., & Cui, X. (2005). Comparative transcriptional profiling of two contrasting rice genotypes under salinity stress during the vegetative growth stage. Plant Physiology, 139(2), 822e835. Wang, J.-W., Wang, L.-J., Mao, Y.-B., Cai, W.-J., Xue, H.-W., & Chen, X.-Y. (2005). Control of root cap formation by microRNA-targeted auxin response factors in Arabidopsis. The Plant Cell, 17(8), 2204e2216. Small RNAs involved in salt stress tolerance of food crops 345 Wang, H., & Wang, H. (2015). The miR156/SPL module, a regulatory hub and versatile toolbox, gears up crops for enhanced agronomic traits. Molecular Plant, 8(5), 677e688. Wang, Q., Yang, Y., Lu, G., Sun, X., Feng, Y., Yan, S., Zhang, H., Jiang, Q., Zhang, H., & Hu, Z. (2020). Genome-wide identification of microRNAs and phased siRNAs in soybean roots under long-term salt stress. Genes & Genomics, 42(11), 1239e1249. Wang, J., Ye, Y., Xu, M., Feng, L., & Xu, L-a (2019). Roles of the SPL gene family and miR156 in the salt stress responses of tamarisk (Tamarix chinensis). BMC Plant Biology, 19(1), 1e11. Xia, K., Ou, X., Tang, H., Wang, R., Wu, P., Jia, Y., Wei, X., Xu, X., Kang, S. H., & Kim, S. K. (2015). Rice microRNA osa-miR1848 targets the obtusifoliol 14a-demethylase gene Os CYP 51G3 and mediates the biosynthesis of phytosterols and brassinosteroids during development and in response to stress. New Phytologist, 208(3), 790e802. Xia, K., Wang, R., Ou, X., Fang, Z., Tian, C., Duan, J., Wang, Y., & Zhang, M. (2012). OsTIR1 and OsAFB2 downregulation via OsmiR393 overexpression leads to more tillers, early flowering and less tolerance to salt and drought in rice. PloS One, 7(1), Article e30039. Xia, G., Xiang, F., Zhou, A., Wang, H., & Chen, H. (2003). Asymmetric somatic hybridization between wheat (Triticum aestivum L.) and Agropyron elongatum (Host) Nevishi. Theoretical and Applied Genetics, 107(2), 299e305. Xie, J., Lei, B., Niu, M., Huang, Y., Kong, Q., & Bie, Z. (2015a). High throughput sequencing of small RNAs in the two Cucurbita germplasm with different sodium accumulation patterns identifies novel microRNAs involved in salt stress response. PloS One, 10(5), Article e0127412. Xie, R., Zhang, Y. Ma, Pan, X., Dong, C., Pang, S., He, S., Deng, L., Yi, S., & Zheng, Y. (2017). Combined analysis of mRNA and miRNA identifies dehydration and salinity responsive key molecular players in citrus roots. Scientific Reports, 7(1), 1e19. Xie, F., Wang, Q., Sun, R., & Zhang, B. (2015). Deep sequencing reveals important roles of microRNAs in response to drought and salinity stress in cotton. Journal of Experimental Botany, 66(3), 789e804. Xing, Y., & Zhang, Q. (2010). Genetic and molecular bases of rice yield. Annual Review of Plant Biology, 61, 421e442. Yaish, M. W., Sunkar, R., Zheng, Y., Ji, B., Al-Yahyai, R., & Farooq, S. A. (2015). A genome-wide identification of the miRNAome in response to salinity stress in date palm (Phoenix dactylifera L.). Frontiers in Plant Science, 6, 946. Yang, Z., Zhu, P., Kang, H., Liu, L., Cao, Q., Sun, J., Dong, T., Zhu, M., Li, Z., & Xu, T. (2020). High-throughput deep sequencing reveals the important role that microRNAs play in the salt response in sweet potato (Ipomoea batatas L.). BMC Genomics, 21(1), 1e16. Yang, W., Fan, T., Hu, X., Cheng, T., & Zhang, M. (2017). Overexpressing osa-miR171c decreases salt stress tolerance in rice. Journal of Plant Biology, 60(5), 485e492. Yang, Y., & Guo, Y. (2018). Elucidating the molecular mechanisms mediating plant saltstress responses. New Phytologist, 217(2), 523e539. Yu, Y., Wu, G., Yuan, H., Cheng, L., Zhao, D., Huang, W., Zhang, S., Zhang, L., Chen, H., & Zhan, J. (2016). Identification and characterization of miRNAs and targets in flax (Linum usitatissimum) under saline, alkaline, and saline-alkaline stresses. BMC Plant Biology, 16(1), 1e13. Yuan, S., Li, Z., Li, D., Yuan, N., Hu, Q., & Luo, H. (2015). Constitutive expression of rice microRNA528 alters plant development and enhances tolerance to salinity stress and nitrogen starvation in creeping bentgrass. Plant Physiology, 169(1), 576e593. 346 Plant Small RNA in Food Crops Yuan, S., Zhao, J., Li, Z., Hu, Q., Yuan, N., Zhou, M., Xia, X., Noorai, R., Saski, C., & Li, S. (2019). MicroRNA396-mediated alteration in plant development and salinity stress response in creeping bentgrass. Horticulture Research, 6(1), 1e13. Zhang, X., Zou, Z., Gong, P., Zhang, J., Ziaf, K., Li, H., Xiao, F., & Ye, Z. (2011). Overexpression of microRNA169 confers enhanced drought tolerance to tomato. Biotechnology Letters, 33(2), 403e409. Zhang, Y., Gong, H., Li, D., Zhou, R., Zhao, F., Zhang, X., & You, J. (2020). Integrated small RNA and Degradome sequencing provide insights into salt tolerance in sesame (Sesamum indicum L.). BMC Genomics, 21(1), 1e12. Zhang, Y., Fang, J., Wu, X., & Dong, L. (2018). Naþ/Kþ balance and transport regulatory mechanisms in weedy and cultivated rice (Oryza sativa L.) under salt stress. BMC Plant Biology, 18(1), 1e14. Zhao, B., Ge, L., Liang, R., Li, W., Ruan, K., Lin, H., & Jin, Y. (2009). Members of miR169 family are induced by high salinity and transiently inhibit the NF-YA transcription factor. BMC Molecular Biology, 10(1), 1e10. Zhao, S., Zhang, Q., Liu, M., Zhou, H., Ma, C., & Wang, P. (2021). Regulation of plant responses to salt stress. International Journal of Molecular Sciences, 22(9), 4609. Zhou, X., Wang, G., Sutoh, K., Zhu, J.-K., & Zhang, W. (2008). Identification of coldinducible microRNAs in plants by transcriptome analysis. Biochimica et Biophysica Acta (BBA)-Gene Regulatory Mechanisms, 1779(11), 780e788. CHAPTER 12 miRNAs perspective in mitigating waterlogging stress in plants Garima Singroha and Pradeep Sharma ICAR-Indian Institute of Wheat and Barley Research, Karnal, Haryana, India 1. Introduction The growing global population necessitates a significant increase in food production. As stated by the UN Population Division, the world’s population will reach 8.3 billion people by 2030. The scientific community’s critical mission in the 21st century is to supply better quality food for an ever-growing population. Furthermore, changing climatic circumstances have a negative impact on agricultural productivity all over the world. Extreme climatic conditions are the leading cause of global crop output loss of more than 50% per year (Singhal et al., 2015). Hydrological fluctuation, such as excessive precipitation causing flooding of farming land is common and occurs in 10%e12% of agricultural regions worldwide, resulting in annual losses of more than $74 billion (He et al., 2020; Shabala, 2011; Voesenek & Sasidharan, 2013). Waterlogging is an important stressor that reduces yield by 20%e25% in barley (Ahmed et al., 2013), 20%e30% in maize (Du et al., 2017), and 20%e50% yield loss in wheat (Manik et al., 2019) as well as harming more than 16% of the world’s rice (Sarkar et al., 2006). Therefore, it is a prerequisite to develop high-yielding stress adaptive crop varieties to meet future food security challenges. Genetic engineering is one such method that is now being utilized all over the world to boost crop output by producing disease and stress-resistant crop types (Lesk et al., 2016). The pleiotropic effects of genetic engineering complicates the genetics of agronomical characteristics since a single trait might be regulated by several genes or vice versa. As a result, genetic engineering that improves one feature may have a detrimental influence on other important traits. Furthermore, agronomic traits like high yield and stress tolerance are also regulated by a network of genes or pathways, making gene selection for desirable trait(s) a demanding process. Manipulation of agronomical attributes to improve crop performance, therefore, involves the employment of Plant Small RNA in Food Crops ISBN 978-0-323-91722-3 https://doi.org/10.1016/B978-0-323-91722-3.00009-9 © 2023 Elsevier Inc. All rights reserved. 347 348 Plant Small RNA in Food Crops precise and particular genetic modulators. MicroRNAs have just evolved as a potential genetic engineering target, and they have been exploited to generate high-yielding and stress-tolerant crop varieties (Djami-Tchatchou et al., 2017; Xu et al., 2019; Zhang, 2015; Zhang & Wang, 2015). MiRNAs are 20e24 nucleotide long, non-coding endogenous regulatory RNAs that govern various biological processes by silencing genes at the transcriptional and post-transcriptional stages (Jones-Rhoades et al., 2006). MiRNAs govern gene expression by both pairing with and cleaving their target mRNAs or by limiting protein translation (Jones-Rhoades et al., 2006; Singroha & Sharma, 2019). They have been recognized as important post-transcriptional regulators of gene expression (Saroha et al., 2017, Singroha et al., 2022). Many reports advocate vital roles for miRNAs in plant’s behavior toward abiotic and biotic stresses such as cold (Bouba et al., 2019; Li & Zhang, 2016; Song et al., 2019; Sunkar et al., 2012), salt (Fouracre & Poethig, 2016; Li & Zhang, 2016), heat (Bouba et al., 2019), drought (Singroha et al., 2021; Xie et al., 2015), oxidative stress (Smoczynska and Szweykowska-Kulinska, 2016), mechanical stress (Bouba et al., 2019), pathogen infection (Du et al., 2011), and submergence (Salvador-Guirao et al., 2018). Recent findings claim that miRNAs are potentially implicated in the regulation of adaptive response to hypoxia (Paul & Chakraborty, 2013) and submergence (Zhai et al., 2013). Transcription factors (TFs) are well-known targets of posttranscriptional regulation, implying that miRNAs could serve as early signaling components, leading to broader changes in gene expression in response to stress. Owing to their crucial role in controlling gene regulation under adverse conditions (miRNAs) have been proposed as promising targets for developing plants that are more resistant to a variety of abiotic stresses (Kumar, 2014). With the development of new platforms for highthroughput sequencing of small RNAs, several miRNAs involved in the response to submergence in plants such as Arabidopsis thaliana (Pegler et al., 2019), Brachypodium distachyon (Jeong et al., 2013), Oryza sativa (BarreraFigueroa et al., 2012; Jin et al., 2017) Nelumbo nucifera as well as Zea mays (Liu et al., 2012, 2019) have been identified. According to miRBase, 325 mature miRNAs were identified in maize with some of them being reported to be responsive to drought (Seeve et al., 2019), submergence (Zhang et al., 2008), or waterlogging (Zhai et al., 2013). In this chapter, we will go over the miRNAs that have been identified so far in various crops that are subjected to waterlogged conditions in plants, as well as their functions. miRNAs perspective in mitigating waterlogging stress in plants 349 2. Waterlogging effect on plant growth and development Waterlogging, flooding and submergence, are all interrelated and exert similar effects on plants growth and development (Fukao et al., 2019). Flooding is described as either waterlogging, which occurs when the water is shallow and merely covers the root. Submergence occurs when plants upper tissues are completely covered under water. Both forms of flooding restrict oxygen absorption from the air into tissues (Lee et al., 2011), leading to hypoxia (21% O2). Waterlogging causes leaf stomata to close, leaf senescence leading to incapability to capture sunlight, and chlorophyll degradation, resulting in a decline in photosynthetic rate (Kuai et al., 2014; Yan et al., 2018). Waterlogging prevents gaseous exchange between soil and atmosphere by clogging soil pores; nevertheless, the oxygen diffusion rate in water is only 1/10,000 of that in air. As a result, oxygen availability in damp soil is strictly limited, ensuing reduced root respiration, reduced root activity, and a lack of energy (van Veen et al., 2014). During hypoxia caused by waterlogging, plants can temporarily continue energy production by using glycolysis and ethanol fermentation. Long-term waterlogging and anaerobic respiration, on the other hand, result in the accumulation of toxic metabolites like lactic acid, ethanol, and aldehydes, as well as an increase in reactive oxygen species (ROSs), particularly hydrogen peroxide, which eventually leads to cell death and plant senescence (Xu et al., 2014; Zhang et al., 2017). Plant hormones can accumulate or degrade quickly if a gaseous exchange is hampered, and this can alter the plant waterlogging tolerance mechanism (Hattori et al., 2009; Kuroha et al., 2018). Although most plants suffer when they are wet, they can respond to the damage caused by such stress by employing a variety of techniques (Doupis et al., 2017; Xu et al., 2016; Yin et al., 2019). Waterlogging is an important limiting factor that severely restrict crop productivity and leads to submersion; hypoxia; and waterlogging stress. Flooding causes submergence and, as a result, raises the groundwater table, resulting in a hypoxic rhizosphere. The anaerobic environment created by the hypoxic situation in the rhizosphere inhibits oxygen intake, resulting in plant mortality (Fukao et al., 2019). 3. miRNA biogenesis in plants Pri-miRNAs (20e24 ntd) fold back on themselves, generating a doublestranded RNA stem-loop structure that DCL1 (DICER-LIKE1) acts on, 350 Plant Small RNA in Food Crops resulting in a miRNA: miRNA* duplex with two nucleotide overhangs at its 3’ end (Li et al., 2021). The active strand involved in regulating target gene expression is known as miRNA, while the opposing strand is known as miRNA*. The nucleus contains the RNA-binding protein HYPONASTIC LEAVES1 (Kurihara et al. 2006), SERRATE (SE; Machida et al., 2011; Yang et al., 2006), DOUBLE RNA BINDING PROTEIN (DRB2; Eamens et al., 2012), and C-TERMINAL DOMAIN PHOSPHATASE LIKE1 (CPL1; Manavella et al., 2012). HYL1 is essential for the precised miRNA processing, whereas SE increases DCL1 activity as well as play a scaffolding role (Dolata et al., 2018; Yang et al., 2014). The HUAENHANCER 1 (HEN1) RNA methyltransferase (located in the nucleus) adds a methyl group to the duplex at the 30 ends (20-O-methylation) of the miRNA: miRNA* duplex (Yu et al., 2005). A nuclear membranelocalized HASTY protein transports the miRNA: miRNA* duplex formed in the nucleus to the cytoplasm. The mature miRNA is subsequently placed into the RNA-induced silencing complex (RISC), where it acts as a lead molecule while the miRNA* degrades rapidly (Bologna et al., 2018). Although the duplex structure, HYL1 and the identity of the 50 base sequence come out to play vital role in miRNA strand assortment for loading into ARGONAUTE 1 (AGO1) (Fang and Qi, 2016). According to one research, the nucleus contains the empty AGO1, but miRNA loading causes a conformational change, revealing the nuclear export signal that indicates its route to the cytoplasm (Bologna et al., 2018). The mature miRNA on AGO1 instructs the RISC to pairwith the corresponding sequence on the target mRNAs (Jones-Rhoades et al., 2006). Plant miRNAs interact with their targets by perfect or near-perfect complementarity, causing cleavage of the target mRNA between the 10th and 11th nucleotides relative to the miRNA’s 50 end. When a miRNA with a higher number of mismatches and bulges aligns with its target miRNA, translation is suppressed (Brodersen et al., 2008; Lanet et al., 2009). 4. Expression and modulation of miRNAs and morphological adaptations microRNAs (miRNAs) are endogenous tiny non-coding RNAs that regulate gene expression after transcription. They’ve been implicated in a variety of biological activities in plants, including development and response to environmental signals. Plant miRNAs recognize target mRNAs via almost perfect base matching and direct them to be cleaved or inhibited miRNAs perspective in mitigating waterlogging stress in plants 351 during translation (Rogers and Chen, 2013). It is expected that when a certain miRNA accumulates, the expression of the target mRNA would decrease, and vice versa. MiRNA expression is highly responsive to environmental signals, allowing them to act as fine-tuned dynamic stress regulators. Furthermore, miRNAs serve as regulatory nodes in complex networks that connect plant development to biotic and abiotic stress responses (Rubio-Somoza and Weigel, 2011). Several miRNAs have been discovered revealing a wide spectrum of biological roles in plants in response to stress. MiRNAs promote and control the balance between the response to flooding stress and development via hormonal signaling pathways. Analysis of cis-regulatory elements in the promoters of floodingresponsive miRNAs revealed that the phytohormones ethylene, GA, ABA, and auxin signal transduction pathways are critical components involving the complex network of flooding response (Liu et al., 2012; Zhang et al., 2008). It has been found that flooding-responsive miRNAs regulate mRNAs that encode transcription factors (TFs) that are involved in imparting flooding adaptation to different plant tissues. In maize roots, for example, waterlogging enhanced the expression of miR159, which silenced two mRNAs encoding GAMYBs i.e. MYB101 and MYB33 homologs (Liu et al., 2012). MiR159 inhibits primary root growth in Arabidopsis via regulating MYB33, MYB101, and MYB65 (Xue et al., 2017). During long-standing waterlogging, adventitious roots develope at the base of the stem or at the internodes of the hypocotyl to promote gaseous exchange and absorption of water and nutrients. The development of adventitious root replaces primary rootsto some extent that die due to hypoxic stress and preserve metabolic cycles for normal growth and development of the plant (Eysholdt Derzsó and Sauter, 2019; Xu et al., 2016). As a result, the upregulation of miR159 in maize roots under waterlogging could be critical for clearing GAMYB-mRNAs and inhibiting main root development. GAMYBs repression may also affect responses to ABA signaling pathways, which incorporates MYB factors (Fig. 12.1). Flooding, on the other hand, has been found to down-regulate miR159 in various tissues, including submerged Lotus seedlings, where degradome and transcriptome research has confirmed multiple GAMYB targets of miR159 that may be involved in GA-mediated petiole elongation (Jin et al., 2017). Tolerant plants, such as Alternanthera philoxeroides, respond quickly to oxygen deprivation by establishing adventitious roots on submerged stem nodes (Ayi et al., 2016). 352 Plant Small RNA in Food Crops Figure 12.1 The expression level of miR164 is upregulated under waterlogging conditions to promote growth of the adventitious roots. Similarly miR159 is upregulated to supress primary roots growth. Auxin signaling is obligatory for causing morphological changes in roots due to the action of miRNAs and auxin response factor TFs (ARF; Meng et al., 2010). As part of a complex phytohormone response network that includes auxins, MiR166 has been found to control root development by targeting HD-ZIP III transcripts (Singh et al., 2017). Overexpression of miR166 in Arabidopsis resulted in downregulation of HD-ZIP III and improved root development in ordinary conditions (Singh et al., 2014). In response to short-term waterlogging, MiR166 was shown to be upregulated in maize roots, where it was shown to trigger down-regulation of its target, the maize HD-ZIP III. MiR166 may take part in the modulation of phytohormone signaling pathways to endorse sideways and adventitious rooting during waterlogging by regulating HD-ZIP III. Under hypoxia stress, however, miR166 was found to be down-regulated in Arabidopsis roots (Moldovan et al., 2010). Because hypoxia stops root growth and is followed by the rapid development of adventitious roots when normoxia is restored, it has been hypothesized that miR166 may play a key role in the pathways that integrate critical levels of hypoxia signals during floodings, such as calcium spikes and an increase in ROS, to miRNAs perspective in mitigating waterlogging stress in plants 353 readdress root growth toward branching or adventitious rooting when normal oxygen levels are restored. MiR166 was shown to be up-regulated in maize roots in response to short-term waterlogging, where it was found to cause down-regulation of its target, the maize HD-ZIP III family member rolled leaf 1 (Zhang et al., 2008). In contrast to short-term stress, long-term waterlogging downregulated miR167 in maize roots, suggesting differential regulation as stress proceeded (Zhai et al., 2013). Experiments showed the presence of two miR167 targets in maize roots, which encode the transcription factors ARF16 and ARF18 (Liu et al., 2012). Based on earlier results that miR167 modulates ARF expression to regulate adventitious and lateral root formation, miR167 regulation of ARFs in maize roots may have a time-scale balancing impact between reducing primary root growth and encouraging adventitious rooting in response to floods (Gutierrez et al., 2009). MiR167 was also found to be up-regulated in the internodes of Alternanthera plants (Li et al., 2017) and Populus seedlings (Ren et al., 2013), but down-regulated in Lotus seedlings under submersion (Jin et al., 2017), implying that it may regulate some of the assorted roles of ARF transcription factors in diverse tissues (e.g., shoot/ petiole elongation). In Arabidopsis roots subjected to hypoxia, which is an outcome of waterlogging stress, miR390 was upregulated (another miRNA involved in the control of auxin response pathways) (Moldovan et al., 2010). Small transacting RNAs (tasiRNA3), which negatively affect the expression of ARF TFs, are created from miR390’s target mRNA, TAS3. MiR390 and tasiRNA3 accumulated at the regions of lateral root initiation to curb ARF expression and eventually increased lateral roots length according to a study of Arabidopsis mutants with different TAS3a levels. Another miRNA involved in auxin response pathways, miR390, was found to be upregulated in roots of Arabidopsis exposed to hypoxia, which is a component of flooding stress (Moldovan et al., 2010). By interacting with its target mRNA, TAS3, which produces small transacting RNAs, miR390 suppresses the development of ARF TFs (tasiRNA3). In Arabidopsis mutants with different levels of TAS3a, MiR390 and tasiRNA3 were shown to concentrate at the sites of lateral root initiation, inhibiting the expression of ARFs and extending the length of lateral roots. ARFs also govern auxin-inducible miR390 expression, providing a feedback loop that retains ARF expression in a fine-temporal and spatial regulation for optimal root development control under oxygen-limited conditions (Marin et al., 2010). Two other miRNAs connected to auxin regulation mechanisms are miR393 and miR164. 354 Plant Small RNA in Food Crops miR393 was downregulated after Lotus seedlings were submerged. As proven by degradome and short RNA sequencing study, their targets are mRNAs encoding TIR/F-BOX, which are enhanced by miR393 downregulation to boost the auxin response in submerged seedlings (Jin et al., 2017). MiR393 was found to be up-regulated in damp maize roots (Liu et al., 2012) and submerged Brachypodium aerial tissue (Franke et al., 2018). However, in response to floods, miR164 was increased in maize roots (Liu et al., 2012). The expression of NAC/NAM domain proteins is suppressed by miR164 in Arabidopsis, allowing for fine regulation of auxin-induced lateral root growth (Guo et al., 2005). In response to waterlogging, upregulation of miR164 in maize roots appears to have a comparable effect on root growth control. Hypoxia increased miR156 expression in Arabidopsis roots, as did submersion in Brachypodium aerial tissues and Lotus seedlings (Franke et al., 2018; Jin et al., 2017; Moldovan et al., 2010). MiR156 has been shown to be involved in regulating a variety of mRNAs that encode members of the SQUAMOSA PROMOTER BINDING PROTEIN-LIKES (SPLs) gene family, which further plays an important role in root growth, shoot branching and maturity and the transition from juvenile to the adult phase, to name a few (Schwarz et al., 2008). SPL10, in Arabidopsis for example, is potrayed as one of the most important repressors of root growth (Yu et al., 2015). Overexpression of miR156 in Arabidopsis under standard conditions increases lateral roots by downregulating SPL10 (Gao et al., 2018). This suggests that the miR156:SPL10 module may play a role in gene regulation. During floods, ethylene is a significant signal that induces metabolic and morphological changes. AP2/ERF (APETALA2/Ethylene Responsive Element) is a large family of ethylene-controlled transcription factors involved in floral organ identity, shoot meristem development, primary and secondary metabolism, and other aspects of plant growth and development (Licausi et al., 2013). MiR172 regulates them post-transcriptionally as well. Prolonged waterlogging in maize roots diminished miR172 expression and accumulated AP2/ERF mRNAs, signifying an increase in ethylene signaling pathways to enhance crown root formation as an adaptive morphological response, according to degradome research (Zhai et al., 2013). Submergence dramatically impacted the levels of several miRNAs that directly altered the level of transcripts encoding some essential components in those phytohormone-regulated networks. miRNAs perspective in mitigating waterlogging stress in plants 355 Several members of the miR159 and miR319 families, for example, formed a subnetwork to regulate the expression of GAMYB genes, which mediate GA signaling in petiole elongation (Gocal et al., 2002). Aside from that, the miR159 and miR319 families are involved in regulating the expression of GAMYB genes, which mediate Gibberellin signaling in petiole elongation (Gocal et al., 2002). Jin et al. (2017) found that submergence caused down-regulation of miR159b-3p, miR159, miR319p, and miR159h-3p, resulting in activation of the GAMYB gene and perhaps promoting elongation of petiole. TIR1 transcripts were also upregulated due to decreased expression of miR393, miR393h, and miR393-5p. TRANSPORT INHIBITOR RESPONSE 1/AUXIN SIGNALING F-box proteins AUXIN/INDOLE-3ACETIC ACID (Aux/IAA) repressors are degraded by F-BOX (TIR1/AFB) auxin receptors, resulting in auxin-regulated responses (Terrile et al., 2012). 5. Conclusion Plant miRNAs have a range of roles in almost all cellular networks at the molecular level, in addition to their core function of gene silencing. Because of their ability to control stress-responsive genes, plant miRNAs are a promising candidate for developing stress-tolerant crop varieties. If researchers have a better understanding of the molecular pathways mediated by miRNA in complex molecular networking systems, they will be able to modify specific agronomical traits in crops. Because a single miRNA in plants can regulate many genes and networks, scientists must select a potential miRNA to target a certain agronomically important trait. To unravel pri-miRNA-mediated regulatory networks for such traits, effective approaches are required. Without a doubt, miRNA-based strategies have enormous promise for crop improvement, fostering future inter-disciplinary collaborations among researchers from various disciplines. Furthermore, before realizing the entire potential of miRNA-based genome editing in agriculture, appropriate laboratory research and controlled field experiments are necessary. However, when using miRNA-based genetic alteration processes, it is vital to be aware of any unexpected consequences that may arise in the future (Table 12.1). 356 Table 12.1 Long non coding RNAs/miRNAs involved in imparting salt tolerance. lncRNA/miRNA Plant species Characteristics References 1 ThSAIR6 Tamarix hispida Xu et al. (2021) 2 AtR8l AtR8lncRNA Arabidopsis thaliana 3 LncRNA973 Gossypium hirsutum 4 Pal_00132209 Populus alba 5 Pal_00184400 Populus alba 6 lnc_388, lnc_973, lnc_253 Gossypium hirsutum 7 DRIR (drought induced long non coding RNA) Arabidopsis thaliana Decreased the contents of H2O2 and enhanced activity of antioxidative enzymes Regulate seed germination in response to salt Increased expression resulted into increased salt tolerance Affect fucosyltransferase or NAC3 and regulates growth under salt stress HKT1 and show differential expression in xylem Regulates tolerance to salt stress Regulates ABA mediated responses to both salt and drought Zhang et al. (2020) Zhang et al. (2019) Ma et al. (2019) Ma et al. (2019) Ding et al. (2018) Qin et al. (2017) Plant Small RNA in Food Crops S.N. 8 TCONS_00116877 Medicago truncatula 9 TCONS_00046739 Medicago truncatula 10 miR156, miR398 Solanum lycopersicum 11 nta-miR156a_ R þ 3, farmiR159_L þ 2_1ss22T, mes-MIR319ep5_2ss12GC19GA miR26, miR05, miR20, miR31, miR11, miR28, miR15, miR14, miR32, miR09, miR22, miR33, miR19, miR24 miR172, miR319, miR408, miR2590 Ipomoea batatas Pennisetum glaucum Shows altered expression under salinity Shinde et al. (2020) Alfalfa Ma et al. (2019) 14 TaemiR408 Triticum aestivum 15 miR164s, mir-36 Zea maize Regulates gene associated with salt tolerance Overexpression resulted in enhanced salt tolerance Up-regulated in leaves under salt treatment 13 Wang et al. (2015) Wang et al. (2015) Cakir et al. (2021) Yan et al. (2020) Bai et al. (2018) Fu et al. (2017) miRNAs perspective in mitigating waterlogging stress in plants 12 Regulates oxidative stress under salt conditions Regulates cytochrome P450 under salt stress Increased expression levels imparted salt tolerance Tissue specific expression under salt stress 357 Continued 358 Table 12.1 Long non coding RNAs/miRNAs involved in imparting salt tolerance.dcont'd lncRNA/miRNA Plant species Characteristics References 16 osa-miR1878, osamiR2863c miR171b, miR167f Oryza sativa Goswami et al. (2017) Parmar et al. (2020) 19 sly-miR156e-5p, slymiRn23b, slymiRn50a miR172 Solanum pimpinellifolium Glycine max 20 miRNVL5 Gossypium hirsutum 21 miR-395 Cucumis sativus 22 miR156/157, miR158, miR166, miR168 and miR408 miR-160 Raphanus sativus Upregulated under salt stress Promotes better adaptability to salt Involved in stress related pathways Improves salt tolerance Regulation of plant stress to salt Up-regulated and regulates ATP sulfurylase Expression was upregulated significantly Up-regulated under salt stress and control auxin response factor (ARF) 17 18 23 Oryza sativa Gossypium raimondii Zhao et al. (2017) Pan et al. (2016) Gao et al. (2016) Li et al. (2016) Sun et al. (2015) Xie et al. (2015) Plant Small RNA in Food Crops S.N. miRNAs perspective in mitigating waterlogging stress in plants 359 References Ahmed, F., Rafii, M. Y., Ismail, M. R., Juraimi, A. S., Rahim, H. A., Asfaliza, R., & Latif, M. A. (2013). Waterlogging tolerance of crops: Breeding, mechanism of tolerance, molecular approaches, and future prospects. BioMed Research International, 2013, 963525. Bai, Q, Wang, X, Chen, X, Shi, G, Liu, Z, Guo, C, & Xiao, K (2018). Wheat miRNA TaemiR408 Acts as an Essential Mediator in Plant Tolerance to Pi Deprivation and Salt Stress via Modulating Stress-Associated Physiological Processes. Front. Plant Sci., 9(499). https://doi.org/10.3389/fpls.2018.00499 Barrera-Figueroa, B. E., Gao, L., Wu, Z., Zhou, X., Zhu, J., Jin, H., Liu, R., & Zhu, J.-K. (2012). High throughput sequencing reveals novel and abiotic stress-regulated microRNAs in the inflorescences of rice. BMC Plant Biology, 12, 132. Bologna, N. G., Iselin, R., Abriata, L. A., Sarazin, A., Pumplin, N., Jay, F., Grentzinger, T., Dal Peraro, M., & Voinnet, O. (2018). Nucleo-cytosolicshuttling of ARGONAUTE1 prompts a revised model of the plantmicroRNA pathway. Molecular Cell, 69, 709e719. Bouba, I., Kang, Q., Luan, Y.-S., & Meng, J. (2019). Predicting miRNA-lncRNA interactions and recognizing their regulatory roles in stress response of plants. Mathematical Biosciences, 312, 67e76. Brodersen, P., Sakvarelidze-Achard, L., Bruun-Rasmussen, M., Dunoyer, P., Yamamoto, Y. Y., Sieburth, L., & Voinnet, O. (2008). Widespread transla-tional inhibition by plant miRNAs and siRNAs. Science, 320, 1185e1190. Çakır, O., Arıkan, B., & Karpuz, B. (2021). Expression Analysis of miRNAs and Their Targets Related to Salt Stress in Solanum lycopersicum H-2274. Biotechnol. Biotechnological Equipment, 35, 283e290. Djami-Tchatchou, A. T., Sanan-Mishra, N., Ntushelo, K., & Dubery, I. A. (2017). Functional roles of microRNAs in agronomically important plantsdpotential as targets for crop improvement and protection. Frontiers of Plant Science, 8, 378. Dolata, J., Taube, M., Bajczyk, M., Jarmolowski, A., Szweykowska-Kulinska, Z., & Bielewicz, D. (2018). Regulation of plant microprocessor functionin shaping microRNA landscape. Frontiers in Plant Science, 9, 753. Doupis, G., Kavroulakis, N., Psarras, G., & Papadakis, I. (2017). Growth photosynthetic performance and antioxidative response of ‘Hass’ and ‘Fuerte’avocado (Persea americana Mill.) plant grown under high soil moisture. Photosynthetica, 55, 655e663. https:// doi.org/10.1007/s11099-016-0679-7 Du, P., Wu, J., Zhang, J., Zhao, S., Zheng, H., Gao, G., Wei, L., & Li, Y. (2011). Viral infection induces expression of novel phased microRNAs from conserved cellular microRNA precursors. PLoS Pathogens, 7, e1002176. Du, H., Zhu, J., Su, H., Huang, M., Wang, H., Ding, S., Zhang, B., Luo, A., Wei, S., Tian, X., & Xu, Y. (2017). Bulked segregant RNA-seq reveals differential expression and SNPs of candidate genes associated with waterlogging tolerance in maize. Frontiers of Plant Science, 8, 1022. https://doi.org/10.3389/fpls.2017.01022 Eamens, A. L., Kim, K. W., Curtin, S. J., & Waterhouse, P. M. (2012). DRB2 isrequired for microRNA biogenesis in Arabidopsis thaliana. Plos One, 7,e35933. https://doi.org/ 10.1371/journal.pone.0035933 Fang, X., & Qi, Y. (2016). RNAi in plants: an argonaute-centered view. PlantCell, 28, 272e285. https://doi.org/10.1105/tpc.15.00920 Fouracre, J. P., & Poethig, R. S. (2016). The role of small RNAs in vegetative shoot development. Current Opinion in Plant Biology, 29, 64e72. Franke, K. R., Schmidt, S. A., Park, S., Jeong, D.-H., Accerbi, M., & Green, P. J. (2018). Analysis of Brachypodium miRNA targets: Evidence for diverse control during stress and conservation in bioenergy crops. BMC Genomics, 19, 547. https://doi.org/10.1186/ s12864-018-4911-7 360 Plant Small RNA in Food Crops Fu, R, Zhang, M, Zhao, Y, He, X, Ding, C, Wang, S, Feng, Y, Song, X, Li, P, & Wang, B (2017). Identification of Salt Tolerance-related microRNAs and Their Targets in Maize (Zea mays L.) Using High-throughput Sequencing and Degradome Analysis. Front. Plant Sci., 8(864). https://doi.org/10.3389/fpls.2017.00864 Fukao, T., Barrera-Figueroa, B. E., Juntawong, P., & Peña-Castro, J. M. (2019). Submergence and waterlogging stress in plants: A review highlighting research opportunities and understudied aspects. Frontiers of Plant Science, 10, 340. https://doi.org/10.3389/ fpls.2019.00340 Gao, S, Yang, L, Zeng, HQ, Zhou, ZS, Yang, ZM, Li, H, Sun, D, Xie, F, & Zhang, B (2016). A cotton miRNA is involved in regulation of plant response to salt stress. Sci Rep., 27(6), 19736. https://doi.org/10.1038/srep19736 Gao, R., Wang, Y., Gruber, M. Y., & Hannoufa, A. (2018). MiR156/SPL10 modulates lateral root development, branching and leaf morphology in Arabidopsis by silencing AGAMOUS-LIKE 79. Frontiers of Plant Science, 8, 2226. https://doi.org/10.3389/ fpls.2017.02226 Goswami, Kavita, Tripathi, Anita, & Sanan-Mishra, Neeti (2017). “Comparative miRomics of Salt-Tolerant and Salt-Sensitive Rice”. Journal of Integrative Bioinformatics, 14(1). https://doi.org/10.1515/jib-2017-0002 Guo, H.-S., Xie, Q., Fei, J.-F., & Chua, N.-H. (2005). MicroRNA directs mRNA cleavage of the transcription factor NAC1 to downregulate auxin signals for Arabidopsis lateral root development. The Plant Cell Online, 17, 1376e1386. https://doi.org/10.1105/ tpc.105.030841 Hattori, Y., Nagai, K., Furukawa, S., Song, X.-J., Kawano, R., Sakakibara, H., Wu, J., Matsumoto, T., Yoshimura, A., Kitano, H., Matsuoka, M., Mori, H., & Ashikari, M. (2009). The ethylene response factors SNORKEL1 and SNORKEL2 allow rice to adapt to deep water. Nature, 460, 1026e1030. https://doi.org/10.1038/nature08258 He, X., Pan, M., Wei, Z., Wood, E. F., & Sheeld, J. (2020). A global drought and flood catalogue from 1950 to 2016. Bulletin of the American Meteorological Society, 101, E508eE535. Jeong, D.-H., Schmidt, S. A., Rymarquis, L. A., Park, S., Ganssmann, M., German, M. A., Accerbi, M., Zhai, J., Fahlgren, N., Fox, S. E., Garvin, D. F., Mockler, T. C., Carrington, J. C., Meyers, B. C., & Green, P. J. (2013). Parallel analysis of RNA ends enhances global investigation of microRNAs and target RNAs of Brachypodium distachyon. Genome Biology, 14, R145. https://doi.org/10.1186/gb-2013-14-12-r145 Jin, Q., Xu, Y., Mattson, N., Li, X., Wang, B., Zhang, X., Jiang, H., Liu, X., Wang, Y., & Yao, D. (2017). Identification of submergence-responsive microRNAs and their targets reveals complex miRNA-mediated regulatory networks in lotus (Nelumbo nucifera Gaertn). Frontiers of Plant Science, 8, 6. https://doi.org/10.3389/fpls.2017.00006 Jones-Rhoades, M. W., Bartel, D. P., & Bartel, B. (2006). MicroRNAs and their regulatory roles in plants. Annual Review of Plant Biology, 57, 19e53. Kuai, J., Liu, Z., Wang, Y., Meng, Y., Chen, B., Zhao, W., Zhou, Z., & Oosterhuis, D. M. (2014). Waterlogging during flowering and boll forming stages affects sucrose metabolism in the leaves subtending the cotton boll and its relationship with boll weight. Plant Science, 223, 79e98. https://doi.org/10.1016/j.plantsci.2014.03.010 Kumar, R. (2014). Role of microRNAs in biotic and abiotic stress responses in crop plants. Applied Biochemistry and Biotechnology, 174, 93e115. Kurihara, Y., Takashi, Y., & Watanabe, Y. (2006). The interaction betweenDCL1 and HYL1 is important for efficient and precise processing ofpri-miRNA in plant microRNA biogenesis. RNA, 12, 206e212. Kuroha, T., Nagai, K., Gamuyao, R., Wang, D. R., Furuta, T., Nakamori, M., Kitaoka, T., Adachi, K., Minami, A., Mori, Y., Sakakibara, H., Wu, J., Ebana, K., Mitsuda, N., Ohme-Takagi, M., & Ashikari, M. (2018). Ethylene-gibberellin signaling underlies the miRNAs perspective in mitigating waterlogging stress in plants 361 adaptation of rice to periodic flooding. Science, 361, 181e186. https://doi.org/10.1126/ science.aat1577 Lanet, E., Delannoy, E., Sormani, R., Floris, M., Crete, P., Voinnet, O., et al. (2009). Biochemical evidence for translational repression by Ara-bidopsis microRNAs. Plant Cell, 21, 1762e1768. Laurent Gutierrez, John D. Bussell, Daniel I. Pacurar, Josèli Schwambach, Monica Pacurar, Catherine Bellini, Phenotypic Plasticity of Adventitious Rooting in Arabidopsis Is Controlled by Complex Regulation of AUXIN RESPONSE FACTOR Transcripts and MicroRNA Abundance. (2009). The Plant Cell, 21(10), 3119-3132. Lee, S. C., Mustroph, A., Sasidharan, R., Vashisht, D., Pedersen, O., Oosumi, T., et al. (2011). Molecular characterization of the submergence response of the Arabidopsis thaliana ecotype Columbia. New Phytol, 190, 457e471. https://doi.org/10.1111/ j.1469-8137.2010.03590.x Lesk, C., Rowhani, P., & Ramankutty, N. (2016). Influence of extreme weather disasters on global crop production. Nature, 529, 84e87. Licausi, F., Ohme-Takagi, M., & Perata, P. (2013). APETALA2/ethylene-responsive factor (AP2/ERF) transcription factors: Mediators of stress responses and developmental programs. New Phytologist, 199, 639e649. https://doi.org/10.1111/nph.12291 Liu, H., Able, A. J., & Able, J. A. (2019). Genotypic performance of Australian durum under single and combined water-deficit and heat stress during reproduction. Scientific Reports, 9, 1e17. Liu, Z., Kumari, S., Zhang, L., Zheng, Y., & Ware, D. (2012). Characterization of miRNAs in response to short-term waterlogging in three inbred lines of Zea mays. PLoS One, 7(6), e39786. https://doi.org/10.1371/journal.pone.0039786 Li, M, & Yu, B (2021). Recent advances in the regulation of plant miRNA biogenesis. RNA Biol., 18(12), 2087e2096. https://doi.org/10.1080/15476286.2021.1899491 Li, C., & Zhang, B. (2016). MicroRNAs in control of plant development. Journal of Cellular Physiology, 231, 303e313. Ma, J, Bai, X, Luo, W, Feng, Y, Shao, X, Bai, Q, Sun, S, Long, Q, & Wan, D (2019). Genome-Wide Identification of Long Noncoding RNAs and Their Responses to Salt Stress in Two Closely Related Poplars. Front Genet., 5(10), 777. https://doi.org/ 10.3389/fgene.2019.00777 Machida, S., Chen, H. Y., & Yuan, Y. A. (2011). Molecular insights into miRNAprocessing by Arabidopsis thaliana serrate. Nucleic Acids Research, 39, 7828e7836. Manavella, P. A., Hagmann, J., Ott, F., Laubinger, S., Franz, M., & Macek, B. (2012). Fastforward genetics identifies plant CPL phosphatases as reg-ulators of miRNA processing factor HYL1. Cell, 151, 859e870. Manik, S. M. N., Pengilley, G., Dean, G., Field, B., Shabala, S., & Zhou, M. (2019). Soil and crop management practices to minimize the impact of waterlogging on crop productivity. Frontiers of Plant Science, 10, 140. Marin, E., Jouannet, V., Herz, A., Lokerse, A. S., Weijers, D., Vaucheret, H., Nussaume, L., Crespi, M. D., & Maizel, A. (2010). Mir390, Arabidopsis TAS3 tasiRNAs, and their AUXIN RESPONSE FACTOR targets define an autoregulatory network quantitatively regulating lateral root growth. The Plant Cell Online, 22, 1104e1117. https:// doi.org/10.1105/tpc.109.072553 Meng, Y, Chen, D, Ma, X, Mao, C, Cao, J, Wu, P, & Chen, M (2010). Mechanisms of microRNA-mediated auxin signaling inferred from the rice mutant osaxr. Plant Signal Behav., 252(4). https://doi.org/10.4161/psb.5.3.10549 Moldovan, D., Spriggs, A., Yang, J., Pogson, B. J., Dennis, E. S., & Wilson, I. W. (2010). Hypoxia-responsive microRNAs and trans-acting small interfering RNAs in Arabidopsis. Journal of Experimental Botany, 61, 165e177. https://doi.org/10.1093/jxb/ erp296 362 Plant Small RNA in Food Crops Parmar, S, Gharat, SA, Tagirasa, R, Chandra, T, Behera, L, Dash, SK, & Shaw, BP (2020). Identification and expression analysis of miRNAs and elucidation of their role in salt tolerance in rice varieties susceptible and tolerant to salinity. PLoS One, 15(4), e0230958. https://doi.org/10.1371/journal.pone.0230958 Paul, P., & Chakraborty, S. (2013). Computational prediction of submergence responsive microRNA and their binding position within the genome of Oryza sativa. Bioinformation, 9, 858. https://doi.org/10.6026/97320630009858 Pegler, JL, Oultram, JMJ, Grof, CPL, & Eamens, AL (2019). Profiling the Abiotic Stress Responsive microRNA Landscape of Arabidopsis thaliana. Plants (Basel), 8(3). https:// doi.org/10.3390/plants8030058. PMID: 30857364; PMCID: PMC6473545. Qiaoli Ayi, Bo Zeng, Jianhui Liu, Siqi Li, Peter M. van Bodegom, Johannes H. C. Cornelissen, (2016). Oxygen absorption by adventitious roots promotes the survival of completely submerged terrestrial plants. Annals of Botany, 118(4), 675e683. https://doi. org/10.1093/aob/mcw051. Qin, T, Zhao, H, Cui, P, Albesher, N, & Xiong, L (2017). A Nucleus-Localized Long NonCoding RNA Enhances Drought and Salt Stress Tolerance. Plant Physiol., 175(3), 1321e1336. https://doi.org/10.1104/pp.17.00574 Ren, Y, Chen, L, Zhang, Y, Kang, X, Zhang, Z, & Wang, Y (2013). Identification and characterization of salt-responsive microRNAs in Populus tomentosa by highthroughput sequencing. Biochimie, 95(4), 743e750. https://doi.org/10.1016/ j.biochi.2012.10.025 Rogers, K, & Chen, X (2013). Biogenesis, turnover, and mode of action of plant microRNAs. Plant Cell, 25(7), 2383e2399. https://doi.org/10.1105/tpc.113.113159 Rubio-Somoza, I, & Weigel, D (2011). MicroRNA networks and developmental plasticity in plants. Trends Plant Sci., 16(5), 258e264. https://doi.org/10.1016/ j.tplants.2011.03.001 Salvador-Guirao, R., Baldrich, P., Weigel, D., Rubio-Somoza, I., & Segundo, B. S. (2018). The MicroRNA miR773 is involved in the Arabidopsis immune response to fungal pathogens. Molecular Plant-Microbe Interactions, 31, 249e259. Sarkar, R. K., Reddy, J. N., Sharma, S. G., & Ismail, A. M. (2006). Physiological basis of submergence tolerance in rice and implications for crop improvement. Current Science, 91, 899e906. Saroha, M., Singroha, G., Sharma, M., Mehta, G., Gupta, O. P., & Sharma, P. (2017). sRNA and epigenetic mediated abiotic stress tolerance in plants. Indian Journal of Plant Physiology, 22, 458e469. Schwarz, S., Grande, A. V., Bujdoso, N., Saedler, H., & Huijser, P. (2008). The microRNA regulated SBP-box genes SPL9 and SPL15 control shoot maturation in Arabidopsis. Plant Molecular Biology, 67, 183e195. https://doi.org/10.1007/s11103-008-9310-z Seeve, C. M., Sunkar, R., Zheng, Y., et al. (2019). Seeve, C.M., Sunkar, R., Zheng, Y. et al. Water-deficit responsive microRNAs in the primary root growth zone of maize. BMC Plant Biol, 19, 447. https://doi.org/10.1186/s12870-019-2037-y Shabala, S. (2011). Physiological and cellular aspects of phytotoxicity tolerance in plants: The role of membrane transporters and implications for crop breeding for waterlogging tolerance. New Phytologist, 190, 289e298. Shinde, H, Dudhate, A, Anand, L, Tsugama, D, Gupta, SK, Liu, S, & Takano, TT (2020). Small RNA sequencing reveals the role of pearl millet miRNAs and their targets in salinity stress responses. South African Journal of Botany, 132, 395e402. https://doi.org/ 10.1016/j.sajb.2020.06.011 Singh, A., Roy, S., Singh, S., et al. (2017). Phytohormonal crosstalk modulates the expression of miR166/165s, target Class III HD-ZIPs, and KANADI genes during root growth in Arabidopsis thaliana. Sci Rep, 7, 3408. https://doi.org/10.1038/s41598-01703632-w miRNAs perspective in mitigating waterlogging stress in plants 363 Singh, A, Singh, S, Panigrahi, KC, Reski, R, & Sarkar, AK (2014). Balanced activity of microRNA166/165 and its target transcripts from the class III homeodomain-leucine zipper family regulates root growth in Arabidopsis thaliana. Plant Cell Rep., 33(6), 945e953. https://doi.org/10.1007/s00299-014-1573-z Singhal, P., Jan, A. T., Azam, M., & Haq, Q. M. R. (2015). Plant abiotic stress: A prospective strategy of exploiting promoters as an alternative to overcome the escalating burden. Frontiers in Life Science, 9, 52e63. Singroha, G, Kumar, S, Gupta, OP, Singh, GP, & Sharma, P (2022). Uncovering the Epigenetic Marks Involved in Mediating Salt Stress Tolerance in Plants. Front Genet., 12(13), 811732. https://doi.org/10.3389/fgene.2022.811732 Singroha, G., & Sharma, P. (2019). Epigenetic modifications in plants under abiotic stress. In R. Meccariello (Ed.), Epigenetics. London: IntechOpen. https://doi.org/10.5772/ intechopen.84455 Singroha, G., Sharma, P., & Sunkur, R. (2021). Current status of microRNA-mediated regulation of drought stress responses in cereals. Physiologia Plantarum, 1e14. https:// doi.org/10.1111/ppl.13451. Available from:. Smoczynska, A., & Szweykowska-Kulinska, Z. (2016). MicroRNA-mediated regulation of flower development in grasses. Acta Biochimica Polonica, 63, 687e692. Song, X., Li, Y., Cao, X., & Qi, Y. (2019). MicroRNAs and their regulatory roles in planteenvironment interactions. Annual Review of Plant Biology, 70, 489e525. Sunkar, R., Li, Y. F., & Jagadeeswaran, G. (2012). Functions of microRNAs in plant stress responses. Trends in Plant Science, 17, 196e203. Terrile, MC, París, R, Calderón-Villalobos, LI, Iglesias, MJ, Lamattina, L, Estelle, M, & Casalongué, CA (2012). Nitric oxide influences auxin signaling through S-nitrosylation of the Arabidopsis TRANSPORT INHIBITOR RESPONSE 1 auxin receptor. Plant Journal, 70(3), 492e500. https://doi.org/10.1111/j.1365-313X.2011.04885.x van Veen, H., Akman, M., Jamar, D. C., Vreugdenhil, D., Kooiker, M., van Tienderen, P., Voesenek, L. A. C. J., Schranz, M. E., & Sasidharan, R. (2014). Group VII Ethylene response factor diversification and regulation in four species from flood-prone environments. Plant, Cell and Environment, 37, 2421e2432. Voesenek, L. A. C. J., & Sasidharan, R. (2013). Ethylene-and oxygen signalling-drive plant survival during flooding. Plant Biology, 15, 426e435. Wang, TZ, Liu, M, Zhao, MG, Chen, R, & Zhang, WH (2015). Identification and characterization of long non-coding RNAs involved in osmotic and salt stress in Medicago truncatula using genome-wide high-throughput sequencing. BMC Plant Biol., 6(15). https://doi.org/10.1186/s12870-015-0530-5 Xie, F., Jones, D. C., Wang, Q., Sun, R., & Zhang, B. (2015). Small RNA sequencing identifies miRNA roles in ovule and fiber development. Plant Biotechnology Journal., 13, 355e369. Xu, J., Hou, Q.-M., Khare, T., Verma, S. K., & Kumar, V. (2019). Exploring miRNAs for developing climate-resilient crops: A perspective review. Science of the Total Environment, 653, 91e104. Xu, X., Ji, J., Ma, X., Xu, Q., Qi, X., & Chen, X. (2016). Comparative proteomic analysis provides insight into the key proteins involved in cucumber (Cucumis sativus L.) adventitious root emergence under waterlogging stress. Frontiers of Plant Science, 7, 1515. https://doi.org/10.3389/fpls.2016.01515 Xu, X., Wang, H., Qi, X., Xu, Q., & Chen, X. (2014). Waterlogging-induced increase in fermentation and related gene expression in the root of cucumber (Cucumis sativus L.). Scientia Horticulturae, 179, 388e395. https://doi.org/10.1016/j.scienta.2014.10.001 Xue, T., Liu, Z., Dai, X., & Xiang, F. (2017). Primary root growth in Arabidopsis thaliana is inhibited by the miR159 mediated repression of MYB33, MYB65 and MYB101. Plant Science, 262, 182e189. 364 Plant Small RNA in Food Crops Yan, M, Nie, H, Wang, Y, Wang, X, Jarret, R, Zhao, J, Wang, H, & Yang, J (2022). Exploring and exploiting genetics and genomics for sweetpotato improvement: Status and perspectives. Plant Communications, 3(5), 2590e3462. https://doi.org/10.1016/j. xplc.2022.100332. ISSN 100332. Yan, K., Zhao, S., Cui, M., Han, G., & Wen, P. (2018). Vulnerability of photosynthesis and photosystem I in Jerusalem artichoke (Helianthus tuberosus L.) exposed to waterlogging. Plant Physiology and Biochemistry, 125, 239e246. https://doi.org/10.1016/ j.plaphy.2018.02.017 Yang, L., Liu, Z., Lu, F., Dong, A., & Huang, H. (2006). SERRATE is a novelnuclear regulator in primary microRNA processing in Arabidopsis. ThePlant, 47, 841e850. Yin, D., Sun, D., Han, Z., Ni, D., Norris, A., & Jiang, C.-Z. (2019). PhERF2, an ethyleneresponsive element-binding factor, plays an essential role in waterlogging tolerance of Petunia. Horticultural Research, 6, 1e11. Yu, N., Niu, Q.-W., Ng, K.-H., & Chua, N.-H. (2015). The role of miR156/SPLs modules in Arabidopsis lateral root development. The Plant Journal, 83, 673e685. https://doi.org/10.1111/tpj.12919 Zhai, L., Liu, Z., Zou, X., Jiang, Y., Qiu, F., Zheng, Y., & Zhang, Z. (2013). Genomewide identification and analysis of microRNA responding to long-term waterlogging in crown roots of maize seedlings. Physiologia Plantarum, 47, 181e193. https://doi.org/ 10.1111/j.1399-3054.2012.01653.x Zhang, B. (2015). MicroRNA: A new target for improving plant tolerance to abiotic stress. Journal of Experimental Botany, 66, 1749e1761. Zhang, P., Lyu, D., Jia, L., He, J., & Qin, S. (2017). Physiological and de novo transcriptome analysis of the fermentation mechanism of Cerasus sachalinensis roots in response to short-term waterlogging. BMC Genomics, 18, 649. https://doi.org/ 10.1186/s12864-017-4055-1 Zhang, B., & Wang, Q. (2015). MicroRNA-based biotechnology for plant improvement. Journal of Cellular Physiology, 230, 1e15. Zhang, Z., Wei, L., Zou, X., Tao, Y., Liu, Z., & Zheng, Y. (2008). Submergenceresponsive MicroRNAs are potentially involved in the regulation of morphological and metabolic adaptations in maize root cells. Annals of Botany, 102(4), 509e519. https://doi.org/10.1093/aob/mcn129 Zhao, G., Yu, H. Y., & Liu, M. M. (2017). Identification of Salt Stress Responsive miRNAs from Solanum lycopersicum and Solanum Pimpinellifolium. Plant Growth Regul., 83, 129e140. https://doi.org/10.1007/s10725-017-0289-9 CHAPTER 13 Molecular mechanisms alleviating drought stress tolerance in crop plants Kolluru Viswanatha Chaitanyaa, Akbar Ali Khan Pathanb and Reddymalla Nikhila Reddyb a Department of Microbiology and Food Science Technology, GITAM Institute of Science, GITAM University, Visakhapatnam, India; bDepartment of Biotechnology, GITAM Institute of Science, GITAM University, Visakhapatnam, India 1. Introduction Crop plants are frequently subjected to harsh environmental conditions, adversely affecting their growth and productivity. Abiotic stress factors such as drought, salinity, high and low temperatures are the major detrimental factors responsible for crop damage. Drought stress is a major limiting factor that causes severe productivity constraints by restricting agriculture production, leading to severe economic losses worldwide every year. Drought stress is one of the main restraining factors for plant growth that hinder stomatal movement, photosynthesis, electron chain transport affecting plant growth and physiological metabolism. Being sessile, plants have developed a wide range of defense mechanisms, including morphological and structural changes, drought-resistant gene expression, synthesis of hormones, and osmoregulatory substances that alleviate drought stress tolerance (Yang et al., 2021). Plants sense the prevailing drought stress conditions and synthesize a set of specialized molecules such as abscisic acid (ABA), Ca2þ, inositol that induces morphological and physiological changes through signaling. Drought stress induces the expression of functional genes, whose products are involved in plant metabolism. Functional gene products such as proline, glycine betaine, LEA proteins, and aquaporins affect the plant state. Regulatory gene products such as calcium-dependent protein kinases (CDPK), Mitogen-activated protein kinases (MAPK) act as transcription factors for regulating the expression of genes that control the morphology and physiology of the plants, enabling the plants to successfully tolerate the brutal drought stress conditions (Li et al., 2020). Drought stress-induced gene expression is regulated at the transcriptional, post-transcriptional, Plant Small RNA in Food Crops ISBN 978-0-323-91722-3 https://doi.org/10.1016/B978-0-323-91722-3.00018-X © 2023 Elsevier Inc. All rights reserved. 365 366 Plant Small RNA in Food Crops and post-translational levels. Among them, gene regulation at the posttranscriptional level plays a pivotal role in the plant response against drought stress. The role of small non-coding RNAs (sRNAs) in posttranscriptional gene regulation during drought stress is elucidated. This chapter will illustrate the molecular responses of crop plants to drought stress and highlights the regulatory events responsible for providing drought stress tolerance. 2. Drought stress tolerance in plants Drought stress inflicts multiple effects on plant growth, adversely damaging its productivity. Plants recognize and respond to the prevailing drought through a variety of signals. Plants are composed of cellular, molecular, and regulatory mechanisms for the short-term responses such as prevention of water loss from the guard cells through transpiration and long-term responses such as acquiring resistance to the drought stress. 2.1 Abscisic acid (ABA) Plants respond to the prevailing drought stress by closing stomata on their leaf surface to prevent transpirational water loss. Phyto hormone Abscisic acid (ABA) acts as an anti transpirant by inducing the stomatal closure (Wilkinson et al., 2012). ABA is a sesquiterpene with a small molecular weight. ABA is synthesized from the beginning of the drying process and is degraded during rehydration after dehydration (Roychoudhury et al., 2013). ABA is synthesized by cyanobacteria, algae, bryophytes, fungus, lichens, and higher plants. Four cyanobacteria species out of eleven have been examined for ABA synthesis under various stress conditions, exhibiting an increase in the ABA levels (Hartung, 2010). Endophytic bacteria in the roots of Helianthus annuus under water deficit conditions were found to synthesize ABA (Forchetti et al., 2007). The C-15 ABA skeleton is present in several biosynthetic precursors, including xanthoxin, abscisic aldehyde, abscisic alcohol, and oxidative catabolites such as phaseic acid, 80 -hydroxyABA, and dihydrophaseic acid. Several stress signals cause an increase in the amount of ABA generated endogenously in the plant system. These may include the activating genes encoding enzymes that produce ABA from b-carotene. ABA substantially alters plant leaf senescence, and senescencerelated gene 113 (SAG113) lowers stomatal conductance, leading in quick water loss in aging leaves, and hence effectively regulates ABA-induced leaf Molecular mechanisms alleviating drought stress tolerance in crop plants 367 senescence. (Govind et al., 2011). Even though light stimulates the stomata, regulation of partial or total stomatal closure is by ABA and high CO2 concentration (Kim et al., 2010). ABA biosynthesis is also critical for root architecture maintenance, as it serves as a mediator of osmotic pressure in roots during drought stress, changing the root architecture in response to environmental stress (Sah et al., 2016). 2.2 ABA biosynthesis under abiotic stress Abscisic acid belongs to the class of metabolites known as isoprenoids or terpenoids. In plastids, ABA is synthesized by a mevalonic-acid-independent mechanism. The mevalonic acid-independent 2-C-methyl-D-erythritol-4phosphate (MEP) pathway is used in plastids to produce ABA, a 15-carbon isoprenoid plant hormone. The 15 carbon atoms in ABA result from the cleavage of C40 carotenoids of the MEP mechanism (Ng et al., 2014). ABA production starts in the plastids with zeaxanthin, a C40 carotenoid, and is completed in the cytosol with abscisic aldehyde oxidized to ABA. The conversion of zeaxanthin and antheraxanthin to trans-violaxanthin in the plastid is mediated by zeaxanthin epoxidase (ZEP), the first step in the ABA production process. In this process, antheraxanthin is produced as an intermediate. After that, all-trans-violaxanthin is converted to 9-cis-violaxanthin or 9-cis-neoxanthin. It is uncertain which enzyme is involved in this process. The 9-cis-epoxy carotenoid dioxygenase (NCED) catalyzes the oxidative cleavage of 9-cis-violaxanthin and 9-cis-neoxanthin, resulting in the synthesis of a C15 product, xanthoxin, and a C25 metabolite. NCED is the main enzyme in ABA biosynthesis, and this reaction is believed to be the rate-limiting step (Seiler et al., 2011). The maize viviparous S14 mutant was used to isolate the ZmNCED gene, and NCED is the main enzyme in ABA production (Tan et al., 1997). Two enzymatic processes convert xanthoxin to ABA in the cytosol. An enzyme belonging to the short-chain dehydrogenase/ reductase (SDR) family converts xanthoxin to the abscisic aldehyde in the first phase. In Arabidopsis thaliana, the gene AtABA2 is responsible (Zhang et al., 2014). The last stage in ABA biosynthesis is the oxidation of the abscisic aldehyde to ABA, mediated by abscisic aldehyde oxidase (AAO) (Fig. 13.1). 2.3 ABA transport Stress-induced ABA production occurs mainly in vascular tissues, although ABA affects various cells, including distant guard cells. Water is collected 368 Plant Small RNA in Food Crops Figure 13.1 ABA biosynthesis in plant cells. form the plants via roots and outflows it through stomata located on the epidermal layer of the terrestrial organs such as stem and leaves. When soil is dried due to drought, ABA accumulates in root tissues and initiates root growth and root branching followed by accelerated absorption of water (Fig. 13.2). Molecular mechanisms alleviating drought stress tolerance in crop plants 369 INITIATION OF ROOT GROWH INITIATION OF ROOT BRANCHING ACCELERATED WATER ABSORPTION AND TRANSMISSION Figure 13.2 ABA function in the roots subjected to drought stress. Stomata play a major role in the absorption and emission of CO2 and O2 throughout photosynthesis and respiration. Regulation of stomatal opening under drought stress is achieved by the integration of environmental signals, facilitating their closure. ABA responses need intercellular transport from ABA-producing cells to facilitate fast dispersion into surrounding tissues. To enhance cell-to-cell ABA transfer, two plasma membrane-related ATP-binding cassette (ABC) transporters were identified. (Kuromori et al., 2010). The movement of ABA across cellular, tissues, and organs is indeed important for the plant’s overall physiologic reaction to stimulus. ABA, a weak acid, may penetrate slowly through cell membrane when protonated. Most ABC transporters are membrane proteins that function as ATP-driven transporters for a wide variety of substrates, including lipids, medicines, heavy metals, and auxin (Kang et al., 2011). AtBCG40, a comprehensive ABC transporter, is an ABA carrier in plant cells. ABCG16 also facilitates ABA tolerance. Using a transport test, Kanno et al. (2012) found an ABA-importing carrier (AIT1) in yeast and insect cells. AIT1 is a member of the NRT1 (nitrate transporter 1) carrier family. AIT1 mutation is little susceptible to ABA during plant growth and after growth, and their stomata remained open, whereas AIT1 upregulation led to a significant in ABA oversensitivity. (Pan et al., 2020). In Arabidopsis, AtDTX50 (Detoxification Efflux Carrier 50) is engaged in ABA transport (Léran et al., 2020). The AtDTX50 mutant had a quicker ABA-actuate stomatal closure, indicating that the guard cells impart drought tolerance had high levels of ABA. AtDTX50 is predominantly found in the stem and root tissues and guard cells of Arabidopsis thaliana. Such findings suggest that AtDTX50 is involved in the ABA diffusion from the cell cytoplasm of vascular and guard cells in the cell membrane (Fig. 13.3). 370 Plant Small RNA in Food Crops STOMATAL CLOSURE LOW LEAF MESOPHYLL TRANSMISSION LOW LEAF WATER DISTRIBUTION INITIATION OF LEAF SENESCENCE REGULATES LEAF GROWTH INITIATION OF CUTICLE WAX DEPOSITION CONTROL OF SHOOT GROWTH Figure 13.3 ABA function in leaves during drought stress. 3. Transcription factors engaged in abiotic stress resistance Transcription factors (TFs) are DNA-binding proteins that control gene responses by engaging with a transcription pre-initiation unit and binding to cis-elements in the promoter domains of the corresponding genes in a sequence-specific way. This interaction is necessary for the re-alignment of gene expression due to the induction or suppression of RNA polymerase. Certain transcription elements interact with their cis-elements to operate as primary controller in plant systems, integrating, balancing, and coordinating hormonal, developmental, and environmental signals (Jaradat et al., 2013). AREBs (ABA-responsive element binding proteins)/ABFs are the key transcription elements in plants’ abiotic stress resistance (ABA-linked gene network). It has been found that severe salt dehydration or ABA application may promote AREB1/ABF2, AREB2/ABF4, and ABF3 in multiple and Molecular mechanisms alleviating drought stress tolerance in crop plants 371 implication of these genes result in improved drought resistance (Yoshida et al., 2010). RD29A and RD29B genes, along with an ABA-responsive promoter, aid in desiccation tolerance (Msanne et al., 2011). RD29A/ COR78/LT178 gene is commonly referred as overexpressed in both ABAdependent and ABA-independent stress conditions. Control of ABAdependent conditions is by ABRE (ABA-responsive element) whereas the regulation of and ABA independent mechanisms is by a cis-acting element DRE (Dehydration Responsive Element) (Khan et al., 2020). ABA has an excessive function in signaling a range of transcription elements engaged in various stressors. An increase in the expression of the NAC transcription factors in the genetically-engineered rice plants is due to ABAmediated signaling (Mathew et al., 2016). A particular transcription factor can regulate the expression of several targeted genes by selectively binding to cis-acting regions in their promoters. Around 7% of the coding motifs in plant genomes are allocated to transcription factor, illustrating the intricacy of transcriptional control. AREBs (ABA-responsive element binding proteins)/ABFs (ABRE binding factors), ABI5 (ABA insensitive 5), MYB (myeloblastosis), MYC (myelocytomatosis), NAC (NAM: no apical meristem; ATAF: Arabidopsis transcription promotion factor; CUC: cupshaped cotyledon), and ERF are transcription elements essential for the control of the ABA-(ethylene response factor) (Yoon et al., 2020). DRE/CRT is the critical cis-acting factor in ABA-responsive or nonresponsive gene expression (Agarwal et al., 2017). ABA-dependent signaling systems are the pathways that facilitate stress adaptation through the activation of at least two different regulons (a set of genes regulated through single kind of TF: AREB/ABF (ABA-responsive element-binding protein/ABA-binding factor) regulon; and MYC/MYB regulon (Li et al., 2019). The bZIP (basic leucine zipper) transcription factor domain includes AREBs/ABFs. The bZIP transcription elements was identified based on their in vitro interactions with ABRE (ABA-responsive element) and regulatory function in ABA and other stress stimuli. Many stress-sensitive genes include a dehydration responsive element (DRE) in their promoter sequence. DREB refers to the proteins that bind to this DRE. TFs from the DREB family have been found in Arabidopsis, rice, mangrove, soybean, and potato, and additional plant species (Li et al., 2018). Drought, salt, and cold stressors all lead to dehydration in plant cells, which causes the upregulation of numerous TFs, consisting of DREB proteins. Arabidopsis and rice, which contain 57 and 52 DREB TFs, have been discovered to react to a wide variety of abiotic stressors that eventually contribute to plant 372 Plant Small RNA in Food Crops dehydration stress (Chai et al., 2020). Arabidopsis DREB2A functions in water and extreme heat adaptations (Singh & Laxmi, 2015). Generical classification of DREB into DREB1 and DREB2 is associated with lesser temperature, dehydration, and reactions to heat stress and excessive salinity, respectively. These two transcription factors essentially followed two distinct signal transduction pathways and were discovered to be constituents of the ethylene-responsive element-binding factor (ERF) class of transcription factors. ERF proteins shared a conserved 58e59 amino acid domain known as the ERF domain, which could interact with two ciselements, the GCC box and the C-repeat CRT/DRE motif (Cui et al., 2021). The isolation of cDNAs encoding DRE binding proteins has facilitated the detection of CBF1 (CRT binding factor 1), DREB1A, and DREB2A from Arabidopsis thaliana using the yeast one-hybrid screening approach. SiDREB2, a gene similar to AuniqueDREB2, was identified in foxtail millet (Setaria italica) and was shown to take part in dehydration stress resistance (Muthamilarasan et al., 2014). DREB1 (CBF) is a member of the AP2/EREBP transcription factor family and is involved in controlling coldresponsive genes. Three primary DREB1 genes have been discovered in Arabidopsis thaliana: DREB1b (CBF1), DREB1c (CBF2), and DREB1a (CBF3). The DREB proteins, through DRE, are critical in regulating gene expression in reactions to a range of abiotic stressors. Since members of the DREB2 subfamily are activated due to dehydration and severe salinity, their involvement in the regulation of stress-responsive gene expression is highly substantial (Singh & Chandra, 2021). List of transcription factors involved in drought stress tolerance is listed in Table 13.1. 4. Non-coding RNAs and drought stress Non-coding RNAs (ncRNA) are vital factors in the modulation of drought stress resistance among crop plants (Yu et al., 2019). The networking of the ncRNAs and their target genes are controlled by various enzymatic and non-enzymatic parts in the cell. High throughout put RNA-sequence analysis and bioinformatics have provided an essential source for studying the differences between stress susceptible and stresstolerant plant species and their varieties, leading to identifying stressinduced gene expression patterns. ncRNAs were classified into lengthy non-coding RNAs (lncRNAs) and shorter non-coding RNAs(sncRNAs). Molecular mechanisms alleviating drought stress tolerance in crop plants 373 Table 13.1 List of transcriptional factors involved in the drought stress tolerance of plants. S.No. Transcription factor Family Plant 1 2 3 4 5 6 7 8 9 10 11 12 13 14 ABF3, 4 FtbZIP5 OSbZIP12, 46 AtbHLH68 PebHLH35 ANAC 019, 055 and 072 OsNAC 5, 6 and 9 TaRNAC1 AtERF74 AhDREB1 ZmDREB2A AtMYB 44, 96 OsWRKY11, 45 TaWRKY1, 33 bZIP bZIP bZIP bHLH bHLH NAC NAC NAC ERF AP2 AP2 MYB WRKY WRKY Arabidopsis thaliana Fagopyrum tataricum Oryza sativa Arabidopsis thaliana Populus euphratica Arabidopsis thaliana Oryza sativa Triticum aestivum Arabidopsis thaliana Arachis hypogaea Zea mays Arabidopsis thaliana Oryza sativa Triticum aestivum 4.1 Long non-coding RNAs Long non-coding RNAs (lncRNAs) are 200 nucleotides in size, involved in genome regulation and epigenetic control. Their evolutionary origins and functional specializations are not identified to date. lncRNAs lack the potentiality to code for a polypeptide but possess a significant biochemical versatility by displaying specific functions. lncRNAs produced in the plants undergo capping, splicing, and poly-adenylation, like that of mRNA. Based on the location and biogenesis, lncRNAs are categorized into Long intergenic ncRNAs (lincRNAs), Transposable Element derived lncRNAs (TE-derived lncRNAs), Intron derived lncRNAs (incRNAs), Natural Antisense Transcripts (NATs), and Circular lncRNAs (circncRNAs) (Lucero et al., 2020). Long intergenic ncRNAs (lincRNAs) also known as long or large intervening cRNAs, and macro RNAs are of 200 nucleotides in length, polyadenylated, weakly spliced, exhibits tissue-specific expression, displaying a trans-regulatory function. lincRNAs are located in gene deserts with at least 5 Kb away from the protein-coding regions (Jha et al., 2020). lincRNAs are further classified into enhancer RNA (eRNA), upstream antisense RNA (uaRNA), promoter-associated long RNA (PALR) also known as promoter upstream transcripts (PROMPTS), and telomeric repeat-containing RNA (TERRA) based on their associations with regulatory regions. Among these, PROMPTS and eRNA are short-lived. Most 374 Plant Small RNA in Food Crops of the lincRNAs are characterized by a rapid turnover rate, posing a challenge in understanding their specific and functional significance. Transposable Element-derived lncRNAs (TE-lncRNAs) are generated from transposable elements. These TE-derived lncRNAs act as precursors for the synthesis of small non-coding RNAs for example microRNA (miRNA) and small interfering RNA (siRNA) (Ali et al., 2021). Association of TE-lncRNA includes abiotic factors like drought was reported in rice, maize, and tomato (Chen et al., 2020). Many TE-lncRNAs are predicted to occur in the maize genome, as more than 85% of its genome is derived from the activity of transposable elements (Lv et al., 2019). Intronderived lncRNAs (incRNAs) originate from the introns of protein-coding genes, comprising either total intronic RNA or partial intronic RNA incRNAs are polyadenylated and are stable. Natural Antisense Transcripts (NATs) are one of the widespread lncRNAs that originate from exon as well as intron regions. These lncRNAs possess cis and trans activity to control gene expression by silencing (Wight & Werner, 2013). The binding of NATs will trigger the synthesis of specific siRNAs, exhibiting a trans mode of action. Circular lncRNAs (circncRNAs) are highly conserved lncRNAs with low abundance. circncRNAs are relatively more stable than linear RNAs and are not degraded easily. They were first characterized in plant viroids as non-polyadenylated circular RNAs. They arise in the nucleus through reverse splicing of exons in pre-mRNAs (Chu et al., 2018). circncRNAs consisting of one or sometimes more than one extra exon is known as extra-exon circular ncRNAs (ee-circncRNAs) and the circncRNAs are derived from the intron region of the gene is known as circular intronic RNAs (circincRNAs). circncRNAs arising from the overlapping regions of intron and exon is known as exon-intron circncRNAs (Qin et al., 2020). circncRNAs are involved in regulating splicing and cell development by acting as target mimics for miRNA, protein scaffolds, and templates for protein translation. circncRNA of exomes regulates cell proliferation (Liu et al., 2015). 4.2 Small non-coding RNAs (sncRNAs) The discovery of small non-coding RNAs and small interfering RNAs has provided the key for a ubiquitous mode of post-transcriptional gene regulation in plants. These small RNAs (sRNAs) are of 20e30 nucleotides in length do not encode proteins but rather control the gene expression by silencing the mRNA post-transcriptionally and guiding them for Molecular mechanisms alleviating drought stress tolerance in crop plants 375 degradation or repressing translation. sRNAs regulate more than 30% of the genes in a cell (Sagar & Xue, 2019). Based on their genomic origin and precursors, sRNAs are divided into MicroRNAs (miRNA), Small Interfering RNAs (siRNAs), trans-acting small interfering RNAs (ta-siRNAs), and natural antisense small interfering RNAs (nat-siRNAs). After incorporating these RNAs into the RISC complex, these sRNAs will control the expression of their target genes by influencing the mRNA levels, chromatin, and methylation of DNA. 4.3 Micro RNA (miRNA) The discovery of the miRNA role in regulating abiotic stress and their target genes have provided new insight regarding the functions of sRNAs in stress responses. miRNAs are the derivatives of single-stranded RNA precursors (pri-miRNA), transcribed from the miRNA genes by RNA polymerase II, processed by DCL1 and other protein factors giving rise to a 20e24 nucleotide long miRNA. It was identified that various plant miRNAs play an important role in generating tolerance to abiotic stress, especially drought. Understanding sRNA-induced stress regulation has provided new tools for improving crop plants’ tolerance against abiotic stress. Modification of miRNA-induced gene controlling mechanisms will assist in plant engineering for improved drought stress resistance (Sunkar et al., 2006). miRNA plays a pivotal role in regulating genes and transcription factors regulates interactions to drought stress. The expression of miRNA will also alter as a result of prevailing water-related stress, making the regulation process more complex (Zhang et al., 2017). Drought tolerance miRNAs were found in rice, arabidopsis, soybean, cowpea, Phaseolus vulgaris, Saccharum species, barley (Sagar & Xue, 2019). Drought stress-related miRNAs are categorized into three classes. (1) miRNAs specific the transcription factors involved in altering genes involved in stress tolerance. miR156, miR159, miR165, miR169, miR171, miR172, miR319, and miR396 belongs to this category. (2) miRNAs are directly associated in the response to stress. miRNAs miR167, miR168, miR393, and miR394 are involved in this function. miR167 targets auxin-responsive factors and miR168 targets ARGONAUTE (Wu & Poethig, 2006). F-box proteins engaged in the resistance to abiotic tolerance are the targets of miR393 and miR394 (Jain et al., 2007). (3) miRNAs such as miR397 and miR408, targeting hydrolase and oxidoreductase genes during their reaction to the abiotic stress conditions (Apel & Hirt, 2004). Drought stress has 376 Plant Small RNA in Food Crops induced the upregulation of miR156, miR159, miR167, miR168, miR171, miR172, miR319, miR393, miR394a, miR395c, miR395e, miR396 and miR397 while miR161, miR168a, miR168b, miR169, miR171a and down regulation of miR319c in Arabidopsis (Liu et al., 2008). More than thirty differingly presented miRNAs were identified in rice subjected to drought stress, comprising 19 novel miRNAs. These 19 miRNAs were significantly suppressed in rice crops under water stress (Zhou et al., 2010). The expression of miRseq13, miR397ab, miR1513c, miR169-3p, and miR166-5p was upregulated in drought-sensitive cultivars of soybean (Kulcheski et al., 2011). miR166 is found to be upregulated in Hordeum vulgare, Triticum aestivum, and Saccharum spp., subjected to drought stress (Agustina et al., 2015). Overexpression of miR474 has been incurred in maize cultivated under water stress. miR474 interacts with proline dehydrogenase, a rate-limiting enzyme responsible for the proline catabolism in the P-5C pathway. The miR474 upregulated by the drought stress is downregulating the target gene for improving the accumulation of proline. The function of miR393 has contributed to the development of antibacterial resistance by TIR1 expression, which has downregulated the auxin signaling followed by the seedling growth during drought stress in rice (Xia et al., 2012). miR159 is also involved in hormonal signaling during dehydration in Arabidopsis (Reyes & Chua, 2007). Reactive oxygen species (ROS) are continually released in plants due to the regular aerobic responses occurring in chloroplasts, mitochondria, and peroxisomes. Levels of ROS are elevated in crops exposed to abiotic stress, like drought (Mittler et al., 2004). As a result of the increasing ROS levels, the plant system consists of a range of antioxidant defense reactions, both enzymatic and non-enzymatic. Superoxide dismutase (SOD) is the pioneering enzyme that superoxide radical into hydrogen peroxide and molecular oxygen. miRNA performs a vital role in the upregulation of antioxidative defense systems in the plants subjected to water stress. Upregulation of two superoxide dismutase genes, CSD1 and CSD2, is dependent on miR398 regulation. miR398 performs two functions in the plants during normal and drought stress conditions. Genome-wide analyses of the miRNAs regulating hydrogen peroxide were studied in rice seedlings (Li et al., 2010). Seven miRNAs, miR169, miR397, miR528, miR827, miR1425, miR319a.2, and miR408-5p were identified to be differentially expressed in rice seedlings treated with hydrogen peroxide, compared with the control. The activity pattern of miRNA in exposure to water stress have been assessed in various plants including Sorghum bicolor, Gossypium hirsutum, Molecular mechanisms alleviating drought stress tolerance in crop plants 377 Oryza rufipogon, Solanum tuberosum, Triticum turgidum, Hordeum vulgare, Cucumis sativus, Triticum aestivum, Solanum lycopersicum, and Elettaria cardamomum (Anjali et al., 2017). Few miRNA such as miR169, miR169g, and miR169n were shown to be controlled in rice due to water stress. The same miR169 was also found to be upregulated in Arabidopsis thaliana under high salinity, demonstrating a variation in response of miRNAs of the same or different plant or different species to different stress conditions. Variations in miRNA expression are also crucial for understanding the tissue-specific regulation within a plant (Ferdous et al., 2016). 4.4 Small interference RNA (siRNA) Synthesis of siRNA occurs through three possible mechanisms. (1) cleavage products of non-coding transcripts are transcribed into dsRNAs by RNAdependent RNA polymerases. (2) dsRNAs formation from the cis-antisense gene pair encoded mRNAs and (3) dsRNAs formed from heterochromatin and DNA repeats. The dsRNA is cleaved into 21e24 nucleotide siRNA by DICER-like (DCL) proteins. The size of the siRNA depends on the catalytic activity of the specific protein. Different DCL proteins cleave dsRNA, producing siRNAs of different sizes. Like miRNA, siRNAs are also transported into ARGONAUTE (AGO) protein containing RNAinduced silencing complex (RISC) for its target regulation at transcriptional and post-transcriptional levels through RNA-directed DNA methylation. Response of siRNAs to drought stress has been seen in the wheat seedlings with a change in the expression of four siRNAs. The expression of siRNA002061_0636_3054.1, 005047_0654_1904.1, siRNA080621_1340_0098.1, and siRNA007927_0100_2975.1 was completely downregulated under drought stress. siRNAs are classified into Transacting siRNA (ta-siRNA), Repeat Associated RNA (ra-siRNA), natural antisense siRNA (nat-siRNA), heterochromatic siRNA (hcsiRNA), and viral siRNA (vi-siRNA). This siRNA classification is based on their site of origin. siRNAs generated from the miRNA-induced mRNA cleavage are trans-acting siRNAs (ta-siRNAs). They form a particular class of siRNAs of 21 nucleotides size, produced by the miRNA processing of a TAS gene transcript. siRNAs derived from the dsRNAs of natural cis-antisense gene pair encoded mRNA are natural antisense transcript produced siRNAs (nat-siRNA). ta-siRNAs belonging to four families were discovered in Arabidopsis. MiR173 recognized transcripts of TAS1 and TAS2, TAS3 378 Plant Small RNA in Food Crops recognized by miR390, TAS4 recognized by miR828. The expression of TAS1, TAS2, and TAS3 was decreased in the Arabidopsis exposure to drought stress (Katiyar et al., 2015). The auxin-responsive factor ta-siRNAARF is involved in the regulation of floral morphogenesis during drought stress (Matsui et al., 2014). Two TAS3 homologs were upregulated in sorghum when exposed to drought stress. Repeat Associated RNA (ra-siRNA) is the derivative of the transposon elements and repetitive DNA, involved in the silencing of retrotransposons and drought stress-responsive factors (Barber et al., 2012). At the transcription level, ra-siRNA act via DNA methylation and histone methylation. They are involved in the methylation of lysine at the ninth position of histone H3, thus causing systemic silencing (Guleria et al., 2011). Natural antisense siRNA (nat-siRNA) originates from the annealed parts of the natural antisense transcripts (NATs), involved in the controlling of stress response, chromatin remodeling, RNA editing, and other biological processes (Lu et al., 2012). Heterochromatic siRNA (hc-siRNA) originates in the intergenic repeats and transposons (Castel & Martienssen, 2013). hc-siRNA recognizes the transcript through base-pair complementarity and further guides the DNA methylation and histone modification machinery to the loci for the transcriptional gene silencing (Azevedo et al., 2011). The role of hc-siRNA has been reported in plants’ response to abiotic stress factors such as drought (Ku et al., 2015). Viral siRNA (v-siRNA) is derived from the viral intermediates of the dsRNA replication for the induction of antiviral specific immunity. These siRNAs are processed from the sense strand of the viral genomes. The role of vi-siRNAs against pathogen attack has been reported in soybean, tomato, and tobacco plants (Zhu et al., 2017). 5. RNAi technology for the improvement of drought stress tolerance in crop plants RNAi technology is proving to be a useful method for counteracting abiotic stress. It is a biological mechanism responsible for the posttranscriptional regulation triggered by double-stranded RNA to prevent the expression of target genes. This technology has been successfully utilized to add desirable properties for improving abiotic stress resistance in crop plants (Dalakouras et al., 2019). RNA-I also has a potential mechanism for identifying and assessing thousands of genes in a genome responsible for crop improvement. This mechanism can efficiently knock down the Molecular mechanisms alleviating drought stress tolerance in crop plants 379 expression of any specific gene in any cell through siRNA. Further, inhibition of the targeted genes can be achieved through siRNA, and loss of function phenotype can also be determined when no mutant alleles are unavailable, leading to gene functional analysis (Adnan et al., 2020). RNA-I technology was applied for the first time for enhancing the anthocyanin pigment in petunia plants by introducing the chalcone synthase gene. Manipulation of the genetic makeup of the crop plants for their improved adaptation to drought stress has been an indispensable tool for ameliorating the productivity, yield, stability, and quality of the plant product. Gene detection techniques and functional genomics analysis have discovered a vast set of genes and gene families, ensuring high production and adjustment to abiotic stress. These genes can be expressed ectopically or delivered into the crop system, which is lacking (Gupta, 2013). The quick response and control of the gene expression will enable the plants to acclimatize to their system to abiotic stress. AtHPR1 promoter driving an RNAi construct downregulates farnesyl transferase in Brassica napus, protecting its produce against drought stress (Wang et al., 2009). Transgenic rice with the receptor for the activated C kinase 1 (RACK) gene inhibited by RNAi has shown improved drought stress resistance (Li et al., 2009). Genetically modified peanut has improved yield and quality under drought stress conditions (Zhao et al., 2007). miRNA expression profiling of drought-stressed rice has shown the downregulation of many drought-responsive genes. Expression of miRNA for the drought stress tolerance has been studied in wild emmer wheat (Triticum dicoccum) and barley using miRNA microarray (Kantar et al., 2010). The expression of the receptor for the activated C kinase 1 (RACK1) gene was suppressed by RNAi in transgenic rice, and the inhibited RACK1 gene activity was used to elucidate its functions in response to drought stress. It was recommended that the RACK1 gene plays role in decreasing the redox system-related tolerance to drought stress. The resistance to drought stress was higher in transgenic rice than nontransgenic ones (Jagtap et al., 2011). RNAi-mediated OsGRXS17 gene suppression has improved the drought stress tolerance in rice (Hu et al., 2017). 6. Conclusions Global crop productivity has become a challenge mainly due to constant changes in the climate, variable weather conditions generating 380 Plant Small RNA in Food Crops environmental stress on plant growth and productivity. Limited water availability and increasing global warming have further enhanced the drought conditions, responsible for a majority of agriculture losses. It is imperative to generate crop plants that can survive under limited water availability, which can be attained by a complete understanding of the plant responses to the drought stress. The discovery of RNA-mediated gene silencing and high throughput RNA sequence analysis supported by computational analysis and functional genomics has identified the role of ncRNAs and their regulations under drought stress conditions. The specific roles of long non-coding, and small non-coding RNA, were identified in crop plants during drought stress. The information available on the long non-coding RNAs is in the infant stage. Small non-coding RNAs such as miRNA and siRNA are effective tools in regulating the plant response to drought stress conditions. Despite the progress rendered in identifying miRNAs and siRNAs as regulating switches for the gene expression, there are still many questions regarding the movement of ncRNAs from the cells produced to the target cells and their protection from the nucleolytic degradation be unanswered. Still, there is a much more to understand regarding the operation of the ncRNAs in different crop plants and their relationship with the plant’s response to the drought stress and communications between different plant organs during drought stress. Such studies will further help in designing plants for their tolerance against drought stress. Acknowledgments The research laboratory of K.V. Chaitanya is funded by grants from the Department of Biotechnology, Govt. of India (No. BT/PR14467/AGR/02/742/2010). References Adnan, M., Fahad, S., Muhammad, Z., Shahen, S., Ishaq, A. M., Subhan, D., & Rahul, D. (2020). Coupling phosphate-solubilizing bacteria with phosphorus supplements improve maize phosphorus acquisition and growth under lime induced salinity stress. Plants, 9, 200. Agarwal, P., Gupta, K., Lopato, S., & Agarwal, P. (2017). Dehydration responsive element binding transcription factors and their applications for the engineering of stress tolerance. Journal of Experimental Botany, 68, 2135e2148. Agustina, G., Dias Lara, I., Mattos Raphael, S., Ferreira Thaís, H., & Marcelo, M. (2015). MicroRNAs and drought responses in sugarcane. Frontiers of Plant Science, 6, 258. Ali, A., Han, K., & Liang, P. (2021). Role of transposable elements in gene regulation in the human genome. Life, 11, 118. Molecular mechanisms alleviating drought stress tolerance in crop plants 381 Anjali, N., Nadiya, F., Thomas, J., & Sabu, K. (2017). Discovery of MicroRNAs in cardamom (Elettaria cardamomum Maton) under drought stress. Dataset Papers in Science, 2017, 1e4. Apel, K., & Hirt, H. (2004). Reactive oxygen species: Metabolism, oxidative stress, and signal transduction. Annual Review of Plant Biology, 55, 373e399. Azevedo, J., Cooke, R., & Lagrange, T. (2011). Taking RISCs with ago hookers. Current Opinion in Plant Biology, 14, 594e600. Barber, W. T., Zhang, W., Win, H., Varala, K. K., Dorweiler, J. E., Hudson, M. E., & Moose, S. P. (2012). Small RNAs in maize hybrids. Proceedings of the National Academy of Sciences, (109), 10444e10449. Castel, S., & Martienssen, R. (2013). RNA interference in the nucleus: Roles for small RNAs in transcription, epigenetics and beyond. Nature Reviews Genetics, 14, 100e112. Chai, M., Cheng, H., Yan, M., Priyadarshani, S., Zhang, M., He, Q., Huang, Y., Chen, F., Liu, L., Huang, X., Lai, L., Chen, H., Cai, H., & Qin, Y. (2020). Identification and expression analysis of the DREB transcription factor family in pineapple (Ananas comosus L. Merr.). PeerJ, 8, e9006. Chen, L., Zhu, Q., & Kaufmann, K. (2020). Long non-coding RNAs in plants: Emerging modulators of gene activity in development and stress responses. Planta, 252, 92. Chu, Q., Bai, P., Zhu, X., Zhang, X., Mao, L., Zhu, Q., Fan, L., & Ye, C. Y. (2018). Characteristics of plant circular RNAs. Briefings in Bioinformatics, 21, 135e143. Cui, M., Haider, M., Chai, P., Guo, J., Du, P., Li, H., Dong, W., Huang, B., Zheng, Z., Shi, L., Zhang, X., & Han, S. (2021). Genome-wide identification and expression analysis of AP2/ERF transcription factor related to drought stress in cultivated peanut (Arachis hypogaea L.). Frontiers in Genetics, 12, 1918. Dalakouras, A., Wassenegger, M., Dadami, E., Ganopoulos, I., Pappas, M., & Papadopoulou, K. (2019). Genetically modified organism-free RNA interference: Exogenous application of RNA molecules in plants. Plant Physiology, 182, 38e50. Ferdous, J., Sanchez-Ferrero, J., Langridge, P., Milne, L., Chowdhury, J., Brien, C., & Tricker, P. J. (2016). Differential expression of microRNAs and potential targets under drought stress in barley. Plant, Cell and Environment, 40, 11e24. Forchetti, G., Masciarelli, O., Alemano, S., Alvarez, D., & Abdala, G. (2007). Endophytic bacteria in sunflower (Helianthus annuus L.): Isolation, characterization, and production of jasmonates and abscisic acid in culture medium. Applied Microbiology and Biotechnology, 76, 1145e1152. Govind, G., Seiler, C., Wobus, U., & Sreenivasulu, N. (2011). Importance of ABA homeostasis under terminal drought stress in regulating grain filling events. Plant Signaling & Behavior, 6, 1228e1231. Guleria, P., Mahajan, M., Bhardwaj, J., & Yadav, S. (2011). Plant Small RNAs: Biogenesis, mode of action and their roles in abiotic stresses. Genomics, Proteomics & Bioinformatics, 9, 183e199. Gupta, B. (2013). Plant abiotic stress: ‘Omics’ approach. Journal of Plant Biochemistry & Physiology, 01, e108. Hartung, W. (2010). The evolution of abscisic acid (ABA) and ABA function in lower plants, fungi and lichen. Functional Plant Biology, 37. Hu, Y., Wu, Q., Peng, Z., Sprague, S., Wang, W., Park, J., Akhunov, E., Jagdish, K. S. V., Nakata, P. A., Cheng, N., Kendal, D. H., Frank, F. W., & Sunghun, P. (2017). Silencing of OsGRXS17 in rice improves drought stress tolerance by modulating ROS accumulation and stomatal closure. Scientific Reports, 7. Jagtap, U., Gurav, R., & Bapat, V. (2011). Role of RNA interference in plant improvement. Naturwissenschaften, 98, 473e492. Jain, M., Nijhawan, A., Arora, R., Agarwal, P., Ray, S., Sharma, P., Sanjay, K., Akhilesh, K. T., & Jitendra, P. K. (2007). F-box proteins in rice. Genome-wide analysis, 382 Plant Small RNA in Food Crops classification, temporal and spatial gene expression during panicle and seed development, and regulation by light and abiotic stress. Plant Physiology, 143, 1467e1483. Jaradat, M., Feurtado, J., Huang, D., Lu, Y., & Cutler, A. (2013). Multiple roles of the transcription factor AtMYBR1/AtMYB44 in ABA signaling, stress responses, and leaf senescence. BMC Plant Biology, 13. Jha, U., Nayyar, H., Jha, R., Khurshid, M., Zhou, M., Mantri, N., & Siddique, K. H. M. (2020). Long non-coding RNAs: Emerging players regulating plant abiotic stress response and adaptation. BMC Plant Biology, 20, 466. Kang, J., Park, J., Choi, H., Burla, B., Kretzschmar, T., Lee, Y., & Martinoia, E. (2011). Plant ABC transporters. The Arabidopsis Book, 9, e0153. Kanno, Y., Hanada, A., Chiba, Y., Ichikawa, T., Nakazawa, M., Matsui, M., Koshiba, T., Yuji, K., & Mitsunori, S. (2012). Identification of an abscisic acid transporter by functional screening using the receptor complex as a sensor. Proceedings of the National Academy of Sciences, 109, 9653e9658. Kantar, M., Lucas, S., & Budak, H. (2010). miRNA expression patterns of Triticum dicoccoides in response to shock drought stress. Planta, 233, 471e484. Katiyar, A., Smita, S., Muthusamy, S., Chinnusamy, V., Pandey, D., & Bansal, K. (2015). Identification of novel drought-responsive microRNAs and trans-acting siRNAs from Sorghum bicolor (L.) Moench by high-throughput sequencing analysis. Frontiers of Plant Science, 6. Khan, I., Ali, A., Khan, H., Baek, D., Park, J., Lim, C. J, Shah, Z., Masood, J., Sang, Y. L., Jose, M. P., Woe, Y. K., & Dae-Jin, Y. (2020). PWR/HDA9/ABI4 complex epigenetically regulates ABA dependent drought stress tolerance in Arabidopsis. Frontiers of Plant Science, 11, 623. Kim, T., Böhmer, M., Hu, H., Nishimura, N., & Schroeder, J. (2010). Guard cell signal transduction network: Advances in understanding abscisic acid, CO2, and Ca2þ signaling. Annual Review of Plant Biology, 61, 561e591. Kulcheski, F., de Oliveira, L., Molina, L., Almerão, M., Rodrigues, F., Marcolino, J., Joice, F. B., Renata, S.-M., Alexandre, L. N., Francismar, C. M.-G., Ricardo, V. A., Leandro, C. N., Marcelo, F. C., Goncalo, A. G. P., & Rogerio, M. (2011). Identification of novel soybean microRNAs involved in abiotic and biotic stresses. BMC Genomics, 12, 307. Kuromori, T., Miyaji, T., Yabuuchi, H., Shimizu, H., Sugimoto, E., Kamiya, A., & Shinozaki, K. (2010). ABC transporter AtABCG25 is involved in abscisic acid transport and responses. Proceedings of the National Academy of Sciences, 107, 2361e2366. Ku, Y., Wong, J., Mui, Z., Liu, X., Hui, J.H.-L., Chan, T.-F., & Lam, H.-M. (2015). Small RNAs in plant responses to abiotic stresses: Regulatory roles and study methods. International Journal of Molecular Sciences, 16, 24532e24554. Léran, S., Noguero, M., Corratgé-Faillie, C., Boursiac, Y., Brachet, C., & Lacombe, B. (2020). Functional characterization of the Arabidopsis abscisic acid transporters NPF4.5 and NPF4.6 in Xenopus oocytes. Frontiers of Plant Science, 11, 144. Li, J., Han, G., Sun, C., & Sui, N. (2019). Research advances of MYB transcription factors in plant stress resistance and breeding. Plant Signaling & Behavior, 14, 1613131. Li, D., Liu, H., Yang, Y., Zhen, P., & Liang, J. (2009). Down-regulated expression of RACK1 gene by RNA interference enhances drought tolerance in rice. Rice Science, 16, 14e20. Li, T., Li, H., Zhang, Y., & Liu, J. (2010). Identification, and analysis of seven H2O2responsive miRNAs and 32 new miRNAs in the seedlings of rice (Oryza sativa L. ssp. indica). Nucleic Acids Research, 39, 2821e2833. Liu, H., Tian, X., Li, Y., Wu, C., & Zheng, C. (2008). Microarray-based analysis of stressregulated microRNAs in Arabidopsis thaliana. RNA, 14, 836e843. Liu, J., Wang, H., & Chua, N. (2015). Long noncoding RNA transcriptome of plants. Plant Biotechnology Journal, 13, 319e328. Molecular mechanisms alleviating drought stress tolerance in crop plants 383 Li, W., Wang, Y., Zhang, Y., Wang, R., Guo, Z., & Xie, Z. (2020). Impacts of drought stress on the morphology, physiology, and sugar content of Lanzhou lily (Lilium davidii var. unicolor). Acta Physiologiae Plantarum, 42. Li, S., Zhao, Q., Zhu, D., & Yu, J. (2018). A DREB-like transcription factor from maize (Zea mays), ZmDREB4.1, plays a negative role in plant growth and development. Frontiers of Plant Science, 9, 395. Lucero, L., Ferrero, L., Fonouni-Farde, C., & Ariel, F. (2020). Functional classification of plant long noncoding RNAs: A transcript is known by the company it keeps. New Phytologist, 229, 1251e1260. Lu, T., Zhu, C., Lu, G., Guo, Y., Zhou, Y., Zhang, Z., Yan, Z., Wenjun, L., Ying, L., Tang, W., Qi, F., & Bin, H. (2012). Strand-specific RNA-seq reveals widespread occurrence of novel cis-natural antisense transcripts in rice. BMC Genomics, 13, 721. Lv, Y., Hu, F., Zhou, Y., Wu, F., & Gaut, B. (2019). Maize transposable elements contribute to long non-coding RNAs that are regulatory hubs for abiotic stress response. BMC Genomics, 20, 864. Mathew, I., Das, S., Mahto, A., & Agarwal, P. (2016). Three rice NAC transcription factors heteromerize and are associated with seed size. Frontiers of Plant Science, 7, 1638. Matsui, A., Mizunashi, K., Tanaka, M., Kaminuma, E., Nguyen, A., Nakajima, M., Dong, V.-N., Tetsuro, T., & Motoaki, S. (2014). tasiRNA-ARF pathway moderates floral architecture in Arabidopsis plants subjected to drought stress. BioMed Research International, 2014, 1e10. Mittler, R., Vanderauwera, S., Gollery, M., & Van Breusegem, F. (2004). Reactive oxygen gene network of plants. Trends in Plant Science, 9, 490e498. Msanne, J., Lin, J., Stone, J., & Awada, T. (2011). Characterization of abiotic stressresponsive Arabidopsis thaliana RD29A and RD29B genes and evaluation of transgenes. Planta, 234, 97e107. Muthamilarasan, M., Khandelwal, R., Yadav, C., Bonthala, V., Khan, Y., & Prasad, M. (2014). Identification and molecular characterization of MYB transcription factor superfamily in C4 model plant foxtail millet (Setaria italica L.). PLoS One, 9, e109920. Ng, L., Melcher, K., Teh, B., & Xu, H. (2014). Abscisic acid perception and signaling: Structural mechanisms and applications. Acta Pharmacologica Sinica, 35, 567e584. Pan, W., You, Y., Shentu, J., Weng, Y., Wang, S., Xu, Q., Liu, H. J., & Du, S. T. (2020). Abscisic acid (ABA)-importing transporter 1 (AIT1) contributes to the inhibition of Cd accumulation via exogenous ABA application in Arabidopsis. Journal of Hazardous Materials, 391, 122189. Qin, T., Li, J., & Zhang, K. (2020). structure, regulation, and function of linear and circular long non-coding RNAs. Frontiers in Genetics, 11, 150. Reyes, J., & Chua, N. (2007). ABA induction of miR159 controls transcript levels of two MYB factors during Arabidopsis seed germination. The Plant Journal, 49, 592e606. Roychoudhury, A., Paul, S., & Basu, S. (2013). Cross-talk between abscisic acid-dependent and abscisic acid-independent pathways during abiotic stress. Plant Cell Reports, 32. Sagar, A., & Xue, B. (2019). Recent advances in machine learning based prediction of RNA-protein interactions. Protein and Peptide Letters, 26, 601e619. Sah, S., Reddy, K., & Li, J. (2016). Abscisic acid and abiotic stress tolerance in crop plants. Frontiers of Plant Science, 7. Seiler, C., Harshavardhan, V., Rajesh, K., Reddy, P., Strickert, M., Rolletschek, H., Uwe, S., Ulrich, W., & Sreenivasulu, N. (2011). ABA biosynthesis and degradation contributing to ABA homeostasis during barley seed development under control and terminal drought-stress conditions. Journal of Experimental Botany, 62, 2615e2632. Singh, K., & Chandra, A. (2021). DREBs-potential transcription factors involve in combating abiotic stress tolerance in plants. Biologia, 76, 3043e3055. 384 Plant Small RNA in Food Crops Singh, D., & Laxmi, A. (2015). Transcriptional regulation of drought response: A tortuous network of transcriptional factors. Frontiers of Plant Science, 6, 895. Sunkar, R., Kapoor, A., & Zhu, J. (2006). Posttranscriptional induction of two Cu/Zn superoxide dismutase genes in arabidopsis is mediated by downregulation of miR398 and important for oxidative stress tolerance. The Plant Cell Online, 18, 2051e2065. Tan, B., Schwartz, S., Zeevaart, J., & McCarty, D. (1997). Genetic control of abscisic acid biosynthesis in maize. Proceedings of the National Academy of Sciences, 94, 12235e12240. Wang, Y., Beaith, M., Chalifoux, M., Ying, J., Uchacz, T., Sarvas, C., Griffiths, R., Kuzma, M., Wan, J., & Huang, Y. (2009). Shoot-specific down-regulation of protein farnesyltransferase (Alpha-subunit) for yield protection against drought in canola. Molecular Plant, 2, 191e200. Wight, M., & Werner, A. (2013). The functions of natural antisense transcripts. Essays in Biochemistry, 54, 91e101. Wilkinson, S., Kudoyarova, G., Veselov, D., Arkhipova, T., & Davies, W. (2012). Plant hormone interactions: Innovative targets for crop breeding and management. Journal of Experimental Botany, 63. Wu, G., & Poethig, R. (2006). Temporal regulation of shoot development in Arabidopsis thaliana by miR156 and its target SPL3. Development, 133, 3539e3547. Xia, K., Wang, R., Ou, X., Fang, Z., Tian, C., Duan, J., Wang, Y., & Zhang, M. (2012). OsTIR1 and OsAFB2 downregulation via OsmiR393 overexpression leads to more tillers, early flowering and less tolerance to salt and drought in rice. PLoS One, 7, e30039. Yang, X., Lu, M., Wang, Y., Wang, Y., Liu, Z., & Chen, S. (2021). Response mechanism of plants to drought stress. Horticulturae, 7, 50. Yoon, Y., Seo, D., Shin, H., Kim, H., Kim, C., & Jang, G. (2020). The role of stressresponsive transcription factors in modulating abiotic stress tolerance in plants. Agronomy, 10, 788. Yoshida, T., Fujita, Y., Sayama, H., Kidokoro, S., Maruyama, K., Mizoi, J., Shinozaki, K., & Yamaguchi-Shinozaki, K. (2010). AREB1, AREB2, and ABF3 are master transcription factors that cooperatively regulate ABRE-dependent ABA signaling involved in drought stress tolerance and require ABA for full activation. The Plant Journal, 61, 672e685. Yu, Y., Zhang, Y., Chen, X., & Chen, Y. (2019). Plant noncoding RNAs: Hidden players in development and stress responses. Annual Review of Cell and Developmental Biology, 35, 407e431. Zhang, H., Zhang, J., Yana, J., Gou, F., Mao, Y., Tange, G., Botella, J. R., & Zhu, J.-K. (2017). Short tandem target mimic rice lines uncover functions of miRNAs in regulating important agronomic traits. Proceedings of the National Academy of Sciences, 114, 5277e5282. Zhang, H., Zhu, H., Pan, Y., Yu, Y., Luan, S., & Li, L. (2014). A DTX/MATE-Type transporter facilitates abscisic acid Efflux and modulates ABA sensitivity and drought tolerance in Arabidopsis. Molecular Plant, 7, 1522e1532. Zhao, B., Liang, R., Ge, L., Li, W., Xiao, H., Lin, H., Ruan, K. C., & Zin, Y. X. (2007). Identification of drought-induced microRNAs in rice. Biochemical and Biophysical Research Communications, 354, 585e590. Zhou, L., Liu, Y., Liu, Z., Kong, D., Duan, M., & Luo, L. (2010). Genome-wide identification and analysis of drought-responsive microRNAs in Oryza sativa. Journal of Experimental Botany, 61, 4157e4168. Zhu, B., Gao, H., Xu, G., Wu, D., Song, S., Jiang, H., Zhu, S., Qi, T., & Xie, D. (2017). Arabidopsis ALA1 and ALA2 mediate RNAi-based antiviral immunity. Frontiers of Plant Science, 8, 422. CHAPTER 14 Grain development and crop productivity: role of small RNA Md Fakhrul Azada, Heshani de Silva Weligodagea, Anuradha Dhingraa, Pranav Dawara and Christopher D. Rock Department of Biological Sciences, Texas Tech University, Lubbock TX, United States 1. Overview The field of sRNAs is dynamic and has generated excitement and breathtaking advances in understanding plant (and animal) growth and development, leveraged by advances in sequencing and gene editing technologies that facilitate reverse genetic tests of function. The resulting progress from economies of scale to elaborate a dedicated microRNA database (miRBase22) (Kozomara et al., 2019), which at present encompasses 80þ plant species, can leverage knowledge to impact not only crop genomics and breeding, but also prospects to elucidate inter-kingdom signaling mechanisms. The inter-kingdom signaling aspect is underscored by the fact that miRNAs are not shared between plant and animal kingdoms (Moran et al., 2017), yet current knowledge of the properties, effectiveness and biological actions of plant miRNAs in animal diets is controversial (Chen & Rechavi, 2022; Zhao et al., 2018). Such questions touch on organic evolution, not limited to plant MIRNAs (Allen et al., 2004, 2005; Lu et al., 2008; Lunardon et al., 2020; Xia et al., 2013, 2015) and potentially leading to the origin of life on Earth (the “RNA World”) (Gilbert, 1986; Ruvkun, 2001). Our focus is translational in scope, therefore does not consider algae or lycophyte lower plants, and attempts to address the question: are pathways and processes controlling plant sRNA regulation of post-transcriptional and transcriptional gene silencing conserved among and/or across clades of flowering plants? For example, some MIRNAs like miR2275 (Taylor et al., 2014), miR444 (Lu et al., 2008; H. Wang et al., 2016), miR528 (Chen et al., 2019; Luján-Soto et al., 2021), miR530 (Patel et al., 2021), and miR1432 (Xia et al., 2013) are (mostly) found in monocots (22% of angiosperm species) yet not dicots a These authors contributed equally. Plant Small RNA in Food Crops ISBN 978-0-323-91722-3 https://doi.org/10.1016/B978-0-323-91722-3.00001-4 © 2023 Elsevier Inc. All rights reserved. 385 386 Plant Small RNA in Food Crops (75%). Phased small interfering RNA (PHAS-RNA) loci producing 21 nt species in reproductive tissues, but not MIRNAs, are far more numerous in grass genomes than other monocots where scant evidence is found for any monocot-wide, conserved and novel miRNAs (Patel et al., 2021). The practical implication of deep knowledge of miRNA and sRNA regulation and mechanisms can present cogent strategies for engineering improvements in food, fiber, and bioenergy production to address sustainability and climate change issues. Examples are recent progress to elucidate plasmodesmatal and exosomal determinants for sRNA (Chekanova et al., 2007) and mRNA intercellular mobility (Kitagawa et al., 2022) and that sRNAs and miRNAs are mobile in the vasculature and can cross graft junctions (Maizel et al., 2020; Molnar et al., 2010; Pant et al., 2008), raising prospects of genetic engineering to create non-genetically modified organism (GMO) scions in vegetative-propagated crops. The United States government has de-regulated Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR-Cas9) editing (Jinek et al., 2012) and related technologies used to create non-transgenic mutant or enhanced-expression alleles of MIRNAs and their targets/effectors (https://www.science.org/ content/article/united-states-relaxes-rules-biotech-crops). This change in policy can leverage applications for molecular breeding of agronomic traits in tomato (Rodríguez-Leal et al., 2017), corn (L. Liu et al., 2021), grapevine (Ren et al., 2022; Sunitha & Rock, 2020), and other crops (Bao et al., 2019). Other examples are facultative apomixis/parthenocarpy (development of a fruit without prior fertilization) in tomato mutants of F-box miRNA biogenesis effector HAWAIIAN SKIRT (Damayanti et al., 2019), and the production of unreduced female gametes in mutants of sRNA pathways, with implications for sRNA as morphogens controlling apomixis/parthenocarpy which could revolutionize plant breeding (Böwer & Schnittger, 2021; Klesen et al., 2020; Olmedo-Monfil et al., 2010; Petrella et al., 2021). There exist large discrepancies between empirical sRNA data in terms of annotation of miRNAs in plants, including gymnosperms (pine nuts are a commodity); of the 80þ species cataloged in miRBase22, a total of 29 (most not of agronomic importance) have genomic coordinates correlated for claimed MIRNA genes that serve as foundations of a phylogenetic approach to plant miRNA complexity. Namely: a basal sister to the eudicots Amborella trichopoda, seven monocots, six Brassicaceae, one Cucurbitaceae, four Fabaceae, one Malvaceae (G. raimondii, Peruvian relative to textile crop G. hirsutum), two Rosaceae (Fragaria vesca, Prunus persica), one Rutaceae (C. sinensis), two Solanaceae (S. lycopersicum, S tuberosum), and one Vitaceae (V. vinifera). The other angiosperm species in Grain development and crop productivity: role of small RNA 387 miRBase may or may not have a reference genome available, either annotated for MIRNAs independent of miRBase or otherwise, making genome-wide analyses and comparisons challenging. The dynamic state of the sRNA field raises numerous issues of sRNA-seq based claims for stable and reliable annotations of miRNA and siRNA-generating genes in plants (Axtell, 2013; Axtell & Meyers, 2018; Brodersen & Voinnet, 2009; Coruh et al., 2014; Taylor et al., 2017), although recent publications and associated databases have captured the phylogenetic diversity of MIRNA and siRNAgenerating loci from 45 angiosperm genomes including many crops (Lunardon et al., 2020) and monocots (Patel et al., 2021). Fig. 14.1 shows annotated MIRNA loci account for only a tiny fraction of the Arabidopsis thaliana genome that actively produces sRNAs (B, left). In contrast, nearly all of the polyA þ RNA-seq is explained by existing gene annotations (B, right). This analysis does not imply a vast amount of unannotated MIRNA loci in the plant kingdom; rather, the majority of expressed plant sRNAs are NOT miRNAs that account for roughly 10% of the sRNAs emanating from plant genomes (C, left). The approach of drawing on model plants and crops with reference genomes and wellannotated MIRNAs has led to a large “annotation gap” between the empirical knowledge of sRNA expression and the annotations of sRNAs. For PHAS loci (A, overlap region), encompassing miR390 targeting TransActing sRNA locus3 (TAS3) and evolutionarily descended superfamily members miR391 and likewise mirR482 22 nt families that also trigger induced secondary siRNAs, these MIRNAs likely evolved by selection of trans-acting siRNAs to target many members of large gene families (Boccara et al., 2014; Patel et al., 2021; Xia et al., 2013, 2015). Thus, secondary phased siRNA production from protein-coding mRNAs may serve as a mechanism to achieve coordinated post-transcriptional repression for many homologous transcripts at once. Viral and bacterial infections correlate with decreased miR482 accumulation and increased target nucleotide bindingleucine-rich repeat (NB-LRR) resistance gene accumulations; miR482 loss results in infection susceptibility (Li et al., 2012; Shivaprasad et al., 2012; Y. Zhang et al., 2016), yet interestingly in Arabidopsis the miR482 family member miR472 has opposite effects on effector-triggered immunity (Boccara et al., 2014; C. Jiang et al., 2020) as did Short Tandem Target Mimic (STTM) knockdown of miR2118b targeting TAS5 in tomato (Canto-Pastor et al., 2019). Although not reviewed here specifically, the PHAS class of sRNA loci are fertile ground for discovery and understanding post-transcriptional gene silencing mechanisms. For example, evolution of 388 Plant Small RNA in Food Crops A Nucleotides with coverage>=0.1 RPM e.g. PHAS loci, transposon-related, IncRNAs? 0.64E6 33.74E6 10.94E6 Small RNA-seq RNA-seq B 0.03E6 11.56E6 26.77E6 Nucleotides with coverage>=0.1 RPM vs. annotated nucleotides small RNA-seq vs. RNA-seq vs. MIRNA hairpins Annotated genes 0.02E6 Genes 1.42E6 32.96E6 MIRNA RNA-seq Small RNA-seq C Fraction of mapped reads overlapping annotated regions RNA-seq vs. Small RNA-seq vs. genes MIRNA hairpins Other Genes Other MIRNA Figure 14.1 The annotation gap: comparison of observed expression data to annotations for sRNAs and polyA þ RNAs in Arabidopsis. (A) Area-proportional Venn diagram showing the extent (number of nts) of significant (defined as a coverage of0.1 read per million) polyA þ RNA (RNA-seq) and sRNA-seq expression in the Arabidopsis genome. (B) Area-proportional Venn diagrams illustrating the overlap between areas of significant sRNAs-seq or RNA-seq expression and annotated regions in Arabidopsis (left: sRNA-seq vs. miRBase20, right: RNA-seq vs. TAIR10 genes including introns). (C) Pie charts illustrating the proportion of aligned sRNA-seq reads overlapping MIRNA annotation (left), or the proportion of RNAs-seq reads overlapping TAIR10 gene annotations including introns (right) for Arabidopsis. (Modified from C. Coruh, S. Shahid, M.J. Axtell, (2014). “Seeing the forest for the trees: Annotating sRNA producing genes in plants,” Current Opinion in Plant Biology, 18: 90. sRNA data from NCBI GEO GSM738731 and GSM738727, and polyA þ RNAs from NCBI GEO GSM946222 and GSM946223.) Grain development and crop productivity: role of small RNA 389 indica rice (Swetha et al., 2022) or functions of “orphan” miRNAs with no known protein-coding targets but when characterized computationally (Guo et al., 2015; J. Liu et al., 2021) demonstrate long non-coding (lnc) PHAS loci may function as a TAS locus with cognate orphan miRNA as elucidated trigger. Future focused efforts can enhance the prospects of applied plant genomics to elucidate miRNA structure/function relationships. We have focused our review on those MIRNA loci claimed to be conserved across at least one flowering plant clade (Patel et al., 2021; Taylor et al., 2014); undoubtedly this is a limitation that may not be justified or borne out as the field matures. Where warranted based primarily on computational predictions by authors, we note those claimed activities of candidate novel miRNAs in crops which remain to be established experimentally. We are interested in miRNA discovery in crops, where annotation in miRBase and the literature remains an issue. We take an empirical approach drawing on thousands of plant miRNAs in miRBase22 and recently published diverse plant MIRNA annotations that meet community standards (Axtell & Meyers, 2018) based on expression (Lunardon et al., 2020). Fig. 14.2 shows results by phylogenomic clustering that “orphan” miR8558 and miR3632 Figure 14.2 Phylogenomic discovery of orphan miRNAs belonging to ancient, deeply conserved MIR482 family, showing bootstrap support for clades. Methods were as described in ref. (Xia et al., 2013). (A) Colored circles correspond to family names, with brown triangles denoting “orphan” miRNAs in miRBase22 (ref (Kozomara et al., 2019)) and/or from published MIRNA datasets from 45 diverse angiosperm plant species (ref (Lunardon et al., 2020)). (B) Conservation profile of miR482-related “orphan” miRNAs actually belonging to respective family, aligned by all mature sequences in cladograms. Position of the core sequences is marked with a box. Asterisks (*) denote absolute conservation with family consensus sequence; o relatively strong sequence conservation. 390 Plant Small RNA in Food Crops in fact belong in the MIR482/472/1448/1859/2118/2948/3633 (Kozomara et al., 2019; Xia et al., 2015) superfamily clade. Similar results have recently been reported for erstwhile miRBase “orphans” miR3267/8585/ 8602/5211/5786 (Attri et al., 2022) which in fact belong to the MIR391/ 1432/3627/4376/5225 superfamily (Xia et al., 2013), with the very first experimental validation supporting miRNA391/1432:target gene evolution via compensatory substitutions from canonical calcium pump target genes in gymnosperms and basal eudicots important for inflorescence architecture diverged to capture, remarkably, novel calcium sensor targets in grasses (Attri et al., 2022). It is noteworthy how the logo conservation for orphans, based on family-wide conservation across species (Fig. 14.2B), extends 50 and 30 beyond the mature species (in capital letters). This is compelling evidence that the “business end” of the hairpin sequence is under purifying selection due to functional slicing of targets by an ARGONAUTE (AGO). Yet when these orphan miRNAs are searched, the algorithm fails to find significant homology to the claimed cognate families in miRBase (data not shown), underscoring the challenge to frame target regulation by claimed “novel” miRNAs. Future work with degradome datasets from myriad species and emerging genomes can assess if purifying selection for maintenance of miRNA:target topology (Fahlgren et al., 2010) and concordant evidences of AGO slicing activity (AddoQuaye et al., 2009) shaped plesiomorphic (i.e. ancient, rather than convergently evolved) diversification of orphan family member functions (Y. Zhang et al., 2016). A recent study on miR394 co-evolution patterns for F-box target effectors of plant shoot apical meristem maintenance defined by cognate module miR394-LEAF CURLING RESPONSIVENESS (LCRs) is a step in this direction (Kumar et al., 2019). 2. Fruit development; case study in Darwin’s “abominable mystery” Plant development, especially flowers and fruits which includes the juvenile-adult transition and induction of floral competence, gives rise to a bewildering complexity of shapes, colors, and functions that Darwin called “an abominable mystery” in his efforts to integrate his theory of evolution with species complexity evidently exploding during the mid Cretaceous (Soltis et al., 2019). The angiosperm radiation was not long ago, relative to arcs of gradual change in the fossil record for other kingdoms of life that Darwin drew upon to deduce evolution was neither rapid, nor saltational. Grain development and crop productivity: role of small RNA 391 Thus, plant model organism study is particularly important for applied crop science-phylogenetics can give perspective on genes (including lnc genes like MIRNAs) with conserved functions across crops. Ovules in flowering plants are seed precursors made up of one or two sheathing integuments surrounding a nucellus in which meiosis, fertilization, and embryo development occur. Ovule, seed, and fruit development are tightly coordinated with pollination and fertilization that promote hormone signaling crucial for fruit set. Studies of events leading to seed and fruit development are principally directed to understanding the female reproductive organ. There is some evidence for specificity of pollen mobile sRNAs (Dukowic-Schulze & van der Linde, 2021) and pollen tube receptors for attractant peptide signals secreted by the megagametophyte that contribute to double fertilization (grain productivity is a function of triploid endosperm development) and interspecies reproductive barriers (X. Zhang et al., 2017; J. Zhang et al., 2021). Disconnecting ovule from carpel development is a long-desired goal in agriculture to generate seedless fruits easier to consume. Extreme oscillations in temperature cause plant sterility and represent a major threat to crop yield around the world in the face of climate change. The developmental process of seed and fruit morphogenesis has been studied extensively at the hormonal and physiological levels, and reviewed recently regarding miRNAs and other lncRNAs (Cedillo-Jimenez et al., 2020; Correa et al., 2018; Dhaka & Sharma, 2021; Rodrigues & Miguel, 2017). Dry fruit examples from dicot and monocot clades are Arabidopsis and corn, and for fleshy fruits tomato, banana, and bromeliads like pineapple, respectively. Given that there are only a handful of plant hormones known to control growth and development, these simple lipophilic molecules presumably “interact” through cross-talk network pathways integrated in time and space with environmental signals. Thus, understanding plant morphogenesis as controlled cell division and elongation (there is no cell migration in plants due to rigid cell walls) requires additional layers of control to generate complexity (think: nodes in networks). It is submitted that sRNAs may be a “missing link” whose importance in complex signaling cross-talk is hinted at by their mechanisms of action: WatsonCrick base complementarity to mediate transcriptional and posttranscriptional regulation of many parallel, and thus independent, protein-coding and TAS RNA targets. Consider for example from a systems perspective the DICER1 genetic names. Before cloning in Arabidopsis demonstrated it encodes a deeply conserved endonuclease gene, later understood to function in miRNA 392 Plant Small RNA in Food Crops biogenesis, names given the locus by geneticists interested in different processes of ovule, embryo, and flower development discounted the pleiotropic nature of dicer phenotypes: short integuments1, embryo defective60, and carpel factory (Robinson-Beers et al., 1992; Schauer et al., 2002). miRNAs are the most well-studied class of the sRNAs, acting as nodes in gene networks whereby mutants can have pleiotropic effects including flower, seed, and fruit development. Various approaches continue to illustrate the role of these riboregulators such as CRISPR-Cas9, STTMs, artificial miRNAs, and Virus Induced Gene Silencing (VIGS). Many aspects related to sRNA research like biogenesis, stability, functional roles in plant development as a whole have been well studied but the mechanistic understanding of such miRNA-mediated gene regulation is still limited, especially in crops. On the basis that translational improvements to crops still relies on deep knowledge from model systems, the following sections focus on work in crops of agronomic importance. We approach the subject by clades of flowering plants nearest to each other phylogenetically: Brassicales-Malvales including citrus and cotton; asterids encompassing the Solanaceae, Daucus/ carrot, Helianthus/sunflower, and Laminales olive and Sesamum indicum plus Ericales kiwifruit; rosids including sister species Vitis and the Fabaceae; and monocots. Genomics has advanced our understanding of “evo-devo”: the evolution of development where phylogenomic analyses largely reinforce the relationships inferred historically by morphometrics. Remarkably, analysis of genes involved in sRNA- and RNA interference provides evidence of over-represented and lost evolutionary signals driving monocot plant adaptation and diversification from magnoliids/eudicots versus gymnosperms (where the megagametophyte develops maternally without fertilization), respectively (Lee et al., 2011). By grouping our coverage phylogenetically, it is anticipated that as knowledge of miRNAs deepens, shared plesiomorphic and diverged families of MIRNAs will be revealed that present unique opportunities for practical applications, not limited to molecular breeding of haplotypes for improved agronomic traits. 3. miRNAs in citrus Citrus is one of the most economically important and nutritious fruit crops cultivated in tropical, sub-tropical and Mediterranean region owing to its high vitamin C, antioxidants, and fiber contents. Despite spectacular genomics insights to citrus evolution (Wu et al., 2018), the reference genome Grain development and crop productivity: role of small RNA 393 still suffers from large numbers of annotated miRNAs assigned to plant families identified elsewhere as exclusive to disparate lineages (Taylor et al., 2017). Here we consider the role of miRNAs in the context of citrus traits of agronomic importance, of which there are many whose physiological basis is less understood, for example ethylene hormone as anti-climacteric inhibitor of ripening, aromas, rind thickness and adherence, and pigmentation. Juice sac granulation is a problem for post-harvest preservation of citrus fruits, and lignin accumulation correlates with juice sac granulation. J. Zhang et al. (2016) hypothesized two miRNA families, MIR397 and MIR828, whose predicted targets laccase17/LAC17, and MYB transcription factors (TFs, discovered in avian viruses causing MYeloBlastosis), respectively, could be causally associated with granulation. Consistent with this hypothesis, authors showed by stem-loop quantitative real-time qRT-PCR that pre-MIR397 and pre-MIR828 had relative expressions in juice sacs significantly negatively correlated with granulation trait, and some predicted targets positively correlated with lignin content. However, the slicing by AGO of those candidate targets remains to be demonstrated. In many species miR408 and miR397b have been shown to regulate lignin content by targeting LAC biosynthetic genes (Gao et al., 2022; S. Huang et al., 2021) and their over-expression (OE) in Arabidopsis increases plant biomass, silique lengths, and overall seed yield (Song et al., 2018; Wang et al., 2014). Dang et al. (2021) predicted but did not establish functionally that novel csi-miRNA-114 from stems, which appears to be homologous to a conserved downstream species in phase with miR169* (Kozomara et al., 2019), might target several plastid-lipid binding homologues proposed to sequester the overaccumulation of carotenoids in flowers and fruit. We explored available degradomes from various tissues (Liu et al., 2014; Wu et al., 2016) for an AGO-mediated slicing signal (Addo-Quaye et al., 2009), but did not verify the candidate miR-114 activity toward predicted targets (data not shown). Their claimed novel csi-miRNA-75 is a miR156 isomiR, whereas claimed csi-miRNA-435 is 24 nt species with no structural evidence for a hairpin or miR* species (Axtell & Meyers, 2018), casting doubt on its veracity since 24 nt miRNA species are rare (Coruh et al., 2014). Xu et al. (2010) profiled miRNAs in a lycopene-rich sweet orange red-flesh mutant and its wild type control and claimed two potential targets: EY752486/geranylgeranyl pyrophosphate synthase (Allen et al., 2005) score ¼ 3.0) by miR167 and lycopene b-cyclase by miR1857e5p, which is not a validated dicot miRNA (and likely the star species, documented only 394 Plant Small RNA in Food Crops in rice (Kozomara et al., 2019)). Despite tantalizing roles for these predicted effectors in carotenoid biosynthesis, available degradome analysis fails to substantiate either (data not shown). The authors also speculated based on target sequence homology that APETALA2 (AP2)-like TFs (viz. EY726563 and TC7810/orange1.1g010454m (Goodstein et al., 2011)) which in other species have a paper trail to transactivation of carotenoid biosynthetic gene phytoene synthase/PSY are regulated by claimed csi-novel03/miR3951a-5p (Kozomara et al., 2019; Taylor et al., 2017) and miR172a, respectively. Our analysis of publicly available degradome datasets does not support those speculated targets (data not shown). Boron (B) toxicity poses a great challenge to citrus production, leading to reductions in both productivity and quality. Huang et al. (2019) profiled miRNAs in B-treated C. sinensis (sweet orange; tolerant) and Capsicum grandis (pummelo; intolerant) roots. Their analysis showed that several miRNAs including miR319, miR171, and miR396 are differentially expressed in response to B toxicity. Known target genes of miR319 and miR171 are MYB and SCARECROW-like TFs respectively, with functions in stem cell maintenance, quiescent center, and endodermis specification. For these validated targets it was observed in root tips that expression was inversely correlated with B-treated root tip effector miRNA changes, suggesting these miRNAs could play important roles in proper absorption of water and nutrients during B-toxicity conditions. Lu et al. (2014) claimed that up-regulation of miR474 (annotated only in poplar otherwise) and downregulation of miR782 and miR843 (annotated only in Arabidopsis otherwise) facilitates the expression of genes involved in defense response (reactive oxygen species) signaling in C. sinensis roots during B-deficiency. These claims, along with others regarding miRNAs functioning in lateral root number, cell transport, and protection against omotic stress remain unsubstantiated, given lack of phylogenetic or annotation/ homology/target prediction and degradome evidences for AGO slicing activities. Liu et al. (2014) claimed three miRNAs, csi-miRN31 (no predicted targets), miR477a-3p (claimed target SEPALLATA1.1 (SEP1)/Cs6g19680 (R. Wang et al., 2020)/orange1.1g026143m (Goodstein et al., 2011), marginally supported by authors’ degradome data) and csi-164a showed highest expression assayed by qPCR, RNA blot, and sRNA libraries made from fruits of sweet orange compared to leaves or flowers. Most notable was functional validation in planta, by degradome sequencing, and RNA ligasemediated rapid amplification of complementary DNA ends (50 RLM-RACE) Grain development and crop productivity: role of small RNA 395 that miR164 targets Cs5g10870/orange1.1g017827m (Goodstein et al., 2011), a “NAC” domain TF (acronym from first cloned members of the family: NO APICAL MERISTEM/Arabidopsis thaliana Activation Factor1/ CUC) which showed anti-concordant expression (down) during fruit ripening when miR164 expression levels peaked. miR827 is a phosphatestarvation induced miRNA that accumulates in citrus fruits and targets two different types of SPX (SYG1/PHO81/XPR1)-domain-containing genes: NITROGEN LIMITATION ADAPTATION (NLA) and PHOSPHATE TRANSPORTER 5 (PHT5), in A. thaliana and O. sativa, respectively (Lin et al., 2018). miR168a, which targets miRNA activity effector AGO1, also showed much higher expression levels in citrus fruit (Liu et al., 2014) which suggests these miRNAs may be important for fruit development (see below for more regarding inorganic phosphate [Pi]-regulated miR827 and miR399). Wu et al. (2016) studied the miRNAome and degradome transcripts between a spontaneous late-ripening sweet orange mutant and its wild-type cultivar L. Osbeck and identified two novel miRNAs, csi-miRN03e3p/ csi-MIR3952-like (Kozomara et al., 2019; Taylor et al., 2017) and csimiRN11 (identical to csi-MIR482e-5p (Kozomara et al., 2019)) which showed very high expression in both mutant and wild type, suggesting their potential relevance for fruit ripening. The degradome-validated target of csi-miRN03 (identical to csi-miRN11 of Liu et al. (2014)), Cs8g13560/ orange1.1g035678m, has homologous domains (E < 10 22; data not shown) to citrus alpha crystalline/HSP20-like chaperones (Goodstein et al., 2011), which is novel and intriguing. The authors documented several wellknown miRNAs, namely miR156, miR159, and miR166 which were differentially expressed during fruit development. These miRNAs target TFs SQUAMOSA-PROMOTER-BINDING-LIKEs (SPLs, a plant-specific DNA-binding domain class of TF), gibberellic acid hormone (GA)-MYBs plus non-canonical NOZZLE/ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motif and a LRR Cs8g05120/orange1.1g016534m (Goodstein et al., 2011) (authors misannotated as polygalacturonase inhibitor1), and homeodomain HD-ZIPIIIs/ATHB TFs, respectively. These degradome-validated target mRNAs were anti-concordantly expressed relative to the miRNA effectors during fruit development, providing strong functional evidence for AGO slicing by the cognate miRNAs as the molecular mechanism of down-regulation by post-transcriptional gene silencing. It was noted by authors that in Arabidopsis, miR156-targeted SPL3 positively and directly regulates the 396 Plant Small RNA in Food Crops MADS-box TF genes AP1, CAULIFLOWER/CAL, FRUITFULL/FUL and the central regulator of flowering LEAFY. MADS is an acronym for first four characterized genes of the deeply conserved TF family: MiniChromosome Maintenance1, Saccharomyces cerevisiae; AGAMOUS, A. thaliana; DEFICIENS, A. majus; and Serum Response Factor, Homo sapiens). FUL is a well-characterized regulator of cell differentiation during the early stages of fruit development, supported by evidences in basal eudicot Papaveraceae species that FULs promote development of the fruit wall during maturation (Pabón-Mora et al., 2012), consistent with a deeply conserved regulatory pathway for plant fruit development mediated by miR156-SPL modules. The authors proposed that miR166d might be involved in the regulation of citrus fruit development through HD-ZIP-III/ ATHB targets known to regulate auxin signaling and development of ovule integuments and vascular bundles (Wu et al., 2016). In the model plant Arabidopsis, miR399 plays an important role in Pi homeostasis by targeting PHO2/UBC24 gene, encoding a E2 ubiquitinconjugating enzyme that negatively affects shoot phosphate content and physically interacts with members of the floral development regulator SEP family. R. Wang et al. (2020) used a miRNA-STTM transgenic approach to demonstrate the importance of PHO2/UBC24 regulation of citrus floral meristem identity genes. In csmiR399a.1-STTM OE plants, the expression of genes involved in starch metabolism and Pi homeostasis was deranged. When grown in Pi-sufficient conditions, CsmiR399a.1-STTM plants had lower total phosphorus content in their leaves than the wild type and showed typical symptoms of Pi deficiency. Moreover, downregulation of miR399a.1 by the STTM transgene resulted in pronounced defects in floral development, inhibition of anther dehiscence, and decreased pollen fertility, demonstrating a miR399-PHO2-UBC24 module influences both reproductive development and male fertility in citrus and likely other species by downregulating SEPs which disrupt the floral meristem identity network. Citrus greening disease or Huanglongbing/HLB “yellow dragon disease” is caused by the fastidious (cannot be cultivated ex vivo its vector or host), phloem-limited Gram-negative bacteria of the genus Candidatus Liberibacter (L. asiaticus; Las). Las is transmitted by psyllids, jumping plant lice. Because of the difficulty studying Las (it must be propagated by grafting. Likely because it is only one of a complex of microbiota associated with vectors), evaluation of target genes/metabolites/factors important for adaption and colonization or possible co-evolution are not yet understood. Grain development and crop productivity: role of small RNA 397 HLB has devastated worldwide citrus production. The severe symptoms of HLB includes premature fruit drop, and production of small, off-flavored fruit with aborted seeds. Zhao et al. (2013) showed that Las-infected citrus trees manifest classical Pi deficiency symptoms. Their sRNA profiling of the infected and control plants showed that miR399 is induced by the Las infection, and authors went on to show that induction of miR399 is associated with the Pi deficiency in Las-infected trees. Authors also demonstrated that application of phosphorous solutions to Las-positive plants significantly reduced HLB symptoms and improved yield in the field. Recently a a-helix-2 domain-containing peptide from Australian finger lime and other HLB-tolerant citrus relatives has been shown to prevent and treat HLB by activating host immunity, which involves host sRNAs (Zhao et al., 2013) and LRR stress response targets (Li et al., 2012; Shivaprasad et al., 2012). This promising stable antimicrobial peptide (SAMP) can be applied by spray to leaves and kills L. crescens, a culturable Liberibacter strain, by ion leakage/lysis and inhibits infections of Las in greenhouse trials (C.Y. Huang et al., 2021). It will be interesting going forward to characterize the miRNAome in SAMP peptide-treated citrus and Las-susceptible plants to better understand the molecular mechanisms of SAMP-triggered plant immunity. 4. miRNAs in Brassica Rapeseed (Brassica napus) is an allotetraploid containing A (B. rapa) and C (B. oleracea) genomes (B. nigra is ancestral diploid source of the B genome) (Prakash et al., 2009) and is one of the major edible oil-producing crops. Rapeseed fruits resemble siliques of closely related diploid model plant Arabidopsis. A hallmark of seed maturation in rapeseed is the deposition of storage compounds such as lipid triacylglycerols and storage proteins. Song et al. (2015) showed that the expression of pre-MIR394 transgene resulted in anti-concordant (reduced) expression of validated target LCR. The authors concluded that miR394 is necessary for proper seed morphology and deposition of storage lipids, glucosinolates, and proteins. Transgenic OE of B. napus MIR394 resulted in delayed flowering and plants developed larger pods and seeds with higher contents of protein and glucosinolates and lower levels of oil accumulation with higher saturated fatty acid composition. Concordantly, transgenic plants with elevated levels of BnLCR due to expression of miR394-resistent mutations in the target site resulted in decreased pod and seed size and oil contents. Moreover, MIR394 OE 398 Plant Small RNA in Food Crops induced the expression of genes coding for TFs controlling fatty acid synthesis and seed maturation BnLEAFY COTYLEDON1 (LEC1), BnLEC2, and BnFUSCA3, whereas LCR OE down-regulated BnLEC2 and BnFUSCA3 (Song et al., 2015). Thus, miR394 likely plays an important role in fruit and seed development, building on prior work demonstrating LCR functions in leaf morphogenesis and shoot stem cell identity in Arabidopsis. Jiang et al. (2014) studied miRNAs in flower buds of male sterile and male fertile lines of Brassica campestris ssp. chinensis. They identified24 known miRNAs, 54 conserved miRNAs, and 25 pairs of novel miRNA/miRNA* with highest expression for miR159a, miR160a, miR171e, miR1885b, and miR5724. The expression level was very low for most of the novel miRNAs except bra-miRn22e3p which was highly abundant between the two genotypes. In another study (J. Jiang et al., 2021) authors identified miRNAs in the flower buds of two dominant genic male sterile (DGMS) and two recessive genic male sterile (RGMS) lines by deep sequencing. Among the differentially expressed miRNAs, a higher abundance for miR158, novel_34, miR159, miR827, miR398, miR166a, and miR167c with highest expression for miR159 was found in the two DGMS and RGMS lines. To further evaluate the role of miR159 in pollen development and fertility, authors made OE MIR159 transgenic Arabidopsis which resulted in plants with decreased seed setting rate along with shortened siliques, supporting a role for miR159 in rapeseed fertility and silique development. Zhao et al. (2012) performed sRNA deep sequencing along with q-PCR and RNA blot analysis of two cultivars of B. napus with high versus low oil content to identify miRNAs and other potential regulatory sRNAs involved in early embryonic development and oil accumulation. Their analysis revealed that miR156, miR167, miR390, miR2111, and a novel miR6029 with a predicted bZIP TF target, as well as the miR390-triggered TAS3-produced ta-siRNAs, were differentially expressed between cultivars. Authors proposed that the miRNA-targeted genes might be involved in regulation of oil content of B. napus seeds in later stages through multiple pathways including auxin signaling (Zhao et al., 2012). The 50 RLM-RACE evidence for claimed novel miR6029 slicing of the bZIP target was non-canonical, with a bulge on the miRNA at 11th nt and apparent slicing at ninth nt. Therefore, it is suggested that publicly available Brassica degradome libraries should be assessed to establish the functional significance of this prospect with practical implications (e.g., NCBI PRJNA Grain development and crop productivity: role of small RNA 399 #s 291,315, 185,138, 322,006, 361,531, 418,924, 508,739, 552,762, 36,430, 742,780, 678,586). Since silique length indirectly influences seed yield in B. napus, Chen et al. (2018) analyzed miRNA profiles in Long Silique and Short Silique lines. Correlation analysis of the 17 differentially expressed miRNAs and their targets suggested that miR159 and miR319 up-regulation promotes cell proliferation, and miR160 regulates auxin signal transduction to control silique length. Additionally, the upregulation of Pi-responsive miR2111, miR399, miR827, and miR408 implicated Pi/copper deficiency that restricted silique development. In Arabidopsisthe OE of copper-responsive MIRNAs miR408 and miR397b, which target LAC gene family members involved in lignin biosynthesis, showed increases in silique size along with higher seed yield (S. Huang et al., 2021; Song et al., 2018; Wang et al., 2014). It was proposed based on MIR160 OE phenotypes in transgenic B. napus of longer siliques that high expression of miR160, and anticoncordant effects on negative Auxin Response Factor (ARF) effector targets ARF10, ARF16, and ARF17 expression in rapeseed promotes auxin response thereby increasing silique length. Based on their study they put forward a model (Fig. 14.3) to show the miRNA-mediated silique development networks involving macro- (Pi) and micro-nutrient (copper, Cu) and cell proliferation via miR160-modulated auxin activity. Consistent with this model, in Arabidopsis expression of a miR160-resistant version of ARF10 target showed a twisted silique phenotype (Liu et al., 2007). As mentioned above in other cases, given the authors’ novel claim miR_11, which is a rare 24 nt species speculated to target ribosomal protein gene S3/ RPS3/BnaCnng27520D with limited degradome evidence (slicing category2 (Addo-Quaye et al., 2009), signal on par with background), therefore other Brassica degradome libraries (see above) can be examined to substantiate that module in their model (Chen et al., 2018) (Fig. 14.3). Huang et al. (2013) performed a comprehensive analysis of miRNAs during seed maturation in B. napus using the available B. rapa. v1 reference genome as surrogate and found that miR156 family was the most abundant in seed followed by the miR159, miR172, miR167 and miR158 families. The high abundance of auxin-related miRNAs i.e., miR166, miR167, miR160 and miR164 suggested that auxin signaling homeostasis is important during seed development. Yao et al. showed miR167 plays an important role in anther dehiscence and maternal control of ovule development in Arabidopsis by fine tuning the expression of AUXIN RESPONSE FACTOR6 and 8 (ARF6/8) during the reproductive phase 400 Plant Small RNA in Food Crops Figure 14.3 The potential miRNA regulation networks for silique development in rapeseed. Arrows show simultaneous effect in the pathway while nail heads represent repression. The dashed lines indicate hypothetical pathways affecting silique development. (From Chen L, Chen L, Zhang X, Liu T, Niu S, Wen J, Yi B, Ma C, Tu J, Fu T et al. (2018). Identification of miRNAs that regulate silique development in Brassica napus. Plant Science, 269:106e117.) (Yao et al., 2019) (for miR167 review see (X. Liu et al., 2021)). Ripoll et al. demonstrated that crosstalk between miR172-AP2 module and miR167ARF6/8 module is responsible for the growth of fruit valve in Arabidopsis (Ripoll et al., 2015). Kamiuchi et al. (2014) showed miR164-CUPSHAPED COTYLEDON1 (CUC1) and CUC2 module is responsible for expression of SHOOT MERISTEMLESS (STMs), a KNOTTED-like homeodomain regulator of shoot meristem maintenance and formation, and for stable positioning of the Carpel margin meristems (CMMs). In addition to all these modules, miR396-GROWTH REGULATING FACTORs (GRFs) module plays an important role in the maintenance and proper functioning of CMMs during the development of Arabidopsis gynoecia. Lee et al. showed spatio-temporal expression of miR396 fine tunes the expression of GRFs which function with GRF-INTERACTING Grain development and crop productivity: role of small RNA 401 Figure 14.4 Summary of miRNA modules associated with carpel patterning that control early (e.g., Carpel Margin Meristem [CMM] establishment and maintenance) and late aspects of Arabidopsis fruit development. miR396 and miR164 modules have important regulatory roles in CMM maintenance. MIR172C is induced by AUXIN RESPONSE FATORS ARF6/8 and FRUITFULL (FUL) specifically in the valves, and this specificity is necessary for proper fruit growth after pollination. miR172-guided APETALA2 (AP2) mRNA cleavage in the valves (but not valve margins) promotes valve growth due to the repression of AP2 growth-blocking activity. Growth is blocked by AP2 in valve margins and replum, where miR172 is not expressed. 1 e stigma, 2 e style, 3 e valve, 4 e valve margin, 5 e replum, 6 e septum, 7 e ovule, 8 e Carpel Margin Meristem (CMM). GRF, GROWTH-REGULATING FACTOR; GIF, GRF-INTERACTING FACTOR; STM, SHOOT MERISTEMLESS; CUC1/2, CUP-SHAPED COTYLEDON1 and 2. (Modified from Correa JPdO, Silva EM, Nogueira FTS. (2018). Molecular control by noncoding RNAs during fruit development: From gynoecium patterning to fruit ripening. Frontiers in Plant Science, 9:1760.) FACTORS (GIFs) in the maintenance of CMMs, meristematic competence, and pluripotency (Lee et al., 2017). Fig. 14.4 summarizes the roles of above-mentioned miRNA modules in the development of the Arabidopsis gynoecium, relevant to consider as a layer on top of Fig. 14.3 model of auxin promotion of silique development in close relative rapeseed. A novel claimed miR5801 in B. napus (nomenclature not adopted by miRBase), was predicted (Allen et al., 2005; Huang et al., 2013 402 Plant Small RNA in Food Crops score ¼ 4.0) to target homologs of DEMETER, required in Arabidopsis for appropriate distribution of DNA methylation marks, endosperm gene imprinting, and seed viability. Notwithstanding the observation by the authors the predicted DEMETER-like target mRNAs showed anticoncordant expressions during seed development, when bna-miR5801 was high, in the absence of degradome validation such predictions have limited significance. Likewise, in the absence of a reference genome Körbes et al. (2012) assembled polyA transcript contig sequences from mRNA-seq libraries of mature versus developing seeds of B. napus as a de novo approach for pre-MIRNA discovery, combined with sRNA-seq to characterize the B. napus miRNAome (miRBase22 presently has w80 annotated B. napus MIRNA loci with genome coordinates). Overall, they reported 13 novel miRNAs in their libraries; some are now traceable by homology in miRBase22 as MIR5654, MIR1511-like, MIR6030, MIR9557, and MIR5375-like, but functions remain to be established. Cabbage leaves overlap a terminal bud; Brussel sprouts are enlarged axillary buds; Rutabagas (Swedish “yellow” turnip, or the swede) and kohlrabi (German or cabbage turnip) are swollen lateral meristems comprising upper root, hypocotyl, and lower stem of diploid B. rapa, B. napus and/or B. oleracea. Kale varieties are variously named for their featured traits of curly-leaf (Scots kale, blue curled kale) and bumpy-leaf. What roles might miRNAs play in the myriad Brassica variety meristem indeterminacies and hybrid heterosis (P. Li et al., 2021)? Plausible candidates might be miR394-LCR (see above), miR319-Teosinte branched 1-Cycloidea 1-Proliferating cell nuclear antigen (TCP) domain TFs (Palatnik et al., 2003), and miR172-AP2 modules. AP2 functions in carpel development by antagonizing AGAMOUS expression that in turn antagonizes AP1 and MADS-box TF homologue CAULIFLOWER/CAL/AGAMOUSLIKE10. The CAL gene was discovered as an enhancer of ap1 mutant phenotype of homeotic transformation of petals to shoots. In Arabidopsis the cal/ap1 double mutant has highly indeterminate floral meristems that resemble broccoli and cauliflower varieties of B. oleracea ssp. italica and botrytis, respectively. In a classic case of serendipity, the subject CAL gene, discovered solely by its mutant phenotype in the model plant Arabidopsis, has undergone positive selection during evolution/domestication to fix a nonsense mutation in exon 5 of BoCAL (Purugganan et al., 2000). In other words, the molecular basis of culinary cauliflower is a gene discovered in Arabidopsis and named after itself. It is tantalizing to speculate which target genes and cognate miRNA effector modules might be causal for the myriad Grain development and crop productivity: role of small RNA 403 agronomic traits of the Brassicaceae and subject to genetic manipulation and molecular breeding for novel and improved agronomic traits and productivity. 5. miRNAs in asterids (Solanaceae) 5.1 Tomato (Solanum lycopersicum) Since a large body of work exists for tomato as a model dicot crop, we approach the subject of miRNA regulation and mechanism of fruit development from a miRNA-centric perspective with focus on the ancient, deeply conserved miRNA families. It remains to be established but is envisioned that sRNA processes/pathways discovered in one species by a reductionist approach can leverage inference and imputation of function translated to other crop species. Where knowledge may be limited, this rationale of evolutionary conservation facilitates experimental designs to test models in the emerging field of systems biology-understanding the bigger picture at the levels of cells, tissues, organism, and ecosystems in which species have evolved. Patterns discussed above for the Rutaceae and Brassicaceae families may hold for nearby relatives Theobroma and cotton. Patterns to be articulated below for the monocots may hold for the bromeliads like pineapple (Patel et al., 2021); and the Solanaceae family findings may hold broadly in the Asterids clade and reinforce or be complementary to findings in the Rosids etc. Juvenility and late flowering in plants are promoted by the miR156SPL module. Cui et al. (2020) showed that a miR156-SPL13- SINGLE FLOWER TRUSS/SFT module regulates the yield and architecture of a tomato plant. They established SPL13, whose transcript is under the negative regulation of miR156, positively effects the expression of SFT, the tomato orthologue of long-range protein signal FLOWERING LOCUS T (FT)/FLORIGEN, by binding directly to its promoter. OE of MIR156 or SPL13 knockdown transgenic experiments resulted in plants with reduced inflorescences and numbers of vegetative branches, and consequently yield. Silva et al. (2014) showed that OE of MIR156b in tomato resulted in the abnormal fruit morphology due to the loss of ovary meristematic tissue identity leading to the formation extra carpels (fused) and presence of the undifferentiated tissue inside post-anthesis ovaries. They also reported that genes associated with meristem maintenance and formation of new organs, LeT6/TKN2 (a KNOTTED-like homeobox class I gene) and GOBLET/ GOB, a NAC under the direct negative regulation of miR164, were 404 Plant Small RNA in Food Crops induced in MIR156b OE lines. Authors also showed reduction in the expression of miR164 in MIR156b OE lines, indicating maintenance of the meristematic tissue present in the ovary, new organ formation, and establishment of organ boundaries is fine-tuned by interaction between miR156 and miR164 modules. Xing et al. (2013) showed similar roles of miR156SPL module in Arabidopsis flower and seed development since seed production was reduced to w60% in MIR156b OE lines and the number was increased to w96% of wild type in spl8-1 p35S:MIR156b lines. They also reported that ovary structure was unaffected in MIR156b OE lines but was completely altered in spl8-1 p35S:MIR156b lines which had altered expression of auxin biosynthesis gene YUCCA4. SPL8 does not have a miR156 binding site as compared to SPL2, 6, 10, 11 and 13 yet is expressed simultaneously with SPL2, 3, 10, 11 and 13 and thus appears to play a redundant role in gynoecium patterning and fruit development through interference with auxin homeostasis/signaling. Karlova et al. (2013) validated at different stages of developing fruits that miR156/157 targets SPL family member COLORLESS NONRIPENING/CNR, intriguingly a positive regulator of AP2a which in turn negatively regulates the expression of CNR, forming a feedback loop by dual effectors with antagonistic functions in fruit ripening and both subject to miRNA-mediated silencing. In addition, AP2a, a validated target of miR172 at breaker stage of fruit development, is also induced by ethylene and yet negatively regulates ethylene biosynthesis and signaling when ethylene biosynthesis is initiated (Karlova et al., 2011), thus forming another homeostatic feedback loop with the autocatalytic hormone that promotes ripening. They also demonstrated miR393 is involved in controlling the expression of target TRANSPORT INHIBITOR RESPONSE1-like auxin receptors during tomato fruit set. Gao et al. showed by chromatin immunoprecipitation and electrophoretic mobility shift assays the direct binding of RIPENING INHIBITOR (RIN), a MADS-box TF master regulator of climacteric ripening, to the promoter of MIR172a (Gao et al., 2015). da Silva et al. (2017) explored the role of miR159-SlGAMYB1 and miR167-SlARF8a modules in the ovule and ovary development and tomato fruit set. They showed ovule development is tightly regulated by auxin, GA, and ethylene which is further fine-tuned by miR159 and miR167 activities. They showed OE of MIR159 and repression of miR167 (induced by OE of MIR159) resulted in aberrant ovule development and production of parthenocarpic (fertilization-independent) fruits. Similar to Grain development and crop productivity: role of small RNA 405 Figure 14.5 Proposed model for the molecular circuit involving miRNA-associated modules and phytohormones in tomato developing ovaries. (From da Silva EM, Silva G, Bidoia DB, da Silva Azevedo M, de Jesus FA, Pino LE, Peres LEP, Carrera E, LópezDíaz I, Nogueira FTS. (2017). microRNA159-targeted SlGAMYB transcription factors are required for fruit set in tomato. The Plant Journal, 92:95e109.) SlMIR159-OE plants that manifested altered auxin signaling markers, SlGAMYB1 was downregulated in ovaries of parthenocarpic mutants with altered responses to GAs and auxin (Fig. 14.5). Similarly Du et al. (2016) also showed that SmARF8 is a key negative regulator of fruit set in eggplant S. melongena. A spontaneous parthenocarpic mutant had low SmARF8 expression, and knockdown by RNA interference in eggplant of SmARF8 also led to the formation of a parthenocarpic fruit, a value-added trait because seeds cause bitterness and browning of flesh. Zhao et al. recently demonstrated miR159 targeting SlGAMYB2 to regulate fruit size by direct repression of the GA biosynthesis gene SlGA3ox2 as a previously unidentified mechanism that controls fruit morphology in tomato (Zhao et al., 2022). miR160 is a class of miRNAs known to target Auxin Response Factors (ARFs) family members and same has been validated by Karlova et al. (2013) who generated degradome evidences to illustrate miR160SlARF10A/B, -16A/B, and -17 interactions. Damodharan et al. (2016) showed abundant expression of miR160 in developing ovaries. Knockdown of miR160 using a STTM160 transgene disrupted the ovary patterning which was marked by thinning of the placenta and increased growth at the proximal end of the ovary upon fertilization, resulting in pear-shaped fruits reminiscent of ovate mutant. STTM160 fruits with 406 Plant Small RNA in Food Crops depleted miR160 resulted in elevated SlARF10A/B and SlARF17 and pleiotropic phenotypes of abnormal floral organ abscission, and leaves, sepals, and petals with diminished blades, demonstrating the positive role of miR160 and repressive roles of cognate ARF10/17 targets in auxin response during ovary patterning, lamina expansion, and floral organ abscission. Four family members of MIR164 have been reported in tomato out of which miR164a/b are conserved in both dicots and monocots, miR164c is conserved in dicots and miR164d is only found in Solanaceae species and has an atypically long pre-MIRNA (Gupta et al., 2021). CRISPR-Cas9 genome editing (Jinek et al., 2012) of MIR164abd loci and STTM experiments revealed typical roles in shoot and floral boundary specification for miR164b-NAC module, versus a specialized role in fruit growth and pericarp/epidermis expansion for miR164a which antagonizes SlNAM2 and SlNAM3 expression in ripening fruit. The sly-miR164dCR edited mutants were phenotypically indistinguishable from wild type. Berger et al. (2009) showed that NAC GOB loss-of-function mutation has reduced shoot apical meristem activity, extended and fused floral organs, and fewer fruit locules whereas a mutagen-induced GOB dominant gain-of-function allele (intact reading frame but disrupted miR164 binding site) manifests higher shoot apical meristem indeterminacy resulting in more cotyledons and extra floral organs and simpler leaves due to secondary leaflet fusion. Hendelman et al. (2013) generated MIR164 OE tomato and further substantiated that miR164-GOB module plays an important role in establishing the organ boundaries in floral meristems. Expression of a miR164-resistant SlNAM2 site-directed mutant could suppress fusion phenotypes and restore floral boundaries in NAM-deficient mutants, strongly supporting that the miR164-SlNAM2 module also participates in flower whorl and sepal organ boundary establishment as well as maintenance after flower formation (when GOB expression is high). In addition to reproductive stage, miR164 is very highly expressed during breaker and subsequent stages of tomato fruit development (Mohorianu et al., 2011). OE of MIR164 had variable effects the duration of different stages of tomato fruit development, for example some events resulted in longer ripening and senescence stages, which could have practical applications such as extended shelf life (Rosas Cárdenas et al., 2017). In this context of climacteric fruit ripening, a claim made based on 50 RLM-RACE during the early discovery phase of the field could be revisited using abundant publicly available degradome data. It was predicted a Grain development and crop productivity: role of small RNA 407 novel miR5301*/miR482e-3p, repressed during fruit maturation stage, targets a membrane-localized glutamate permease/SGN-U585460/Solyc04g082290.4.1 (Goodstein et al., 2011) which might be causal for the distinctive and economically important trait of “umami” taste (Mohorianu et al., 2011) (“essence of deliciousness” in Japanese: refers to the core fifth taste other than sweet, sour, bitter and salty) (Li et al., 2002). As a denizen of west Texas for many years, the senior author grew to appreciate the southern US traditional dish “fried green tomatoes.” Green tomatoes are abundant in the southern US fall season when first frost kills the plant. Typically, tomato fruit set in many southern home gardens from late spring until early Fall is nil until maximum daytime temperatures drop below the non-permissive temperature of 32 C, above which pollen development and germination fails. Differentially expressed miRNAs have been characterized in developing heat-stressed tomato anthers (Keller et al., 2020). Clepet et al. (2021) showed cold stress induces miR166 that causes down-regulation of target parthenocarpic fruit 1/SlHB15A/PF1 which they discovered by cloning a pf1 loss of function mutant they isolated from a mutagenized population screened in non-permissive hot temperatures. Non-fertilization-based fruit set in pf1 and allelic pat mutant causes defects in ovule development and aberrant fruit set, reducing yields but correlating with parthenocarpy and improved yields under cold conditions in the greenhouse and field. Authors showed SlHB15A/PF1 mRNA expression in shoot apical meristem, vascular elements, and ovule primordia and outer cell layer of the ovule integument, mimicking patterns seen for homologous targets of miR166 required for integument development in Arabidopsis (Kelley et al., 2009) and also adaxial cell fate of leaves (Kidner & Martienssen, 2004) and radial vascular patterning (Kim et al., 2005). Transcriptomic profiling in unpollinated pf1 abnormal ovules, compared with wild type pollinated ovules, suggested that SlHB15A directly inhibits auxin signaling and induces ethylene-associated genes, preventing fruit set in the absence of fertilization. The miR166-SlHB15A/PF1 module adds an extra layer of homeostatic regulation to development of ovules and consequent parthenocarpy in tomato controlled by dosage-sensitive haploinsufficient expression of PF1 leading to aborted integument. Plants harboring a miRNA166-resistant SlHB15a allele developed normal ovules and were unable to set parthenocarpic fruit under cold conditions. Based on their genetic and molecular results authors synthesized a model for ovule development tightly regulated by auxin, GA, and ethylene cross-talk downstream of the miR166-PF1 node (Fig. 14.6). da Silva and Nogueira 408 Plant Small RNA in Food Crops Figure 14.6 Working model explaining how the miR166eSlHB15 regulatory module controls ovule development and fruit set. (A) In the absence of fertilization, PF1 inhibits auxin and activates ethylene signaling, maintaining the ovary in a growth-arrest phase until pollination takes place. (B) Ovule fertilization relieves the inhibition; accumulation of auxin and inhibition of ethylene signaling lead to fruit set. (C) pf1 lossof-function alleles induce aberrant ovules that mimic WT pollinated ovules. (D) In Pf1/ pf1 heterozygote plants, under cold conditions, the overexpression of miR166 knocks down PF1 mRNA to threshold, leading to aberrant ovules and fruit set. (From Clepet C, Devani RS, Boumlik R, Hao Y, Morin H, Marcel F, Verdenaud M, Mania B, Brisou G, Citerne S et al. (2021). The miR166eSlHB15A regulatory module controls ovule development and parthenocarpic fruit set under adverse temperatures in tomato. Molecular Plant, 14:1185e1198.) (2021) elaborated on this work as the first example of a recessive dosage sensitivity by a miRNA. Since the miR166 module is highly conserved, there is potential for engineered parthenocarpy and molecular breeding of haplotypes in the Solanaceae and possibly other fruit crops as a pivotal strategy to expand agricultural cultivation areas, mitigate yield losses due to non-permissive temperature fluctuations caused by climate change, and reduce risks to food security in the future. BLIND/BL and FISTULATA/FIS genes were described originally as “A” class mutants of Petunia hybrida and Antirrinum majus (snapdragon), respectively, that resulted in homeotic transformation of petals and sepals Grain development and crop productivity: role of small RNA 409 into antheroid structures due to expansion of the “C” class developmental domain in outer floral organ whorls according to the “ABC model” of flower development (Coen & Meyerowitz, 1991). The ABC model was promulgated from phenotypes of Arabidopsis and Antirrinum majus (snapdragon) mutants of the Brassicaceae order and Asterid clade of eudicots, respectively, and is the classic framework for understanding floral development and evolution by MADS-box TF pairwise interactions. The squamosa mutant of Antirrhinum majus (snapdragon) is cognate, along with ap1 of Arabidopsis, of the A class MADS TF genes. However other than a role of miR172 to regulate A class gene AP2 in Arabidopsis (a member of the Rosid clade), how the A-function is encoded in other species and nonrosid clades like Asterids petunia and snapdragon is not well understood, where evidence for C class antagonism by some A class genes is lacking, contrary to the ABC model. Cartolano et al. (2007) found in P. hybrida and A. majus respectively, partial A-function blind and fistulata mutants encode loss of function alleles of MIR169 family members that target a subset of NUCLEAR FACTOR-YA (NF-YA) domain TFs. The de-repression of NFeYAs in the bl and fis mutants leads to expansion of C function genes into perianth domains normally limited to fourth inner whorl domains specifying carpel organ identity (Morel et al., 2017). This additional C function regulatory layer by a miR169-NF-YA module is unrelated to the miR172-AP2 module for C function patterning in Arabidopsis. Yet its characterization in tomato can help explain why de-repressed A-class TF expression in CRISPR-Cas9 edited miR172CR genome-edited mutant alleles of family members had minimal effects on the fourth whorl of tomato flower development (Lin et al., 2021), consistent with expression profiles for miR169 expression in flowers and fruits yet low in leaves (Moxon et al., 2008). The mechanistic understanding of roles played by miR169 in fruit development is still limited, but roles in abiotic stress adaptation have been demonstrated that vary by species. MIR169 OE conferred drought tolerance in tomato (Zhang et al., 2011), but on the other hand in rice miR169 is induced by salt and drought stresses yet down-regulated by drought and nitrogen deficiency in Arabidopsis (Zhao et al., 2009, 2011). It remains to be elucidated how and why speciesspecific differences in miR169-NF-YA modules affect stress adaptation networks. Kravchik et al. (2019) showed miR171 is expressed differentially during fruit developmental stages in tomato with its highest expression in the flowering stage. They found that the expression of miR171 is important for 410 Plant Small RNA in Food Crops maintaining pollen viability and pollen development. W. Huang et al. (2017) showed miR171-SlGRAS24 TF module is important for the regulation various agronomic traits like plant height, flowering time, leaf architecture, lateral branch number, root length, fruit set and development. miR172 isoforms are encoded by 11 genes in soybean (Glycine max) (Lunardon et al., 2020). miR172-AP2a module interacts with miR156CNR module and regulates the ripening of the tomato fruit, as discussed above (and further below). miR172 module targets euphyllophyte AP2 TFs which consist of TARGET OF EAT/TOE-type and AP2-type clades. In Solanaceous species A-class functions, as described by the elaborated ABCDE model of flower development in Arabidopsis, are fulfilled by AP2type and TOE-type clades. Thus, miR172-AP2 module plays an important role in the regulation of flower development by keeping the determinacy of floral meristem in check (Morel et al., 2017; Zhao et al., 2007), and appears conserved across different families like Solanaceae, Brassicaceae, Vitaceae etc. CRISPR-Cas9 edited miR172dCR tomato mutations resulted in the development of sepaloids in the second and third floral whorl of tomato flowers (Lin et al., 2021), interpreted as antagonism of B- and C-class genes by up-regulated A-class genes, expanding their domain according to the ABC model. This is in contrast to above-mentioned finding that effect of induced A-function gene expression by genome-edited miR172CR null alleles did not affect the fourth whorl pistil development of the flower (Lin et al., 2021). miR396 is known to negatively regulate a large GRF family. GRFs are plant-specific TFs conserved in all Embryophytes and are absent in fungi and green algae. GIF-GRF complexes are known to participate in cotyledon, root, leaf, flower, and seed development (Debernardi et al., 2014; Hewezi et al., 2012; Lee et al., 2017; Liang et al., 2013; Liu et al., 2012; Rodriguez et al., 2010). Cao et al. (2016) knocked down miR396 ab expression via STTM approach which resulted in tomato transgenics with increased growth of floral buds, floral organs (especially sepals via increased epidermal cell size and numbers), fruit weights, and seeds via increased SlGRF expressions compared to wild type. Despite limited mechanistic understanding of roles of miR396-GRF module in fruit development, eight GRFs out of the 13-member GRF family have been validated as targets of miR396 ab identified via degradome analysis in tomato (Karlova et al., 2013). In lettuce (Lactuca sativa, an asterid) OE of LsaGRF5 exhibited larger leaves, while smaller leaves were observed in LsaMIR396a OE lines, in which LsaGRF5 target was down-regulated (B. Zhang et al., 2021). Grain development and crop productivity: role of small RNA 411 miR1917 is a miRNA so far only found in tomato and plays an important role in regulating ethylene responses. Y. Wang et al. (2018) validated a Solanaceae-specific miR1917 by in planta transient assays and site-directed mutagenesis of the binding site in the target mRNA (Moxon et al., 2008). This newly identified miRNA regulates ethylene signaling by targeting CONSTITUTIVE TRIPLE RESPONSE 4 (SlCTR4) 30 -UTR, a repressor of ethylene signaling that can physically interact with ethylene receptors (Zhong et al., 2008). miR1917-mediated SlCTR4 repression resulted in accelerated pedicel abscission and fruit ripening due to increased ethylene responses. Yang et al. (2020) showed suppression of miR1917 via STTM approach, which resulted in increased size and weight of the tomato fruit and seed size. A working model of post-transcriptional and hormonal control can be synthesized which integrates lnc1459/1840/2155, miR1917CTR4, and engineered STTM-miR1917 modules for agronomic improvement of tomato, drawing on knowledge of miR156-CNR and miR172-AP2 modules (Fig. 14.7) (Correa et al., 2018; Yang et al., 2020). Karlova et al. (2013) reported a predicted candidate target of miR1917, Sesquiterpene synthase2/Solyc06g059920.1.1a with limited degradome support (slicing category1), which can be further scrutinized using other degradome libraries and improved algorithms (Addo-Quaye et al., 2009) for independent evidence to support a target enzymatic function in fruit traits such as aroma. Zhu et al. reported 490 and 187 lncRNAs whose expression were significantly up- and down-regulated, respectively, in the MADS TF ripening inhibitor (rin) mutant of tomato (Zhu et al., 2015). The novel intergenic lncRNA1459, lncRNA1840, and lncRNA2155 are significantly down-regulated in rin fruit, and wild type expressions correlate with ripening stages. VIGS silencing of either lncRNA1459 or lncRNA1840 and RIN significantly delayed ripening of the tomato fruit two weeks after infiltration. The group went on to generate a CRISPR-Cas9 edited lncRNA1459 and lncRNA2155 mutants which manifest repressed tomato fruit ripening. Authors subsequently showed by chromatin immunoprecipitation-sequencing, and in vitro, RIN binds to the lncRNA2155 promoter (Li et al., 2018; Yu et al., 2019). Even though mechanistic knowledge of how lncRNA1459 and lncRNA2155 positively regulate fruit ripening is limited, repression of the genes involved in ethylene production and lycopene accumulation (Yu et al., 2019) and derangement of ripening-related genes including 81 up-regulated and 31 down-regulated lncRNAs in lncRNA1459-edited tomato mutants support these lncRNAs play essential roles in fleshy fruit ripening (Li et al., 2018) and warrant further study. 412 Plant Small RNA in Food Crops Figure 14.7 Non-coding RNA networks associated with and fruit ripening. Graphic shows the accumulation of miRNA-cleaved transcripts of COLORLESS NON-RIPENING (CNR) and APETALA2 (AP2) through four stages of fruit development/ripening: 5 days after pollination (5 DAP), Mature green (MG), Breaker (Br), and Red ripe (RR; seven days post-Br). mRNA cleaved product accumulation occurs in the Br stage, coinciding with an ethylene peak production. lnc1840, lnc1459, lnc2155: long noncoding RNAs. Black lines in the transcriptional networks denote direct regulation, whereas gray lines denote indirect regulation. Question mark denotes that is uncertain if CNR forms a complex with MADS-box TF RIPENING INHIBITOR/RIN. SlCTR4, tomato CONSTITUTIVE TRIPLE RESPONSE 4. (Modified from Correa JPdO, Silva EM, Nogueira FTS. (2018). Molecular control by non-coding RNAs during fruit development: From gynoecium patterning to fruit ripening. Frontiers in Plant Science, 9:1760.) 5.2 Potato (Solanum tuberosum L.) miR156 participates in the regulation of tuber formation, which like flowering in plants is a photoperiod-dependent process. Bhogale et al. (2013) studied the expression profile of pre-MIR156 by qRT-PCR in potato leaves, stems and stolons in response to inductive light conditions. They showed expression of miR156 is significantly higher in leaves and stems during long-day (tuber non-inductive) than short-day conditions, and eight-fold induced in stolons under short-day (tuber inductive) conditions. They also showed by mobility assays in heterografting experiments Grain development and crop productivity: role of small RNA 413 that miR156 is a phloem-transmissible signal and OE of MIR156 resulted in the formation of arial tubers from axillary meristems under short-day conditions. Authors proposed that controlled spatiotemporal expression of miR156 along with the tuber inductive conditions regulate tuber formation. Formation of homeotic (arial) tubers in tomato was shown by EviatarRibak et al. (2013) who ectopically expressed LONELY GUY, a cytokinin (a plant hormone critical for source-sink signaling) zeatin riboside activating enzyme. They concluded cytokinin primes the ectopic activity of miR156 in OE tomato lines as positive regulator of juvenility to extend tuber forming potential to distal axillary buds in both potato and tomato. A recent review highlights the roles of photoperiod and epigenetic mechanisms via polycomb group proteins to control miRNA156, phytohormone metabolism/transport/signaling, and key tuberization genes through histone modifications to govern tuber development (Kondhare et al., 2021). miR172 affects the timing of tuberization in potato in addition to flowering time and development. Martin et al. (2009) showed OE of MIR172 promotes tuberization through a graft-transmissible signal under inductive and non-inductive day length conditions. Authors proposed a PHTYOCHROMEB/PHYB-mediated miR172-RAP1-BEL5 model, whereby PHYB represses miR172 in long days, which in short days via RELATED TO AP2/RAP1 target antagonism results in up-regulation of stolon tuberization effector mRNA BELLRINGER1-like5/BEL5 (a KNOTTED-like homeodomain gene). Bhogale et al. (2013) characterized the genetic interactions between miR156-SPL stolon tuberization module and miR172 tuberization-timing modules in potato. OE of miR156 resulted in reduced levels of miR172 and reduced tuber yields. Authors showed miR172 expression is positively regulated by StSPL9 binding the MIR172 promoter; miR156-resistant silent mutations in target SPL9 OE lines exhibited increased miR172 levels under a short-day photoperiod, demonstrating that miR156 and miR172 act in concert to regulate potato tuberization. A role for cytokinin in promoting timing of phase transition to flowering in Arabidopsis by the opposing actions of the miR156-SPL and miR172-AP2 modules has recently been demonstrated (Werner et al., 2021), and may be important for crops like potato. Santin et al. (2017) suggested, based on results of agroinfiltration transient assays in Nicotiana benthamiana, that a gene correlated with hormoneregulated tuber formation Calcium Dependent Protein Kinase 1/CDPK1 expression is negatively regulated by miR390. This novel claim should be evaluated with publicly available degradome datasets for potato (NCBI 414 Plant Small RNA in Food Crops PRJNA #398663, 335,760, 685,865) and sweet potato (#592001) given the miR390: target homology was relatively weak (Allen et al., 2005 score ¼ 6.5) and miR390 is otherwise known only for its evolutionarily conserved slicing of TAS3. 5.3 Capsicum (Capsicum annuum) Similar trends for miRNA expression (as that of tomato, including anticoncordant expression of validated targets) for miR156, miR159, miR160, miR164 and miR172 were reported by Lopez-Ortiz et al. (2021) from profiling results of two domestic and two wild Capsicum species during flowering and stages of fruit development. Since the mechanistic understanding of how miRNA networks interact with gene networks is a fertile ground for discovery, authors claimed Capsicum-specific miR06, miR10, and miR11 were differentially up-regulated, whereas miR13 and miR15 were down-regulated during fruit development. miR06 was predicted to target two methylene tetrahydrofolate reductase genes (CA05g20110, CA12g03850) involved in folate metabolism as potential effectors of vitamin B in pepper fruits. miR23 and miR24 (latter homologous (Kozomara et al., 2019) to a bat transposon/efu-mir-9277 (Platt et al., 2014), so likely not a bona fide miRNA) were predicted to regulate 1-aminocyclopropane-1-carboxylate oxidase gene (ACA03g05550) involved in ethylene biosynthesis. A publicly available degradome dataset (NCBI PRJNA398663) covers Solanaceous crops tomato, potato, and pepper (Seo et al., 2018), making the above speculative claims traceable to evidences of AGO slicing rather than homology-based predictions. 6. miRNAs in other asterids Oleaceae, Sesame, and Actinidiaceae Carbone et al. (2019) showed miR156, miR159, miR166 and miR168 were the most differentially expressed miRNAs in olive (Olea europaea L.) drupe epi-mesocarp tissue at onset and completion of ripening stages between cultivars that differ in accumulation of anthocyanins: “Leucocarpa” and “Cassanese.” The expression of these conserved miRNAs is consistent with observations in tomato yet mechanistic understanding for miRNA functions in olive is lacking. It has been suggested a portable qRT-PCR field instrument could be deployed for screening olive groves for host transcriptomic markers (Martinelli et al., 2019), including phosphateregulated host miRNAs, to detect Xylella fastidiosa infections, causal agent Grain development and crop productivity: role of small RNA 415 of Sudden Olive Decline disease as has been shown for citrus pathogen Las/ HLB (Zhao et al., 2013). In principle, the commercial value of sesame (Sesamum indicum L.) oil could be impacted by cogent insights into miRNA-target interactions involved in fatty acid metabolism. Toward this end, Y.P. Zhang et al. (2021) characterized at a cursory level sRNAs (novel miRNAs claimed from identified sRNA species not in miRBase, which does not meet community standards for MIRNA annotation (Axtell, 2013; Axtell & Meyers, 2018; Brodersen & Voinnet, 2009; Coruh et al., 2014; Taylor et al., 2017)) expressed during sesame seed development. Authors concluded that fatty acid biosynthesis in sesame is regulated by novel miRNAs. However, evidence presented is not compelling, with poor Allen (Allen et al., 2005) scores and poor degradome categories/statistics (AddoQuaye et al., 2009), such that claimed target slicing does not stand up to post-publication review despite some good control degradome evidences to support canonical targets of conserved miRNAs. The case frames a hard lesson for students of the fast-moving miRNA field (Taylor et al., 2017). miR164-NAC is a highly conserved well-studied module which is known to regulate various physiological aspects of plant development like root development, organ boundary formation, cell death, and senescence. Gupta et al. (2021) identified a novel regulatory aspect of miR164-NAC involved in the ripening process of several fruit crops including kiwifruit (Actinidia arguta) (J. Wang et al., 2020). Another important aspect of the kiwifruit is the fruit color which is often affected by anthocyanin accumulation. Y. Li et al. (2019) showed that miR858 negatively effects the accumulation of anthocyanin by targeting AaMYBC1 TF, which is a positive regulator of anthocyanin biosynthesis consistent with results in MIR858-OE and knockdown experiments in transgenic tomato (Jia et al., 2015) and Arabidopsis (Sharma et al., 2016; Y. Wang et al., 2016). 7. miRNAs in rosids The rosids belong to a large group which comprise more than one-fourth of w369,000 extant angiosperm species. Fruit is defined as “the mature ovary of a flowering plant that is edible”. On the basis of this definition, the three Rosaceae model plants: peach, apple, and strawberry develop three different fruit types: drupe/berries, pome, and achene, respectively (Seymour et al., 2013). Drupes of peaches (Prunus persica) are characterized by a lignified endocarp (stone or pit), which envelopes seeds, and an epicarp 416 Plant Small RNA in Food Crops tissue that surrounds fleshy and juicy mesocarp. Apple (Malus domestica) and pear (genus Pyrus) are “false fruits”/pomes, in which seeds are leathery, surrounded by a tough membrane core, and the floral receptacle is the fleshy edible tissue (named cortex) and pericarp is absent. Strawberry (genus Fragaria) achenes, defined as a true fruit, is localized on an accessory structure comprised of a fleshy receptacle. Despite the plethora of fruit tissue patterns in rosids, the critical steps driving fruit set to ripening are common to the different fruit models (Farinati et al., 2017). Vitis (grapevine) berry development is divided into stage I, fruit set stage involving berry growth through cell division; stage II/lag phase, when a pause in cell division and berry growth coincides with seed embryo differentiation; and stage III/veraison, which involves color change and softening of berries with sugar and solute acids accumulations. There are several ancient/conserved miRNAs involved in distinct phases of fruit development in grape, pear, apple, peach and strawberry species. Two highly conserved miRNA families, miR156 and miR172, show an inversely correlated expression during distinct stages of grape berry development. Expression of miR156 decreases in grapevine young berries whereas it is upregulated in mature berries in which target of miR156, SPL9, is decreased (Belli Kullan et al., 2015; Cui et al., 2018; Su et al., 2021). miR156 is the largest miRNA family in pear (Wu et al., 2014). M.M. Wang et al. (2021) reported that a novel-miR156 (most homologous to mdm-MIR11008 (Kaja et al., 2014) in miRBase22) is down-regulated in “Qinguan” apple treated with ethylene receptor inhibitor 1methylcyclopropene. On the basis of anti-concordant up-regulation in the experimental material, a predicted target MdERF118 Ethylene Response Factor was claimed effector of climacteric ripening. However, scrutiny of the novel miRNA sequence that forms the hairpin stem is not complementary to the claimed target sequence (data not shown). Full red strawberry fruits exhibit up-regulation of miR156, which increases anthocyanin accumulation by down-regulation of SPL targets (J. Wang et al., 2020). Peach miR156 is claimed to target the SPL homologue colorless non-ripening (CNR) (Shi et al., 2017), a gene identified genetically in tomato and validated target of miR156/157 (Karlova et al., 2013; Moxon et al., 2008). miR172 is a known positive regulator of flowering stage and a negative regulator of AP2-like floral repressors, which interacts genetically with the miR156-SPL module described above for potato and tomato. Interestingly, a naturally occurring transposon-induced hypomophic miR172 allele of Grain development and crop productivity: role of small RNA 417 apple was claimed to have been selected for in wild apple by large mammals prior to domestication (Yao et al., 2015), consistent with results that OE of MIRNA172 in transgenic apple significantly reduces fruit size (Zhou et al., 2021). miR396-GRF is another conserved module that regulates cell expansion. miR396 is expressed highly in young fruits of grape, pear, and apple and which gradually decreases at ripening stage (Mica et al., 2009; Wu et al., 2014; Xia et al., 2012). Regulation of lignin synthesis pathways in pear fruits by miRNAs is of particular interest given the unique fruit trait of sclerified “stone cells” that give the fruit a “gritty” texture and may contribute to astringency. Wu et al. (2014) showed miR397-LAC and miR408-LAC modules exhibits anti-concordant expression (low miRNA, high LAC target mRNAs) early in fruit development, but evidence for claimed role of miR396 in sugar metabolism by non-canonical targeting Glucose-6-phosphate dehydrogenase/G6PDH based on homology predictions is lacking. miR4414 is a less-conserved miRNA annotated in miRBase22 only for gymnosperm Picea abies, and two Fabaceae species. Notwithstanding, this claimed miR4414 in pear, Pyrus bretschneideri (identical to miRBase22annotated csi-miR156j-5p) was found to highly express in early developmental stages of pear fruit and to decline during maturation. The star species miR4414a* (nearly identical in miRBase22 to mdm-miR319c-3p and csimiR156j-3p) was predicted (no evidence provided) to target the mRNA for Dihydrolipoyl transacetylase (Pbr015262.1), a transporter associated with tricarboxylic acid cycle and pyruvate metabolism (Wu et al., 2014). In peach, high throughput sequencing of sRNAs likewise revealed higher relative expression of miR4414/csi-miR156j-5p in flowers compared to young stems and leaves, despite its relative low abundance as compared to other conserved miRNAs (Luo et al., 2013). The functional significance of miR4414 remains to be established. miR2950 is found (Kozomara et al., 2019) in gymnosperms, Amborella sister to angiosperms, and rosids including grape, curcurbits, malvids, and Ziziphus jujube (Chinese date, buckthorn family) (Kong et al., 2021) but has no known function related to development of fruits. In grapevine, this miRNA has been shown to target chlorophyllase/VIT_07s0151g00250 gene in response to bacterial and viral pathogen attacks including Flavescence dorée (FD) (Chitarra et al., 2018). It was speculated (no evidence provided) for Chinese date jujube (Ziziphus jujuba) that miR2950 might target ARF4/11/16 (miR167 is the canonical effector of Arabidopsis 418 Plant Small RNA in Food Crops ortholog AtARF6, predicted targets ZjARF4/5/7). Consistent with the notion, ZjARF4 expression inversely correlated with later stages of healthy flower development when infected by phytoplasma, causing phyllody symptoms whereby floral organs abnormally developed into leaf-like structures (Ma et al., 2020). It is interesting (with the caveat cause and effect must be proven consistent with a model of divergent evolution) how regulatory roles of the same miRNA in different plant species can differ in fruit development. See above the case of miR169 canonical NF-YA targets potentially functioning in floral homeotic gene regulation in the Asterids, and possibly the sister Solanaceous plant clade, but not the Brassicas (Cartolano et al., 2007). Several members of miR169 (miR169a, miR169f, miR169r, miR169x) are expressed significantly higher in mature berries compared to green berries in stage I of grape development (Paim Pinto et al., 2016). It is plausible, as mentioned above a role in abiotic stress responses, that miR169 may protect mature berries from dehydration stress. However, in Arabidopsis there is evidence for miR169 non-canonical targeting of an effector of flowering/ phenology, JASMONATE-ZIM-DOMAIN PROTEIN 4/JAZ4 (Gyula et al., 2018), a transcriptional co-repressor that interacts with miR172 target TOE1 to relieve the repression effect of TOE1 on FLORIGEN/ FLOWERING TIME (Zhai et al., 2015). Sunitha et al. (2019) validated by degradome slicing evidence the conservation of JAZ3_1 slicing in grape by vvi-miR169x, supporting a more deeply conserved miR169 role in rosid flower development than previously recognized. miR397 in grape has been claimed to impact fruit storage safening in 40 day-sulfur dioxide fumigation-treated stored berries via predicted noncanonical antagonistic regulation of LOX, lipoxygenase (Xue et al., 2018). However, the evidence is lacking LOX is a bona fide miR397 target in grapevine; anti-concordant miR397-LOX target expression assays of test fruit by qRT-PCR were not statistically significant, nor was slicing of LOX mRNA by miR397 tested. In contrast miR397 in pear is more prominently expressed at young fruit stage and targets canonical/validated LAC genes (Wu et al., 2014) as mentioned above. Similarly, miR398 expression trends down during pear development and has the highest expression in floral buds (Wu et al., 2014). Two members of miR398 family, miR398bc have shown to be upregulated after debagging of cultivar “Granny Smith” apple indicating light inducibility (Qu et al., 2016). miR398 canonical targets are Cu/Zn superoxide dismutase mRNA transcripts CSD1/CSD2, and interestingly CCS1, the Grain development and crop productivity: role of small RNA 419 copper chaperone for superoxide dismutase, which delivers copper to CSD1 and CSD2 apoproteins (Kruszka et al., 2012). miR398 has intriguing non-canonical slicing activities in Arabidopsis (Brousse et al., 2014) and grape (Sunitha et al., 2019): not only blue copper protein/plantacyanin transcripts with substantial loops/bulges in the essential “seed region” (Fahlgren et al., 2010) of miRNA target recognition (nt 2e13), but also large atypical bulges in grape CCS1 and novel peptide methionine sulfoxide reductase (Sunitha et al., 2019) transcripts. Such disparate redox enzyme targets may be missing links (Lee et al., 2014) that point to convergent evolution of miR398 to target another non-canonical gene transcript involved in redox homeostasis: cytochrome c oxidaseVb (Kruszka et al., 2012; Sunitha et al., 2019; Sunkar & Zhu, 2004). The structure-function aspects of miR398 non-canonical slicing activities in plants warrants further study. Broader conservation, e.g. in a rosid genus like Carya (hickory (Z. Sun et al., 2020), pecan) and rice (see below) could establish a paradigm for understanding convergent evolution of miRNA targets important for stress adaptations, in distinction from convergent evolution by different miRNAs targeting TFs that control hormone signaling (Allen et al., 2005) and flowering/phenology (Cartolano et al., 2007; Gyula et al., 2018). Qu et al. (2016) claimed without evidence HBCT (anthranilate Nhydroxycinnamoyl and benzoyltransferase) gene is target of miR164. Strawberry miR164 negatively regulates NACs and plays a critical role in delaying fruit senescence during low temperature storage. Zheng et al. (2019) cloned an ethyl methanesulfonate-induced mutant gene, deeply serrated (des), in the woodland strawberry Fragaria vesca that has wrinkled leaves with deeper serrations, serrated petals, and deformed carpels. Authors showed the causative mutation in fve-MIR164a mature sequence and that OE of FveMIR164A rescued the phenotypes of des/fvemir164a mutant, concluding miR164a functions in specification of leaf and floral organ morphology via the posttranscriptional regulation of CUP SHAPED COTYLEDON2 (Zheng et al., 2019). Three NACs are negatively modulated by miRNA164 during strawberry fruit senescence when they accumulate anti-concordant with miR164 decrease (Xu et al., 2013). OE of FveMIR390a delays flowering in woodland strawberry by negatively regulating ARF4 through miR390-TAS3-ARF4 pathway, and miR390 is induced in strawberry by exogenous application of hormones GA, methyl jasmonate, and auxin (Dong et al., 2022). In viticulture GA is used as a spray treatment to maximize cluster expansion during fruit growth. miR159 is a prominently expressed miRNA 420 Plant Small RNA in Food Crops in early fruit development and is involved in control of flowering time by negative regulation of GAMYB TFs during transition from vegetative to reproductive phase (Tang & Chu, 2017). Degradome analysis validated NOZZLE/SPOROCYTELESS-like/VIT_19s0014g01700, MYB65/ VIT_13s0067g01630, and MYB101/VIT_19s0090g00590 (Pagliarani et al., 2017) as targets of miR159 in grapevine (Pantaleo et al., 2010; Sunitha et al., 2019), similarly to citrus (Wu et al., 2016) and Arabidopsis where SPOROCYTELESS is a non-canonical miR159 target implicated in male fertility (Alves-Junior et al., 2009). The GAMYB TFs work in apposition with abscisic acid (ABA) stress hormone signaling processes to promote seed germination (Reyes & Chua, 2007) and bud dormancy in apple (Garighan et al., 2021), but in grape ABA promotes berry ripening and miR159 modulates floral development and GA-induced parthenocarpy (C. Wang et al., 2018). miR159 is differentially expressed in both “Kyoho” and “Fengzao” grape berries, an early ripening bud mutant of control Kyoho (Guo et al., 2018). In “Nanguo” pear, miR159 and some other miRNAs such as miR160, miR395, miR399, miR535 show a negative correlation of their expression to cognate target gene expressions, which was hypothesized to control aroma weakening of fruits upon storage under cold conditions (Shi et al., 2019). miR169 is differentially up-regulated during dormancy induction and down-regulated during the dormancy release periods in Japanese apricot (Prunus mume), and its target NF-YA synergizes with GA signal transduction suppressor PmRGA-LIKE2 to activate dormancy release induced by GA4 (Gao et al., 2021). Similar to miR159, miR319 is also a GA-responsive miRNA that targets some TCP TFs involved in seed and berry development. miR396 is also GA-responsive in grape (J. Han et al., 2014), where it targets GRFs but also two non-canonical targets Vv-bZIP05/VIT_02s0012g02250 and a coiled coil protein gene VIT_07s0191g00220 (Sunitha et al., 2019). Anticoncordant miRNA:target correlated expression profiles in berries support these miRNAs acting as negative regulators during seed and berry development (W. Wang et al., 2020). TCP4 targeted by miR319 in apple may be important for the browning inhibition by H2S treatment of fresh-cut apples mediated through reactive oxygen species, phenylpropanoid, and lipid metabolism pathways (Chen et al., 2020). miR477 is another GAresponsive miRNA in grape claimed to target VvGA-INSENSITVE1/ GAI1, a DELLA-motif transcriptional repressor subject to ubiquitinmediated targeted proteolysis highly expressed in green berries to maintain the balance of GA content (W. Wang et al., 2020). Furthermore, Grain development and crop productivity: role of small RNA 421 miR477b-3p has been reported to have high expression in “Fengzao” grape mutant (Guo et al., 2018), supporting possible involvement in ripening. miR477 targets GRAS family TFs in grape (Chitarra et al., 2018), one of them being VvGAI1 (W. Wang et al., 2020). A recent report claimed miR477 in mulberry (Morus atropurpurea) may function to target an antisense lncRNA-ABCB19AS which acts in fruits as a positive regulator of anthocyanin accumulation (Dong et al., 2021). GRAS TFs are known repressors of differentiation of axillary meristems and canonical targets of miR171 and contribute to disease resistance and abiotic stress responses (Grimplet et al., 2016). Interestingly, miR159 acts as a ripening regulator by targeting FaGAMYB, which plays crucial roles during the transition of strawberry receptacle from development to ripening (Csukasi et al., 2012). It is noteworthy that miR395, which targets ATP Sulfurylase 1/APS1 and Low affinity sulfate transporter/AST68 (Sunitha et al., 2019), increases at veraison and declines when grape berries reach the maturation phase, signifying sulfur metabolism is important during ripening (Belli Kullan et al., 2015). Consistent with this notion, flavonoids and derived condensed tannins and stilbenes accumulate under sulfur-deprived conditions (Tavares et al., 2013). Elevated expression of miR395 at veraison may create a sulfurlimited environment by downregulating sulfate transporters (Guillaumie et al., 2011) in mature berries leading to accumulation of phenolic compounds. miR395a-i are light-induced upon debagging of cultivar “Granny Smith” apple. Moreover, the expressions of miR395d-3p and a speculated target MdWRKY26 TF were anti-concordantly expressed, respectively, by 1-methylcyclopropene (an ethylene inhibitor) treatment during postharvest storage (M.M. Wang et al., 2021), supporting a plausible functional role for this non-canonical miR395 target, contingent upon degradome validation going forward. Fruit development and ripening typically results in attractive red color formation by anthocyanins. A few MYB TFs, targets of TAS4-30 D4(-) tasiRNA, are known regulators of fruit development. VvMYBA6 and A7 TFs in grape are involved in bioflavonoid synthesis including anthocyanin production in vegetative and fruit tissues of grape and are under a tight control of miR828/TAS4 auto regulatory loop (Rock, 2013). These MYBs and their upstream regulator, HYPOCOTYL5/HY5 accumulate during early berry development upon exposure to UV-B radiation, supporting a role berries in response to radiation stress (Sunitha et al., 2019). miR828 also directly targets numerous other MYBs, like MYB114/ VIT_09s0002g01380/PAL3 affecting proanthocyanidin and flavonol 422 Plant Small RNA in Food Crops biosynthesis in grapevine (Sunitha et al., 2019; Tirumalai et al., 2019). Similar to grape, miR828-triggered TAS4 targets MYBs that regulate anthocyanin synthesis during apple fruit development (Xia et al., 2012). It was recently reported that up-regulation of miR3627 and miR4376 are associated with anthocyanin accumulation in grapevine berries (Owusu Adjei et al., 2021). An interesting role of miR172 in flavonoid biosynthesis was elucidated recently in apple by Ding et al. (2022) (Fig. 14.8) who demonstrated Figure 14.8 A simplified model for miR172-AP2 regulation of flavonoid biosynthesis. miR172 targets the mRNA of AP2-like genes to repress gene expression by inhibiting translation or initiating mRNA degradation. AP2s-like proteins enhance the expression of anthocyanin pathway genes (ANSs, DFR, UFTG and others) through binding the promotor of MYB10 to enhance its transcription and also binding MYB10 protein that is a known activator of anthocyanin biosynthesis. (From Ding T, Tomes S, Gleave AP, Zhang H, Dare AP, Plunkett B, Espley RV, Luo Z, Zhang R, Allan AC et al. (2022). MicroRNA172 targets APETALA2 to regulate flavonoid biosynthesis in apple (Malus domestica). Horticultural Research, 9:uhab007, doi:10.1093/hr/uhab1007.) Grain development and crop productivity: role of small RNA 423 MIR172 OE in transiently transformed tissues, and stably transformed transgenic Arabidopsis, show a reduction in red coloration (and flavonoids in Arabidopsis) via suppression of AP2-1a TF, and concordant reductions in MdMYB10 and its transcriptional targets. Enhanced petal anthocyanin accumulation was observed in transgenic tobacco MdAP2_1 OE lines, whereas in apple and cherry fruits transfected with a MdAP2_1a VIGS construct manifested reduced anthocyanin accumulation. Anthocyanin content and RNA abundances of anthocyanin biosynthetic genes could be partially restored by expressing a synonymous mutant of MdAP2_1a which had been engineered to lack the miR172 target sequence. MdAP2_1a bound directly to the promoter and protein sequences of MdMYB10 in yeast and tobacco to enhance MdMYB10 promotor activity. The authors conclude that miR172 negatively affects flavonoid and anthocyanin biosynthesis via antagonism of downstream transactivation of the promoter of MdMYB10, a positive regulator that interacts with AP2-1a to promote anthocyanin biosynthetic gene transcription (Ding et al., 2022). Interestingly, miR156 target SPL9 was shown in Arabidopsis to negatively regulate anthocyanin accumulation through destabilization of a PRODUCTION OF ANTHOCYANIN PIGMENT1/MYB75-bHLH/ TT8-WD40 transcriptional activation complex (Gou et al., 2011), whereas in grapevine repression of SPL2/9/10/16 positively regulates dihydroflavonol-4-reductase transcription and anthocyanin content (Cui et al., 2018). This is evident by distinct expression profiles of miR156 during different stages of fruit development, where miR156/535 family members are highly expressed and the expression of target SPLs are anti-concordantly decreased in mature fruits such as grape (Pantaleo et al., 2010, 2016). Regulation of SPL genes by miR535, a divergent branch of the miR156 family (Carra et al., 2009), may occur in the absence of miR156/157 expression or these miRNAs may interact for fine-tuning the expression of grapevine targets (Pagliarani et al., 2017). Up-regulation of miR535 may control low levels of SPLs, expected at berry maturation stages (Sunitha et al., 2019). A novel miRNA, miR73 was claimed to regulate strawberry fruit ripening via targeting ABA INSENSITIVE5 TF transcript to negatively affect ABA signaling involved in fruit ripening (D. Li et al., 2016). HERCULES1 (HERK1) encoding a receptor-like kinase, which likely contributes to strawberry fruit size determination, was claimed to be targeted by a novel miRNA, Fa_novel6 (D. Li et al., 2019). Additional evidence to substantiate novel miRNA functions in berry development are warranted and appropriate going forward. 424 Plant Small RNA in Food Crops Besides size/shape/colors of flowers, which Darwin speculated could account for elevated angiosperm evolutionary rates driven by coevolution with pollinators (Soltis et al., 2019), unique flavors are key qualities of fruit evolved by plants as enticements to attract animal seed dispersers. The sugars, organic acids and specific metabolite compositions determine fruit flavors. Flavonoids, stilbenes, and polyphenolics (tannins) are components of fruit that impart color, aromas, and flavor enhancers associated with purported nutraceutical benefits (e.g., antioxidant and anti-inflammatory properties) and in-mouth tactile sensations (astringency). In fully ripened strawberry fruits, a positive correlation was observed between soluble solids content and P content (Cao et al., 2015). miR399 targets PHOSPHATE OVERACCUMULATOR2 (PHO2) to regulate Pi homeostasis. Transgenic OE of MIR399a in woodland strawberry increased Pi levels and sugar content which substantially enhanced fruit flavor (Wang et al., 2017). A comprehensive analysis of pear miRNAome has identified nine miRNAs (miR1132, miR1318, miR2635, miR394a, miR396b, miR5077, miR5500, miR825* and miR952b) that may play a role in sugar and acid metabolism during the formation of fruit flavor (Wu et al., 2014). “Melting” and stony (hard) flesh fruit varieties of peach have distinct flavors. The up-regulation of miR171 in melting varieties and down-regulation in stony hard type flesh is claimed to attribute to the texture and flavor of peach (Kong et al., 2021). 8. miRNAs in monocots In monocot species like rice, maize, and wheat plant height and grain size are vital for yield, and miRNAs indeed play key roles in grain development and more broadly in shoot apex, the ultimate source of assimilate for grain filling (Ma et al., 2021). Advances in sequencing technologies have leveraged molecular genetic characterization of many agronomic traits known previously only from breeding as quantitative trait loci. OE of MIR156 in rice leads to reductions in secondary branching, grain number/panicle, and increase in tiller number (Xie et al., 2006). Similarly, OE of MIR156 in the bread wheat cultivar Kenong199 leads to severe defect in the spikelet formation and higher tiller number (Liu et al., 2017). Results further demonstrate that strigolactone signaling repressor DWARF53 (D53) physically interacts with the N-terminal domains of the miR156-regulated SPL3/17 proteins, revealing a potential association between the miR156SPL module and strigolactone branching hormones involved in wheat Grain development and crop productivity: role of small RNA 425 spikelet development and tillering. Two studies on semi-dominant quantitative trait loci, harboring point mutations- Ideal Plant Architecture1 (IPA1) and WEALTHY FARMER’S PANICLE (WFP), showed these mutants had perturbed miR156-directed regulation of SPL14 leading to the formation of an “ideal” plant with enhanced grain yield, lodging resistance, and reduced tiller number (Jiao et al., 2010; Miura et al., 2010). The miR156-SPL16 module has significant impact on panicle branching, endosperm cell division and grain width/yield and quality (low chalkiness) in rice, where a major semi-dominant quantitative trait locus GRAIN WIDTH8 that has a loss of function allele in Basmati rice cultivars was shown orthologous to SPL16 (Wang et al., 2012). mir156 insertion and deletion mutations generated by multiplex CRISPR-Cas9 enhanced seed dormancy by suppressing the GA pathway through de-represssion of IPA1, which directly regulates multiple GA pathway genes (Miao et al., 2019). Molecular marker-assisted selection of the Basmati quantitative trait locus qgw8 and Iranian gw8 Amol miR156-resistant haplotypes facilitated breeding of elite new indica varieties with substantially improved grain quality and high yields due to increased panicle branching. Among the 19 SPLs identified in rice, SPL14 promotes panicle branching and SPL13 and SPL16 regulates grain size and shape (Si et al., 2016; Wang et al., 2015). A recent report demonstrated the utility of CRISPR-Cas9 editing of all 19 SPL genes in rice which caused defects in panicle size, plant height, and grain length supporting important roles of the miR156-SPL module in plant architecture and grain size (M. Jiang et al., 2020). Moreover, OE of SPL7/8 in Panicum virgatum (switchgrass) promotes flowering whereas down-regulation using artificial miRNA and RNAi knockdown transgenes led to extra internodes, decreased panicle numbers, increased plant height and reversion of inflorescence/rachis meristems to vegetative shoot apical meristems. Taken together, the results demonstrate the miR156-SPL7/8 module regulates phase transition and flowering by directly up-regulating SEP3 and MADS32 (Gou et al., 2019). In Brachypodium distachyon, a T-DNA mutant of SPL9 (target of miR156) showed reduced spike length and growth. In addition to this, several SPLs had elevated expression during spikelet initiation which showed differential transcript abundance during spikelet development under high temperature stress (Tripathi et al., 2020), suggesting temperature sensitivity is subject to multi-layered regulation of SPLs in Brachypodium. Overall, miR156 expression increases linearly with the advancement of seed development in maize (Jin et al., 2015), rice (Peng et al., 2013, 2014), 426 Plant Small RNA in Food Crops wheat (Meng et al., 2013), and barley (Bai et al., 2017) with respective anticoncordant decreases in SPL expression supporting SPLs as key effectors of early seed development to promote width and number of grains. As grain development progresses the SPL expression decreases and lengthens the developmental window for grain filling (Jin et al., 2015). miR535 shows high sequence similarity to miR156 and miR529 in many species including monocots, however, it is less conserved than miR156 family members. Like miR156, miR535 targets SPL7/12/16 in rice and OE of MIR535 led to shorter panicles, fewer panicle branching and increase in grain length (not width) (M. Sun et al., 2019). Similarly, miR529 (found mostly in monocots but also gymnosperms, consistent with an ancient lineage from miR156) targets SPLs to control tiller, panicle architecture and grain size (Jeong et al., 2011; Yan et al., 2021). During banana fruit development, miR156 shows decreased expression with anti-concordant increased expression of target SPLs in fruits (Chai et al., 2015). PP2A-B (Protein phosphatase 2A B subunit) was predicted as non-canonical target of miR156 in banana, but authors did discount this speculation since its expression assayed by qRTPCR failed to follow anti-concordant patterns observed for miR156 induction in fruits in response to climacteric hormone ethylene or miR156 repression in response to ethylene inhibitor treatments (Bi et al., 2015). As described in above sections in general, increasing levels of miR172 promote the development of adult vegetative leaf and inflorescence meristem features, acting in opposition to miR156, which is abundant early in the lifecycle and acts to promote juvenile characteristics such as shoot and tiller production (Fornara & Coupland, 2009; Poethig, 2009). Mutations in miR172 target gene AP2-5 have significant effects on spike development and the “free-threshing” character associated with polymorphism in the pleiotropic Q allele of wild species of wheat, proposed to have been selected for during domestication (Debernardi et al., 2017). In Tasselseed6 dominant mutants of maize, null mutation in the IDS1 (INDETERMINATE SPIKELET 1), an ortholog of AP2-5, within the miR172/tasselseed4 target site lead to defects in floral meristems and lack of pistil abortion in the tassels (Chuck et al., 2007). SUPERNUMERARY BRACT is TDNA insertion mutant in a miR172 AP2-like target in rice (Zhu et al., 2009). In barley cultivated varieties, the HvAP2-2L alleles have impaired miR172-mediated repression resulting in compact spikes and cleistogamous flowers (do not open and thus self-pollinate) (Houston et al., 2013; Nair et al., 2010). These authors proposed the miR172-AP2 module controls timing of elongation of the internodes along the axis of the spike affecting Grain development and crop productivity: role of small RNA 427 morphological diversity and grain yields. The findings shed light on how two-row and six-row barley have arisen independently three times during domestication as HvAP2 haplotypes, and suggest the miR172-AP2 module may be important for grain quality in temperate Triticaceae tribe members wheat and potentially rye (Secale cereale) (G. Li et al., 2021). In maize kernels miR156 peaks in expression at 7 DAP pollination, whereas miR172 has the lowest expression at this stage when endosperm completes cell differentiation at the transition from juvenile to seed maturation phase of protein and starch accumulation (M. Xin et al., 2015). Transgenic rice expressing STTM-miR172 had a novel phenotype of defective culms (jointed stems) resulting in enclosed panicles (H. Zhang et al., 2017). This study demonstrated the utility and potential of STTMs for creating novel breeding haplotypes and revealing cryptic functions of long-studied miRNAs, where phenotypes are likely masked by genetic redundancy inherent in miRNA modules and allopolyploid crops. Barley embryos (source of malt for beer and whisky) are a classic model for study of GA and ABA hormone interactions that respectively promote or antagonize signaling pathways for germination as alternate fates of seeds maturing to a quiescent state (Finkelstein et al., 2002). miR159 is expressed in embryos and restricted expression to that tissue allows expression of target MYBs in the endosperm to program expression of target starch hydrolytic enzymes, followed by programmed cell death (Huang et al., 2013). Both these processes are associated with GA action and within the embryo, miR159 is specifically expressed in hypocotyl and cotyledons in barley suppressing GA processes to allow ABA-associated maturation to be maximized. GA antagonizes ABA and promotes germination and release of ABA-mediated seed dormancy, suggesting that miR159 restricts GAspecific effects in the embryo. miR319 is evolutionarily related to miR159 and also targets GAMYBs in banana fruits as key regulator of anther and stamen development (Chai et al., 2015). Similar to prior-mentioned studies in tomato (Damodharan et al., 2016), in date palm Phoenix dactylifera L. miR160 targets five ARFs expressed at low levels during fruit development (C. Xin et al., 2015). It is intriguing that some miRNAs typically found in dicot clades (Taylor et al., 2014): miR391/miR5225/4376, miR828, and miR1507 are apparently extant in date palms (Patel et al., 2021; C. Xin et al., 2015) and other basal monocots like lilies (Trillium camshatensese) (Rock, 2013), which raises questions about evolutionary forces potentially shaping development of monocots with fleshy fruits (Rock, 2020). In rice grains, miR160 has low 428 Plant Small RNA in Food Crops expression leading to accumulation of target ARFs during the grain filling stage (Yi et al., 2013). Expressing miR160-resistant transgene version of ARF18 leads to lesser and smaller seeds where starch accumulation is significantly affected, suggesting miR160 is essential for seed development in rice (Huang et al., 2016). ARF6 and ARF8 are targeted by miR167, which is preferentially expressed in rice seeds (Xue et al., 2009). OsGH3.2, a rice indole-3-acetic acid (IAA)-conjugating enzyme, is positively regulated by miR167, which in turn regulates the cellular-free levels of auxin IAA (Yang et al., 2006). Similar to B. napus (Wang et al., 2014), rice seeds are rich in miR167 abundance suggesting a role of miR167 and OsGH3.2 in regulating the auxin levels during seed development in rice. A recent study showed OsMIR167a OE lines, and T-DNA insertion mutants of target ARFs osarf12/osarf17 and osarf12/osarf25, showed larger tiller angle phenotypes with disrupted auxin distribution in axillary buds (Li et al., 2020). In wheat grains, miR164 targets NAC TFs to regulate senescence and nitrogen re-mobilization, and improve zinc, iron, and protein content in the grains (Uauy et al., 2006; Zuluaga & Sonnante, 2019). The expression of miR164 increases during wheat grain development (R. Han et al., 2014) and decreases during the later stages in maize when targets increase anticoncordantly (D. Li et al., 2016). OE of an engineered NAC2 silent allele resistant to miR164-mediated AGO slicing led to plants having better architecture, longer panicles, and more grains suggesting NAC2 is critical for grain number determination (Jiang et al., 2018). Recent work in maize demonstrated anti-concordant expression evidence for a non-canonical miR164 target ABCG26 transporter shown by CRISPR-Cas9 editing to be essential for pollen viability and anther wall and cuticle lipid deposition (Y. Jiang et al., 2021). miR168 has been reported to be the most abundant miRNA in monocot plants like rice, barley (Puchta et al., 2021), wheat (Y.F. Li et al., 2019), and Brachypodium distachyon, but not the ayurvedic medicinal ginger, Elettaria cardamomum (Nadiya et al., 2019) whereas in barley seeds miR168 is expressed at low levels in the endosperm. High levels of miR168 in monocots are not due to a large portion of endosperm present in the seeds, but possibly due to the significant regulatory differences between the dicots and the monocots. Monocots have a greater number of AGO family members as compared to the dicots, functions of which are yet poorly understood. OsAGO18, a member of a new AGO clade that is conserved in monocots, was confirmed to be a target of miR168a by cell-based Grain development and crop productivity: role of small RNA 429 cleavage assay. Characterization of CRISPR-Cas9 edited OsMIR168a knockout mutants manifested pleiotropic phenotypes of more tillers, early maturation, and smaller spikelets and seeds (Zhou et al., 2022). In maize AGO18b may function in regulation of spikelet meristems by primarily binding to 21-nt PHAS-RNAs/miRNAs with a 50 -uridine (W. Sun et al., 2019). In rice AGO17 is expressed in reproductive tissue and the OE of OsAGO17 in rice transgenics led to higher yield and robust growth (Pachamuthu et al., 2021). Silencing AGO17 in rice resulted in reduced fertility, panicle length and poor growth suggesting OsAGO17 plays an essential role in reproductive development, knowledge that could lead to cogent strategies to engineer grain yields in rice and grain crops. In maize miR159, miR164, miR166, miR171, miR390, miR393 and miR529 accumulate at high levels during the early stages of grain development between four and six DAP (D. Li et al., 2016). In wheat seeds, the abundance of miR169 decreases across seed development from five-day old seed to 20-day old seed, suggesting it and NF-YA targets play a role in seed development (R. Han et al., 2014). Similarly in maize kernels nine different miR169 family members had dynamic expression profiles in embryo, endosperm, placenta, pedicel, and basal embryo transfer layer, supporting roles for NF-YA targets in kernel development and nutrient transport (Xing et al., 2017). As documented in above sections, the best-defined miRNA roles in regulating phase changes in the shoot meristem of dicots and monocots are miR156 and miR172 modules, with differences observed across species for miR169 modules impacting flower development distinctly in asterids and rosids. It is plausible that miR169-NF-YA modules may have evolved a distinct node impacting flower and grain development in monocots. Interestingly in this context it was shown OE in barley floral meristems of MIR171, which targets SCARECROW-LIKE GRAS TFs, manifests pleiotropic phenotypes of branching defects, increased vegetative phytomers, and late flowering by down-regulating at least one target HvSCL/ GRAS proposed to act upstream of the miR156-SPL module (Curaba et al., 2013). miR390 triggers the production of TAS3 siRNAs which in turn leads to tasiRNA-programed AGO slicing of ARF3/4 TFs controlling leaf polarity specification, gynoecium patterning/morphogenesis, self-incompatibility, and cytokinin signaling (Zhang et al., 2018). M. Xin et al. (2015) suggested miR390-TAS3-ARF3/4 module may play a role in endosperm maturation as miR390-TAS3 are highly expressed in maize kernels up to five DAP but 430 Plant Small RNA in Food Crops its expression is barely detectable in endosperm at 7e15 DAP, when ARF3 was speculated to promote precocious entrance of the maize kernel to the adult stage. A recent study in rice shows that tasi-ARF-mediated target cleavage primarily occurs on polysomes. The authors also identified two additional targets of tasi-ARFs which are involved in heavy metal response along with several TAS3-derived phasiRNAs which might act in trans to direct cleavage of several targets, especially on polysomes (Luo et al., 2021). Reproductive 21 nt lnc PHAS transcripts are bound by polysomes, and high-frequency cleavage of 21 nt PHAS precursors by miR2118 and 24 nt PHAS precursors by miR2275 is associated with membrane-bound polysomes in maize and rice (Yang & You et al., 2021). The biogenesis of 21-nt reproductive phasiRNAs in maize is largely dependent on the HD-ZIPIV TF OUTER CELL LAYER4 by production of PHAS precursor transcripts, expression of miR2118 that modulates PHAS processing, and accumulation of 21-nt phasiRNAs (Yadava et al., 2021). A recent study in rice (Yang & Zhao et al., 2021) showed through STTM sequestering of miR396 that grain size, tillers, and high panicle density were modulated indirectly via miR408, an embryo-specific miRNA which positively regulates grain size and photosynthesis through its cognate target PHYTOCYANIN (J.P. Zhang et al., 2017). CRISPRCas9 engineered mutations in MIR408 led to smaller grain which could be complemented by large grain phenotypes in STTM-miR396 transgenic rice, also phenocopied by OE of miR396 target OsGRF8 (Yang & Zhao et al., 2021) suggesting miR396 regulates grain size by regulation of embryo-specific miR408 expression. In maize grains, miR396 expression increased initially and decreased during grain filling when GRF targets anticoncordantly increased (Jin et al., 2015; D. Li et al., 2016; Zhang et al., 2015); similar results have been reported for rice (He et al., 2017) and wheat (R. Han et al., 2014; Li et al., 2015; Meng et al., 2013; Sun et al., 2014; Yu et al., 2021). In orchard grass (Dactylis glomerata L.), miR396 was shown to participate in vernalization by regulating plant morphogenesis, transmembrane transport and plant hormones (Feng et al., 2018). In sugarcane (Saccharum officinarum), during the ripening period miR396 target GRFs regulate the process of leaf shedding via plant pathogen interaction and plant hormone signaling (Li et al., 2017). miR397 is expressed in high levels in young grains and panicles, and OE studies demonstrated a positive effect grain numbers and seed size in rice by targeting LAC, whose laccase-like product is involved in sensitivity of plants to brassinosteroid hormones (Zhang et al., 2013). miR397 is up-regulated Grain development and crop productivity: role of small RNA 431 by moderate drought, leading to a decrease in target OsLAC and grain filling of inferior spikelets (Teng et al., 2022). miR397 enhances brassinosteroid signaling (but not accumulation) and cell division in endosperm indirectly by downregulating target LACs associated with increased grain size, more rice panicle branching, and higher grain productivity. Modulation of miR397 could improve grain yields in not only monocots like sorghum and switchgrass, but possibly also dicots. However, a study in maize showed that expression of miR397 is maintained at low level during grain filling when the relative abundance of the a candidate oxidoreductase LAC increases slowly, suggesting miR397 in corn might function in early reproductive stages, however later LAC expression could function to supply energy or adapt seeds to desiccation stress during grain filling (Jin et al., 2015). OE of copper deficiency-induced MIR397 in banana resulted in enhanced biomass (Patel et al., 2019). Copper homeostasis is vital for proper development as it regulates cell wall metabolism, ethylene perception, photosynthetic/respiratory electron transport, and protection from oxidative stress. miR408 in rice targets UCL8/uclacyanin which is localized to the cytoplasm, unlike other plastocyanins, and affects photosynthesis, abundance of plastocyanins, and grain yield (J.P. Zhang et al., 2017). Consistent with this finding, Pan et al. (2018) found OE of MIR408 in Arabidopsis, tobacco, and rice led to enhanced vegetative growth and enlarged seeds. In wheat and possibly barley, miR408 has a non-canonical target TIMING OF CAB EXPRESSION1/ TOC1 which regulates flowering time; OE of MIR408 or transgenic RNA interference knockdown of TaTOC1s resulted in wheat plants heading approximately one week earlier due to down regulation of TaTOC-A1/B1/-D1 (Zhao et al., 2016). Optimal anthesis timing is vital for wheat cultivation to avoid high temperatures during development. Genetic manipulation of miR408 could facilitate control of heading timing in Triticaceae. On the other hand, wheat miR408 has been claimed to target three non-canonical genes (in addition to canonical copper-binding targets) that encode proteins involved in microtubule organization, protein-protein interaction, and cyclic-AMP signal transduction on the basis targets showed anti-concordant expressions in response to elevated miR408 expression by Pi starvation and salt stress (Bai et al., 2018). Degradome analyses to validate these claims are suggested. miR398, which targets canonical genes Cu/Zn superoxide dismutase2/ Os07g46990 and CCS/Os04g48410 (Li et al., 2010) also regulates panicle length, grain size and grain number in rice. Silencing MIR398 in rice plants 432 Plant Small RNA in Food Crops led to reduction in grain number, grain length/number and smaller panicles (H. Zhang et al., 2017). Another monocot-specific miRNA which may modulate grain yield in rice is miR1432, which was claimed to target AcylCoA thioesterase involved in biosynthesis of medium-chain fatty acids (Zhao et al., 2019). Free fatty acid molecules make membranes more fluid to facilitate membrane vesicle-transport and hormone signaling. Suppression of miR1432 led to significant improvement in grain weight, leading authors to suggest miR1432 might regulate grain filling through regulating auxin transport and ABA signaling to enhance sucrose to starch metabolism (Zhao et al., 2019). In rice, miR530 is a negative regulator of rice grain yield as it affects cell expansion and division in spikelet hulls by targeting PL3 or PLUS3 containing domain, also potentially a conserved target in grapevine VIT_05s0020g04860 based on degradome evidence (Sunitha et al., 2019). OE of MIR530 in rice and OsPL3 down regulation by CRISPR-Cas9 editing decreased the size of the seeds and inhibited panicle branching, whereas blocking osa-miR530 by STTM expression increased grain yield (W. Sun et al., 2020). miR530 plays a vital role in rice grain yield where its expression is activated when TF phytochrome-interacting factor-like15/ PIL15 accumulates and binds to the G-box element in the MIR530 promoter. Increased expression of miR530 suppresses panicle branching and seed enlargement by downregulating its target PL3 leading to substantial loss of yield (W. Sun et al., 2020). The miR530-PIL15 module is a promising new target for breeding high-yielding rice. Auxin receptors TRANSPORT INHIBITOR-RESISTANT1/TIR1 and AFB2 are targeted by miR393. In rice this module regulates coleoptile elongation and stomatal development via modulation of auxin signaling during seed germination and seedling establishment under water submergence, providing perspective on direct sowing of rice seeds in flooded paddy fields (Guo et al., 2016). A monocot-specific miR444 targets TF OsMADS57 which subsequently negatively regulates the expression of DWARF14 to regulate outgrowth of axillary buds in rice. An activationtagged mutant osmads57-1 and OsMADS57-OE lines showed increased tillers, whereas OsMADS57 antisense lines had fewer tillers. OE of MIR444 resulted in reduced transcript levels of OsMADS57 and reduced tillering. OsMADS57 interaction with TCP domain TFTEOSINTE BRANCHED1reduces the repression of DWARF14 by OsMADS57, leading to the balanced expression of DWARF14 for tillering (Guo et al., 2013). Grain development and crop productivity: role of small RNA 433 Epigenetic modification is essential for germ cell development not only in mammals, but also plants. A study of maize developing pollen identified several novel miRNAs whose targets were related to chromatin assembly and disassembly. In addition to this, targets of miR820 and miR827 were also confirmed to regulate genome methylation by targeting DNA cytosine methyltransferase and methyl-CpG binding domain genes, respectively, suggesting miRNAs can modulate the epigenetic regulation of chromatin involved in rice pollen development (Wei et al., 2011). Long noncoding RNAs (lncRNA) are a diverse class of transcripts emerging as players in post-transcriptional regulation including fruit ripening and yield (Q. Chen et al., 2021; Y. Chen et al., 2021; Di Marsico et al., 2022; Meng et al., 2021; J. Wang et al., 2021; Zhao et al., 2020). It is recently shown that Panicum virgatum (switchgrass, a perennial species) has several INDUCED BY PHOSPHATE STARVATION1 (IPS1)-like lncRNAs that act as endogenous target mimics of miR399 by competing for binding to multiple canonical targets in the 50 -UTR regions of UBC24/ PHO2 homologs and PHO84 Pi transporter coding sequences. The endogenous target mimic IPS1s have a critical, central 3-nt mismatched loop that allows binding to miR399 but prevents miR399-guided lncRNA cleavage. In rice, two RING-finger ubiquitin E3 ligases SDEL1 and SDEL2 regulate the degradation of SPX4, a Pi sensor that transactivates IPS1, MIR399, and MIR827 promoters, for Pi homeostasis (Ruan et al., 2019). Like AtIPS1 in annual Arabidopsis, the three switchgrass IPS1-like transcripts are also highly induced under P-deficiency stress in shoot and root (Ding et al., 2021). A dominant repressor, Iw1 (INHIBITOR of WAX1), was identified as a recently evolved MIRNA gene which targets WAX1CARBOXYLESTERASE (D. Huang et al., 2017). Interestingly, this MIRNA gene arose as an inverted duplication of its target gene suggesting a vital role for this MIRNA regulating an agronomically important trait related to stress tolerance. A study in rice identified 21-mer phased clusters preferentially expressed in inflorescence, flanked by 22-nt motifs which was offset by 12 nts from the main phase. The authors identified a new miRNA family conserved across maize and rice specifically expressed in developing reproductive tissues suggesting these novel sRNAs play a significant role in monocot reproductive development. Another recent study claimed OE of a novel rice-specific MIR5506 causes pleiotropic abnormalities of the palea (upper bracts), various numbers of floral organs, and spikelet indeterminacy (Z. Chen et al., 2021). 434 Plant Small RNA in Food Crops 9. Conclusions and future prospects The advent of various genomic tools and sequencing technologies over the past two decades has revealed the complexity of organisms and at the same time the largely conserved nature of the plant sRNA World. This review touches two different aspects of sRNAs (mostly miRNA) significance as relates to crops. Most miRNA research to date has primarily focused on the spatiotemporal expression profiles of miRNAs and their targets in different plant tissues to frame the complexity of miRNA-mediated developmental programs. miRNAs represent nodes for regulatory networks mediating myriad aspects of crop yields; these modules interact with and form hierarchical layers with other miRNA/sRNAs and signaling pathways like phytohormones to integrate temporal/environmental modulation of growth and development. First aspect touches upon the challenges of sRNA research, which includes mis-annotation of miRNAs, lack of functional evidence for novel miRNA activities, lack of experimental evidence for novel/non-canonical miRNA-target interactions, and the extent to which plant miRNAs can act at other levels of the molecular dogma (transcriptional and translational) other than at the post-transcriptional level. These limitations of sRNA research have made it difficult to appreciate the complexity and practical potential of the RNA World for a better integration of systems biology toward crop improvement. Given that miRNAs are integral to plant growth and development, it must be considered when translating basic knowledge to crop improvement there may be undesirable pleiotropic side effects when the expression of miRNAs are manipulated. The second aspect is focused on the roles of miRNAs in fruit development and other physiological features directly or indirectly linked to the fruit as sink for assimilate from vegetative organs. From the evolutionary perspective and evidences that show ancient plant miRNA functions and mechanisms of action for cell fate and cellular homeostasis are largely conserved, the question arises: how accurate or justified are predictions based on orthology of miRNAs across model organisms to translate to similar processes in crops, versus speciation and neo-functionalization of miRNAs? Table 14.1 summarizes knowledge gained in crops for the evolutionarily conserved miRNAs. These higher confidence miRNAtarget modules can serve as foundation for cogent strategies to deploy emerging genetic engineering and breeding tools applied to and drawn from testable crop models (Fig. 14.3e14.8) to impact crop productivity. In the future, research challenges are to address the functional relevance of Table 14.1 Representative functional miRNAs characterized as affecting crop yields. miRNA/lncRNA miR156-7/535/529 Agronomic trait affected Plant species Target gene family 1. Fruit development 2. Timing of flowering 3. Seed development and maturation 4. Yield and architecture of the plant; ovary and fruit development; fruit ripening 5. Gynoecium patterning; anthocyanin accumulation 6. Tuber formation 1. Citurs 2. Arabidopsis SPLs Validated target? Transgenic evidence? References/Figs 2. SPL3 1. NA 2. NA Fig. 7; 1,2: Wu et al. (2016) 3. NA 3: Zhao et al. (2012) 4. Yes (RNAi suppression of SPL13 and overexpression of miR156a); OE of MIR156b 5. Yes (spl8 mutant and OE of miR156); OE of miR156 4: Cui et al. (2020), Karlova et al. (2013) 6: Bhogale et al. (2013) 7: Lopez-Ortiz et al. (2021) 8: Carbone et al. (2019) 9: Cui et al. (2018), Belli et al. (2015), Su et al. (2021) 3. B. napus 4. Tomato 4. SPL13; COLORLESS NONRIPENING/CNR (miR156/157) 5. Arabidopsis 5. SPL8;SPL9 6. Potato 6. SPL9 6. OE of MIR156 7. Fruit devt.(miR156/157) 8. Regulation of color transition in olive 9. Berry devt.; anthocyanin biosynthesis 7. Capsicum 7. CA03g12170 7. NA 10. Fruit development 11. Secondary branching, grain number/panicle, and increase in tiller number; panicle size, plant height, and grain length 10. Peach 11. .Rice 8. Cassanese cv 9. Grape 8. NA 9. SPL9 11. SPL16; All 19 SPLs 9. NA; yes (vvmiR156b/c/d and VvSPL9 overexpression) 10. NA 11. Yes (OE of MIR156); CRISPR 5: Xing et al. (2013), Gou et al. (2011) 10: Shi et al. (2017) 11: Xie et al. (2006), Wang et al. (2012) Continued Table 14.1 Representative functional miRNAs characterized as affecting crop yields.dcont'd miRNA/lncRNA miR159/319 Agronomic trait affected Validated target? Plant species Transgenic evidence? References/Figs Target gene family 12. Tiller and spikelet formation 12. Bread wheat 12. SPL3/17 12. Yes (OE of MIR156) 12: Liu et al. (2017) 13. Flowering, panicle numbers, increased plant height 13. Switchgrass 13. SPL7/8 13. OE of SPL7/8; down-regulation using artificial miRNA and RNAi knockdown transgenes 13: M. Jiang et al. (2020) miR535 14. Aroma weakening 14. Pear 14. NA 14: Shi et al. (2019) 15. Berry maturation 15. Grape 15. NA 16. Panicle branching and grain length miR529 17. Tiller, panicle architecture and grain size 1. Fruits development; 16. Rice 14. 9 s-lipoxygenase (LOX2S) 15. SPL12/ GSVIVT00017032001 16. SPL7/12/16 16. Yes (OE of MIR535) 15: Sunitha et al. (2019) 16: M. Sun et al. (2019) 17. OsSPL2, OsSPL17 and OsSPL18 17. Yes (miR529aMIMIC) 17: Yan et al. (2021), Jeong et al. (2011) 1. NOZZLE/ ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motif, LRR Cs8g05120/ orange1.1g016534 m; 1. NA Figs. 3 and 5; 1: Wu et al. (2016) 2. NA 2: Jiang et al. (2014), Huang et al. (2013) 3: J. Jiang et al. (2021) 2. Flower buds, seed development; 3. Flower buds, seed fertility, silique development; 17. Rice 1. Citrus; 2. Brassica campestris; 3. Brasscia napus GA-MYB TFs; Teosinte branched 1Cycloidea 1-Proliferating cell nuclear antigen (TCP) domain TFs Teosinte branched 1Cycloidea 1-Proliferating cell nuclear antigen (TCP) domain TFs 3. Dominant genic male sterile (DGMS) and recessive genic male sterile (RGMS) lines and OE lines (Yes) 4. Silique length 4. B. napus 5. Ovule/ovary development 6. Fruit size 7. Flowering and fruit development 8. Ripening stages in drupe epi-mesocarp tissue stage 9,10. Male fertility 5. Tomato 4. Long silique and short silique lines (No) 5. OE SlmiR159 lines (Yes) 6. Tomato 7. Capsisum 8. Olive (Olea europaea L.) 9. Grapevine 10. Arabidopsis 11. Floral development and GAinduced parthenocarpy 11,12. Grape, Kyoho’ and ‘Fengzao’ cv. 13. Aroma weakening of fruits upon storage under cold conditions 14. Transition of receptacle from development to ripening 15. Seed germination/ maturation 13. ‘Nanguo’ pear 16. Grain development 9. NOZZLE/ SPOROCYTELESSlike/ VIT_19s0014g01700, MYB65/ VIT_13s0067g01630, and MYB101/ VIT_19s0090g00590 10. SPOROCYTELESS 4: Chen et al. (2018) 5: da Silva et al. (2017) 6: Zhao et al. (2021) 7: Lopez-Ortiz et al. (2021) 8: Carbone et al. (2019) 9: Pagliarani et al. (2017), Pantaleo et al. (2010), Sunitha et al. (2019) 10: Alves-Junior et al. (2009) 11: C. Wang et al. (2018), Ruvkun (2001), Guo et al. (2018) 13: Shi et al. (2019) 14. Strawberry 14: Csukasi et al. (2012) 15. Barley 15: Finkelstein et al. (2002) 16. Maize 16: D. Li et al. (2016) Continued Table 14.1 Representative functional miRNAs characterized as affecting crop yields.dcont'd miRNA/lncRNA Agronomic trait affected miR160 17. Proper absorption of water and nutrients during B-toxicity conditions 18. Browning inhibition by H2S treatment of fresh-cut apples mediated through reactive oxygen species, phenylpropanoid, and lipid metabolism pathways 19. Regulator of anther and stamen development 1. Pollen development Validated target? Plant species Transgenic evidence? References/Figs Target gene family miR319 2. Silique length 17. C. sinensis (sweet orange) and C. grandis (pummelo) 17: Huang et al. (2019) 18. Apple 18: Chen et al. (2020) 19. Banana 19: Chai et al. (2015) 1. Brassica campestris ssp. chinensis 2. Brassica napus 3. Seed development 4. Ovary patterning, lamina expansion, and floral organ abscission 3. Brassica napus 4. Tomato 5. Flowering and fruit development 6. Aroma 7. Fruit development 8. Seed development 5. Capsicum 6. Grape 7. Date palm 8. Rice Auxin Response Factor (ARF) 2. ARF10, ARF16, and ARF17 2. MIR160 OE 4. SlARF10A/B, -16A/B, and -17 4. Degradome evidence, knockdown of miR160 using an STTM160 transgene 8. ARF18 8. miR160-resistant transgene version of ARF18 Fig. 3; 1. Jiang et al. (2014) 2. Chen et al. (2018) 3. Huang et al. (2013) 4. Karlova et al. (2013), Damodharan et al. (2016) 5. Lopez-Ortiz et al. (2021) 6. Guo et al. (2018) 7. C. Xin et al. (2015) 8. Yi et al. (2013) miR164 miR165/166 1. Fruit ripening 1. Sweet orange 2. CMM maintenance; timing of fruit devt (CUC1) 3. Reproductive development; fruit growth; leaflet boundaries (GOB); organ boundaries in floral meristems (GOB); floral boundaries (SINMA2); timing of fruit devt (GOB) 4. Fruit devt 2. Arabidopsis 2. CUC1 and CUC2 3. Tomato 3. GOBLET/GOB; SlNAM2 and SlNAM3 3. Yes (OE of MIR164a); slmir164bCR and STTM; GOB loss-ofand gain-of function mutation/OE of miR164; MIR164 OE; OP:MIR164 4. Capsicum 4. CA06g18770 4. NA 5. Kiwifruit 6. Apple 5. NAC6/7 6. HBCT 5. Yes (STTM164) 7. Woodland strawberry Fragaria vesca 1. Citrus 7. CUC1;NAC87,38 7. Yes (overexpressing FveMIR164A); NA 5. Fruit ripening 6. Protection against high light stress 7. Leaf and floral organ morphology; fruit senescence 1. Fruits development; 1. NAC TF 1. Cs5g10870 HD-ZIP-IIIs/ATHB TFs 2. Brasscia napus 3. Seed development 4. Development of ovules and consequent parthenocarpy 3. Brasscia napus 4. Tomato 1. Transient expression assay 2. NA; OE of miR164 2. Dominant genic male sterile (DGMS) and recessive genic male sterile (RGMS) lines and OE lines (Yes) 4. PARTHENOCARPIC FRUIT 1/SlHB15A/PF1 4. miRNA166resistant SlHB15a (Yes) Fig. 4; 1: Liu et al. (2014) 2: Kamiuchi et al. (2014), Rosas Cárdenas et al. (2017) 3: Silva et al. (2014), Gupta et al. (2021), Berger et al. (2009), Hendelman et al. (2013), Rosas Cárdenas et al. (2017) 4: Lopez-Ortiz et al. (2021) 5: Gupta et al. (2021) 6 Qu et al. (2016) 7: Zheng et al. (2019), Xu et al. (2013) Fig. 6; 1: Wu et al. (2016) 2: J. Jiang et al. (2021) 3: Huang et al. (2013) 4: Clepet et al. (2021) Continued Table 14.1 Representative functional miRNAs characterized as affecting crop yields.dcont'd miRNA/lncRNA miR167 miR168 miR169 Agronomic trait affected Validated target? Plant species Transgenic evidence? References/Figs Target gene family 5: Carbone et al. (2019) 5. Ripening stages in drupe epi-mesocarp tissue stage 5. Olive (Olea europaea L.) 6. Grain development 1. Fruit development 6. Maize 1. Orange 2. Seed development, seed oil content 2. B. napus 3. Anther dehiscence and maternal control of ovule development, growth of fruit valve 4. Ovule and ovary development and tomato fruit set 5. Seed development, axillary bud development 3. A. thaliana 3. AUXIN RESPONSE FACTOR6 and 8 4. Tomato 4. SlARF8a 5. Rice 5. osarf12/osarf17 and osarf12/osarf25 Fruit ripening 1. Flower development Citrus 1. P. hybrida and A. majus 2. Water loss through leaves and hypersensitivity to drought 2. Arabidopsis Auxin Response Factor (ARF) AGOs 1. NUCLEAR FACTOR-YA (NFYA) domain TFs 6: D. Li et al. (2016) Figs. 4 and 5; 1: Xu et al. (2010) 1. EY752486/ geranylgeranyl pyrophosphate synthase AGO1 2: J. Jiang et al. (2021), Zhao et al. (2012), Huang et al. (2013) 3: Yao et al. (2019), Ripoll et al. (2015) 4: da Silva et al. (2017) 4. Repression of miR167 (induced by OE of MIR159) 5. OsMIR167a OE, T-DNA insertion mutants of target ARFs osarf12/osarf17 and osarf12/osarf25 NA 1. blind and fistulata mutants (No) Liu et al. (2014) 1: Cartolano et al. (2007) 2. OEmiR169 (Yes) 2: Zhao et al. (2011) 5: Huang et al. (2016), Xue et al. (2009), Li et al. (2020) 3. Conferred drought tolerance 3. Tomato 4. Berry development 4. Grape 5. Flower development 5. Arabidopsis/Grape 6. Seed dormancy 6. Japanese apricot (Prunus mume) 7. Wheat 7. Seed development miR170/171 8. Kernel development and nutrient transport 1. Response to boron toxicity, stem cell maintenance, quiescent center, and endodermis specification 2. Pollen development 3. Plant height, flowering time, leaf architecture, lateral branch number, root length, fruit set, and development, pollen viability and pollen development 4. Texture and flavor 5. Grain development 6. Vegetative development 3. OEmiR169 (Yes) 4: Paim Pinto et al. (2016) 5: Gyula et al. (2018), Sunitha et al. (2019) 5. JASMONATEZIM-DOMAIN PROTEIN 4/JAZ4 6: Gao et al. (2021) 7: R. Han et al. (2014) 8: Xing et al. (2017) 8. Maize 1. Citrus 2. Brassica campestris 3. Tomato 4. Peach 5. Maize 6. Barley 3: Zhang et al. (2011), Zhao et al. (2009, 2011) 1: Huang et al. (2019) SCARECROW-like TFs 2: Jiang et al. (2014) 3. Kravchik et al. (2019), W. Huang et al. (2017) 3. SlGRAS24 TF 6. HvSCL/GRAS 6. OE of miR171 4: Kong et al. (2021) 5: D. Li et al. (2016) 6: Curaba et al. (2013) Continued Table 14.1 Representative functional miRNAs characterized as affecting crop yields.dcont'd miRNA/lncRNA miR172 miR390 Agronomic trait affected 1. Growth of fruit valve seed maturation; flower development 2. Carpel development 3. Fruit ripening; organ identity and number 4. Flower development 5. Timing of tuberization 6. Flowering time and fruit development 7. Fruit size; flavonoid biosynthesis (AP2-1a) Validated target? Plant species Target gene family 1. Arabidopsis 1. AP2 2. NA 3. NA; CRISPR-Cas9 targeting of miR172 4. TOE type AP2 5. Potato 4. BEN 4. NA 5. RAP1 (RELATED TO APETALA2 1) 5. Yes (OE of MIR172) 6. NA 6. Capsicum 7. Apple 8. Spike devt. 8. Wheat 9. Grain yields 1. Regulation of oil content 2. Hormone-regulated tuber formation 9. Barley 1. Brassica napus 3. Delayed flowering 3. Woodland strawberry 4. Maize 5. Maize 4. Grain development 5. Endosperm maturation 1. NA; OE of AP2m3 2. Chinese cabbage 3. Tomato 4. Petunia 2. Nicotiana benthamiana (transient assay) Transgenic evidence? 7. AP2-1a TF 7. MdMYB10 8. AP2-5 9. HvAP2 7. OE of MIRNA172; tobacco MdAP2_1 OE lines/MdAP2_1a VIGS 8. Yes (mimic of miR172, MIM172) 9. NA 1. Trans-Acting sRNA locus3 (TAS3) References/Figs Figs. 4, 7 and 8; 1: Ripoll et al. (2015), Zhao et al. (2007) 2: P. Li et al. (2021) 3: Karlova et al. (2013), Lin et al. (2021) 4: Morel et al. (2017) 5: Martin et al. (2009) 6: Lopez-Ortiz et al. (2021) 7: Zhou et al. (2021), Ding et al. (2022) 8: Debernardi et al. (2017) 9: Nair et al. (2010) 1; Zhao et al. (2012) 2: Santin et al. (2017) 2. Calcium Dependent Protein Kinase 1/CDPK1 (needs to be validated) 3. OE FveMIR390a (Yes) 3: Dong et al. (2022) 4: D. Li et al. (2016) 5: M. Xin et al. (2015) miR391/173/ 1432/1509/3627/ 4376/5225/7122 miR393 miR394 miR395 miR396 1: Zhao et al. (2019), Attri et al. (2022) 1. miR1432 1. Rice 1. Acyl-CoA thioesterase Grain yield in rice 2. miR4376 2. Grape 2: Owusu Adjei et al. (2021) Anthocyanin accumulation 3. miR5225 3. Date palm 1. Fruit set 1. Tomato 3: Patel et al. (2021), C. Xin et al. (2015) 1: Karlova et al. (2013) 2. Grain development 3. Seed germination and seedling establishment 1. Proper seed morphology and deposition of storage lipids, glucosinolates, and proteins/fruit and seed development 2. Sugar and acid metabolism during the formation of fruit flavor 1. Aroma weakening of fruits upon storage under cold conditions, ripening 2. Maize 3. Rice 1. Grape ATP sulfurylases (APS), sulfate transporter 1. Maintenance of CMMs, meristematic competence, and pluripotency; growth of floral buds, floral organs 1. Tomato 1. GRF 1. TRANSPORT INHIBITOR RESPONSE1-like auxin receptors 2. Cyclin like F-Box 3. TIR1 and AFB2 1. Brassica napus 1. LEAF CURLING RESPONSIVENESS (LCR) 1. NA 2. NA 3. Yes (OE of miR393) 2: D. Li et al. (2016) 3: Guo et al. (2016) 1. OE of B. napus MIR394; miR394resistent mutations transgenic (Yes) 1: Song et al. (2015) 2. Pear 2: S. Huang et al. (2021) 1. Sulfate transporters, MdWRKY26 TF 1. NA; STTM 1. Shi et al. (2019), Belli Kullan et al. (2015), Tavares et al. (2013), Guillaumie et al. (2011) Fig. 4; 1: Lee et al. (2017), Cao et al. (2016) Continued Table 14.1 Representative functional miRNAs characterized as affecting crop yields.dcont'd miRNA/lncRNA miR397 miR398 Transgenic evidence? References/Figs 2. GRF5 2. OE of LsaGRF5 and LsaMIR396a OE 2: B. Zhang et al. (2021) 3. Grape 3. VvGIB1B 3. NA 4. Pbr006726.1 5. GRF 6. OsGRF8 4,5. NA 6. Grain size 4. Pear 5. Apple 6. Rice 7. Vernalization 1. Granulation 7. Orchard grass 1. Citrus 2. Plant biomass, silique lengths, and overall seed yield 2. Arabidopsis 3. Fruit development 4. Fruit storage safening 3. Pear 4. Grape 3: Mica et al. (2009), W. Wang et al. (2020) 4: Wu et al. (2014); 5: Xia et al. (2012) 6: Yang and Zhao et al. (2021) 7: Feng et al. (2018) 1: J. Zhang et al. (2016) 2: S. Huang et al. (2021), Song et al. (2018), Wang et al. (2014) 3: Wu et al. (2014) 4: Xue et al. (2018) 5. Grain numbers and seed size; grain filling 6. Grain filling 7. Biomass 5. Rice Validated target? Agronomic trait affected Plant species 2. Leaf size regulation 2. Lettuce 3 Cell expansion; seed and berry devt. 4,5 Cell expansion 1. Pollen development 2. Development of fruits and flower buds 3. Light perception 4. Panicle length, grain size, and grain number Target gene family 7. GRF 1. laccase17/LAC17 2. OEmiR397 (Yes) 4. LOX, lipoxygenase (not statistically significant) 5. OEmiR397 (Yes) 6. Maize 7. Banana 1. Brassica campestris ssp. Chinensis 2. Pear 3. Apple 4. Rice 6. Yes (STTMmiR396) 7. NA 7. OE of copper deficiency-induced MIR397 (Yes) CSD1, CSD2, CCS1, BCBP 5: Zhang et al. (2013), Teng et al. (2021) 6: Jin et al. (2015) 7: Patel et al. (2019) 1: J. Jiang et al. (2021) 2: Wu et al. (2014) 4. Cu/Zn superoxide dismutase2/ Os07g46990 and CCS/Os04g48410 4. Silencing MIR398 3: Qu et al. (2016) 4: Li et al. (2010) miR399 1. Floral development 1. Citrus 1. PHO2/UBC24 1. Yes (STTM) 2. Woodland strawberry 1. Arabidopsis 2. PHO2 miR408 2. Sugar content and fruit flavor 1. Biomass, silique lengths, and overall seed yield 2. Yes (OE of MIR399a) 1. OEmiR408 (Yes) 2. Silique development 3. Fruit development 4. Grain size and photosynthesis 5. Regulates grain size 2. Brassica napus 6. Photosynthesis, abundance of plastocyanins, and grain yield 7. Enhanced vegetative growth and enlarged seeds 8. Regulates flowering time Outgrowth of axillary buds 2. Long silique and short silique lines (No) 3. Pear 4. Rice 4. PHYTOCYANIN 5. Rice 5. UCL8/uclacyanin 6. Rice 6. TIMING OF CAB EXPRESSION1/ TOC1 5. CRISPR-Cas9 engineered mutations in MIR408 (Yes) Fig. 3; 1:S. Huang et al. (2021), Song et al. (2018), Wang et al. (2014) 2: Chen et al. (2018) 3: Wu et al. (2014) 4: J.P. Zhang et al. (2017) 5: Yang and Zhao et al. (2021) 6: J.P. Zhang et al. (2017) 7. Arabidopsis, tobacco, and rice 7. OEmiR408 (Yes) 7: Pan et al. (2018) 8. Wheat, barley 8. OEmiR408 (Yes) 8: Zhao et al. (2016) 9. Wheat miR444 1. LAC Fig. 3; 1: R. Wang et al. (2020) 2: Wang et al. (2017) 1. Rice 9. Targets involved in microtubule organization, proteinprotein interaction, and cyclic-AMP signal transduction (no degradome evidence) OsMADS57 9: Bai et al. (2018) Continued Table 14.1 Representative functional miRNAs characterized as affecting crop yields.dcont'd miRNA/lncRNA miR474/782/843 miR477 Agronomic trait affected 1. ROS scavenging and osmoprotection (adaptive strategy to B-deficiency; affect fruit production72) 1. Fruit development 2. Maintain the balance of GA content 3. Ripening miR482/472/2118 Validated target? Transgenic evidence? References/Figs 1. C. sinensis 1. PDH 1. NA 1: Lu et al. (2014) 1. Citrus 1. SEPALLATA1.1 (SEP1)/Cs6g19680/ orange1.1g02614 m (marginally supported by authors’ degradome data) Plant species 2. Green berries 3. Grape 4. Positive regulator of anthocyanin accumulation miR482 4. Mulberry (Morus atropurpurea) 1. Response to viral and bacterial infection. 1. All 2. Fruit maturation 2. Tomato miR472 3. Effector-triggered immunity 3. Arabidopsis Target gene family 1: Liu et al. (2014), Goodstein et al. (2011), R. Wang et al. (2020) 2. VvGAINSENSITVE1/GAI1 3. GRAS family TFs (VvGAI1) 2: W. Wang et al. (2020) 3: Guo et al. (2018), Chitarra et al. (2018), W. Wang et al. (2020) 4: Dong et al. (2021) 4. Anti-sense lncRNA-ABCB19AS Fig. 2; nucleotide bindingleucine-rich repeat (NBLRR) 2. Glutamate permease/ SGN-U585460/ Solyc04g082290.4.1 2. 50 RLM-RACE 1: Li et al. (2012), Shivaprasad et al. (2012), Y. Zhang et al. (2016) 2: Goodstein et al. (2011) 3: Boccara et al. (2014), C. Jiang et al. (2020) miR2118 miR528 miR530 miR820 miR827 miR828 4. phasiRNA generation 4. Rice and maize 4. Trans-acting sRNA locus5 TAS5 1. Flowering time 1. Rice 1. Zinc-finger transcription factor gene 2. Maize seed imbibition and germiation 1. Negative regulator of rice grain yield 1. OsRFI2 1. NA 2. Maize 2. MATE, bHLH, and SOD1a 2. NA 1. Rice 1. PL3 or PLUS3 containing domain and VIT_05s0020g04860 (in grape) 2. Decreased the size of the seeds and inhibited panicle branching; increased grain yield 2. Rice Genome methylation (pollen development) 1. Phosphate (Pi) transport and storage 1. Granulation 1. Rice 2. Bioflavonoid synthesis 3. Affecting proanthocyanidin and flavonol biosynthesis 4. Anthocyanin synthesis during apple fruit development 2. Grape 1. Arabidopsis thaliana and Oryza sativa 1. Citrus 3. Grape 4. Apple DNA cytosine methyltransferase 1. NLA and PHT5 2: Luján-Soto et al. (2021) 1: Sunitha et al. (2019) 2. OE and downregulation (CRISPR-cas9); and blocking osa-miR530 by STTM expression (Yes) DNA cytosine methyltransferase 1. SPX 4: Sunitha and Rock (2020), Yang and You et al. (2021), Yadava et al. (2021), CantoPastor et al. (2019) 1: Chen et al. (2019) 2: W. Sun et al. (2020) 1: Wei et al. (2011) 1. NA 1. MYB transcription factors [TFs] 2. VvMYBA6 Fig. 3; 1: Silva et al. (2014) 1: J. Zhang et al. (2016) 2: Rock (2013) 3. MYB114/ VIT_09s0002g01380/ PAL3 4. TAS4 targets MYBs 3: Sunitha et al. (2019), Tirumalai et al. (2019) 4: Xia et al. (2012) Continued Table 14.1 Representative functional miRNAs characterized as affecting crop yields.dcont'd miRNA/lncRNA miR825/952/1132/ 1318/2635/5077/5500 miR858 miR1432 miR1511/5375/5654/ 6030/9557 miR1885/5724 miR1917 Agronomic trait affected Validated target? Plant species Transgenic evidence? References/Figs Target gene family 5,6 Potentially shaping development of monocots with fleshy fruits 5. Date palm 6. Lilies (Trillium camshatensese) 5: Patel et al. (2021), C. Xin et al. (2015), Rock (2020) 6: Rock (2013, 2020) 1. Sugar and acid metabolism during the formation of fruit flavor 1. Anthocyanin biosynthesis and fruit coloring 2. Anthocyanin biosynthesis 3. Anthocyanin biosynthesis 1. Pear 1: Wu et al. (2014) 1. Biosynthesis of medium-chain fatty acids Functions remain to be established 1. Disease resistance 1. Regulating ethylene responses 1. Kiwifruit 1. MYBC1 1. NA 1: Y. Li et al. (2019) 2. Tomato 2. SlMYB7-like and SlMYB48-like transcripts 3. CHS, CHI, F3H, and FLS1; MYBL2 2. Yes (STTM858) 2: Jia et al. (2015) 3. Yes (miR858OX/ Target mimic, MIM858); STTM,miR858ab 3: Sharma et al. (2016), Y. Wang et al. (2016) 3. Arabidopsis 3. R2R3-MYB 1. Rice 1. Acyl-CoA thioesterase 1: Zhao et al. (2019) Körbes et al. (2012) 1. Brassica campestris ssp. chinensis 1. Tomato 1. TIR-NBS-LRR 1. CONSTITUTIVE TRIPLE RESPONSE 4 (SlCTR4) 30 -UTR 1. Validated by planta transient assays and site-directed mutagenesis of the binding site in the target mRNA 1. NA 1: Jiang et al. (2014) 1. Suppression of miR1917 via STTM approachdincreased size and weight of the tomato fruit and seed size. (Yes) Fig. 7; 1: Moxon et al. (2008), Y. Wang et al. (2018), Zhong et al. (2008), Yang et al. (2020), Correa et al. (2018) 2. Tomato miR2111 miR2950 miR4414 miR5506 miR5801 miR6029 lnc1459/1840/2155 1. Early embryonic development and oil accumulation, regulation of silique length 1. Photosynthesis 2. Flower development 1. Expressed in early developmental stages of pear fruit and to decline during maturation 2. High expression in flowers Vegetative development, floral organ formation 1. Seed maturation Regulation of oil content of B. napus seeds in later stages through multiple pathways including auxin signaling 1. Fruit ripening 2: Karlova et al. (2013) 2. Sesquiterpene synthase2/ Solyc06g059920.1.1a (limited degradome support) 1. B. napus Fig. 3; 1: Zhao et al. (2012), Chen et al. (2018) 1. Grape 1. Chlorophyllase 2. Chinese date jujube (Ziziphus jujuba 1. Pear, Pyrus bretschneideri 2. ARF 1. Dihydrolipoyl transacetylase (Pbr015262.1) 1. VIT_07s0151 g00250 2. ARF4 1. NA 2. NA 1: Chitarra et al. (2018) 2: Ma et al. (2020) 1: Wu et al. (2014) (No evidence provided) 2. Peach 2; Luo et al. (2013) 1. Rice 1: Z. Chen et al. (2021) 1. B.-napus Brassica napus 1. Tomato DEMETER bZIP TF 1. NA Validated by 50 RLMRACE 1: Huang et al. (2013) 1: Zhao et al. (2012) Fig. 7 450 Plant Small RNA in Food Crops novel species-specific miRNAs and lncRNAs. How pervasive (where demonstration of conservation across species is key) are plant non-canonical miRNA targets? Are some animal miRNA features and mechanisms of action actually operating in plants, where Watson-Crick base complementarity has been the paradigm that has driven progress to date? Will there be another paradigm shift in the future that changes our understanding of the Small RNA World (Chen & Rechavi, 2022; Zhao et al., 2018)? References Addo-Quaye, C., Miller, W., & Axtell, M. J. (2009). CleaveLand: A pipeline for using degradome data to find cleaved small RNA targets. Bioinformatics, 25, 130e131. Allen, E., Xie, Z. X., Gustafson, A. M., & Carrington, J. C. (2005). MicroRNA-directed phasing during trans-acting siRNA biogenesis in plants. Cell, 121, 207e221. Allen, E., Xie, Z., Gustafson, A. M., Sung, G. H., Spatafora, J. W., & Carrington, J. C. (2004). Evolution of microRNA genes by inverted duplication of target gene sequences in Arabidopsis thaliana. Nature Genetics, 36, 1282e1290. Alves-Junior, L., Niemeier, S., Hauenschild, A., Rehmsmeier, M., & Merkle, T. (2009). Comprehensive prediction of novel microRNA targets in Arabidopsis thaliana. Nucleic Acids Research, 37, 4010e4021. Attri, K., Zhang, Z., Singh, A., Sharrock, R. A., & Xie, Z. (2022). Rapid sequence and functional diversification of a miRNA superfamily targeting calcium signaling components in seed plants. New Phytologist, 235, 1082e1095. Axtell, M. J. (2013). Classification and comparison of small RNAs from plants. Annual Review of Plant Biology, 64, 137e159. Axtell, M. J., & Meyers, B. C. (2018). Revisiting criteria for plant microRNA annotation in the era of big data. The Plant Cell, 30, 272e284. Bai, B., Shi, B., Hou, N., Cao, Y., Meng, Y., Bian, H., Zhu, M., & Han, N. (2017). microRNAs participate in gene expression regulation and phytohormone cross-talk in barley embryo during seed development and germination. BMC Plant Biology, 17, 150. Bai, Q., Wang, X., Chen, X., Shi, G., Liu, Z., Guo, C., & Xiao, K. (2018). Wheat miRNA tae-miR408 acts as an essential mediator in plant tolerance to Pi deprivation and salt stress via modulating stress-associated physiological processes. Frontiers in Plant Science, 9, 499. Bao, A., Burritt, D. J., Chen, H., Zhou, X., Cao, D., & Tran, L. P. (2019). The CRISPR/ Cas9 system and its applications in crop genome editing. Critical Reviews in Biotechnology, 39, 321e336. Belli Kullan, J., Paim Pinto, D. L., Bertolini, E., Fasoli, M., Zenoni, S., Tornielli, G. B., Pezzotti, M., Meyers, B. C., Farina, L., Pè, M. E., & Mica, E. (2015). miRVine: a microRNA expression atlas of grapevine based on small RNA sequencing. BMC Genomics, 16, 393. Berger, Y., Harpaz-Saad, S., Brand, A., Melnik, H., Sirding, N., Alvarez, J. P., Zinder, M., Samach, A., Eshed, Y., & Ori, N. (2009). The NAC-domain transcription factor GOBLET specifies leaflet boundaries in compound tomato leaves. Development, 136, 823e832. Bhogale, S., Mahajan, A. S., Natarajan, B., Rajabhoj, M., Thulasiram, H. V., & Banerjee, A. K. (2013). MicroRNA156: A potential graft-transmissible microRNA that modulates plant architecture and tuberization in Solanum tuberosum ssp. andigena. Plant Physiology, 164, 1011e1027. Grain development and crop productivity: role of small RNA 451 Bi, F. C., Meng, X. C., Ma, C., & Yi, G. J. (2015). Identification of miRNAs involved in fruit ripening in Cavendish bananas by deep sequencing. BMC Genomics, 16. Boccara, M., Sarazin, A., Thiébeauld, O., Jay, F., Voinnet, O., Navarro, L., & Colot, V. (2014). The Arabidopsis miR472-RDR6 silencing pathway modulates PAMP- and effector-triggered immunity through the post-transcriptional control of disease resistance genes. PLoS Pathogens, 10, e1003883. Böwer, F., & Schnittger, A. (2021). How to switch from mitosis to meiosis: Regulation of germline entry in plants. Annual Review of Genetics, 55, 427e452. Brodersen, P., & Voinnet, O. (2009). Revisiting the principles of microRNA target recognition and mode of action. Nature Reviews Molecular Cell Biology, 10, 141e148. Brousse, C., Liu, Q., Beauclair, L., Deremetz, A., Axtell, M. J., & Bouché, N. (2014). A non-canonical plant microRNA target site. Nucleic Acids Research, 42, 5270e5279. Canto-Pastor, A., Santos, B. A. M. C., Valli, A. A., Summers, W., Schornack, S., & Baulcombe, D. C. (2019). Enhanced resistance to bacterial and oomycete pathogens by short tandem target mimic RNAs in tomato. Proceedings of the National Academy of Sciences of the United States of America, 116, 2755e2760. Cao, F., Guan, C., Dai, H., Li, X., & Zhang, Z. (2015). Soluble solids content is positively correlated with phosphorus content in ripening strawberry fruits. Scientia Horticulturae, 195, 183e187. Cao, D., Wang, J., Ju, Z., Liu, Q., Li, S., Tian, H., Fu, D., Zhu, H., Luo, Y., & Zhu, B. (2016). Regulations on growth and development in tomato cotyledon, flower and fruit via destruction of miR396 with short tandem target mimic. Plant Science, 247, 1e12. Carbone, F., Bruno, L., Perrotta, G., Bitonti, M. B., Muzzalupo, I., & Chiappetta, A. (2019). Identification of miRNAs involved in fruit ripening by deep sequencing of Olea europaea L. transcriptome. PLoS One, 14, e0221460. Carra, A., Mica, E., Gambino, G., Pindo, M., Moser, C., Pe, M. E., & Schubert, A. (2009). Cloning and characterization of small non-coding RNAs from grape. The Plant Journal, 59, 750e763. Cartolano, M., Castillo, R., Efremova, N., Kuckenberg, M., Zethof, J., Gerats, T., Schwarz-Sommer, Z., & Vandenbussche, M. (2007). A conserved microRNA module exerts homeotic control over Petunia hybrida and Antirrhinum majus floral organ identity. Nature Genetics, 39, 901e905. Cedillo-Jimenez, C. A., Feregrino-Perez, A. A., Guevara-González, R. G., & CruzHernández, A. (2020). MicroRNA regulation during the tomato fruit development and ripening: A review. Scientia Horticulturae, 270, 109435. Chai, J., Feng, R., Shi, H., Ren, M., Zhang, Y., & Wang, J. (2015). Bioinformatic identification and expression analysis of banana microRNAs and their targets. PLoS One, 10, e0123083. Chekanova, J. A., Gregory, B. D., Reverdatto, S. V., Chen, H., Kumar, R., Hooker, T., Yazaki, J., Li, P., Skiba, N., Peng, Q., Alonso, J., Brukhin, V., Grossniklaus, U., Ecker, J. R., & Belostotsky, D. A. (2007). Genome-wide high-resolution mapping of exosome substrates reveals hidden features in the Arabidopsis transcriptome. Cell, 131, 1340e1353. Chen, Y., Cheng, C., Feng, X., Lai, R., Gao, M., Chen, W., & Wu, R. (2021). Integrated analysis of lncRNA and mRNA transcriptomes reveals the potential regulatory role of lncRNA in kiwifruit ripening and softening. Scientific Reports, 11, 1671. Chen, L., Chen, L., Zhang, X., Liu, T., Niu, S., Wen, J., Yi, B., Ma, C., Tu, J., Fu, T., & Shen, J. (2018). Identification of miRNAs that regulate silique development in Brassica napus. Plant Science, 269, 106e117. Chen, Z., Li, Y., Li, P., Huang, X., Chen, M., Wu, J., Wang, L., Liu, X., & Li, Y. (2021). MircroRNA profiles of early rice inflorescence revealed a specific miRNA5506 452 Plant Small RNA in Food Crops regulating development of floral organs and female mega gametophyte in rice. International Journal of Molecular Sciences, 22, 6610. Chen, C., Liu, C., Jiang, A., Zhao, Q., Zhang, Y., & Hu, W. (2020). miRNA and degradome sequencing identify miRNAs and their target genes involved in the browning inhibition of fresh-cut apples by hydrogen sulfide. Journal of Agricultural and Food Chemistry, 68, 8462e8470. Chen, C., Liu, Y., & Xia, R. (2019). Jack of many trades: The multifaceted role of miR528 in monocots. Molecular Plant, 12, 1044e1046. Chen, Q., Liu, K., Yu, R., Zhou, B., Huang, P., Cao, Z., Zhou, Y., & Wang, J. (2021). From “dark matter” to “star”: Insight into the regulation mechanisms of plant functional long non-coding RNAs. Frontiers in Plant Science, 12, 650926. Chen, X., & Rechavi, O. (2022). Plant and animal small RNA communications between cells and organisms. Nature Reviews Molecular Cell Biology, 23, 185e203. Chitarra, W., Pagliarani, C., Abbà, S., Boccacci, P., Birello, G., Rossi, M., Palmano, S., Marzachì, C., Perrone, I., & Gambino, G. (2018). miRVIT: A novel miRNA database and its application to uncover Vitis responses to Flavescence dorée infection. Frontiers in Plant Science, 9, 1034. Chuck, G., Meeley, R., Irish, E., Sakai, H., & Hake, S. (2007). The maize tasselseed4 microRNA controls sex determination and meristem cell fate by targeting Tasselseed6/ indeterminate spikelet1. Nature Genetics, 39, 1517e1521. Clepet, C., Devani, R. S., Boumlik, R., Hao, Y., Morin, H., Marcel, F., Verdenaud, M., Mania, B., Brisou, G., Citerne, S., Mouille, G., Lepeltier, J. C., Koussevitzky, S., Boualem, A., & Bendahmane, A. (2021). The miR166eSlHB15A regulatory module controls ovule development and parthenocarpic fruit set under adverse temperatures in tomato. Molecular Plant, 14, 1185e1198. Coen, E. S., & Meyerowitz, E. M. (1991). The war of the whorls: Genetic interactions controlling flower development. Nature, 353, 31e37. Correa, JPdO., Silva, E. M., & Nogueira, F. T. S. (2018). Molecular control by non-coding RNAs during fruit development: From gynoecium patterning to fruit ripening. Frontiers in Plant Science, 9, 1760. Coruh, C., Shahid, S., & Axtell, M. J. (2014). Seeing the forest for the trees: Annotating small RNA producing genes in plants. Current Opinion in Plant Biology, 18, 87e95. Csukasi, F., Donaire, L., Casañal, A., Martínez-Priego, L., Botella, M. A., MedinaEscobar, N., Llave, C., & Valpuesta, V. (2012). Two strawberry miR159 family members display developmental-specific expression patterns in the fruit receptacle and cooperatively regulate Fa-GAMYB. New Phytologist, 195, 47e57. Cui, M., Wang, C., Zhang, W., Pervaiz, T., Haider, M. S., Tang, W., & Fang, J. (2018). Characterization of Vv-miR156:Vv-SPL pairs involved in the modulation of grape berry development and ripening. Molecular Genetics and Genomics, 293, 1333e1354. Cui, L., Zheng, F., Wang, J., Zhang, C., Xiao, F., Ye, J., Li, C., Ye, Z., & Zhang, J. (2020). miR156a-targeted SBP-Box transcription factor SlSPL13 regulates inflorescence morphogenesis by directly activating SFT in tomato. Plant Biotechnology Journal, 18, 1670e1682. Curaba, J., Talbot, M., Li, Z., & Helliwell, C. (2013). Over-expression of microRNA171 affects phase transitions and floral meristem determinancy in barley. BMC Plant Biology, 13, 6. Damayanti, F., Lombardo, F., Masuda, J-i, Shinozaki, Y., Ichino, T., Hoshikawa, K., Okabe, Y., Wang, N., Fukuda, N., Ariizumi, T., & Ezura, H. (2019). Functional disruption of the tomato putative ortholog of HAWAIIAN SKIRT results in facultative parthenocarpy, reduced fertility and leaf morphological defects. Frontiers in Plant Science, 10, 1234. Grain development and crop productivity: role of small RNA 453 Damodharan, S., Zhao, D., & Arazi, T. (2016). A common miRNA160-based mechanism regulates ovary patterning, floral organ abscission and lamina outgrowth in tomato. The Plant Journal, 86, 458e471. Dang, T., Lavagi-Craddock, I., Bodaghi, S., & Vidalakis, G. (2021). Next-generation sequencing identification and characterization of microRNAs in dwarfed citrus trees infected with Citrus Dwarfing Viroid in high-density plantings. Frontiers in Microbiology, 12, 980. Debernardi, J. M., Lin, H., Chuck, G., Faris, J. D., & Dubcovsky, J. (2017). MicroRNA172 plays a crucial role in wheat spike morphogenesis and grain threshability. Development, 144, 1966e1975. Debernardi, J. M., Mecchia, M. A., Vercruyssen, L., Smaczniak, C., Kaufmann, K., Inze, D., Rodriguez, R. E., & Palatnik, J. F. (2014). Post-transcriptional control of GRF transcription factors by microRNA miR396 and GIF co-activator affects leaf size and longevity. The Plant Journal, 79, 413e426. Dhaka, N., & Sharma, R. (2021). MicroRNA-mediated regulation of agronomically important seed traits: A treasure trove with shades of grey! Critical Reviews in Biotechnology, 41, 594e608. Di Marsico, M., Paytuvi Gallart, A., Sanseverino, W., & Aiese Cigliano, R. (2022). GreeNC 2.0: A comprehensive database of plant long non-coding RNAs. Nucleic Acids Research, 50, D1442eD1447. Ding, N., Huertas, R., Torres-Jerez, I., Liu, W., Watson, B., Scheible, W.-R., & Udvardi, M. (2021). Transcriptional, metabolic, physiological and developmental responses of switchgrass to phosphorus limitation. Plant, Cell and Environment, 44, 186e202. Ding, T., Tomes, S., Gleave, A. P., Zhang, H., Dare, A. P., Plunkett, B., Espley, R. V., Luo, Z., Zhang, R., Allan, A. C., Zhou, Z., Wang, H., Wu, M., Dong, H., Liu, C., Liu, J., Yan, Z., & Yao, J. L. (2022). MicroRNA172 targets APETALA2 to regulate flavonoid biosynthesis in apple (Malus domestica). Horticultural Research, 9. https:// doi.org/10.1093/hr/uhab1007. uhab007. Dong, X., Guan, Y., Zhang, Z., & Li, H. (2022). miR390-tasiRNA3-ARF4 pathway is involved in regulating flowering time in woodland strawberry. Plant Cell Reports, 41, 921e934. Dong, X., Liu, C., Wang, Y., Dong, Q., Gai, Y., & Ji, X. (2021). MicroRNA profiling during mulberry (Morus atropurpurea Roxb) fruit development and regulatory pathway of miR477 for anthocyanin accumulation. Frontiers in Plant Science, 12, 687364. Du, L., Bao, C., Hu, T., Zhu, Q., Hu, H., He, Q., & Mao, W. (2016). SmARF8, a transcription factor involved in parthenocarpy in eggplant. Molecular Genetics and Genomics, 291, 93e105. Dukowic-Schulze, S., & van der Linde, K. (2021). Oxygen, secreted proteins and small RNAs: Mobile elements that govern anther development. Plant Reproduction, 34, 1e19. Eviatar-Ribak, T., Shalit-Kaneh, A., Chappell-Maor, L., Amsellem, Z., Eshed, Y., & Lifschitz, E. (2013). A cytokinin-activating enzyme promotes tuber formation in tomato. Current Biology, 23, 1057e1064. Fahlgren, N., & Carrington, J. C. (2010). miRNA target prediction in plants. In B. C. Meyers, & P. J. Green (Eds.), Plant microRNAs (Vol 592, pp. 51e57). Humana Press. Farinati, S., Rasori, A., Varotto, S., & Bonghi, C. (2017). Rosaceae fruit development, ripening and post-harvest: An epigenetic perspective. Frontiers in Plant Science, 8, 1247. Feng, G., Xu, L., Wang, J., Nie, G., Bushman, B. S., Xie, W., Yan, H., Yang, Z., Guan, H., Huang, L., & Zhang, X. (2018). Integration of small RNAs and transcriptome sequencing uncovers a complex regulatory network during vernalization and heading stages of orchardgrass (Dactylis glomerata L.). BMC Genomics, 19, 727. 454 Plant Small RNA in Food Crops Finkelstein, R. R., Gampala, S. S. L., & Rock, C. D. (2002). Abscisic acid signaling in seeds and seedlings. The Plant Cell, 14, S15eS45. Fornara, F., & Coupland, G. (2009). Plant phase transitions make a SPLash. Cell, 138, 625e627. Gao, Y., Feng, B., Gao, C., Zhang, H., Wen, F., Tao, L., Fu, G., & Xiong, J. (2022). The evolution and functional roles of miR408 and its targets in plants. International Journal of Molecular Sciences, 23, 530. Gao, C., Ju, Z., Cao, D., Zhai, B., Qin, G., Zhu, H., Fu, D., Luo, Y., & Zhu, B. (2015). MicroRNA profiling analysis throughout tomato fruit development and ripening reveals potential regulatory role of RIN on microRNAs accumulation. Plant Biotechnology Journal, 13, 370e382. Gao, J., Ni, X., Li, H., Hayat, F., Shi, T., & Gao, Z. (2021). miR169 and PmRGL2 synergistically regulate the NF-Y complex to activate dormancy release in Japanese apricot (Prunus mume Sieb. et Zucc.). Plant Molecular Biology, 105, 83e97. Garighan, J., Dvorak, E., Estevan, J., Loridon, K., Huettel, B., Sarah, G., Farrera, I., Leclercq, J., Grynberg, P., Coiti Togawa, R., Mota do Carmo Costa, M., Costes, E., & Andrés, F. (2021). The identification of small RNAs differentially expressed in apple buds reveals a potential role of the miR159-MYB regulatory module during dormancy. Plants, 10, 2665. Gilbert, W. (1986). Origin of life: The RNA world. Nature, 319, 618. Goodstein, D. M., Shu, S., Howson, R., Neupane, R., Hayes, R. D., Fazo, J., Mitros, T., Dirks, W., Hellsten, U., Putnam, N., & Rokhsar, D. S. (2011). Phytozome: A comparative platform for green plant genomics. Nucleic Acids Research, 40, D1178eD1186. Gou, J.-Y., Felippes, F. F., Liu, C.-J., Weigel, D., & Wang, J.-W. (2011). Negative regulation of anthocyanin biosynthesis in Arabidopsis by a miR156-targeted SPL transcription factor. The Plant Cell, 23, 1512e1522. Gou, J., Tang, C., Chen, N., Wang, H., Debnath, S., Sun, L., Flanagan, A., Tang, Y., Jiang, Q., Allen, R. D., & Wang, Z. Y. (2019). SPL7 and SPL8 represent a novel flowering regulation mechanism in switchgrass. New Phytologist, 222, 1610e1623. Grimplet, J., Agudelo-Romero, P., Teixeira, R. T., Martinez-Zapater, J. M., & Fortes, A. M. (2016). Structural and functional analysis of the GRAS gene gamily in grapevine indicates a role of GRAS proteins in the control of development and stress responses. Frontiers in Plant Science, 7, 353. Guillaumie, S., Fouquet, R., Kappel, C., Camps, C., Terrier, N., Moncomble, D., Dunlevy, J., Davies, C., Boss, P., & Delrot, S. (2011). Transcriptional analysis of late ripening stages of grapevine berry. BMC Plant Biology, 11, 165. Guo, F., Han, N., Xie, Y., Fang, K., Yang, Y., Zhu, M., Wang, J., & Bian, H. (2016). The miR393a/target module regulates seed germination and seedling establishment under submergence in rice (Oryza sativa L.). Plant, Cell and Environment, 39, 2288e2302. Guo, D.-L., Li, Q., Lv, W.-Q., Zhang, G.-H., & Yu, Y.-H. (2018). MicroRNA profiling analysis of developing berries for “Kyoho” and its early-ripening mutant during berry ripening. BMC Plant Biology, 18, 285. Guo, Q. L., Qu, X. F., & Jin, W. B. (2015). PhaseTank: genome-wide computational identification of phasiRNAs and their regulatory cascades. Bioinformatics, 31, 284e286. Guo, S., Xu, Y., Liu, H., Mao, Z., Zhang, C., Ma, Y., Zhang, Q., Meng, Z., & Chong, K. (2013). The interaction between OsMADS57 and OsTB1 modulates rice tillering viaDWARF14. Nature Communications, 4, 1566. Gupta, S. K., Vishwakarma, A., Kenea, H. D., Galsurker, O., Cohen, H., Aharoni, A., & Arazi, T. (2021). CRISPR/Cas9 mutants of tomato MICRORNA164 genes uncover their functional specialization in development. Plant Physiology, 187, 1636e1652. Gyula, P., Baksa, I., Tóth, T., Mohorianu, I., Dalmay, T., & Szittya, G. (2018). Ambient temperature regulates the expression of a small set of sRNAs influencing plant development through NF-YA2 and YUC2. Plant, Cell and Environment, 41, 2404e2417. Grain development and crop productivity: role of small RNA 455 Han, J., Fang, J., Wang, C., Yin, Y., Sun, X., Leng, X., & Song, C. (2014). Grapevine microRNAs responsive to exogenous gibberellin. BMC Genomics, 15, 1e17. Han, R., Jian, C., Lv, J., Yan, Y., Chi, Q., Li, Z., Wang, Q., Zhang, J., Liu, X., & Zhao, H. (2014). Identification and characterization of microRNAs in the flag leaf and developing seed of wheat (Triticum aestivum L.). BMC Genomics, 15, 289. Hendelman, A., Stav, R., Zemach, H., & Arazi, T. (2013). The tomato NAC transcription factor SlNAM2 is involved in flower-boundary morphogenesis. Journal of Experimental Botany, 64, 5497e5507. He, Z., Zeng, J., Ren, Y., Chen, D., Li, W., Gao, F., Cao, Y., Luo, T., Yuan, G., Wu, X., Liang, Y., Deng, Q., Wang, S., Zheng, A., Zhu, J., Liu, H., Wang, L., Li, P., & Li, S. (2017). OsGIF1positively regulates the sizes of stems, leaves, and grains in rice. Frontiers in Plant Science, 8, 1730-1730. Hewezi, T., Maier, T. R., Nettleton, D., & Baum, T. J. (2012). The Arabidopsis microRNA396-GRF1/GRF3 regulatory module acts as a developmental regulator in the reprogramming of root cells during cyst nematode infection. Plant Physiology, 159, 321e335. Houston, K., McKim, S. M., Comadran, J., Bonar, N., Druka, I., Uzrek, N., Cirillo, E., Guzy-Wrobelska, J., Collins, N. C., Halpin, C., Hansson, M., Dockter, C., Druka, A., & Waugh, R. (2013). Variation in the interaction between alleles of HvAPETALA2 and microRNA172 determines the density of grains on the barley inflorescence. Proceedings of the National Academy of Sciences of the United States of America, 110, 16675e16680. Huang, C.-Y., Araujo, K., Sánchez, J. N., Kund, G., Trumble, J., Roper, C., Godfrey, K. E., & Jin, H. (2021). A stable antimicrobial peptide with dual functions of treating and preventing citrus Huanglongbing. Proceedings of the National Academy of Sciences of the United States of America, 118. e2019628118. Huang, D., Feurtado, J. A., Smith, M. A., Flatman, L. K., Koh, C., & Cutler, A. J. (2017). Long noncoding miRNA gene represses wheat b-diketone waxes. Proceedings of the National Academy of Sciences of the United States of America, 114, E3149eE3158. Huang, D., Koh, C., Feurtado, J. A., Tsang, E., & Cutler, A. (2013). MicroRNAs and their putative targets in Brassica napus seed maturation. BMC Genomics, 14, 140. Huang, J.-H., Lin, X.-J., Zhang, L.-Y., Wang, X.-D., Fan, G.-C., & Chen, L.-S. (2019). MicroRNA sequencing revealed citrus adaptation to long-term boron toxicity through modulation of root development by miR319 and miR171. International Journal of Molecular Sciences, 20, 1422. Huang, J., Li, Z., & Zhao, D. (2016). Deregulation of the OsmiR160 target gene OsARF18 causes growth and developmental defects with an alteration of auxin signaling in rice. Scientific Reports, 6, 29938. Huang, W., Peng, S., Xian, Z., Lin, D., Hu, G., Yang, L., Ren, M., & Li, Z. (2017). Overexpression of a tomato miR171 target gene SlGRAS24 impacts multiple agronomical traits via regulating gibberellin and auxin homeostasis. Plant Biotechnology Journal, 15, 472e488. Huang, S., Zhou, J., Gao, L., & Tang, Y. (2021). Plant miR397 and its functions. Functional Plant Biology, 48, 361e370. Jeong, D.-H., Park, S., Zhai, J., Gurazada, S. G. R., De Paoli, E., Meyers, B. C., & Green, P. J. (2011). Massive analysis of rice small RNAs: Mechanistic implications of regulated microRNAs and variants for differential target RNA cleavage. The Plant Cell, 23, 4185e4207. Jiang, D., Chen, W., Dong, J., Li, J., Yang, F., Wu, Z., Zhou, H., Wang, W., & Zhuang, C. (2018). Overexpression of miR164b-resistant OsNAC2 improves plant architecture and grain yield in rice. Journal of Experimental Botany, 69, 1533e1543. Jiang, C., Fan, Z., Li, Z., Niu, D., Li, Y., Zheng, M., Wang, Q., Jin, H., & Guo, J. (2020). Bacillus cereus AR156 triggers induced systemic resistance against Pseudomonas syringae pv. tomato DC3000 by suppressing miR472 and activating CNLs-mediated basal immunity in Arabidopsis. Molecular Plant Pathology, 21, 854e870. 456 Plant Small RNA in Food Crops Jiang, M., He, Y., Chen, X., Zhang, X., Guo, Y., Yang, S., Huang, J., & Traw, M. B. (2020). CRISPR-based assessment of genomic structure in the conserved SQUAMOSA promoter-binding-like gene clusters in rice. The Plant Journal, 104, 1301e1314. Jiang, Y., Li, Z., Liu, X., Zhu, T., Xie, K., Hou, Q., Yan, T., Niu, C., Zhang, S., Yang, M., Xie, R., Wang, J., Li, J., An, X., & Wan, X. (2021). ZmFAR1 and ZmABCG26 regulated by microRNA are essential for lipid metabolism in maize anther. International Journal of Molecular Sciences, 22. Jiang, J., Lv, M., Liang, Y., Ma, Z., & Cao, J. (2014). Identification of novel and conserved miRNAs involved in pollen development in Brassica campestris ssp. chinensis by highthroughput sequencing and degradome analysis. BMC Genomics, 15, 146. Jiang, J., Xu, P., Li, Y., Li, Y., Zhou, X., Jiang, M., Zhang, J., Zhu, J., Wang, W., & Yang, L. (2021). Identification of miRNAs and their target genes in genic male sterility lines in Brassica napus by small RNA sequencing. BMC Plant Biology, 21, 520. Jiao, Y., Wang, Y., Xue, D., Wang, J., Yan, M., Liu, G., Dong, G., Zeng, D., Lu, Z., Zhu, X., Qian, Q., & Li, J. (2010). Regulation of OsSPL14 by OsmiR156 defines ideal plant architecture in rice. Nature Genetics, 42, 541e544. Jia, X. Y., Shen, J., Liu, H., Li, F., Ding, N., Gao, C. Y., Pattanaik, S., Patra, B., Li, R. Z., & Yuan, L. (2015). Small tandem target mimic-mediated blockage of microRNA858 induces anthocyanin accumulation in tomato. Planta, 242, 283e293. Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. (2012). A programmable dual-RNAeguided DNA endonuclease in adaptive bacterial immunity. Science, 337, 816e821. Jin, X., Fu, Z., Lv, P., Peng, Q., Ding, D., Li, W., & Tang, J. (2015). Identification and characterization of microRNAs during maize grain filling. PLoS One, 10, e0125800. Kaja, E., Szczesniak, M. W., Jensen, P. J., Axtell, M. J., McNellis, T., & Makałowska, I. (2014). Identification of apple miRNAs and their potential role in fire blight resistance. Tree Genetics & Genomes, 11, 812. Kamiuchi, Y., Yamamoto, K., Furutani, M., Tasaka, M., & Aida, M. (2014). The CUC1 and CUC2 genes promote carpel margin meristem formation during Arabidopsis gynoecium development. Frontiers in Plant Science, 5, 165. Karlova, R., Rosin, F. M., Busscher-Lange, J., Parapunova, V., Do, P. T., Fernie, A. R., Fraser, P. D., Baxter, C., Angenent, G. C., & de Maagd, R. A. (2011). Transcriptome and metabolite profiling show that APETALA2a is a major regulator of tomato fruit ripening. The Plant Cell, 23, 923e941. Karlova, R., van Haarst, J. C., Maliepaard, C., van de Geest, H., Bovy, A. G., Lammers, M., Angenent, G. C., & de Maagd, R. A. (2013). Identification of microRNA targets in tomato fruit development using high-throughput sequencing and degradome analysis. Journal of Experimental Botany, 64, 1863e1878. Keller, M., Schleiff, E., & Simm, S. (2020). miRNAs involved in transcriptome remodeling during pollen development and heat stress response in Solanum lycopersicum. Scientific Reports, 10, 10694. Kelley, D. R., Skinner, D. J., & Gasser, C. S. (2009). Roles of polarity determinants in ovule development. The Plant Journal, 57, 1054e1064. Kidner, C. A., & Martienssen, R. A. (2004). Spatially restricted microRNA directs leaf polarity through ARGONAUTE1. Nature, 428, 81e84. Kim, J., Jung, J.-H., Reyes, J. L., Kim, Y.-S., Kim, S.-Y., Chung, K.-S., Kim, J. A., Lee, M., Lee, Y., Narry Kim, V., Chua, N.-H., & Park, C.-M. (2005). MicroRNA-directed cleavage of ATHB15 mRNA regulates vascular development in Arabidopsis inflorescence stems. The Plant Journal, 42, 84e94. Kitagawa, M., Wu, P., Balkunde, R., Cunniff, P., & Jackson, D. (2022). An RNA exosome subunit mediates cell-to-cell trafficking of a homeobox mRNA via plasmodesmata. Science, 375, 177e182. Grain development and crop productivity: role of small RNA 457 Klesen, S., Hill, K., & Timmermans, M. C. P. (2020). Small RNAs as plant morphogens. Current Topics in Developmental Biology, 137, 455e480. Kondhare, K. R., Kumar, A., Patil, N. S., Malankar, N. N., Saha, K., & Banerjee, A. K. (2021). Development of aerial and belowground tubers in potato is governed by photoperiod and epigenetic mechanism. Plant Physiology, 187, 1071e1086. Kong, X., He, M., Guo, Z., Zhou, Y., Chen, Z., Qu, H., & Zhu, H. (2021). microRNA regulation of fruit development, quality formation and stress response. Fruit Research, 1, 5. Körbes, A. P., Machado, R. D., Guzman, F., Almerão, M. P., de Oliveira, L. F. V., LossMorais, G., Turchetto-Zolet, A. C., Cagliari, A., dos Santos Maraschin, F., MargisPinheiro, M., & Margis, R. (2012). Identifying conserved and novel microRNAs in developing seeds of Brassica napus using deep sequencing. PLoS One, 7, e50663. Kozomara, A., Birgaoanu, M., & Griffiths-Jones, S. (2019). miRBase: from microRNA sequences to function. Nucleic Acids Research, 47, D155eD162. Kravchik, M., Stav, R., Belausov, E., & Arazi, T. (2019). Functional characterization of microRNA171 family in tomato. Plants, 8, 10. Kruszka, K., Pieczynski, M., Windels, D., Bielewicz, D., Jarmolowski, A., SzweykowskaKulinska, Z., & Vazquez, F. (2012). Role of microRNAs and other sRNAs of plants in their changing environments. Journal of Plant Physiology, 169, 1664e1672. Kumar, A., Gautam, V., Kumar, P., Mukherjee, S., Verma, S., & Sarkar, A. K. (2019). Identification and co-evolution pattern of stem cell regulator miR394s and their targets among diverse plant species. BMC Evolutionary Biology, 19, 55. Lee, E. K., Cibrian-Jaramillo, A., Kolokotronis, S.-O., Katari, M. S., Stamatakis, A., Ott, M., Chiu, J. C., Little, D. P., Stevenson, D. W., McCombie, W. R., Martienssen, R. A., Coruzzi, G., & DeSalle, R. (2011). A functional phylogenomic view of the seed plants. PLoS Genetics, 7, e1002411. Lee, S.-J., Lee, B. H., Jung, J.-H., Park, S. K., Song, J. T., & Kim, J. H. (2017). Growthregulating factor and GRF-interacting factor specify meristematic cells of gynoecia and anthers. Plant Physiology, 176, 717e729. Lee, S.-H., Li, C.-W., Koh, K. W., Chuang, H.-Y., Chen, Y.-R., Lin, C.-S., & Chan, M.T. (2014). MSRB7 reverses oxidation of GSTF2/3 to confer tolerance of Arabidopsis thaliana to oxidative stress. Journal of Experimental Botany, 65, 5049e5062. Li, Y., Cui, W., Wang, R., Lin, M., Zhong, Y., Sun, L., Qi, X., & Fang, J. (2019). MicroRNA858-mediated regulation of anthocyanin biosynthesis in kiwifruit (Actinidia arguta) based on small RNA sequencing. PLoS One, 14, e0217480. Li, R., Fu, D., Zhu, B., Luo, Y., & Zhu, H. (2018). CRISPR/Cas9-mediated mutagenesis of lncRNA1459 alters tomato fruit ripening. The Plant Journal, 94, 513e524. Li, M., Liang, Z., He, S., Zeng, Y., Jing, Y., Fang, W., Wu, K., Wang, G., Ning, X., Wang, L., Li, S., Tan, H., & Tan, F. (2017). Genome-wide identification of leaf abscission associated microRNAs in sugarcane (Saccharum officinarum L.). BMC Genomics, 18, 754. Li, Y., Li, J., Chen, Z., Wei, Y., Qi, Y., & Wu, C. (2020). OsmiR167a-targeted auxin response factors modulate tiller angle via fine-tuning auxin distribution in rice. Plant Biotechnology Journal, 18, 2015e2026. Li, D., Liu, Z., Gao, L., Wang, L., Gao, M., Jiao, Z., Qiao, H., Yang, J., Chen, M., Yao, L., Liu, R., & Kan, Y. (2016). Genome-wide identification and characterization of microRNAs in developing grains of Zea mays L. PLoS One, 11, e0153168. Li, T., Ma, L., Geng, Y., Hao, C., Chen, X., & Zhang, X. (2015). Small RNA and degradome sequencing reveal complex roles of miRNAs and their targets in developing wheat grains. PLoS One, 10, e0139658. Li, D., Mou, W., Luo, Z., Li, L., Limwachiranon, J., Mao, L., & Ying, T. (2016). Developmental and stress regulation on expression of a novel miRNA, Fan-miR73 and its target ABI5 in strawberry. Scientific Reports, 6, 28385. 458 Plant Small RNA in Food Crops Li, D., Mou, W., Xia, R., Li, L., Zawora, C., Ying, T., Mao, L., Liu, Z., & Luo, Z. (2019). Integrated analysis of high-throughput sequencing data shows abscisic acid-responsive genes and miRNAs in strawberry receptacle fruit ripening. Horticultural Research, 6, 26. Li, F., Pignatta, D., Bendix, C., Brunkard, J. O., Cohn, M. M., Tung, J., Sun, H., Kumar, P., & Baker, B. (2012). MicroRNA regulation of plant innate immune receptors. Proceedings of the National Academy of Sciences of the United States of America, 109, 1790e1795. Li, X., Staszewski, L., Xu, H., Durick, K., Zoller, M., & Adler, E. (2002). Human receptors for sweet and umami taste. Proceedings of the National Academy of Sciences of the United States of America, 99, 4692e4696. Li, P., Su, T., Zhang, D., Wang, W., Xin, X., Yu, Y., Zhao, X., Yu, S., & Zhang, F. (2021). Genome-wide analysis of changes in miRNA and target gene expression reveals key roles in heterosis for Chinese cabbage biomass. Horticultural Research, 8, 39. Li, G., Wang, L., Yang, J., He, H., Jin, H., Li, X., Ren, T., Ren, Z., Li, F., Han, X., Zhao, X., Dong, L., Li, Y., Song, Z., Yan, Z., Zheng, N., Shi, C., Wang, Z., Yang, S., … Wang, D. (2021). A high-quality genome assembly highlights rye genomic characteristics and agronomically important genes. Nature Genetics, 53, 574e584. Li, Y.-F., Wei, K., Wang, M., Wang, L., Cui, J., Zhang, D., Guo, J., Zhao, M., & Zheng, Y. (2019). Identification and temporal expression analysis of conserved and novel microRNAs in the leaves of winter wheat grown in the field. Frontiers in Genetics, 10, 779. Li, Y.-F., Zheng, Y., Addo-Quaye, C., Zhang, L., Saini, A., Jagadeeswaran, G., Axtell, M. J., Zhang, W., & Sunkar, R. (2010). Transcriptome-wide identification of microRNA targets in rice. The Plant Journal, 62, 742e759. Liang, G., He, H., Li, Y., Wang, F., & Yu, D. (2013). Molecular mechanism of microRNA396 mediating pistil development in Arabidopsis. Plant Physiology, 164, 249e258. Lin, W., Gupta, S. K., Arazi, T., & Spitzer-Rimon, B. (2021). MIR172dis required for floral organ identity and number in tomato. International Journal of Molecular Sciences, 22, 4659. Lin, W.-Y., Lin, Y.-Y., Chiang, S.-F., Syu, C., Hsieh, L.-C., & Chiou, T.-J. (2018). Evolution of microRNA827 targeting in the plant kingdom. New Phytologist, 217, 1712e1725. Liu, J., Cheng, X., Liu, P., & Sun, J. (2017). miR156-targeted SBP-box transcription factors interact with DWARF53 to regulate TEOSINTE BRANCHED1 and BARREN STALK1 expression in bread wheat. Plant Physiology, 174, 1931e1948. Liu, L., Gallagher, J., Arevalo, E. D., Chen, R., Skopelitis, T., Wu, Q., Bartlett, M., & Jackson, D. (2021). Enhancing grain-yield-related traits by CRISPReCas9 promoter editing of maize CLE genes. Native Plants, 7, 287e294. Liu, X., Huang, S., & Xie, H. (2021). Advances in the regulation of plant development and stress response by miR167. Frontiers in Bioscience, 26, 655e665. Liu, J., Hua, W., Yang, H. L., Zhan, G. M., Li, R. J., Deng, L. B., Wang, X. F., Liu, G. H., & Wang, H. Z. (2012). The BnGRF2 gene (GRF2-likegene from Brassica napus) enhances seed oil production through regulating cell number and plant photosynthesis. Journal of Experimental Botany, 63, 3727e3740. Liu, J., Liu, X., Zhang, S., Liang, S., Luan, W., & Ma, X. (2021). TarDB: An online database for plant miRNA targets and miRNA-triggered phased siRNAs. BMC Genomics, 22, 348. Liu, P. P., Montgomery, T. A., Fahlgren, N., Kasschau, K. D., Nonogaki, H., & Carrington, J. C. (2007). Repression of AUXIN RESPONSE FACTOR10 by microRNA160 is critical for seed germination and post-germination stages. The Plant Journal, 52, 133e146. Grain development and crop productivity: role of small RNA 459 Liu, Y., Wang, L., Chen, D., Wu, X., Huang, D., Chen, L., Li, L., Deng, X., & Xu, Q. (2014). Genome-wide comparison of microRNAs and their targeted transcripts among leaf, flower and fruit of sweet orange. BMC Genomics, 15, 695. Lopez-Ortiz, C., Peña-Garcia, Y., Bhandari, M., Abburi, V. L., Natarajan, P., Stommel, J., Nimmakayala, P., & Reddy, U. K. (2021). Identification of miRNAs and their targets involved in flower and fruit development across domesticated and wild Capsicum species. International Journal of Molecular Sciences, 22, 4866. Lu, C., Jeong, D. H., Kulkarni, K., Pillay, M., Nobuta, K., German, R., Thatcher, S. R., Maher, C., Zhang, L., Ware, D., Liu, B., Cao, X., Meyers, B. C., & Green, P. J. (2008). Genome-wide analysis for discovery of rice microRNAs reveals natural antisense microRNAs (nat-miRNAs). Proceedings of the National Academy of Sciences of the United States of America, 105, 4951e4956. Lu, Y.-B., Yang, L.-T., Qi, Y.-P., Li, Y., Li, Z., Chen, Y.-B., Huang, Z.-R., & Chen, L.-S. (2014). Identification of boron-deficiency-responsive microRNAs in Citrus sinensis roots by illumina sequencing. BMC Plant Biology, 14, 123. Luján-Soto, E., Juárez-González, V. T., Reyes, J. L., & Dinkova, T. D. (2021). microRNA zma-miR528 versatile regulation on target mRNAs during maize somatic embryogenesis. International Journal of Molecular Sciences, 22, 5310. Lunardon, A., Johnson, N. R., Hagerott, E., Phifer, T., Polydore, S., Coruh, C., & Axtell, M. J. (2020). Integrated annotations and analyses of small RNA-producing loci from 47 diverse plants. Genome Research, 30, 497e513. Luo, X., Gao, Z., Shi, T., Cheng, Z., Zhang, Z., & Ni, Z. (2013). Identification of miRNAs and their target genes in peach (Prunus persica L.) using high-throughput sequencing and degradome analysis. PLoS One, 8, e79090. Luo, L., Yang, X., Guo, M., Lan, T., Yu, Y., Mo, B., Chen, X., Gao, L., & Liu, L. (2021). TRANS-ACTING SIRNA3-derived short interfering RNAs confer cleavage of mRNAs in rice. Plant Physiology, 188, 347e362. Ma, X., Denyer, T., Javelle, M., Feller, A., & Timmermans, M. C. P. (2021). Genome-wide analysis of plant miRNA action clarifies levels of regulatory dynamics across developmental contexts. Genome Research, 31, 811e822. Ma, F., Huang, J., Yang, J., Zhou, J., Sun, Q., & Sun, J. (2020). Identification, expression and miRNA targeting of auxin response factor genes related to phyllody in the witches’ broom disease of jujube. Gene, 746, 144656. Maizel, A., Markmann, K., Timmermans, M., & Wachter, A. (2020). To move or not to move: Roles and specificity of plant RNA mobility. Current Opinion in Plant Biology, 57, 52e60. _ Martin, A., Adam, Hln, Díaz-Mendoza, M., Zurczak, M., González-Schain, N. D., & Suárez-López, P. (2009). Graft-transmissible induction of potato tuberization by the microRNA miR172. Development, 136, 2873e2881. Martinelli, F., Marchese, A., Giovino, A., Marra, F. P., Della Noce, I., Caruso, T., & Dandekar, A. M. (2019). In-field and early detection of Xylella fastidiosa infections in olive using a portable instrument. Frontiers in Plant Science, 9, 2007. Meng, F., Liu, H., Wang, K., Liu, L., Wang, S., Zhao, Y., Yin, J., & Li, Y. (2013). Development-associated microRNAs in grains of wheat (Triticum aestivum L.). BMC Plant Biology, 13, 140. Meng, X., Li, A., Yu, B., & Li, S. (2021). Interplay between miRNAs and lncRNAs: Mode of action and biological roles in plant development and stress adaptation. Computational and Structural Biotechnology Journal, 19, 2567e2574. Miao, C., Wang, Z., Zhang, L., Yao, J., Hua, K., Liu, X., Shi, H., & Zhu, J.-K. (2019). The grain yield modulator miR156 regulates seed dormancy through the gibberellin pathway in rice. Nature Communications, 10, 3822. Mica, E., Piccolo, V., Delledone, M., Ferrarini, A., Pezzotti, M., Casati, C., Del Fabbro, C., Valle, G., Policriti, A., Morgante, M., Pesole, G., Pe, M. E., & Horner, D. S. (2009). High throughput approaches reveal splicing of primary microRNA transcripts and tissue specific expression of mature microRNAs in Vitis vinifera. BMC Genomics, 10, 558. 460 Plant Small RNA in Food Crops Miura, K., Ikeda, M., Matsubara, A., Song, X. J., Ito, M., Asano, K., Matsuoka, M., Kitano, H., & Ashikari, M. (2010). OsSPL14 promotes panicle branching and higher grain productivity in rice. Nature Genetics, 42. 545-U102. Mohorianu, I., Schwach, F., Jing, R. C., Lopez-Gomollon, S., Moxon, S., Szittya, G., Sorefan, K., Moulton, V., & Dalmay, T. (2011). Profiling of short RNAs during fleshy fruit development reveals stage-specific sRNAome expression patterns. The Plant Journal, 67, 232e246. Molnar, A., Melnyk, C. W., Bassett, A., Hardcastle, T. J., Dunn, R., & Baulcombe, D. C. (2010). Small silencing RNAs in plants are mobile and direct epigenetic modification in recipient cells. Science, 328, 872e875. Moran, Y., Agron, M., Praher, D., & Technau, U. (2017). The evolutionary origin of plant and animal microRNAs. Nature Ecology and Evolution, 1, 0027. Morel, P., Heijmans, K., Rozier, F., Zethof, J., Chamot, S., Bento, S. R., VialetteGuiraud, A., Chambrier, P., Trehin, C., & Vandenbussche, M. (2017). Divergence of the floral A-function between an asterid and a rosid species. The Plant Cell, 29, 1605e1621. Moxon, S., Jing, R. C., Szittya, G., Schwach, F., Pilcher, R. L. R., Moulton, V., & Dalmay, T. (2008). Deep sequencing of tomato short RNAs identifies microRNAs targeting genes involved in fruit ripening. Genome Research, 18, 1602e1609. Nadiya, F., Anjali, N., Thomas, J., Gangaprasad, A., & Sabu, K. K. (2019). Deep sequencing identified potential miRNAs involved in defence response, stress and plant growth characteristics of wild genotypes of cardamom. Plant Biology, 21, 3e14. Nair, S. K., Wang, N., Turuspekov, Y., Pourkheirandish, M., Sinsuwongwat, S., Chen, G., Sameri, M., Tagiri, A., Honda, I., Watanabe, Y., Kanamori, H., Wicker, T., Stein, N., Nagamura, Y., Matsumoto, T., & Komatsuda, T. (2010). Cleistogamous flowering in barley arises from the suppression of microRNA-guided HvAP2 mRNA cleavage. Proceedings of the National Academy of Sciences of the United States of America, 107, 490e495. Olmedo-Monfil, V., Duran-Figueroa, N., Arteaga-Vazquez, M., Demesa-Arevalo, E., Autran, D., Grimanelli, D., Slotkin, R. K., Martienssen, R. A., & Vielle-Calzada, J. P. (2010). Control of female gamete formation by a small RNA pathway in Arabidopsis. Nature, 464, 628e632. Owusu Adjei, M., Zhou, X., Mao, M., Rafique, F., & Ma, J. (2021). MicroRNAs roles in plants secondary metabolism. Plant Signaling & Behavior, 16, 1915590. Pabón-Mora, N., Ambrose, B. A., & Litt, A. (2012). Poppy APETALA1/FRUITFULL orthologs control flowering time, branching, perianth identity, and fruit development. Plant Physiology, 158, 1685e1704. Pachamuthu, K., Swetha, C., Basu, D., Das, S., Singh, I., Sundar, V. H., Sujith, T. N., & Shivaprasad, P. V. (2021). Rice-specific argonaute 17 controls reproductive growth and yield-associated phenotypes. Plant Molecular Biology, 105, 99e114. Pagliarani, C., Vitali, M., Ferrero, M., Vitulo, N., Incarbone, M., Lovisolo, C., Valle, G., & Schubert, A. (2017). The accumulation of miRNAs differentially modulated by drought stress is affected by grafting in grapevine. Plant Physiology, 173, 2180e2195. Paim Pinto, D. L., Brancadoro, L., Dal Santo, S., De Lorenzis, G., Pezzotti, M., Meyers, B. C., Pè, M. E., & Mica, E. (2016). The influence of genotype and environment on small RNA profiles in grapevine berry. Frontiers in Plant Science, 7, 1459. Palatnik, J. F., Allen, E., Wu, X. L., Schommer, C., Schwab, R., Carrington, J. C., & Weigel, D. (2003). Control of leaf morphogenesis by microRNAs. Nature, 425, 257e263. Pan, J., Huang, D., Guo, Z., Kuang, Z., Zhang, H., Xie, X., Ma, Z., Gao, S., Lerdau, M. T., Chu, C., & Li, L. (2018). Overexpression of microRNA408 enhances photosynthesis, growth, and seed yield in diverse plants. Journal of Integrative Plant Biology, 60, 323e340. Grain development and crop productivity: role of small RNA 461 Pantaleo, V., Szittya, G., Moxon, S., Miozzi, L., Moulton, V., Dalmay, T., & Burgyan, J. (2010). Identification of grapevine microRNAs and their targets using high-throughput sequencing and degradome analysis. The Plant Journal, 62, 960e976. Pantaleo, V., Vitali, M., Boccacci, P., Miozzi, L., Cuozzo, D., Chitarra, W., Mannini, F., Lovisolo, C., & Gambino, G. (2016). Novel functional microRNAs from virus-free and infected Vitis vinifera plants under water stress. Scientific Reports, 6, 20167. Pant, B. D., Buhtz, A., Kehr, J., & Scheible, W.-R. (2008). MicroRNA399 is a longdistance signal for the regulation of plant phosphate homeostasis. The Plant Journal, 53, 731e738. Patel, P., Mathioni, S. M., Hammond, R., Harkess, A. E., Kakrana, A., Arikit, S., Dusia, A., & Meyers, B. C. (2021). Reproductive phasiRNA loci and DICER-LIKE5, but not microRNA loci, diversified in monocotyledonous plants. Plant Physiology, 185, 1764e1782. Patel, P., Yadav, K., Srivastava, A. K., Suprasanna, P., & Ganapathi, T. R. (2019). Overexpression of native Musa-miR397 enhances plant biomass without compromising abiotic stress tolerance in banana. Scientific Reports, 9, 16434. Peng, T., Sun, H., Du, Y., Zhang, J., Li, J., Liu, Y., Zhao, Y., & Zhao, Q. (2013). Characterization and expression patterns of microRNAs involved in rice grain filling. PLoS One, 8, e54148. Peng, T., Sun, H., Qiao, M., Zhao, Y., Du, Y., Zhang, J., Li, J., Tang, G., & Zhao, Q. (2014). Differentially expressed microRNA cohorts in seed development may contribute to poor grain filling of inferior spikelets in rice. BMC Plant Biology, 14, 196. Petrella, R., Cucinotta, M., Mendes, M. A., Underwood, C. J., & Colombo, L. (2021). The emerging role of small RNAs in ovule development, a kind of magic. Plant Reproduction, 34, 335e351. Platt, R. N., 2nd, Vandewege, M. W., Kern, C., Schmidt, C. J., Hoffmann, F. G., & Ray, D. A. (2014). Large numbers of novel miRNAs originate from DNA transposons and are coincident with a large species radiation in bats. Molecular Biology and Evolution, 31, 1536e1545. Poethig, R. S. (2009). Small RNAs and developmental timing in plants. Current Opinion in Genetics & Development, 19, 374e378. Prakash, S., Bhat, S. R., Quiros, C. F., Kirti, P. B., & Chopra, V. L. (2009). Brassica and its close allies: Cytogenetics and evolution. In Plant breeding reviews (pp. 21e187). Puchta, M., Groszyk, J., Małecka, M., Koter, M. D., Niedzielski, M., RakoczyTrojanowska, M., & Boczkowska, M. (2021). Barley seeds miRNome stability during long-term storage and aging. International Journal of Molecular Sciences, 22, 4315. Purugganan, M. D., Boyles, A. L., & Suddith, J. I. (2000). Variation and selection at the CAULIFLOWER floral homeotic gene accompanying the evolution of domesticated Brassica oleracea. Genetics, 155, 855e862. Qu, D., Yan, F., Meng, R., Jiang, X., Yang, H., Gao, Z., Dong, Y., Yang, Y., & Zhao, Z. (2016). Identification of microRNAs and their targets associated with fruit-bagging and subsequent sunlight re-exposure in the “Granny Smith” apple exocarp using highthroughput sequencing. Frontiers in Plant Science, 7, 27. Ren, C., Li, H., Liu, Y., Li, S., & Liang, Z. (2022). Highly efficient activation of endogenous gene in grape using CRISPR/dCas9-based transcriptional activators. Horticultural Research, 9. uhab037, online ahead of print. Reyes, J. L., & Chua, N. H. (2007). ABA induction of miR159 controls transcript levels of two MYB factors during Arabidopsis seed germination. The Plant Journal, 49, 592e606. Ripoll, J. J., Bailey, L. J., Mai, Q.-A., Wu, S. L., Hon, C. T., Chapman, E. J., Ditta, G. S., Estelle, M., & Yanofsky, M. F. (2015). microRNA regulation of fruit growth. Native Plants, 1, 15036. 462 Plant Small RNA in Food Crops Robinson-Beers, K., Pruitt, R. E., & Gasser, C. S. (1992). Ovule development in wild-type Arabidopsis and two female-sterile mutants. The Plant Cell, 4, 1237e1249. Rock, C. D. (2013). Trans-acting small interfering RNA4: Key to nutraceutical synthesis in grape development? Trends in Plant Science, 18, 601e610. Rock, C. (2020). A role for MIR828 in pineapple fruit development. F1000Research, 9, 16. Rodrigues, A. S., & Miguel, C. M. (2017). The pivotal role of small non-coding RNAs in the regulation of seed development. Plant Cell Reports, 36, 653e667. Rodríguez-Leal, D., Lemmon, Z. H., Man, J., Bartlett, M. E., & Lippman, Z. B. (2017). Engineering quantitative trait variation for crop improvement by genome editing. Cell, 171, 470e480. e478. Rodriguez, R. E., Mecchia, M. A., Debernardi, J. M., Schommer, C., Weigel, D., & Palatnik, J. F. (2010). Control of cell proliferation in Arabidopsis thaliana by microRNA miR396. Development, 137, 103e112. Rosas Cárdenas, F. D. F., Ruiz Suárez, Y., Cano Rangel, R. M., Luna Garcia, V., González Aguilera, K. L., Marsch Martínez, N., & De Folter, S. (2017). Effect of constitutive miR164 expression on plant morphology and fruit development in Arabidopsis and tomato. Agronomy, 7, 48. Ruan, W., Guo, M., Wang, X., Guo, Z., Xu, Z., Xu, L., Zhao, H., Sun, H., Yan, C., & Yi, K. (2019). Two RING-finger ubiquitin E3 ligases regulate the degradation of SPX4, an internal phosphate sensor, for phosphate homeostasis and signaling in rice. Molecular Plant, 12, 1060e1074. Ruvkun, G. (2001). Molecular biology: Glimpses of a tiny RNA world. Science, 294, 797e799. Santin, F., Bhogale, S., Fantino, E., Grandellis, C., Banerjee, A. K., & Ulloa, R. M. (2017). Solanum tuberosum StCDPK1 is regulated by miR390 at the posttranscriptional level and phosphorylates the auxin efflux carrier StPIN4 in vitro, a potential downstream target in potato development. Physiologia Plantarum, 159, 244e261. Schauer, S. E., Jacobsen, S. E., Meinke, D. W., & Ray, A. (2002). DICER-LIKE1: Blind men and elephants in Arabidopsis development. Trends in Plant Science, 7, 487e491. Seo, E., Kim, T., Park, J. H., Yeom, S. I., Kim, S., Seo, M. K., Shin, C., & Choi, D. (2018). Genome-wide comparative analysis in Solanaceous species reveals evolution of microRNAs targeting defense genes in Capsicum spp. DNA Research, 25, 561e575. Seymour, G. B., Chapman, N. H., Chew, B. L., & Rose, J. K. C. (2013). Regulation of ripening and opportunities for control in tomato and other fruits. Plant Biotechnology Journal, 11, 269e278. Sharma, D., Tiwari, M., Pandey, A., Bhatia, C., Sharma, A., & Trivedi, P. K. (2016). MicroRNA858 is a potential regulator of phenylpropanoid pathway and plant development. Plant Physiology, 171, 944e959. Shi, M., Hu, X., Wei, Y., Hou, X., Yuan, X., Liu, J., & Liu, Y. (2017). Genome-wide profiling of small RNAs and degradome revealed conserved regulations of miRNAs on auxin-responsive genes during fruit enlargement in peaches. International Journal of Molecular Sciences, 18, 2599. Shivaprasad, P. V., Chen, H. M., Patel, K., Bond, D. M., Santos, B., & Baulcombe, D. C. (2012). A microRNA superfamily regulates nucleotide binding site-leucine-rich repeats and other mRNAs. The Plant Cell, 24, 859e874. Shi, F., Zhou, X., Yao, M. M., Tan, Z., Zhou, Q., Zhang, L., & Ji, S. J. (2019). miRNAs play important roles in aroma weakening during the shelf life of ’Nanguo’ pear after cold storage. Food Res internatl (Ottawa, Ont), 116, 942e952. Si, L., Chen, J., Huang, X., Gong, H., Luo, J., Hou, Q., Zhou, T., Lu, T., Zhu, J., Shangguan, Y., Chen, E., Gong, C., Zhao, Q., Jing, Y., Zhao, Y., Li, Y., Cui, L., Fan, D., Lu, Y..., & Han, B (2016). OsSPL13controls grain size in cultivated rice. Nature Genetics, 48, 447e456. Grain development and crop productivity: role of small RNA 463 da Silva, E. M., & Nogueira, F. T. S. (2021). Guarding tomato fruit setting in adverse temperatures through the miRNA166-SlHB15A regulatory module. Molecular Plant, 14, 1046e1048. da Silva, E. M., Silva, G., Bidoia, D. B., da Silva Azevedo, M., de Jesus, F. A., Pino, L. E., Peres, L. E. P., Carrera, E., López-Díaz, I., & Nogueira, F. T. S. (2017). microRNA159-targeted SlGAMYB transcription factors are required for fruit set in tomato. The Plant Journal, 92, 95e109. Silva, GFFe, Silva, E. M., da Silva Azevedo, M., Guivin, M. A. C., Ramiro, D. A., Figueiredo, C. R., Carrer, H., Peres, L. E. P., & Nogueira, F. T. S. (2014). microRNA156-targeted SPL/SBP box transcription factors regulate tomato ovary and fruit development. The Plant Journal, 78, 604e618. Soltis, P. S., Folk, R. A., & Soltis, D. E. (2019). Darwin review: Angiosperm phylogeny and evolutionary radiations. Proceedings of the Royal Society B: Biological Science, 286, 20190099. Song, J. B., Shu, X. X., Shen, Q., Li, B. W., Song, J., & Yang, Z. M. (2015). Altered fruit and seed development of transgenic rapeseed (Brassica napus) over-expressing microRNA394. PLoS One, 10, e0125427. Song, Z., Zhang, L., Wang, Y., Li, H., Li, S., Zhao, H., & Zhang, H. (2018). Constitutive expression of miR408 improves biomass and seed yield in Arabidopsis. Frontiers in Plant Science, 8. Sun, W., Chen, D., Xue, Y., Zhai, L., Zhang, D., Cao, Z., Liu, L., Cheng, C., Zhang, Y., & Zhang, Z. (2019). Genome-wide identification of AGO18b-bound miRNAs and phasiRNAs in maize by cRIP-seq. BMC Genomics, 20, 656. Sun, F., Guo, G., Du, J., Guo, W., Peng, H., Ni, Z., Sun, Q., & Yao, Y. (2014). Wholegenome discovery of miRNAs and their targets in wheat (Triticum aestivum L.). BMC Plant Biology, 14, 142. Sunitha, S., Loyola, R., Alcalde, J. A., Arce-Johnson, P., Matus, J. T., & Rock, C. D. (2019). The role of UV-B light on small RNA activity during grapevine berry development. G3, 9, 769e787. Sunitha, S., & Rock, C. D. (2020). CRISPR/Cas9-mediated targeted mutagenesis of TAS4 and MYBA7 loci in grapevine rootstock 101-14. Transgenic Research, 29, 355e367. Sunkar, R., & Zhu, J.-K. (2004). Novel and stress-regulated microRNAs and other small RNAs from Arabidopsis. The Plant Cell, 16, 2001e2019. Sun, M., Shen, Y., Li, H., Yang, J., Cai, X., Zheng, G., Zhu, Y., Jia, B., & Sun, X. (2019). The multiple roles of OsmiR535 in modulating plant height, panicle branching and grain shape. Plant Science, 283, 60e69. Sun, Z., Shu, L., Zhang, W., & Wang, Z. (2020). Cca-miR398 increases copper sulfate stress sensitivity via the regulation of CSD mRNA transcription levels in transgenic Arabidopsis thaliana. PeerJ, 8. e9105-e9105. Sun, W., Xu, X. H., Li, Y., Xie, L., He, Y., Li, W., Lu, X., Sun, H., & Xie, X. (2020). OsmiR530 acts downstream of OsPIL15 to regulate grain yield in rice. New Phytologist, 226, 823e837. Su, Z., Wang, X., Xuan, X., Sheng, Z., Jia, H., Emal, N., Liu, Z., Zheng, T., Wang, C., & Fang, J. (2021). Characterization and action mechanism analysis of VvmiR156b/c/dVvSPL9 module responding to multiple-hormone signals in the modulation of grape berry color formation. Foods, 10, 896. Swetha, C., Narjala, A., Pandit, A., Tirumalai, V., & Shivaprasad, P. V. (2022). Degradome comparison between wild and cultivated rice identifies differential targeting by miRNAs. BMC Genomics, 23, 53. Tang, J., & Chu, C. (2017). MicroRNAs in crop improvement: Fine-tuners for complex traits. Native Plants, 3, 17077. Tavares, S., Vesentini, D., Fernandes, J. C., Ferreira, R. B., Laureano, O., Ricardo-DaSilva, J. M., & Amâncio, S. (2013). Vitis vinifera secondary metabolism as affected by sulfate depletion: Diagnosis through phenylpropanoid pathway genes and metabolites. Plant Physiology and Biochemistry, 66, 118e126. 464 Plant Small RNA in Food Crops Taylor, R. S., Tarver, J. E., Foroozani, A., & Donoghue, P. C. (2017). MicroRNA annotation of plant genomesdDo it right or not at all. BioEssays, 39. Taylor, R. S., Tarver, J. E., Hiscock, S. J., & Donoghue, P. C. J. (2014). Evolutionary history of plant microRNAs. Trends in Plant Science, 19, 175e182. Teng, Z., Chen, Y., Yuan, Y., Peng, Y., Yi, Y., Yu, H., Yi, Z., Yang, J., Peng, Y., Duan, M., Zhang, Y., & Ye, N. (2022). Identification of microRNAs regulating grain filling of rice inferior spikelets in response to moderate soil drying post-anthesis. Crop Journal, 10, 962e971. Tirumalai, V., Swetha, C., Nair, A., Pandit, A., & Shivaprasad, P. V. (2019). miR828 and miR858 regulate VvMYB114 to promote anthocyanin and flavonol accumulation in grapes. Journal of Experimental Botany, 70, 4775e4792. Tripathi, R. K., Overbeek, W., & Singh, J. (2020). Global analysis of SBP gene family in Brachypodium distachyonreveals its association with spike development. Scientific Reports, 10, 15032. Uauy, C., Distelfeld, A., Fahima, T., Blechl, A., & Dubcovsky, J. (2006). A NAC gene regulating senescence improves grain protein, zinc, and iron content in wheat. Science, 314, 1298e1301. Wang, W., Bai, Y., Koilkonda, P., Guan, L., Zhuge, Y., Wang, X., Liu, Z., Jia, H., Wang, C., & Fang, J. (2020). Genome-wide identification and characterization of gibberellin metabolic and signal transduction (GA MST) pathway mediating seed and berry development (SBD) in grape (Vitis vinifera L.). BMC Plant Biology, 20, 384. Wang, R., Fang, Y.-N., Wu, X.-M., Qing, M., Li, C.-C., Xie, K.-D., Deng, X.-X., & Guo, W.-W. (2020). The miR399-Cs UBC24 module regulates reproductive development and male fertility in citrus. Plant Physiology, 183, 1681e1695. Wang, J., Feng, Y., Ding, X., Huo, J., & Nie, W.-F. (2021). Identification of long noncoding RNAs associated with tomato fruit expansion and ripening by strand-specific paired-end RNA sequencing. Horticulturae, 7, 522. Wang, H., Jiao, X., Kong, X., Hamera, S., Wu, Y., Chen, X., Fang, R., & Yan, Y. (2016). A signaling cascade from miR444 to RDR1 in rice antiviral RNA silencing pathway. Plant Physiology, 170, 2365e2377. Wang, C., Jogaiah, S., Zhang, W., Abdelrahman, M., & Fang, J. G. (2018). Spatio-temporal expression of miRNA159 family members and their GAMYB target gene during the modulation of gibberellin-induced grapevine parthenocarpy. Journal of Experimental Botany, 69, 3639e3650. Wang, S., Li, S., Liu, Q., Wu, K., Zhang, J., Wang, S., Wang, Y., Chen, X., Zhang, Y., Gao, C., Wang, F., Huang, H., & Fu, X. (2015). The OsSPL16-GW7 regulatory module determines grain shape and simultaneously improves rice yield and grain quality. Nature Genetics, 47, 949e954. Wang, M.-M., Li, T.-X., Wu, Y., Song, S.-W., Bai, T.-H., Jiao, J., Song, C.-H., & Zheng, X.-B. (2021). Genome-wide identification of microRNAs involved in the regulation of fruit ripening in apple (Malus domestica). Scientia Horticulturae, 289, 110416. Wang, Y., Wang, Y., Song, Z., & Zhang, H. (2016). Repression of MYBL2 by both microRNA858a and HY5 leads to the activation of anthocyanin biosynthetic pathway in Arabidopsis. Molecular Plant, 9, 1395e1405. Wang, W. Q., Wang, J., Wu, Y. Y., Li, D. W., Allan, A. C., & Yin, X. R. (2020). Genome-wide analysis of coding and non-coding RNA reveals a conserved miR164NACregulatory pathway for fruit ripening. New Phytologist, 225, 1618e1634. Wang, S., Wu, K., Yuan, Q., Liu, X., Liu, Z., Lin, X., Zeng, R., Zhu, H., Dong, G., Qian, Q., Zhang, G., & Fu, X. (2012). Control of grain size, shape and quality by OsSPL16 in rice. Nature Genetics, 44, 950e954. Grain development and crop productivity: role of small RNA 465 Wang, Y., Zhang, J., Cui, W., Guan, C., Mao, W., & Zhang, Z. (2017). Improvement in fruit quality by overexpressing miR399a in woodland strawberry. Journal of Agricultural and Food Chemistry, 65, 7361e7370. Wang, C.-Y., Zhang, S., Yu, Y., Luo, Y.-C., Liu, Q., Ju, C., Zhang, Y.-C., Qu, L.-H., Lucas, W. J., Wang, X., & Chen, Y.-Q. (2014). MiR397b regulates both lignin content and seed number in Arabidopsis via modulating a laccase involved in lignin biosynthesis. Plant Biotechnology Journal, 12, 1132e1142. Wang, Y., Zou, W., Xiao, Y., Cheng, L., Liu, Y., Gao, S., Shi, Z., Jiang, Y., Qi, M., Xu, T., & Li, T. (2018). MicroRNA1917 targets CTR4 splice variants to regulate ethylene responses in tomato. Journal of Experimental Botany, 69, 1011e1025. Wei, L. Q., Yan, L. F., & Wang, T. (2011). Deep sequencing on genome-wide scale reveals the unique composition and expression patterns of microRNAs in developing pollen of Oryza sativa. Genome Biology, 12, R53. Werner, S., Bartrina, I., & Schmülling, T. (2021). Cytokinin regulates vegetative phase change in Arabidopsis thaliana through the miR172/TOE1-TOE2 module. Nature Communications, 12, 5816. Wu, G. A., Terol, J., Ibanez, V., López-García, A., Pérez-Román, E., Borredá, C., Domingo, C., Tadeo, F. R., Carbonell-Caballero, J., Alonso, R., Curk, F., Du, D., Ollitrault, P., Roose, M. L., Dopazo, J., Gmitter, F. G., Rokhsar, D. S., & Talon, M. (2018). Genomics of the origin and evolution of Citrus. Nature, 554, 311e316. Wu, J., Wang, D., Liu, Y., Wang, L., Qiao, X., & Zhang, S. (2014). Identification of miRNAs involved in pear fruit development and quality. BMC Genomics, 15, 953. Wu, J., Zheng, S., Feng, G., & Yi, H. (2016). Comparative analysis of miRNAs and their target transcripts between a spontaneous late-ripening sweet orange mutant and its wildtype using small RNA and degradome sequencing. Frontiers in Plant Science, 7, 1416. Xia, R., Meyers, B. C., Liu, Z., Beers, E. P., Ye, S., & Liu, Z. (2013). MicroRNA super families descended from miR390 and their roles in secondary small interfering RNA biogenesis in Eudicots. The Plant Cell, 25, 1555e1572. Xia, R., Xu, J., Arikit, S., & Meyers, B. C. (2015). Extensive families of miRNAs and PHAS loci in Norway spruce demonstrate the origins of complex phasiRNA networks in seed plants. Molecular Biology and Evolution, 32, 2905e2918. Xia, R., Zhu, H., An, Y-q, Beers, E., & Liu, Z. (2012). Apple miRNAs and tasiRNAs with novel regulatory networks. Genome Biology, 13, R47. Xie, K., Wu, C., & Xiong, L. (2006). Genomic organization, differential expression, and interaction of SQUAMOSA promoter-binding-like transcription factors and microRNA156 in rice. Plant Physiology, 142, 280e293. Xin, C., Liu, W., Lin, Q., Zhang, X., Cui, P., Li, F., Zhang, G., Pan, L., Al-Amer, A., Mei, H., Al-Mssallem, I. S., Hu, S., Al-Johi, H. A., & Yu, J. (2015). Profiling microRNA expression during multi-staged date palm (Phoenix dactylifera L.) fruit development. Genomics, 105, 242e251. Xin, M., Yang, G., Yao, Y., Peng, H., Hu, Z., Sun, Q., Wang, X., & Ni, Z. (2015). Temporal small RNA transcriptome profiling unraveled partitioned miRNA expression in developing maize endosperms between reciprocal crosses. Frontiers in Plant Science, 6, 744. Xing, S., Salinas, M., Garcia-Molina, A., Höhmann, S., Berndtgen, R., & Huijser, P. (2013). SPL8 and miR156-targeted SPL genes redundantly regulate Arabidopsis gynoecium differential patterning. The Plant Journal, 75, 566e577. Xing, L., Zhu, M., Zhang, M., Li, W., Jiang, H., Zou, J., Wang, L., & Xu, M. (2017). High-throughput sequencing of small RNA transcriptomes in maize kernel identifies miRNAs involved in embryo and endosperm development. Genes, 8, 385. 466 Plant Small RNA in Food Crops Xu, Q., Liu, Y. L., Zhu, A. D., Wu, X. M., Ye, J. L., Yu, K. Q., Guo, W. W., & Deng, X. X. (2010). Discovery and comparative profiling of microRNAs in a sweet orange red-flesh mutant and its wild type. BMC Genomics, 11, 246. Xu, X., Yin, L., Ying, Q., Song, H., Xue, D., Lai, T., Xu, M., Shen, B., Wang, H., & Shi, X. (2013). High-throughput sequencing and degradome analysis identify miRNAs and their targets involved in fruit senescence of Fragaria ananassa. PLoS One, 8, e70959. Xue, M., Yi, H., & Wang, H. (2018). Identification of miRNAs involved in SO2 preservation in Vitis vinifera L. by deep sequencing. Environmental and Experimental Botany, 153, 218e228. Xue, L.-J., Zhang, J.-J., & Xue, H.-W. (2009). Characterization and expression profiles of miRNAs in rice seeds. Nucleic Acids Research, 37, 916e930. Yadava, P., Tamim, S., Zhang, H., Teng, C., Zhou, X., Meyers, B. C., & Walbot, V. (2021). Transgenerational conditioned male fertility of HD-ZIP IV transcription factor mutant ocl4: Impact on 21-nt phasiRNA accumulation in pre-meiotic maize anthers. Plant Reproduction, 34, 117e129. Yan, Y., Wei, M., Li, Y., Tao, H., Wu, H., Chen, Z., Li, C., & Xu, J. H. (2021). MiR529a controls plant height, tiller number, panicle architecture and grain size by regulating SPL target genes in rice (Oryza sativa L.). Plant Science, 302, 110728. Yang, J. H., Han, S. J., Yoon, E. K., & Lee, W. S. (2006). Evidence of an auxin signal pathway, microRNA167-ARF8-GH3, and its response to exogenous auxin in cultured rice cells. Nucleic Acids Research, 34, 1892e1899. Yang, T., Wang, Y., Liu, H., Zhang, W., Chai, M., Tang, G., & Zhang, Z. (2020). MicroRNA1917-CTR1-like protein kinase 4 impacts fruit development via tuning ethylene synthesis and response. Plant Science, 291, 110334. Yang, X., You, C., Wang, X., Gao, L., Mo, B., Liu, L., & Chen, X. (2021). Widespread occurrence of microRNA-mediated target cleavage on membrane-bound polysomes. Genome Biology, 22, 15. Yang, X., Zhao, X., Dai, Z., Ma, F., Miao, X., & Shi, Z. (2021). OsmiR396/growth regulating factor modulate rice grain size through direct regulation of embryo-specific miR408. Plant Physiology, 186, 519e533. Yao, X., Chen, J., Zhou, J., Yu, H., Ge, C., Zhang, M., Gao, X., Dai, X., Yang, Z.-N., & Zhao, Y. (2019). An essential role for miRNA167 in maternal control of embryonic and seed development. Plant Physiology, 180, 453e464. Yao, J. L., Xu, J., Cornille, A., Tomes, S., Karunairetnam, S., Luo, Z., Bassett, H., Whitworth, C., Rees-George, J., Ranatunga, C., Snirc, A., Crowhurst, R., de Silva, N., Warren, B., Deng, C., Kumar, S., Chagné, D., Bus, V. G., Volz, R. K..., & Gleave, A. P. (2015). A microRNA allele that emerged prior to apple domestication may underlie fruit size evolution. The Plant Journal, 84, 417e427. Yi, R., Zhu, Z., Hu, J., Qian, Q., Dai, J., & Ding, Y. (2013). Identification and expression analysis of microRNAs at the grain filling stage in rice (Oryza sativa L.) via deep sequencing. PLoS One, 8, e57863. Yu, Y., Sun, F., Chen, N., Sun, G., Wang, C. Y., & Wu, D. X. (2021). MiR396 regulatory network and its expression during grain development in wheat. Protoplasma, 258, 103e113. Yu, T., Tzeng, D. T. W., Li, R., Chen, J., Zhong, S., Fu, D., Zhu, B., Luo, Y., & Zhu, H. (2019). Genome-wide identification of long non-coding RNA targets of the tomato MADS box transcription factor RIN and function analysis. Annals of Botany, 123, 469e482. Zhai, Q., Zhang, X., Wu, F., Feng, H., Deng, L., Xu, L., Zhang, M., Wang, Q., & Li, C. (2015). Transcriptional mechanism of jasmonate receptor COI1-mediated delay of flowering time in Arabidopsis. The Plant Cell, 27, 2814e2828. Zhang, X., Liu, W., Nagae, T. T., Takeuchi, H., Zhang, H., Han, Z., Higashiyama, T., & Chai, J. (2017). Structural basis for receptor recognition of pollen tube attraction peptides. Nature Communications, 8, 1331. Grain development and crop productivity: role of small RNA 467 Zhang, K., Shi, X., Zhao, X., Ding, D., Tang, J., & Niu, J. (2015). Investigation of miR396 and growth-regulating factor regulatory network in maize grain filling. Acta Physiologiae Plantarum, 37, 28. Zhang, B., Tong, Y., Luo, K., Zhai, Z., Liu, X., Shi, Z., Zhang, D., & Li, D. (2021). Identification of growth-regulating factor transcription factors in lettuce (Lactuca sativa) genome and functional analysis of LsaGRF5 in leaf size regulation. BMC Plant Biology, 21, 485. Zhang, J., Wang, M., Cheng, F., Dai, C., Sun, Y., Lu, J., Huang, Y., Li, M., He, Y., Wang, F., & Fan, B. (2016). Identification of microRNAs correlated with citrus granulation based on bioinformatics and molecular biology analysis. Postharvest Biology and Technology, 118, 59e67. Zhang, K., Wang, R., Zi, H., Li, Y., Cao, X., Li, D., Guo, L., Tong, J., Pan, Y., Jiao, Y., Liu, R., Xiao, L., & Liu, X. (2018). AUXIN RESPONSE FACTOR3 regulates floral meristem determinacy by repressing cytokinin biosynthesis and signaling. The Plant Cell, 30, 324e346. Zhang, Y., Xia, R., Kuang, H., & Meyers, B. C. (2016). The diversification of plantNBSLRRdefense genes directs the evolution of microRNAs that target them. Molecular Biology and Evolution, 33, 2692e2705. Zhang, J., Yue, L., Wu, X., Liu, H., & Wang, W. (2021). Function of small peptides during male-female crosstalk in plants. Frontiers in Plant Science, 12. Zhang, J.-P., Yu, Y., Feng, Y.-Z., Zhou, Y.-F., Zhang, F., Yang, Y.-W., Lei, M.-Q., Zhang, Y.-C., & Chen, Y.-Q. (2017). miR408 regulates grain yield and photosynthesis via a phytocyanin protein. Plant Physiology, 175, 1175e1185. Zhang, Y.-C., Yu, Y., Wang, C.-Y., Li, Z.-Y., Liu, Q., Xu, J., Liao, J.-Y., Wang, X.-J., Qu, L.-H., Chen, F., Xin, P., Yan, C., Chu, J., Li, H.-Q., & Chen, Y.-Q. (2013). Overexpression of microRNA OsmiR397 improves rice yield by increasing grain size and promoting panicle branching. Nature Biotechnology, 31, 848e852. Zhang, Y.-P., Zhang, Y.-Y., Thakur, K., Zhang, F., Hu, F., Zhang, J.-G., Wei, P.-C., & Wei, Z.-J. (2021). Integration of miRNAs, degradome, and transcriptome omics uncovers a complex regulatory network and provides insights into lipid and fatty acid synthesis during sesame seed development. Frontiers in Plant Science, 12, 709197. Zhang, H., Zhang, J., Yan, J., Gou, F., Mao, Y., Tang, G., Botella, J. R., & Zhu, J.-K. (2017). Short tandem target mimic rice lines uncover functions of miRNAs in regulating important agronomic traits. Proceedings of the National Academy of Sciences of the United States of America, 114, 5277e5282. Zhang, X., Zou, Z., Gong, P., Zhang, J., Ziaf, K., Li, H., Xiao, F., & Ye, Z. (2011). Overexpression of microRNA169 confers enhanced drought tolerance to tomato. Biotechnology Letters, 33, 403e409. Zhao, Y., Cong, L., & Lukiw, W. J. (2018). Plant and animal microRNAs (miRNAs) and their potential for inter-kingdom communication. Cellular and Molecular Neurobiology, 38, 133e140. Zhao, M., Ding, H., Zhu, J.-K., Zhang, F. S., & Li, W. X. (2011). Involvement of miR169 in the nitrogen-starvation responses in Arabidopsis. New Phytologist, 190, 906e915. Zhao, B., Ge, L., Liang, R., Li, W., Ruan, K., Lin, H., & Jin, Y. (2009). Members of miR169 family are induced by high salinity and transiently inhibit the NF-YA transcription factor. BMC Molecular Biology, 10, 29. Zhao, X. Y., Hong, P., Wu, J. Y., Chen, X. B., Ye, X. G., Pan, Y. Y., Wang, J., & Zhang, X. S. (2016). The tae-miR408-mediated control of Ta_TOC1 genes transcription is required for the regulation of heading time in wheat. Plant Physiology, 170, 1578e1594. 468 Plant Small RNA in Food Crops Zhao, L., Kim, Y., Dinh, T. T., & Chen, X. (2007). miR172 regulates stem cell fate and defines the inner boundary of APETALA3 and PISTILLATA expression domain in Arabidopsis floral meristems. The Plant Journal, 51, 840e849. Zhao, Z., Liu, D., Cui, Y., Li, S., Liang, D., Sun, D., Wang, J., & Liu, Z. (2020). Genomewide identification and characterization of long non-coding RNAs related to grain yield in foxtail millet [Setaria italica (L.) P. Beauv.]. BMC Genomics, 21, 853. Zhao, Y. F., Peng, T., Sun, H. Z., Teotia, S., Wen, H. L., Du, Y. X., Zhang, J., Li, J. Z., Tang, G. L., Xue, H. W., & Zhao, Q. Z. (2019). miR1432-OsACOT(acyl-CoA thioesterase) module determines grain yield via enhancing grain filling rate in rice. Plant Biotechnology Journal, 17, 712e723. Zhao, H., Sun, R., Albrecht, U., Padmanabhan, C., Wang, A., Coffey, M. D., Girke, T., Wang, Z., Close, T. J., Roose, M., Yokomi, R. K., Folimonova, S., Vidalakis, G., Rouse, R., Bowman, K. D., & Jin, H. (2013). Small RNA profiling reveals phosphorus deficiency as a contributing factor in symptom expression for citrus Huanglongbing disease. Molecular Plant, 6, 301e310. Zhao, P., Wang, F., Deng, Y., Zhong, F., Tian, P., Lin, D., Deng, J., Zhang, Y., & Huang, T. (2022). Sly-miR159 regulates fruit morphology by modulating GA biosynthesis in tomato. Plant Biotechnology Journal, 20, 833e845. Zhao, Y.-T., Wang, M., Fu, S.-X., Yang, W.-C., Qi, C.-K., & Wang, X.-J. (2012). Small RNA profiling in two Brassica napus cultivars identifies microRNAs with oil production- and development-correlated expression and new small RNA classes. Plant Physiology, 158, 813e823. Zheng, G., Wei, W., Li, Y., Kan, L., Wang, F., Zhang, X., Li, F., Liu, Z., & Kang, C. (2019). Conserved and novel roles of miR164-CUC2 regulatory module in specifying leaf and floral organ morphology in strawberry. New Phytologist, 224, 480e492. Zhong, S., Lin, Z., & Grierson, D. (2008). Tomato ethylene receptoreCTR interactions: Visualization of NEVER-RIPE interactions with multiple CTRs at the endoplasmic reticulum. Journal of Experimental Botany, 59, 965e972. Zhou, J., Zhang, R., Jia, X., Tang, X., Guo, Y., Yang, H., Zheng, X., Qian, Q., Qi, Y., & Zhang, Y. (2022). CRISPR-Cas9 mediated OsMIR168 a knockout reveals its pleiotropy in rice. Plant Biotechnology Journal, 20, 310e322. Zhou, Z., Zhu, Y., Zhang, H., Zhang, R., Gao, Q., Ding, T., Wang, H., Yan, Z., & Yao, J. L. (2021). Transcriptome analysis of transgenic apple fruit overexpressing microRNA172 reveals candidate transcription factors regulating apple fruit development at early stages. PeerJ, 9, e12675. Zhu, Q. H., Upadhyaya, N. M., Gubler, F., & Helliwell, C. A. (2009). Over-expression of miR172 causes loss of spikelet determinacy and floral organ abnormalities in rice (Oryza sativa). BMC Plant Biology, 9, 149. Zhu, B., Yang, Y., Li, R., Fu, D., Wen, L., Luo, Y., & Zhu, H. (2015). RNA sequencing and functional analysis implicate the regulatory role of long non-coding RNAs in tomato fruit ripening. Journal of Experimental Botany, 66, 4483e4495. Zuluaga, D. L., & Sonnante, G. (2019). The use of nitrogen and its regulation in cereals: Structural genes, transcription factors, and the role of miRNAs. Plants, 8, 294. SECTION 3 Applications and future scope 469 This page intentionally left blank CHAPTER 15 Food crops improvement: comparative biotechnological approaches Ting Shi College of Horticulture, Nanjing Agricultural University, Nanjing, China 1. Introduction Over the last century, crops have been improved through classical breeding techniques, and many varieties of crops have been developed worldwide (Knight, 2003; Tester & Langridge, 2010). However, due to genetic erosion, genetic resistance, and reproductive barriers, traditional breeding is limited in improving crops and usually takes a long time (Maxted & Guarino, 2006). Therefore, there is an urgent need for new breeding and biotechnology-assisted crop improvement to obtain new plant traits. Many molecular marker-assisted breeding technologies, such as marker-assisted selection (MAS) (Collard et al., 2005; Ribaut & Hoisington, 1998), marker-assisted backcross breeding (Neeraja et al., 2007; Yi et al., 2009), marker-assisted gene pyramiding (Chukwu et al., 2019; Jiang et al., 2012; Ye & Smith, 2010), marker-assisted recurrent selection (MARS), genomic selection (GS) (Heffner et al., 2009; Wang, Xu, et al., 2018) or genomewide selection (GWS) (Wang et al., 2020), play an essential role in crop improvement. Advances in plant genetic engineering (genetic transformation (Gao, 2021; Miflin, 2000) and genome editing (Abdallah et al., 2015; Khatodia et al., 2016; Rodríguez-Leal et al., 2017; Xu et al., 2019; Zhang, Massel, et al., 2018)) have made it possible to transfer genes into crops from unrelated plants and even non-plant organisms (Gao, 2021; Xu et al., 2019; Zhang, Massel, et al., 2018). These biotechnological approaches are an excellent option to improve crops with significant commercial properties such as increased biotic stress resistance (Khatodia et al., 2016) or abiotic stress tolerance (Basso et al., 2019; Zafar et al., 2020), nutrient (Kim, 2020), and quality (Ku & Ha, 2020). MicroRNAs (miRNAs) are a class of endogenous small RNAs that negatively regulate gene expression at post-transcriptional levels (Bartel, Plant Small RNA in Food Crops ISBN 978-0-323-91722-3 https://doi.org/10.1016/B978-0-323-91722-3.00015-4 © 2023 Elsevier Inc. All rights reserved. 471 472 Plant Small RNA in Food Crops 2004; Jones-Rhoades et al., 2006). With the application of next-generation deep sequencing (Fahlgren et al., 2007; Song et al., 2010; Wang, Pan, et al., 2014) and advanced bioinformatics (Jones-Rhoades & Bartel, 2004; Rhoades et al., 2002), miRNA-related research has expanded to nonmodel plant species (Gao et al., 2012), and the number of identified miRNAs has increased dramatically over the past few years. miRNAs play a crucial role in almost all biological and metabolic processes, providing a unique strategy for plant improvement. Here, we focus on the application and future direction of miRNAs in plant breeding. Plant architecture greatly impacts on crop development, yield, and stress resistance (Guo, Chen, Herrera-Estrella, et al., 2020). A complex interaction of regulatory networks comprising miRNAs, phytohormones, and essential transcription factors (TFs) results in the creation of plant architecture (Farinati et al., 2020; Guo, Chen, Chen, et al., 2020; Karlova et al., 2013; Molesini et al., 2012; Rosas Cárdenas et al., 2017; Wang, Xu, et al., 2018). Regulation of plant growth and development by targeting miRNAs will significantly promote plant growth and further increase plant yield, which is especially important for food crops improvement (Miao et al., 2020; Sun et al., 2019; Tang et al., 2017; Zhang et al., 2019; Zhang & Wang, 2015). Plant tolerance to abiotic and biotic stresses was also significantly enhanced by regulating the expression of individual miRNAs (Basso et al., 2019; Kang et al., 2020; Zafar et al., 2020). We outline the evidence for the involvement of miRNAs in plant development, growth, and response to environmental stress through transgenic techniques and other functional analyses. Both endogenous and artificial miRNAs can be used as essential tools to improve plant biomass and respond to biotic and biotic stresses. These point to potential directions for utilizing miRNA-based knowledge for plant improvement. 2. miRNA-based biotechnology for food crop improvement 2.1 Transgenes and food crop improvement Transgenesis is the insertion of one or more genes from a non-plant organism or a donor plant that is sexually incompatible with the receiving plant into a recipient plant (Mujjassim et al., 2019; Pandey et al., 2019). In response to public concerns about transgenic crop safety, intragenesis and cisgenesis are two transformation approaches that have been explored as alternatives to transgenic crop production. To address these concerns while Food crops improvement: comparative biotechnological approaches 473 also maintaining environmentally sound and efficient plant production (Mujjassim et al., 2019). Cisgenesis is the process of genetically modifying a recipient plant using a natural gene from a sexually compatible crossable plant (Basso et al., 2019). Conversely, intragenesis allows for the design of cassettes combining specific genetic elements from plants belonging to the same sexual compatibility gene pool (Basso et al., 2019; Mujjassim et al., 2019). Several plant species have successfully transferred one or more MIR genes between uncrossable plant species or between crossable or the same species (Basso et al., 2019) (Table 15.1). For example, strong constitutive, mainly CaMV 35S (Geng et al., 2020), native tissue-specific (Manavella, Koenig, Rubio-Somoza, et al., 2012) or stress-inducible (Kang et al., 2020) promoters were usually used to overexpress MIR genes. However, utilizing any of these approaches to manipulate MIR genes, particularly overexpression driven by strong promoters, led to undesirable features, including pleiotropic phenotypes (Curaba et al., 2013; Jia, Ding, et al., 2015), which might be overcome using specific promoters. The participation of miRNAs in many complicated regulatory networks explains this conclusion (Jones-Rhoades et al., 2006). In transgenic melon (Cucumis melo) plants with overexpressing pre-cmemiR393 (cme-miR393-OE), fruit ripening was delayed compared to nontransgenic fruits, which provides evidence that miRNA regulates melon fruit ripening and provide potential targets to improve the horticultural traits of melon fruit (Bai et al., 2020). Over-expression of miR394 altered the fatty acid (FA) composition by increasing several FA species in rapeseed (Brassica napus). This change was accompanied by the induction of genes coding for transcription factors of FA synthesis including LEAFY COTYLEDON1 (BnLEC1), BnLEC2, and FUSCA3 (FUS3). These results suggest that BnmiR394 is involved in rapeseed fruit and seed development (Song et al., 2015). TaemiR408 is a miRNA family member of wheat (Triticum aestivum). It overexpressed in tobacco lines and displayed higher stress tolerance, biomass, and photosynthetic behavior than wild type under both Pi deprivation and salt treatments. Which are strongly associated with increased P accumulation upon Pi deprivation and raised osmolytes under salt stress (Bai et al., 2018). In comparison to wild-type controls, OsamiR393a transgenic creeping bentgrass (Agrostis stolonifera L.) overexpressing rice pri-miR393a had fewer but longer tillers, improved drought stress tolerance associated with reduced stomata density and denser cuticles, improved salt stress tolerance associated with increased potassium uptake, 474 Plant Small RNA in Food Crops Table 15.1 Transgenes and food crop improvement. Plant source Nicotiana tabacum Arabidopsis thaliana Arabidopsis thaliana Brassica campestris Oryza sativa Oryza sativa Arabidopsis thaliana Arabidopsis thaliana Arabidopsis thaliana Arabidopsis thaliana Solanum lycopersicum miRNA miR156 miR156 miR156 miR158a miR160a and miR398b miR164 miR164 miR165/166 miR167c miR169a miR169c Arabidopsis thaliana Arabidopsis thaliana miR169d Medicago truncatula miR171h miR171a Transgenic plants Function in transgenic plants Nicotiana tabacum Arabidopsis thaliana Arabidopsis thaliana Brassica campestris Oryza sativa Drought and salt tolerance Flowering times Stress tolerance Oryza sativa Arabidopsis thaliana Arabidopsis thaliana Arabidopsis thaliana Arabidopsis thaliana Solanum lycopersicum Arabidopsis thaliana Arabidopsis thaliana Medicago truncatula Pleiotropic phenotypes Resistance to Magnaporthe oryzae Root growth and yield traits Leaf longevity Plant development Somatic embryo formation Drought stress Stomatal conductance and transpiration rate Early flowering Plant development Root symbiosis with Sinorhizobium meliloti References Kang et al. (2020) Huo et al. (2016) Cui et al. (2014) Ma et al. (2017) Li et al. (2014a, 2014b) Geng et al. (2020) Kim et al. (2009) Jia, Ding, et al. (2015) Su et al. (2016) Li et al. (2008) Zhang, Zou, Zhang, et al. (2011) Xu et al. (2014) Manavella, Koenig, RubioSomoza, et al. (2012) Hofferek et al. (2014) Food crops improvement: comparative biotechnological approaches 475 Table 15.1 Transgenes and food crop improvement.dcont'd Plant source miRNA Transgenic plants Function in transgenic plants Glycine max miR172a Arabidopsis thaliana Glycine max miR172c Oryza sativa miR319a Arabidopsis thaliana Oryza sativa Oryza sativa miR319b Oryza sativa Oryza sativa miR390 Oryza sativa Oryza sativa miR393a Brassica napus Arabidopsis thaliana miRNA394 Creeping bentgrass Brassica napus Brassica napus Early flowering phenotype Tolerance to salinity Pleiotropic phenotypes Tolerance to cold Susceptibility to cadmium Drought stress tolerance Fruit and seed development Leaf morphology miR395d Poncirus trifoliata miR396b Citrus limon Tolerance to cold Oryza sativa miR396c Arabidopsis thaliana Oryza sativa miR396f Oryza sativa Arabidopsis thaliana miR397 Arabidopsis thaliana Oryza sativa miR398 Oryza sativa Arabidopsis thaliana miR399f Arabidopsis thaliana Arabidopsis thaliana Wheat miR408 Cicer arietinum Nicotiana tabacum Less tolerance to salinity and alkali stress Resistance to Dickeya zeae Tolerance to chilling and freezing stresses More sensitive to environmental stress Tolerance to salt stress and exogenous ABA Tolerance to drought stress Plant tolerance to Pi deprivation and salt stress miR408 References Wang, Sun, et al. (2016) Li, Wang, et al. (2016) Zhou et al. (2013) Wang, Sun, et al. (2014) Ding et al. (2016) Zhao, Yuan, et al. (2019) Song et al. (2015) Huang et al. (2010) Zhang et al. (2016) Gao et al. (2010) Li et al. (2019) Dong and Pei (2014) Lu et al. (2010) Baek et al. (2016) Hajyzadeh et al. (2015) Bai et al. (2018) Continued 476 Plant Small RNA in Food Crops Table 15.1 Transgenes and food crop improvement.dcont'd Plant source Triticum aestivum Arabidopsis thaliana miRNA miR444a miR778 Transgenic plants Function in transgenic plants Nicotiana tabacum Arabidopsis thaliana Improves the plant development Roots development References Gao et al. (2016) Wang, ZengJ, et al. (2015) and enhanced heat stress tolerance associated with induced expression of small heat-shock protein (Zhao, Yuan, et al., 2019). 2.2 RNA interference (RNAi) and food crop improvement RNA silencing pathways convert the sequence information in long, double-stranded RNA into w21-nt RNA signaling molecules such as small interfering RNAs (siRNAs) and microRNAs (miRNAs). siRNAs and miRNAs provide specificity to protein effector complexes that repress mRNA transcription or translation or catalyze mRNA destruction. RNA interference (RNAi) is a strong technique for gene silencing in plants and other organisms (Rajam, 2020). Identification of the target gene(s), hairpin cassette (gene cloned in sense and antisense orientation flanked a spacer or intron), plant transformation, and ultimately screening and evaluating the attributes are all part of the RNAi mediated gene silencing technique. (Kamthan et al., 2015) (Fig. 15.1). This method has also seen a number of possible applications in agriculture, including the production of crops that are resistant to biotic pests and abiotic stresses (Li et al., 2008; Yang et al., 2013), nutritional quality improvement (Kamthan et al., 2015; Rajam, 2020), delayed ripening (Chen, Kong, et al., 2015), modification of flowering time (Chuck et al., 2011), alteration of plant architecture (Qiao et al., 2007). RNAi has been utilized in the ripening of tomato fruit with enhanced lycopene and b-carotene contents, which are highly beneficial for human health (Sun, Yuan, et al., 2012). The SlNCED1 gene encoding 9-cisepoxycarotenoid dioxygenase (NCED) (Sun, Sun, et al., 2012), a key enzyme in the ABA biosynthesis, was suppressed in tomato plants by Food crops improvement: comparative biotechnological approaches 477 Figure 15.1 RNA interference (RNAi) in the plants. AGO, ARGONAUTE protein; dsRNA, double strands RNA; RISC, RNA-induced silencing complex. (Fig. 1 was created using Figdraw (www.figdraw.com).) transformation with an RNAi construct driven by a fruit-specific E8 promoter (Sun, Yuan, et al., 2012). The fruit of RNAi lines displayed deep red coloration compared with the pink color of control fruit. By boosting the transcription of genes involved in ethylene synthesis during ripening, the decrease in endogenous ABA in these transgenics resulted in an increase in ethylene. RNAi in combination with fruit specific promoter has also been used to suppress an endogenous DET1 gene in tomato (Davuluri et al., 2005), a photomorphogenesis regulatory gene involved in the repression of several light controlled signaling pathways (Levin et al., 2003). miR156 is one of the most conserved miRNA families in plants, and it targets SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) TF genes (Rhoades et al., 2002). RNAi suppression of CsSPL3 and CsSPL14 enhances somatic embryogenesis capability in citrus Fortunella hindsii, which suggests that miR156-SPL modules enhance the somatic embryogenesis potential of citrus callus (Long et al., 2018). RNAi has been applied successfully to develop drought-tolerant crops. GmNFYA3 encodes the NFYA subunit of the NFeY complex in soybeans (Glycine max L.), which is the target of miR169. Overexpression of GmNFYA3 resulted in Arabidopsis with reduced leaf water loss and enhanced drought tolerance (Ni et al., 2013). MdSE (serrate) acts as a negative regulator of apple (Malus  domestica) drought resistance by regulating the expression levels of MdMYB88 and MdMYB124, and miRNAs. MdSE RNAi transgenic 478 Plant Small RNA in Food Crops plants are more sensitive to ABA-induced stomatal closure, which reveals that MdSE regulates the biogenesis of miRNAs in apple under drought (Li et al., 2020). 2.3 Artificial microRNAs and food crop improvement The artificial MIR gene (amiRNA) strategy was developed to generate specific miRNAs and efficiently silence target genes (Lunardon et al., 2021; Sablok et al., 2011) (Table 15.2). These amiRNAs have a conserved secondary folded structure, like typical pre-miRNAs. Nonetheless, the original miRNA-5p:miRNA-3p sequences were replaced by engineered miRNAs targeting specific mRNAs. Therefore, amiRNA can target any mRNA with higher specificity than strategies based on dsRNA overexpression or Table 15.2 Artificial microRNAs and food crop improvement. Transgenic plants Function in transgenic plants amiR-24 Nicotiana tabacum Artificial miRNA against PDS gene Triticum aestivum Artificial and engineered Hvu-miR171 targeting viral genes OsMIR390based amiRNAs Nicotiana benthamiana and Hordeum vulgare Efficient amiR-24 targeting chitinase gene from Helicoverpa armigera, improving plant tolerance to caterpillar Efficient downregulation of Ta-miR156 and Ta-miR166 and overexpression of miR156 or artificial miRNA (amiRNA) targeting phytoene desaturase gene (amiR-PDS) Resistance to wheat dwarf virus amiRNA amiRPPO1-4 Brachypodium distachyon Solanum tuberosum Reddish coloration of lignified tissues such as tillers, internodes and nodes Oxidative browning References Agrawal et al. (2015) Jian et al. (2017) Kis et al. (2016) Carbonell et al. (2015) Chi et al. (2014) Food crops improvement: comparative biotechnological approaches 479 siRNA accumulation (Basso et al., 2019; Lunardon et al., 2021). PreamiRNA treatment often targets a single amiRNA of known sequence, thereby helping to avoid off-target effects. In some cases, the systemic movement of these mature non-RNAs is restricted (Carlsbecker et al., 2010) and the ability to generate secondary siRNAs from non-RNA sequences is very limited (Manavella, Koenig, & Weigel, 2012). In addition, amiRNAs are stable and heritable (Basso et al., 2019). The main disadvantage of this strategy is the selection of backbone or pre-amino RNA sequences for efficient silencing without any off-target effects (Carbonell et al., 2015). For primary target specificity, mature non-target genes must have low sequence similarity to non-target genes (Zhang, Zhang, et al., 2018). Similar to the overexpression of MIR genes, negative regulation of the accumulation of some specific miRNAs can achieve desirable agronomic traits. Endogenous target mimicry (eTM), another natural mechanism involved in regulating miRNA accumulation, controls several biological processes in plants (Karakülah et al., 2016). These eTMs are myriad long noncoding RNAs (lncRNAs) or circular noncoding RNAs (circRNAs) transcribed from the genome, often differentially expressed under stress or other adverse conditions (Liu et al., 2022). It acts as a natural sponge and is primarily used to rapidly fine-tune plant responses to new conditions or miRNAs in adaptation. To replicate this mechanism, an artificial short tandem target mimic (STTM) strategy was developed to modulate miRNA accumulation and improve desirable agronomic traits (Teotia et al., 2016). This strategy is based on transient or constitutive overexpression of engineered lncRNAs with high nucleotide sequence identity to target mRNAs (Reichel et al., 2015). Likewise, overexpression of synthetic circRNAs has also been considered as a potential alternative for miRNA regulation in plants. These STTMs contain two or more conserved binding sites for specific target miRNAs, but have a three-nucleotide mismatch at the miRNA cleavage site, which prevents its cleavage, while the miRNA remains hybridized and biologically inactive. Therefore, STTM isolates miRNAs from endogenous target mRNAs, leading to their upregulation (Franco-Zorrilla et al., 2007). Several STTMs targeting MIR genes in crops have been designed and constitutively expressed as transgenes for comprehensive functional analysis of miRNAs (Peng et al., 2018; Zhang et al., 2017). In addition to controlling endogenous gene expression and studying the role of novel MIR genes, an amiRNA strategy has been successfully used to 480 Plant Small RNA in Food Crops knock out genes in pests, nematodes, viruses, and other plant pathogens (Kis et al., 2016; Wagaba et al., 2016; Yogindran & Rajam, 2021). In tobacco, overexpression of an engineered amiRNA is resistant to Helicoverpa armigera (Agrawal et al., 2015). A novel artificial and endogenous miRNA and siRNA overexpression system based on viral satellite DNA vectors for functional analysis in plants yields promising results in the overexpression of tobacco endogenous or artificial MIR genes, siRNA, and trans-acting-siRNA in Nicotiana benthamiana (Ju et al., 2017). amiRNA-mediated strategy can be applied to the development of insect-resistant crops. The amiRNA of striped stem borer endogenous miRNA has significant application potential in developing striped stem borer-resistant rice (Liu et al., 2022). STTM strategy was used to silence miR482b in tomatoes and increase tomato resistance to Phytophthora infestans (Jiang et al., 2018). amiRNAs can be used to suppress closely related members of a highly conserved multi-gene family (Basso et al., 2019; Chi et al., 2014) in potatoes. This approach was used for breeding low-browning crops using small DNA inserts (Chi et al., 2014). 2.4 Genome editing 2.4.1 Genome editing Genome editing technology is the most recent of the genetic alteration technologies. Specific genes and other genetic elements can be stably altered, knocked out, or replaced using genome editing technologies. This method uses a sequence-specific nuclease (SSN) to make precise gene knockout and knock-in edits, or synthetic oligonucleotides to make exact point mutations in the target DNA region (Ku & Ha, 2020; Songstad et al., 2017). Synthetic oligonucleotides (RNA/DNA chimeric oligonucleotides or single-stranded DNA oligonucleotide molecules of 20e100 nucleotides) have lately been used for targeted editing, such as the generation of custom single nucleotide polymorphisms (SNPs). Oligonucleotide-Directed Mutagenesis is the name for this method (ODM) (Kim, 2020). In ODM, a plant cell receives an oligonucleotide that is identical to the target DNA sequence but has the desired mismatch (es). During cell division, the mismatch (es) between the oligonucleotide and target DNA sequence are recognized by the plant’s native repair system, which uses the oligonucleotide as a template to repair the cell’s own DNA through homologydirected pairing, resulting in the direct incorporation of site-specific point mutations/SNPs, typically 1e3 nucleotides, into a gene of interest (Sauer Food crops improvement: comparative biotechnological approaches 481 et al., 2016). Tissue-culture procedures can then be used to identify the plant cell with the desired mutation and regenerate it into a whole plant. In addition to ODM, three additional forms of SSN are being used to alter genomes. Zinc-Finger Nucleases (ZFNs), Transcription ActivatorLike Effector Nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeat-associated endonucleases (CRISPR/Cas) are the SSNs in question. SSNs work by binding to a specific target DNA sequence in the genome and causing double-stranded breaks (DSBs) in a specific genomic region (Kumar et al., 2020). DSBs are typically repaired using either an error-prone endogenous repair mechanism (Non-homologous end joining/NHEJ) or a homologous DNA repair template introduced externally (Homology-directed repair/HDR). This can be used to make changes at the target site, such as gene knockout (insertion/deletion resulting in frame shift mutations) by NHEJ or precision targeted sequence replacement/substitution (point mutations, targeted gene replacement, or site-specific gene insertion) using HDR (Kim & Kim, 2014). These various DSB repair outcomes can be divided into three categories. ZFNs and TALENs were the first generation targeted genome editing tools among the three designer nuclease systems. ZFN and TALEN are made by combining two separate protein domains, namely a sequence of DNAbinding domains [either zinc-finger domains (for ZFN) or TALE domains (for TALEN)] with a synthetic FokI endonuclease domain (Kumar et al., 2020; Songstad et al., 2017). 2.4.2 CRISPR/Cas9 gene-editing Recently, the clustered regularly interspaced short palindromic repeats/ CRISPR-associated protein-9 nuclease (CRISPR/Cas9), CRISPR/Cpf1 or CRISPR/Csm1 systems, a new class of nucleases guided by RNA (guide RNA), have been Optimized for plant genome editing (Osakabe & Osakabe, 2017; Sami et al., 2021). CRISPR/Cas system has three types, CRISPR/Cas9 belongs to type II, because type I and type III require the participation of a variety of Cas proteins, more complex and not widely used, but CRISPR/Cas9 system only needs a Cas protein that is, Cas9 involvement, simple to control, so it is widely used. The CRISPR/Cas9 system consists of a single guide RNA (sgRNA) and a Cas9 protein with endonuclease activity, sgRNA needs to have a sequence that matches the target gene, and the Cas9 protein needs to have a nuclear localization signal. By designing sequence-specific guide RNA, the Cas9 protein is recruited to a specific location in the genome, the Cas9 protein performs the function of 482 Plant Small RNA in Food Crops nucleic acid endonuclease to cause DSBs, and the cell will cause mutations or deletions of bases in the process of initiating repair, so that the function of the target gene is lost, and the purpose of gene orientation editing is achieved (Chang et al., 2016; Osakabe & Osakabe, 2017). At present, the system has been widely used in gene editing of rice (Jiang et al., 2013; Toda & Okamoto, 2020), corn (Chilcoat et al., 2017; Liu et al., 2021), tomato (Chandrasekaran et al., 2021; Reem & Van Eck, 2019) and other plants (Chilcoat et al., 2017; Osakabe et al., 2018; Zhou et al., 2020), and has achieved good results, which is an effective targeted gene editing method. Exogenous DNA sequences for gene editing are easily removed from the edited plants in subsequent generations, converting them into GMOfree strains, helping to dispel public concerns about the hidden dangers of transgenes. Similar to coding genes, the use of CRISPR/Cas9 gene editing technology can be knocked out or base replacement of specific non-coding gene miRNA, knockout and replacement sequences can be miRNA seed sequences, stem ring structure sequences or promoter sequences, replacement seed sequences can affect its recognition of the target, replacement stem ring structure can affect its biosynthesis, replacement promoter can affect its expression patterns. Different family members of the same miRNA may have different expression patterns and have different regulatory functions during plant growth and development, and the CRISPR/Cas9 system can be specifically studied for each member of the miRNA gene family, which is not achieved by STTM technology. However, due to the short sequence of miRNAs, some miRNAs may be difficult to design sgRNAs that meet the conditions on their sequences, so it is generally possible to select suitable locations in the upstream and downstream regions near the mature miRNA sequence to design two sgRNA sites, and when the two sites are edited at the same time, the mature miRNA sequences between them will be deleted, but this puts forward higher requirements for the editing efficiency of the CRISPR/ Cas9 system. At present, CRISPR-Cas9 technology has been proven to be an effective means of editing plant miRNAs, which will greatly expand the application of miRNAs in the field of crop molecular breeding (Table 15.3). CRISPR/Cas9 successfully targets soybean miR1514 and miR1509 by gene knockout transiently expressing a CRISPR/Cas9 vector containing Cas9 nuclease and gRNAs (Jacobs et al., 2015). Food crops improvement: comparative biotechnological approaches 483 Table 15.3 miRNAs related gene-editing technologies in plants. Editing method Plants miRNA Arabidopsis thaliana miR160 and miR390 Solanum lycopersicum miR164 Oryza sativa miR168a CRISPR/ Cas9 Pleiotropy Oryza sativa miR396 Solanum lycopersicum miR482b and miR482c CRISPR/ Cas9 CRISPR/ Cas9 Oryza sativa miR408 and miR528 miR815a/b/c and miR820a/b/c Plant development Enhanced tomato resistance to Phytophthora infestans Salt stress TALEN CRISPR/ Cas9 CRISPR/ Cas9 CRISPR/ Cas9 Function References Plant development Bi et al. (2020) Fruit growth Gupta et al. (2021) Zhou et al. (2022) Lin et al. (2021) Hong et al. (2021) Zhou et al. (2017, 2021) 3. miRNAs roles in food crops improvement MiRNAs are important regulators of plant growth and development processes, and there is growing evidence that miRNAs play a key role in regulating crop traits. MiRNA and its target genes have many influences on crop traits, and play a key role in regulating crop architecture, flowering period, fertility, yield, stress resistance, quality, and other aspects. 3.1 Crop architecture Plant architecture has a big impact on crop development, yield, and stress resistance (Guo, Chen, Herrera-Estrella, et al., 2020). A complex interaction of regulatory networks comprising miRNAs, phytohormones, and essential transcription factors (TFs) results in the creation of plant architecture (Farinati et al., 2020; Guo, Chen, Chen, et al., 2020; Karlova et al., 2013; Molesini et al., 2012; Rosas Cárdenas et al., 2017; Wang, Xu, et al., 2018). Regulation of plant growth and development by targeting miRNAs 484 Plant Small RNA in Food Crops Table 15.4 miRNAs associated with crop architecture and development. miRNA Plants Function References miR156 Oryza sativa Plant architecture miR156b Glycine max miR156 Zea may Shoot architecture and yield Delay flowering miR159 Oryza sativa Male sterility miR159 Triticum aestivum Arabidopsis thaliana Triticum aestivum Arabidopsis thaliana and tomato Triticum aestivum Solanum lycopersicum Solanum lycopersicum Solanum lycopersicum Arabidopsis thaliana Male sterility Jiao et al. (2010) Sun et al. (2019) Xie et al. (2020) Tsuji et al. (2006) Wang, Sun, et al. (2012) Reyes and Chua (2007) Feng et al. (2014) Rosas Cárdenas et al. (2017) miR159 miR164 miR164 miR164 miR167 miR168 miR171 miR172 miR172 Oryza sativa miRNA393 Oryza sativa miR393 Oryza sativa miR396e and miR396f miR396d Oryza sativa Oryza sativa Seed development Resistance to stripe rust Plant morphology and fruit development Seed development Flower development and male infertility Delay flowering Regulating gibberellin and auxin homeostasis Early flowering Increased expression of anthocyanin synthesis genes and earlier flowering Flag leaf inclination and primary and crown root growth Early flowering Increase grain size and modulate shoot architecture Plant architecture Han et al. (2014) Liu et al. (2014) Xian et al. (2014) Huang et al. (2017) Wollmann et al. (2010), Zhao et al. (2007) Lee et al. (2014) Bian et al. (2012) Zhao, Yuan, et al. (2019) Miao et al. (2020) Tang et al. (2017) Food crops improvement: comparative biotechnological approaches 485 Table 15.4 miRNAs associated with crop architecture and development.dcont'd miRNA Plants Function References miR397 Oryza sativa miR399 Fragaria vesca Enlarge the grain and promote panicle branching Fruit quality miR408 Zea may miR444 Oryza sativa miR529 Arabidopsis Early panicle extraction time Antiviral RNA silencing pathway Plant tillering/branching miR1432 Oryza sativa Regulator of grains Zhang et al. (2013) Wang et al. (2017) Bai et al. (2018) Wang, Jiao, et al. (2016) Guo, Chen, HerreraEstrella, et al. (2020) Zhao, Peng, et al. (2019) miR1867 Seed development Oryza sativa Male fertile miR2118 miR4376 Solanum lycopersicum Fruit development Kim and Zhang (2018) Wang et al. (2011) (Table 15.4) will significantly promote plant growth and further increase plant yield, which is especially important for food crop improvement (Zhang & Wang, 2015). Plant architecture and resistance are controlled by miRNAs such as miR156s/miR529s and miR396s (Miao et al., 2020; Tang et al., 2017; Zhang et al., 2019), which operate as master regulators (Sun et al., 2019). The plant type of grasses is determined mainly by their branching patterns, wherein tillers occur during the vegetative growth period and panicle branches occur during the reproductive growth stage and their patterns are closely related to yield. In rice, miR156, miR529 and miR172 synergistically regulated their branching patterns (Wang, Sun, et al., 2015). Among them, miR156 and miR529 regulate tillering and panicle branching of rice by targeting genes of the SPL (SQUAMOSA PROMOTER BINDING PROTEIN LIKE) family, while miR172 regulates tillering and panicle branching of rice by targeting genes of the AP2 (APETALA2) family. miR156 and miR529 can increase the number of tillers in rice, but they will also make its panicles smaller, the number of paniclelets decreases, and they negatively regulate the activity of 486 Plant Small RNA in Food Crops inflorescence meristem and the onset of panicle branching. miR172 has nothing to do with tillering numbers, but it also reduces the number of spikelets, which negatively regulates the transition from flowers to spikelets. MiR156 in wheat has a similar effect to miR156 in rice, and also plays a role in promoting tillering and inhibiting the formation of spikelets, and its miR529 and miR172 may also play a similar role to that in rice (Wang, Sun, et al., 2015). miR393 regulates tillering numbers in rice by targeting two auxin receptor genes, TIR1 (TRANSPORT INHIBITOR RESPONSE 1) and AFB2 (AUXIN SIGNALING F-BOX 2) (Bian et al., 2012). miR444 inhibits tillers, the mechanism of which is: miR444 inhibits the expression of MADS57, and MADS57 is the expression inhibitor of D14 (Dwarf14), in the case of miR444 overdose, the expression of D14 increases, thereby inhibiting the tillering of rice. In addition, the interaction of TB1 (TEOSINTE BRANCHED1) with MADS57 will reduce the inhibition of D14 expression by MADS57 and increase the expression of D14 (Wang, Jiao, et al., 2016). Therefore, the TB1 gene is also a negative regulator of tillering in rice. miR164 by targeting NAC2 (NAC-REGULATED SEED MORPHOLOGY 2) negatively regulated panicle size, overexpressing miR164 will reduce panicle length and grain yield, while over-expressing the target mimic-resistant target mimic will increase panicle length and grain yield (Feng et al., 2014). MiR398 and miR172 positively regulate the size of the spikes, and when they are silenced, the spikes become shorter (Li et al., 2014b). Among them, the miR398 silent strain becomes shorter, the grain becomes less and lighter, and the phenomenon of late flowers is produced, while the over-expression results in the opposite, while the miR172 silent strain only makes the panicle shorter, but the grain becomes denser. Both miR156 and miR396 have the effect of regulating multiple traits, and they both negatively regulate plant height (Jiao et al., 2010). In addition to the two of them, miR171 is also a negative regulator of plant height, and in tomato lines that overexpress miR171, plants are dwarfed and fruit yields are reduced (Huang et al., 2017). 3.2 Crop flowering Flowering is a very critical event in the life cycle of higher plants (Srikanth & Schmid, 2011). The transition of plants from vegetative growth to flowering is tightly controlled, which is related to their reproduction success. To ensure flowering and fruiting under the most favorable conditions, plants have evolved complex regulatory networks that integrate Food crops improvement: comparative biotechnological approaches 487 endogenous and environmental signals, of which miRNAs are an important regulator (Table 15.4). The expression of miR172 was very low during vegetative growth, and the expression gradually increased with the flowering process. Overexpression of miR172 inhibits the translation of AP2, resulting in the early flowering of plants (Wollmann et al., 2010; Zhao et al., 2007). Taking rice miR172 as an example, Hd3a (Heading date 3a) and RFT1 (Rice Flowering Locus T 1) encode the anthocyanin synthesis gene of rice, which can promote rice flowering. Ehd1 (Early heading date 1) is a positive regulator upstream of Hd3a and RFT1. miR172 is to inhibit the expression of two members of the AP2 family, IDS1 (INDETERMINATE SPIKELET 1) and SNB (SUPERNUMERARY BRACT), which increases the expression of Ehd1, resulting in increased expression of anthocyanin synthesis genes and an earlier flowering period of rice (Lee et al., 2014). In rice and maize overexpression of miR156, plant flowering was delayed. In addition, miR156, which over-expresses corn in switchgrass, can also delay its flowering period (Jiao et al., 2010; Xie et al., 2020). In addition to miR156 and miR172, there are other miRNAs in crops play a role in regulating flowering. Early flowering occurred in rice strains that overexpressed miR393, indicating that it could bring flowering earlier (Zhao, Yuan, et al., 2019). In tomatoes, the silent miR168 can delay its flowering time (Xian et al., 2014). Overexpression of miR408 in maize has early panicle extraction time (Bai et al., 2018). 3.3 Crop fertility Crop fertility is related to whether they can produce offspring, which is one of the key traits in the reproductive process of plants (Tester & Langridge, 2010). However, in crop breeding research, it is usually necessary to breed hybrid varieties of crops through male sterile strains, and use the advantages of hybridization to increase crop yields (Kim & Zhang, 2018). There have been fewer reports of male fertile miRNAs (Table 15.4) regulating crops, one of the most important of which is miR2118. Under long-day conditions, miR2118 in the photoperiod-sensitive male sterile (PSMS) strain of rice produces a series of 21 nt phasiRNAs by targeting PMS1T, a long chain of non-coded RNA, so that this phasiRNA accumulates specifically in PSMS lines of rice, and these phasiRNAs may act on the Rf gene, which in turn regulates PSMS (Fan et al., 2016). Another miRNA in rice that affects male fertility is miR159. According to studies, miR159 affects the 488 Plant Small RNA in Food Crops development of rice anthers, and overexpression of miR159 in rice can cause its flower development deformities, and there is no pollen inside the stamens (Tsuji et al., 2006). In addition, the overexpression of wheat’s miR159 in rice, that is, tae-miR159, can also lead to male sterility in rice (Wang, Sun, et al., 2012). In addition, overexpression of miR167 in tomatoes can cause defects in flower development and male infertility. Its target genes are auxin response factor ARF6 (Auxin Response Factor 6) and ARF8 (Auxin Response Factor 8). The role of ARF6 and ARF8 in Arabidopsis thaliana is to promote inflorescence stem elongation and late stamens pistil development (Liu et al., 2014). 3.4 Crop seed/fruit development Seeds are the basis of plant growth and an important means of production in agricultural production, and miRNAs also play an essential role in seed development (Table 15.4). miRNAs can regulate seed development in various ways, such as signal transduction (ABA, auxin, rapeseed sterols, etc.), starch synthesis, antioxidant effects, sugar conversion, and cell growth. In germination seeds, miR159 can negatively regulate the positive regulators of ABA signaling MYB33 and MYB101, indicating that miR159 may regulate seed development through the ABA signaling pathway (Reyes & Chua, 2007). In addition, miR159 was expressed higher in inferior rice grains than dominant grains (Peng et al., 2014), and ABA treatment of rice during the filling stage could accelerate cell division, increase cell number, and increase grouting rate, thereby increasing the weight of inferior grains (Zhang et al., 2012). This suggests that miR159 may have affected seed grouting by regulating seed transduction to ABA signaling. MiR164 was on the rise in developing wheat seeds (Han et al., 2014), miR167 was highly expressed in maize seeds (Kang et al., 2012), and their target genes were all genes associated with auxin signaling pathways (Jones-Rhoades & Bartel, 2004), indicating that they may regulate seed development through auxin pathways. Overexpression of miR397 in rice will enlarge the grain and promote panicle branching, thereby increasing rice yield. The target gene of miR397 is LACcase encoded laccase-like protein, which is involved in rapesesterol signal transduction, indicating that miRNA397 is likely to regulate seed development through the rapeseed sterol signal transduction pathway (Zhang et al., 2013). miR156 exercises its functions through its target gene SPL family genes, such as OsSPL14 to regulate grain yield (Jiao et al., 2010), and OsSPL13 Food crops improvement: comparative biotechnological approaches 489 and OsSPL16 to control grain size, quality and shape. OsSPL13 can positively regulate cell size in the chaff, thereby increasing rice grain length and yield (Jiao et al., 2010; Wang, Wu, et al., 2012). OsSPL16 is a positive regulator of cell proliferation, which can promote cell differentiation and grain filling, thereby increasing the width and yield of grains (Wang, Wu, et al., 2012). Like miR156, miR396 is also a negative regulator of grain yield, passed by miR396-OsGRF4 (GROWTH-REGULATING). Factor 4)-OsGIF1 (GRF-interacting factors 1) mode regulates grain size (Li, Gao, et al., 2016). In rice overexpressing miR396, both grain size and weight decreased, indicating that miR396 is a negative regulator of grain length and width, and it regulates grain size by inhibiting cell expansion. In addition, OsGRF4 can interact with the activator OsGIF1, and increasing the expression of OsGIF1 will also increase the size of the grain. In addition, miR1432 is also a negative regulator of grains, and the total yield of grains increases in plants that inhibit miR1432. In tomatoes, miR156, miR157, miR168 and miR4376 were reported to be involved in regulating fruit development. Both miR156 and miR157 targeted genes of the SPL family, LeSPL-CNR, which were involved in regulating the maturation of tomato fruits. Among them, miR157 was associated with the onset of fruit ripening, while miR156 was associated with softening after fruit ripening (Chen, Kong, et al., 2015). MiR168 regulates fruit initiation and development by targeting AGO1 (Xian et al., 2014); miR4376 regulates fruit development by targeting ACA10 (autoinhibited Ca2þ-ATPase 10), a Ca2þ-ATPase (Wang et al., 2011). Overexpressing miR399 in strawberries can increase glucose, fructose and soluble substances, thereby improving fruit quality (Wang et al., 2017). In addition, miR408, miR1867, etc. may participate in the regulation of seed development through antioxidant, sugar conversion, starch synthesis and other pathways (Sun, 2012; Wang et al., 2017). MiRNAs that coordinate to regulate seed fruit development should have functional redundancy to ensure normal seed and fruit development. The development of seeds and fruits is related to the success of plant reproduction, and there must be a complex and delicate regulatory network behind it, and the miRNAs involved in the regulation of this process and their target genes are an important part of it. Among these miRNAs and target genes, some may be upstream and downstream relationships, some may be synergistic or antagonistic, and the specific regulatory networks have yet to be further revealed. 490 Plant Small RNA in Food Crops 3.5 Crop stress resistance traits Abiotic and biotic environmental stresses are major factors limiting plant growth, yield and sustainable agriculture. Common abiotic stresses include drought, high salinity, high and low temperature, nutrient deficiencies, hypoxia, and pollutants (Osakabe & Osakabe, 2017). Global climate change has exacerbated these constraints, especially drought and high salinity, making marginal lands more vulnerable to drought and brackish conditions. Therefore, breeding new plant varieties to improve plant tolerance to environmental stress is necessary to maximize plant biomass and crop yield. This is based on how well we understand plant responses to stress at the molecular level. Although several protein-coding genes have been identified for controlling plant responses to environmental abiotic stresses over the past few decades, knowledge about the regulatory mechanisms of plant responses to abiotic stresses is still limited, and engineering tools are needed to enable crops to adapt to harsh environments. miRNA is the main regulator of crop response to stress, it has become a genetic tool with great potential, can be used to understand the adaptation mechanism of crop stress at the molecular level, and used in genetic engineering to improve crop stress resistance (Table 15.5). MiRNAs responding to temperature stress have been reported in crops such as rice (Yang et al., 2013), wheat (Xin et al., 2010), barley (Kruszka et al., 2014), cotton (Wang, Liu, et al., 2016) and tobacco (Chen, Luan, & Zhai, 2015). Among them, miR167, miR319, miR396, miR444, etc. respond to cold stress, miR319 and miR396 respond to cold stress by reactive oxygen species (ROS) levels, and the cold stress tolerance of overexpressed plants of miR319 and miR396 is enhanced (Chen, Luan, & Zhai, 2015) MiRNAs that respond to thermal stress include miR159, miR160, miR166, miR167, etc., among which overexpression of miR159 in wheat will make it more sensitive to thermal stress. Overexpressing miR169 in tomatoes will make its pores less open, thereby increasing its drought resistance (Zhang, Zou, Gong, et al., 2011). Overexpressing miR827 in barley can also improve its drought resistance (Ferdous et al., 2017). NAC transcription factors are plant-specific transcription factors that are critical not only in plant development but also in abiotic stress responses (Nakashima et al., 2012). Overexpression of the stress-responsive NAC (SNAC) gene in southern Greater drought tolerance was shown in mustard and rice plants. Studies of the drought/salinity-induced transcription factor Food crops improvement: comparative biotechnological approaches 491 Table 15.5 miRNAs associated with crop stress resistance. miRNA Plants Function References miR159 Triticum aestivum Arachis hypogaea Solanum lycopersicum Oryza sativa Solanum lycopersicum Gossypium hirsutum Nicotiana tabacum Glycine max Thermal stress Wang, Sun, et al. (2012) Salt stress Tang et al. (2022) Cold stress Wang, Shi, et al. (2018) Cold stress Drought resistance Jiao et al. (2010) Zhang, Zou, Gong, et al. (2011) Xie et al. (2015) miR160 miR166 miR167 miR169 miR172 miR172 miR319 miR 393 miR 394 miR396 miR398 miR444 miR528 miR827 miR858 miR1848 Arabidopsis thaliana Arabidopsis thaliana Arabidopsis thaliana Oryza sativa Triticum aestivum Zea may Barley Arabidopsis thaliana Oryza sativa Drought and salinity stress Drought and salinity stress Cold stress Frazier et al. (2011) Salt resistance Chen, Luan, and Zhai (2015) Chen, Hu, et al. (2015) Drought resistance Basso et al. (2019) Cold stress Chen, Luan, and Zhai (2015) Basso et al. (2019) Drought and salinity stress Nitrogen-starvation stress Lodging resistance Drought resistance Fiber growth Wax biosynthesis Gao et al. (2016) Sun et al. (2018) Ferdous et al. (2017) Guan et al. (2014) Xia et al. (2015) AP2 (Mizoi et al., 2012) are also targets of the miRNA miR172. Drought and salinity stress altered the expression of miR 172 in cotton (Wang et al., 2013) and tobacco (Frazier et al., 2011). It suggests that miRNAs may play a key role in plant responses to abiotic stresses, and regulating miRNA expression may improve plant tolerance to environmental stresses. Drought and high salinity stress are two major constraints on plant growth and agricultural productivity worldwide. Although many genes are induced by these two types of stress, some of these genes are overexpressed by transgenic technology to improve the drought and salt tolerance of 492 Plant Small RNA in Food Crops plants; There was very little or no increase in tolerance (Bartels & Sunkar, 2005; Sunkar et al., 2012). One of the main reasons is that the plant response to environmental stress is a complex mechanism in which many genes, including protein-coding genes, transcription factors, and small RNAs, participate in this gene network. Therefore, recently discovered miRNAs may play an important cross-linking role in this process. Insects and diseases are two major biotic stresses that limit plant growth, development, and biomass and yield. Over the long evolutionary process, host plants have developed complex defense mechanisms that include rapid changes in the levels of genes, hormones, and metabolites. Recent studies have also shown that miRNAs also play an important role in this defense mechanism. 3.6 Other traits MiR159 in rice regulates joint pulling (Tsuji et al., 2006) and miR1848 regulates the wax biosynthesis (Xia et al., 2015). In maize, miR164 is involved in regulating the growth of lateral roots (Li et al., 2012), miR166 is involved in deciding leaf polarity (Juarez et al., 2004), miR528 is involved in regulating its lodging resistance by regulating lignin synthesis (Sun et al., 2018). Cotton miR828 and miR858 regulates fiber growth (Guan et al., 2014), miR319 in Chinese cabbage affects its glomerating properties by regulating cell differentiation in leaves (Mao et al., 2014). Tobacco miR395 is involved in regulating the homeostasis of sulfates in vivo (Yuan et al., 2016). miR858 in tomatoes is involved in regulating the accumulation of anthocyanins (Jia, Shen, et al., 2015). Constitutive expression of AtmiR156b in B. napus resulted in enhanced levels of seed lutein and bcarotene and a 2-fold increase in the number of flowering shoots (Wei et al., 2010). It is of great significance for the growth and development of plants and the formation of stress resistance traits. The mechanism of miRNA was studied, the key target genes and downstream gene regulatory networks were revealed, and combined with the means of genetic engineering, genetic transformation and molecular design breeding could effectively change the traits of crops, providing basic research materials and theoretical support for improving the important traits of crops. 4. Future perspectives In the past decade, significant progress has been made in studing plant miRNAs. Currently, miRNAs are one of the important gene regulators Food crops improvement: comparative biotechnological approaches 493 that control plant growth, development and response to environmental abiotic and biotic stresses New miRNA-based biotechnology has emerged for improving plant growth, development and tolerance to abiotic and biotic stresses, thereby targeting yield and quality as well as plant tolerance. However, plant miRNA-based biotechnology is still in its infancy, and many mysteries remain to be solved in order to utilize miRNA biology better to improve plant yield, quality, and tolerance to environmental biotic and abiotic stresses. The nutritional value of human vegetable food is one of the focuses of agricultural plant breeding, playing an increasing role in the prevention of various human diseases associated with malnutrition. Over the past few years, tremendous efforts have been made to improve the nutritional value of human plant foods and livestock feed. Controlling spatial and temporal expression patterns is important. For many crops, tissues eaten as food (mainly seeds) are different from those that control plant growth and ductility (mainly roots and shoots). In many cases, however, genes that control specific traits do not operate in a tissue-specific manner, but rather function in all or most plant organs. Therefore, mutations in a given gene that favor improving seed quality are often harmful to the growth of other plant organs. At present, miRNAs have become a research hotspot in the field of molecular biology. A large number of miRNAs have been found and identified in plants. With the continuous advancement of research methods, more and more plant miRNAs biological functions and molecular mechanisms of action will gradually be elucidated, and these miRNAs are involved in plant growth and development, metabolism and stress response, and other processes, indicating that miRNAs and their target genes have the potential to be applied in crop trait improvement. Some miRNAs that affect important agronomic traits (such as yield, plant type, fertility, etc.) of crops have been discovered, such as miR156 regulating tiller count, plant height, flowering period and grain size (Wang, Sun, et al., 2015). miR396 regulating leaf angle, plant height, and grain size (Li, Gao, et al., 2016). miR2118 regulating fertility (Fan et al., 2016). Molecular design breeding technology based on miRNA can develop excellent crop varieties by increasing crop stress tolerance, increasing crop yield, and improving quality. In addition, the theory of miRNA can also be applied to crop hybrid breeding. It was found that the overall expression of miRNAs was down-regulated compared to their parents in hybrid varieties such as rice and wheat (Fang et al., 2013; Li et al., 2014a). Studying miRNAs associated with heterosis will help crop breeders select the best combination of hybrid 494 Plant Small RNA in Food Crops offspring to produce commercially available F1 hybrid varieties (Tang & Chu, 2017). There are also some breeding-related miRNAs, such as OsamiR2118 (Fan et al., 2016), indicating that miRNAs can also be used in the breeding of male sterile lines. In addition, the application of CRISPR/Cas9 gene editing technology to crop miRNAs and their target genes (Jacobs et al., 2015; Zhou et al., 2017) makes it possible to obtain genetic materials with improved traits and no transgenic components. miRNAs have broad application prospects in the molecular breeding and improvement of crop traits. References Abdallah, N. A., Prakash, C. S., & McHughen, A. G. (2015). Genome editing for crop improvement: Challenges and opportunities. GM Crops & Food, 6, 183e205. Agrawal, A., Rajamani, V., Reddy, V. S., Mukherjee, S. K., & Bhatnagar, R. K. (2015). Transgenic plants over-expressing insect-specific microRNA acquire insecticidal activity against Helicoverpa armigera: An alternative to Bt-toxin technology. Transgenic Research, 24, 791e801. Baek, D., Chun, H. J., Kang, S., Shin, G., Park, S. J., Hong, H., Kim, C., Kim, D. H., Lee, S. Y., & Kim, M. C. (2016). A role for Arabidopsis miR399f in salt, drought, and ABA signaling. Molecular Cell, 39, 111. Bai, S., Tian, Y., Tan, C., Bai, S., Hao, J., & Hasi, A. (2020). Genome-wide identification of microRNAs involved in the regulation of fruit ripening and climacteric stages in melon (Cucumis melo). Horticulture Research, 7. Bai, Q., Wang, X., Chen, X., Shi, G., Liu, Z., Guo, C., & Xiao, K. (2018). Wheat miRNA TaemiR408 acts as an essential mediator in plant tolerance to Pi deprivation and salt stress via modulating stress-associated physiological processes. Frontiers of Plant Science, 9, 499. Bartel, D. P. (2004). MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell, 116, 281e297. Bartels, D., & Sunkar, R. (2005). Drought and salt tolerance in plants. Critical Reviews in Plant Sciences, 24, 23e58. Basso, M. F., Ferreira, P. C. G., Kobayashi, A. K., Harmon, F. G., Nepomuceno, A. L., Molinari, H. B. C., & Grossi-de-Sa, M. F. (2019). MicroRNAs and new biotechnological tools for its modulation and improving stress tolerance in plants. Plant Biotechnology Journal, 17, 1482e1500. Bian, H., Xie, Y., Guo, F., Han, N., Ma, S., Zeng, Z., Wang, J., Yang, Y., & Zhu, M. (2012). Distinctive expression patterns and roles of the miRNA393/TIR1 homolog module in regulating flag leaf inclination and primary and crown root growth in rice (Oryza sativa). New Phytologist, 196, 149e161. Bi, H., Fei, Q., Li, R., Liu, B., Xia, R., Char, S. N., Meyers, B. C., & Yang, B. (2020). Disruption of miRNA sequences by TALENs and CRISPR/Cas9 induces varied lengths of miRNA production. Plant Biotechnology Journal, 18, 1526e1536. Carbonell, A., Fahlgren, N., Mitchell, S., Cox, K. L., Jr., Reilly, K. C., Mockler, T. C., & Carrington, J. C. (2015). Highly specific gene silencing in a monocot species by artificial microRNAs derived from chimeric miRNA precursors. The Plant Journal, 82, 1061e1075. Food crops improvement: comparative biotechnological approaches 495 Carlsbecker, A., Lee, J. Y., Roberts, C. J., Dettmer, J., Lehesranta, S., Zhou, J., Lindgren, O., Moreno-Risueno, M. A., Vatén, A., Thitamadee, S., Campilho, A., Sebastian, J., Bowman, J. L., Helariutta, Y., & Benfey, P. N. (2010). Cell signalling by microRNA165/6 directs gene dose-dependent root cell fate. Nature, 465, 316e321. Chandrasekaran, M., Boopathi, T., & Paramasivan, M. (2021). A status-quo review on CRISPR-Cas9 gene editing applications in tomato. International Journal of Biological Macromolecules, 190, 120e129. Chang, H., Yi, B., Ma, R., Zhang, X., Zhao, H., & Xi, Y. (2016). CRISPR/cas9, a novel genomic tool to knock down microRNA in vitro and in vivo. Scientific Reports, 6, 22312. Chen, Z., Hu, L., Han, N., Hu, J., Yang, Y., Xiang, T., Zhang, X., & Wang, L. (2015). Overexpression of a miR393-resistant form of transport inhibitor response protein 1 (mTIR1) enhances salt tolerance by increased osmoregulation and Naþ exclusion in Arabidopsis thaliana. Plant and Cell Physiology, 56, 73e83. Chen, W., Kong, J., Lai, T., Manning, K., Wu, C., Wang, Y., Qin, C., Li, B., Yu, Z., & Zhang, X. (2015). Tuning LeSPL-CNR expression by SlymiR157 affects tomato fruit ripening. Scientific Reports, 5, 1e6. Chen, L., Luan, Y., & Zhai, J. (2015). Sp-miR396a-5p acts as a stress-responsive genes regulator by conferring tolerance to abiotic stresses and susceptibility to Phytophthora nicotianae infection in transgenic tobacco. Plant Cell Reports, 34, 2013e2025. Chi, M., Bhagwat, B., Lane, W. D., Tang, G., Su, Y., Sun, R., Oomah, B. D., Wiersma, P. A., & Xiang, Y. (2014). Reduced polyphenol oxidase gene expression and enzymatic browning in potato (Solanum tuberosum L.) with artificial microRNAs. BMC Plant Biology, 14, 62. Chilcoat, D., Liu, Z. B., & Sander, J. (2017). Use of CRISPR/Cas9 for crop improvement in maize and soybean. Progress in Molecular Biology and Translational Science, 149, 27e46. Chuck, G. S., Tobias, C., Sun, L., Kraemer, F., Li, C., Dibble, D., Arora, R., Bragg, J. N., Vogel, J. P., & Singh, S. (2011). Overexpression of the maize Corngrass1 microRNA prevents flowering, improves digestibility, and increases starch content of switchgrass. Proceedings of the National Academy of Sciences, 108, 17550e17555. Chukwu, S. C., Rafii, M. Y., Ramlee, S. I., Ismail, S. I., Oladosu, Y., Okporie, E., Onyishi, G., Utobo, E., Ekwu, L., & Swaray, S. (2019). Marker-assisted selection and gene pyramiding for resistance to bacterial leaf blight disease of rice (Oryza sativa L.). Biotechnology & Biotechnological Equipment, 33, 440e455. Collard, B. C., Jahufer, M., Brouwer, J., & Pang, E. (2005). An introduction to markers, quantitative trait loci (QTL) mapping and marker-assisted selection for crop improvement: The basic concepts. Euphytica, 142, 169e196. Cui, L. G., Shan, J. X., Shi, M., Gao, J. P., & Lin, H. X. (2014). The miR156-SPL9-DFR pathway coordinates the relationship between development and abiotic stress tolerance in plants. The Plant Journal, 80, 1108e1117. Curaba, J., Talbot, M., Li, Z., & Helliwell, C. (2013). Over-expression of microRNA171 affects phase transitions and floral meristem determinancy in barley. BMC Plant Biology, 13, 1e10. Davuluri, G. R., Van Tuinen, A., Fraser, P. D., Manfredonia, A., Newman, R., Burgess, D., Brummell, D. A., King, S. R., Palys, J., & Uhlig, J. (2005). Fruit-specific RNAimediated suppression of DET1 enhances carotenoid and flavonoid content in tomatoes. Nature Biotechnology, 23, 890e895. Ding, Y., Ye, Y., Jiang, Z., Wang, Y., & Zhu, C. (2016). MicroRNA390 is involved in cadmium tolerance and accumulation in rice. Frontiers of Plant Science, 7, 235. Dong, C.-H., & Pei, H. (2014). Over-expression of miR397 improves plant tolerance to cold stress in Arabidopsis thaliana. Journal of Plant Biology, 57, 209e217. 496 Plant Small RNA in Food Crops Fahlgren, N., Howell, M. D., Kasschau, K. D., Chapman, E. J., Sullivan, C. M., Cumbie, J. S., Givan, S. A., Law, T. F., Grant, S. R., Dangl, J. L., & Carrington, J. C. (2007). High-throughput sequencing of Arabidopsis microRNAs: Evidence for frequent birth and death of MIRNA genes. PLoS One, 2, e219. Fang, R., Li, L., & Li, J. (2013). Spatial and temporal expression modes of MicroRNAs in an elite rice hybrid and its parental lines. Planta, 238, 259e269. Fan, Y., Yang, J., Mathioni, S. M., Yu, J., Shen, J., Yang, X., Wang, L., Zhang, Q., Cai, Z., & Xu, C. (2016). PMS1T, producing phased small-interfering RNAs, regulates photoperiod-sensitive male sterility in rice. Proceedings of the National Academy of Sciences, 113, 15144e15149. Farinati, S., Forestan, C., Canton, M., Varotto, S., & Bonghi, C. (2020). microRNA regulation of fruit development. In Plant microRNAs (pp. 75e98). Springer. Feng, H., Duan, X., Zhang, Q., Li, X., Wang, B., Huang, L., Wang, X., & Kang, Z. (2014). The target gene of tae-miR164, a novel NAC transcription factor from the NAM subfamily, negatively regulates resistance of wheat to stripe rust. Molecular Plant Pathology, 15, 284e296. Ferdous, J., Whitford, R., Nguyen, M., Brien, C., Langridge, P., & Tricker, P. J. (2017). Drought-inducible expression of Hv-miR827 enhances drought tolerance in transgenic barley. Functional & Integrative Genomics, 17, 279e292. Franco-Zorrilla, J. M., Valli, A., Todesco, M., Mateos, I., Puga, M. I., Rubio-Somoza, I., Leyva, A., Weigel, D., García, J. A., & Paz-Ares, J. (2007). Target mimicry provides a new mechanism for regulation of microRNA activity. Nature Genetics, 39, 1033e1037. Frazier, T. P., Sun, G., Burklew, C. E., & Zhang, B. (2011). Salt and drought stresses induce the aberrant expression of microRNA genes in tobacco. Molecular Biotechnology, 49, 159e165. Gao, C. (2021). Genome engineering for crop improvement and future agriculture. Cell, 184(6), 1621e1635. Gao, P., Bai, X., Yang, L., Lv, D., Li, Y., Cai, H., Ji, W., Guo, D., & Zhu, Y. (2010). Overexpression of osa-MIR396c decreases salt and alkali stress tolerance. Planta, 231, 991e1001. Gao, S., Guo, C., Zhang, Y., Zhang, F., Du, X., Gu, J., & Xiao, K. (2016). Wheat microRNA member TaMIR444a is nitrogen deprivation-responsive and involves plant adaptation to the nitrogen-starvation stress. Plant Molecular Biology Reporter, 34, 931e946. Gao, Z., Shi, T., Luo, X., Zhang, Z., Zhuang, W., & Wang, L. (2012). High-throughput sequencing of small RNAs and analysis of differentially expressed microRNAs associated with pistil development in Japanese apricot. BMC Genomics, 13, 371. Geng, Y., Jian, C., Xu, W., Liu, H., Hao, C., Hou, J., Liu, H., Zhang, X., & Li, T. (2020). miR164-targeted TaPSK5 encodes a phytosulfokine precursor that regulates root growth and yield traits in common wheat (Triticum aestivum L.). Plant Molecular Biology, 104, 615e628. Guan, X., Pang, M., Nah, G., Shi, X., Ye, W., Stelly, D. M., & Chen, Z. J. (2014). miR828 and miR858 regulate homoeologous MYB2 gene functions in Arabidopsis trichome and cotton fibre development. Nature Communications, 5, 1e14. Guo, W., Chen, L., Chen, H., Yang, H., You, Q., Bao, A., Chen, S., Hao, Q., Huang, Y., & Qiu, D. (2020). Overexpression of GmWRI1b in soybean stably improves plant architecture and associated yield parameters, and increases total seed oil production under field conditions. Plant Biotechnology Journal, 18, 1639e1641. Guo, W., Chen, L., Herrera-Estrella, L., Cao, D., & Tran, L. P. (2020). Altering plant architecture to improve performance and resistance. Trends in Plant Science, 25, 1154e1170. Food crops improvement: comparative biotechnological approaches 497 Gupta, S. K., Vishwakarma, A., Kenea, H. D., Galsurker, O., Cohen, H., Aharoni, A., & Arazi, T. (2021). CRISPR/Cas9 mutants of tomato MICRORNA164 genes uncover their functional specialization in development. Plant Physiology, 187, 1636e1652. Hajyzadeh, M., Turktas, M., Khawar, K. M., & Unver, T. (2015). miR408 overexpression causes increased drought tolerance in chickpea. Gene, 555, 186e193. Han, R., Jian, C., Lv, J., Yan, Y., Chi, Q., Li, Z., Wang, Q., Zhang, J., Liu, X., & Zhao, H. (2014). Identification and characterization of microRNAs in the flag leaf and developing seed of wheat (Triticum aestivum L.). BMC Genomics, 15, 1e14. Heffner, E. L., Sorrells, M. E., & Jannink, J.-L. (2009). Genomic selection for crop improvement. Hofferek, V., Mendrinna, A., Gaude, N., Krajinski, F., & Devers, E. A. (2014). MiR171h restricts root symbioses and shows like its target NSP2 a complex transcriptional regulation in Medicago truncatula. BMC Plant Biology, 14, 1e16. Hong, Y., Meng, J., He, X., Zhang, Y., Liu, Y., Zhang, C., Qi, H., & Luan, Y. (2021). Editing miR482b and miR482c simultaneously by CRISPR/Cas9 enhanced tomato resistance to Phytophthora infestans. Phytopathology, 111, 1008e1016. Huang, W., Peng, S., Xian, Z., Lin, D., Hu, G., Yang, L., Ren, M., & Li, Z. (2017). Overexpression of a tomato miR171 target gene Sl GRAS 24 impacts multiple agronomical traits via regulating gibberellin and auxin homeostasis. Plant Biotechnology Journal, 15, 472e488. Huang, S. Q., Xiang, A. L., Che, L. L., Chen, S., Li, H., Song, J. B., & Yang, Z. M. (2010). A set of miRNAs from Brassica napus in response to sulphate deficiency and cadmium stress. Plant Biotechnology Journal, 8, 887e899. Huo, H., Wei, S., & Bradford, K. J. (2016). Delay of germination1 (DOG1) regulates both seed dormancy and flowering time through microRNA pathways. Proceedings of the National Academy of Sciences of the United States of America, 113, E2199eE2206. Jacobs, T. B., LaFayette, P. R., Schmitz, R. J., & Parrott, W. A. (2015). Targeted genome modifications in soybean with CRISPR/Cas9. BMC Biotechnology, 15, 16. Jia, X., Ding, N., Fan, W., Yan, J., Gu, Y., Tang, X., Li, R., & Tang, G. (2015). Functional plasticity of miR165/166 in plant development revealed by small tandem target mimic. Plant Science, 233, 11e21. Jiang, H., Feng, Y., Bao, L., Li, X., Gao, G., Zhang, Q., Xiao, J., Xu, C., & He, Y. (2012). Improving blast resistance of Jin 23B and its hybrid rice by marker-assisted gene pyramiding. Molecular Breeding, 30, 1679e1688. Jiang, N., Meng, J., Cui, J., Sun, G., & Luan, Y. (2018). Function identification of miR482b, a negative regulator during tomato resistance to Phytophthora infestans. Horticultural Research, 5, 9. Jiang, W., Zhou, H., Bi, H., Fromm, M., Yang, B., & Weeks, D. P. (2013). Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice. Nucleic Acids Research, 41, e188. Jian, C., Han, R., Chi, Q., Wang, S., Ma, M., Liu, X., & Zhao, H. (2017). Virus-based microRNA silencing and overexpressing in common wheat (Triticum aestivum L.). Frontiers of Plant Science, 8, 500. Jiao, Y., Wang, Y., Xue, D., Wang, J., Yan, M., Liu, G., Dong, G., Zeng, D., Lu, Z., & Zhu, X. (2010). Regulation of OsSPL14 by OsmiR156 defines ideal plant architecture in rice. Nature Genetics, 42, 541e544. Jia, X., Shen, J., Liu, H., Li, F., Ding, N., Gao, C., Pattanaik, S., Patra, B., Li, R., & Yuan, L. (2015). Small tandem target mimic-mediated blockage of microRNA858 induces anthocyanin accumulation in tomato. Planta, 242, 283e293. Jones-Rhoades, M. W., & Bartel, D. P. (2004). Computational identification of plant microRNAs and their targets, including a stress-induced miRNA. Molecular Cell, 14, 787e799. 498 Plant Small RNA in Food Crops Jones-Rhoades, M. W., Bartel, D. P., & Bartel, B. (2006). MicroRNAs and their regulatory roles in plants. Annual Review of Plant Biology, 57, 19e53. Juarez, M. T., Kui, J. S., Thomas, J., Heller, B. A., & Timmermans, M. C. (2004). microRNA-mediated repression of rolled leaf1 specifies maize leaf polarity. Nature, 428, 84e88. Ju, Z., Cao, D., Gao, C., Zuo, J., Zhai, B., Li, S., Zhu, H., Fu, D., Luo, Y., & Zhu, B. (2017). A viral satellite DNA vector (TYLCCNV) for functional analysis of miRNAs and siRNAs in plants. Plant Physiology, 173, 1940e1952. Kamthan, A., Chaudhuri, A., Kamthan, M., & Datta, A. (2015). Small RNAs in plants: Recent development and application for crop improvement. Frontiers of Plant Science, 6, 208. Kang, T., Yu, C.-Y., Liu, Y., Song, W.-M., Bao, Y., Guo, X.-T., Li, B., & Zhang, H.-X. (2020). Subtly manipulated expression of ZmmiR156 in tobacco improves drought and salt tolerance without changing the architecture of transgenic plants. Frontiers of Plant Science, 10, 1664. Kang, M., Zhao, Q., Zhu, D., & Yu, J. (2012). Characterization of microRNAs expression during maize seed development. BMC Genomics, 13, 1e11. Karakülah, G., Yücebilgili Kurto
glu, K., & Unver, T. (2016). PeTMbase: A database of plant endogenous target mimics (eTMs). PLoS One, 11, e0167698. Karlova, R., van Haarst, J. C., Maliepaard, C., van de Geest, H., Bovy, A. G., Lammers, M., Angenent, G. C., & de Maagd, R. A. (2013). Identification of microRNA targets in tomato fruit development using high-throughput sequencing and degradome analysis. Journal of Experimental Botany, 64, 1863e1878. Khatodia, S., Bhatotia, K., Passricha, N., Khurana, S., & Tuteja, N. (2016). The CRISPR/ cas genome-editing tool: Application in improvement of crops. Frontiers of Plant Science, 7, 506. Kim, S.-G. (2020). The way to true plant genome editing. Nature Plants, 6, 736e737. Kim, H., & Kim, J.-S. (2014). A guide to genome engineering with programmable nucleases. Nature Reviews Genetics, 15, 321e334. Kim, J. H., Woo, H. R., Kim, J., Lim, P. O., Lee, I. C., Choi, S. H., Hwang, D., & Nam, H. G. (2009). Trifurcate feed-forward regulation of age-dependent cell death involving miR164 in Arabidopsis. Science, 323, 1053e1057. Kim, Y.-J., & Zhang, D. (2018). Molecular control of male fertility for crop hybrid breeding. Trends in Plant Science, 23, 53e65. Kis, A., Tholt, G., Ivanics, M., Várallyay, É., Jenes, B., & Havelda, Z. (2016). Polycistronic artificial miRNA-mediated resistance to Wheat dwarf virus in barley is highly efficient at low temperature. Molecular Plant Pathology, 17, 427e437. Knight, J. (2003). Crop improvement: A dying breed. Nature, 421, 568e571. Kruszka, K., Pacak, A., Swida-Barteczka, A., Nuc, P., Alaba, S., Wroblewska, Z., Karlowski, W., Jarmolowski, A., & Szweykowska-Kulinska, Z. (2014). Transcriptionally and post-transcriptionally regulated microRNAs in heat stress response in barley. Journal of Experimental Botany, 65, 6123e6135. Ku, H.-K., & Ha, S.-H. (2020). Improving nutritional and functional quality by genome editing of crops: Status and perspectives. Frontiers of Plant Science, 11, 1514. Kumar, K., Gambhir, G., Dass, A., Tripathi, A. K., Singh, A., Jha, A. K., Yadava, P., Choudhary, M., & Rakshit, S. (2020). Genetically modified crops: Current status and future prospects. Planta, 251, 91. Lee, Y.-S., Lee, D.-Y., Cho, L.-H., & An, G. (2014). Rice miR172 induces flowering by suppressing OsIDS1 and SNB, two AP2 genes that negatively regulate expression of Ehd1 and florigens. Rice, 7, 1e13. Food crops improvement: comparative biotechnological approaches 499 Levin, I., Frankel, P., Gilboa, N., Tanny, S., & Lalazar, A. (2003). The tomato dark green mutation is a novel allele of the tomato homolog of the DEETIOLATED1 gene. Theoretical and Applied Genetics, 106, 454e460. Li, X., Chen, P., Xie, Y., Yan, Y., Wang, L., Dang, H., Zhang, J., Xu, L., Ma, F., & Guan, Q. (2020). Apple SERRATE negatively mediates drought resistance by regulating MdMYB88 and MdMYB124 and microRNA biogenesis. Horticulture Research, 7. Li, S., Gao, F., Xie, K., Zeng, X., Cao, Y., Zeng, J., He, Z., Ren, Y., Li, W., & Deng, Q. (2016). The OsmiR396c-OsGRF4-OsGIF1 regulatory module determines grain size and yield in rice. Plant Biotechnology Journal, 14, 2134e2146. Li, J., Guo, G., Guo, W., Guo, G., Tong, D., Ni, Z., Sun, Q., & Yao, Y. (2012). miRNA164-directed cleavage of ZmNAC1 confers lateral root development in maize (Zea mays L.). BMC Plant Biology, 12, 1e14. Li, W., Jia, Y., Liu, F., Wang, F., Fan, F., Wang, J., Zhu, J., Xu, Y., Zhong, W., & Yang, J. (2019). Integration analysis of small RNA and degradome sequencing reveals microRNAs responsive to Dickeya zeae in resistant rice. International Journal of Molecular Sciences, 20, 222. Li, A., Liu, D., Wu, J., Zhao, X., Hao, M., Geng, S., Yan, J., Jiang, X., Zhang, L., & Wu, J. (2014a). mRNA and small RNA transcriptomes reveal insights into dynamic homoeolog regulation of allopolyploid heterosis in nascent hexaploid wheat. The Plant Cell Online, 26, 1878e1900. Lin, Y., Zhu, Y., Cui, Y., Chen, R., Chen, Z., Li, G., Fan, M., Chen, J., Li, Y., Guo, X., Zheng, X., Chen, L., & Wang, F. (2021). Derepression of specific miRNA-target genes in rice using CRISPR/Cas9. Journal of Experimental Botany, 72, 7067e7077. Li, Y., Lu, Y.-G., Shi, Y., Wu, L., Xu, Y.-J., Huang, F., Guo, X.-Y., Zhang, Y., Fan, J., & Zhao, J.-Q. (2014b). Multiple rice microRNAs are involved in immunity against the blast fungus Magnaporthe oryzae. Plant Physiology, 164, 1077e1092. Li, W.-X., Oono, Y., Zhu, J., He, X.-J., Wu, J.-M., Iida, K., Lu, X.-Y., Cui, X., Jin, H., & Zhu, J.-K. (2008). The Arabidopsis NFYA5 transcription factor is regulated transcriptionally and posttranscriptionally to promote drought resistance. The Plant Cell Online, 20, 2238e2251. Liu, L., Gallagher, J., Arevalo, E. D., Chen, R., Skopelitis, T., Wu, Q., Bartlett, M., & Jackson, D. (2021). Enhancing grain-yield-related traits by CRISPR-Cas9 promoter editing of maize CLE genes. Native Plants, 7, 287e294. Liu, H., Shen, E., Wu, H., Ma, W., Chen, H., & Lin, Y. (2022). Trans-kingdom expression of an insect endogenous microRNA in rice enhances resistance to striped stem borer Chilo suppressalis. Pest Management Science, 78, 770e777. Liu, N., Wu, S., Van Houten, J., Wang, Y., Ding, B., Fei, Z., Clarke, T. H., Reed, J. W., & Van Der Knaap, E. (2014). Down-regulation of AUXIN RESPONSE FACTORS 6 and 8 by microRNA 167 leads to floral development defects and female sterility in tomato. Journal of Experimental Botany, 65, 2507e2520. Li, W., Wang, T., Zhang, Y., & Li, Y. (2016). Overexpression of soybean miR172c confers tolerance to water deficit and salt stress, but increases ABA sensitivity in transgenic Arabidopsis thaliana. Journal of Experimental Botany, 67, 175e194. Long, J.-M., Liu, C.-Y., Feng, M.-Q., Liu, Y., Wu, X.-M., & Guo, W.-W. (2018). miR156-SPL modules regulate induction of somatic embryogenesis in citrus callus. Journal of Experimental Botany, 69, 2979e2993. Lu, Y., Feng, Z., Bian, L., Xie, H., & Liang, J. (2010). miR398 regulation in rice of the responses to abiotic and biotic stresses depends on CSD1 and CSD2 expression. Functional Plant Biology, 38, 44e53. Lunardon, A., Kariuki, S. M., & Axtell, M. J. (2021). Expression and processing of polycistronic artificial microRNAs and trans-acting siRNAs from transiently introduced 500 Plant Small RNA in Food Crops transgenes in Solanum lycopersicum and Nicotiana benthamiana. The Plant Journal, 106, 1087e1104. Ma, Z., Jiang, J., Hu, Z., Lyu, T., Yang, Y., Jiang, J., & Cao, J. (2017). Over-expression of miR158 causes pollen abortion in Brassica campestris ssp. chinensis. Plant Molecular Biology, 93, 313e326. Manavella, P. A., Koenig, D., Rubio-Somoza, I., Burbano, H. A., Becker, C., & Weigel, D. (2012). Tissue-specific silencing of Arabidopsis SU(VAR)3-9 HOMOLOG8 by miR171a. Plant Physiology, 161, 805e812. Manavella, P. A., Koenig, D., & Weigel, D. (2012). Plant secondary siRNA production determined by microRNA-duplex structure. Proceedings of the National Academy of Sciences of the United States of America, 109, 2461e2466. Mao, Y., Wu, F., Yu, X., Bai, J., Zhong, W., & He, Y. (2014). MicroRNA319a-targeted Brassica rapa ssp. pekinensis TCP genes modulate head shape in Chinese cabbage by differential cell division arrest in leaf regions. Plant Physiology, 164, 710e720. Maxted, N., & Guarino, L. (2006). Genetic erosion and genetic pollution of crop wild relatives. In Genetic erosion and pollution assessment methodologies (pp. 35e45). Proceedings of PGR Forum Workshop. Miao, C., Wang, D., He, R., Liu, S., & Zhu, J. K. (2020). Mutations in MIR 396e and MIR 396f increase grain size and modulate shoot architecture in rice. Plant Biotechnology Journal, 18, 491e501. Miflin, B. (2000). Crop improvement in the 21st century. Journal of Experimental Botany, 51, 1e8. Mizoi, J., Shinozaki, K., & Yamaguchi-Shinozaki, K. (2012). AP2/ERF family transcription factors in plant abiotic stress responses. Biochimica et Biophysica Acta (BBA)dGene Regulatory Mechanisms, 1819, 86e96. Molesini, B., Pii, Y., & Pandolfini, T. (2012). Fruit improvement using intragenesis and artificial microRNA. Trends in Biotechnology, 30, 80e88. Mujjassim, N. E., Mallik, M., Rathod, N. K. K., & Nitesh, S. (2019). Cisgenesis and intragenesis a new tool for conventional plant breeding: A review. Journal of Pharmacognosy and Phytochemistry, 8, 2485e2489. Nakashima, K., Takasaki, H., Mizoi, J., Shinozaki, K., & Yamaguchi-Shinozaki, K. (2012). NAC transcription factors in plant abiotic stress responses. Biochimica et Biophysica Acta (BBA)dGene Regulatory Mechanisms, 1819, 97e103. Neeraja, C. N., Maghirang-Rodriguez, R., Pamplona, A., Heuer, S., Collard, B. C., Septiningsih, E. M., Vergara, G., Sanchez, D., Xu, K., & Ismail, A. M. (2007). A marker-assisted backcross approach for developing submergence-tolerant rice cultivars. Theoretical and Applied Genetics, 115, 767e776. Ni, Z., Hu, Z., Jiang, Q., & Zhang, H. (2013). GmNFYA3, a target gene of miR169, is a positive regulator of plant tolerance to drought stress. Plant Molecular Biology, 82, 113e129. Osakabe, Y., Liang, Z., Ren, C., Nishitani, C., Osakabe, K., Wada, M., Komori, S., Malnoy, M., Velasco, R., Poli, M., Jung, M. H., Koo, O. J., Viola, R., & Nagamangala Kanchiswamy, C. (2018). CRISPR-Cas9-mediated genome editing in apple and grapevine. Nature Protocols, 13, 2844e2863. Osakabe, Y., & Osakabe, K. (2017). Genome editing to improve abiotic stress responses in plants. Progress in Molecular Biology and Translational Science, 149, 99e109. Pandey, K., Dangi, R., Prajapati, U., Kumar, S., Maurya, N. K., Singh, A. V., Pandey, A. K., Singh, J., & Rajan, R. (2019). Advance breeding and biotechnological approaches for crop improvement: A review. International Journal of Chemical Studies, 7, 837e841. Peng, T., Qiao, M., Liu, H., Teotia, S., Zhang, Z., Zhao, Y., Wang, B., Zhao, D., Shi, L., Zhang, C., Le, B., Rogers, K., Gunasekara, C., Duan, H., Gu, Y., Tian, L., Nie, J., Food crops improvement: comparative biotechnological approaches 501 Qi, J., Meng, F., … Tang, G. (2018). A resource for inactivation of MicroRNAs using short tandem target mimic technology in model and crop plants. Molecular Plant, 11, 1400e1417. Peng, T., Sun, H., Qiao, M., Zhao, Y., Du, Y., Zhang, J., Li, J., Tang, G., & Zhao, Q. (2014). Differentially expressed microRNA cohorts in seed development may contribute to poor grain filling of inferior spikelets in rice. BMC Plant Biology, 14, 1e17. Qiao, F., Yang, Q., Wang, C.-L., Fan, Y.-L., Wu, X.-F., & Zhao, K.-J. (2007). Modification of plant height via RNAi suppression of OsGA20ox2 gene in rice. Euphytica, 158, 35e45. Rajam, M. V. (2020). RNA silencing technology: A boon for crop improvement. Journal of Biosciences, 45, 1e5. Reem, N. T., & Van Eck, J. (2019). Application of CRISPR/Cas9-Mediated gene editing in tomato. Methods in Molecular Biology, 1917, 171e182. Reichel, M., Li, Y., Li, J., & Millar, A. A. (2015). Inhibiting plant microRNA activity: Molecular SPONGEs, target MIMICs and STTMs all display variable efficacies against target microRNAs. Plant Biotechnology Journal, 13, 915e926. Reyes, J. L., & Chua, N. H. (2007). ABA induction of miR159 controls transcript levels of two MYB factors during Arabidopsis seed germination. The Plant Journal, 49, 592e606. Rhoades, M. W., Reinhart, B. J., Lim, L. P., Burge, C. B., Bartel, B., & Bartel, D. P. (2002). Prediction of plant microRNA targets. Cell, 110, 513e520. Ribaut, J.-M., & Hoisington, D. (1998). Marker-assisted selection: New tools and strategies. Trends in Plant Science, 3, 236e239. Rodríguez-Leal, D., Lemmon, Z. H., Man, J., Bartlett, M. E., & Lippman, Z. B. (2017). Engineering quantitative trait variation for crop improvement by genome editing. Cell, 171, 470e480. e478. Rosas Cárdenas, F. D. F., Ruiz Suárez, Y., Cano Rangel, R. M., Luna Garcia, V., González Aguilera, K. L., Marsch Martínez, N., & De Folter, S. (2017). Effect of constitutive miR164 expression on plant morphology and fruit development in Arabidopsis and tomato. Agronomy, 7, 48. Sablok, G., Pérez-Quintero, A. L., Hassan, M., Tatarinova, T. V., & López, C. (2011). Artificial microRNAs (amiRNAs) engineeringdOn how microRNA-based silencing methods have affected current plant silencing research. Biochemical and Biophysical Research Communications, 406, 315e319. Sami, A., Xue, Z., Tazein, S., Arshad, A., He Zhu, Z., Ping Chen, Y., Hong, Y., Tian Zhu, X., & Jin Zhou, K. (2021). CRISPR-Cas9-based genetic engineering for crop improvement under drought stress. Bioengineered, 12, 5814e5829. Sauer, N. J., Mozoruk, J., Miller, R. B., Warburg, Z. J., Walker, K. A., Beetham, P. R., Schopke, C. R., & Gocal, G. F. (2016). Oligonucleotide-directed mutagenesis for precision gene editing. Plant Biotechnology Journal, 14, 496e502. Song, J. B., Shu, X. X., Shen, Q., Li, B. W., Song, J., & Yang, Z. M. (2015). Altered fruit and seed development of transgenic rapeseed (Brassica napus) over-expressing microRNA394. PLoS One, 10, e0125427. Songstad, D. D., Petolino, J. F., Voytas, D. F., & Reichert, N. A. (2017). Genome editing of plants. Critical Reviews in Plant Sciences, 36, 1e23. Song, C., Wang, C., Zhang, C., Korir, N., Yu, H., Ma, Z., & Fang, J. (2010). Deep sequencing discovery of novel and conserved microRNAs in trifoliate orange (Citrus trifoliata). BMC Genomics, 11, 431. Srikanth, A., & Schmid, M. (2011). Regulation of flowering time: All roads lead to Rome. Cellular and Molecular Life Sciences, 68, 2013e2037. Su, Y. H., Liu, Y. B., Zhou, C., Li, X. M., & Zhang, X. S. (2016). The microRNA167 controls somatic embryogenesis in Arabidopsis through regulating its target genes ARF6 and ARF8. Plant Cell, Tissue and Organ Culture, 124, 405e417. 502 Plant Small RNA in Food Crops Sun, G. (2012). MicroRNAs and their diverse functions in plants. Plant Molecular Biology, 80, 17e36. Sunkar, R., Li, Y.-F., & Jagadeeswaran, G. (2012). Functions of microRNAs in plant stress responses. Trends in Plant Science, 17, 196e203. Sun, Q., Liu, X., Yang, J., Liu, W., Du, Q., Wang, H., Fu, C., & Li, W.-X. (2018). MicroRNA528 affects lodging resistance of maize by regulating lignin biosynthesis under nitrogen-luxury conditions. Molecular Plant, 11, 806e814. Sun, L., Sun, Y., Zhang, M., Wang, L., Ren, J., Cui, M., Wang, Y., Ji, K., Li, P., Li, Q., Chen, P., Dai, S., Duan, C., Wu, Y., & Leng, P. (2012). Suppression of 9-cis-epoxycarotenoid dioxygenase, which encodes a key enzyme in abscisic acid biosynthesis, alters fruit texture in transgenic tomato. Plant Physiology, 158, 283e298. Sun, Z., Su, C., Yun, J., Jiang, Q., Wang, L., Wang, Y., Cao, D., Zhao, F., Zhao, Q., Zhang, M., Zhou, B., Zhang, L., Kong, F., Liu, B., Tong, Y., & Li, X. (2019). Genetic improvement of the shoot architecture and yield in soya bean plants via the manipulation of GmmiR156b. Plant Biotechnology Journal, 17, 50e62. Sun, L., Yuan, B., Zhang, M., Wang, L., Cui, M., Wang, Q., & Leng, P. (2012). Fruitspecific RNAi-mediated suppression of SlNCED1 increases both lycopene and bcarotene contents in tomato fruit. Journal of Experimental Botany, 63, 3097e3108. Tang, J., & Chu, C. (2017). MicroRNAs in crop improvement: Fine-tuners for complex traits. Nature plants, 3, 1e11. Tang, Y., Du, G., Xiang, J., Hu, C., Li, X., Wang, W., Zhu, H., Qiao, L., Zhao, C., & Wang, J. (2022). Genome-wide identification of auxin response factor (ARF) gene family and the miR160-ARF18-mediated response to salt stress in peanut (Arachis hypogaea L.). Genomics, 114, 171e184. Tang, Y., Liu, H., Guo, S., Wang, B., Li, Z., Chong, K., & Xu, Y. (2017). OsmiR396d affects gibberellin and brassinosteroid signaling to regulate plant architecture in rice. Plant Physiology, 176, 946e959. Teotia, S., Singh, D., Tang, X., & Tang, G. (2016). Essential RNA-based technologies and their applications in plant functional genomics. Trends in Biotechnology, 34, 106e123. Tester, M., & Langridge, P. (2010). Breeding technologies to increase crop production in a changing world. Science, 327, 818e822. Toda, E., & Okamoto, T. (2020). CRISPR/Cas9-Based genome editing using rice zygotes. Curr Protoc Plant Biol, 5, e20111. Tsuji, H., Aya, K., Ueguchi-Tanaka, M., Shimada, Y., Nakazono, M., Watanabe, R., Nishizawa, N. K., Gomi, K., Shimada, A., & Kitano, H. (2006). GAMYB controls different sets of genes and is differentially regulated by microRNA in aleurone cells and anthers. The Plant Journal, 47, 427e444. Wagaba, H., Patil, B. L., Mukasa, S., Alicai, T., Fauquet, C. M., & Taylor, N. J. (2016). Artificial microRNA-derived resistance to Cassava brown streak disease. Journal of Virological Methods, 231, 38e43. Wang, Y., Itaya, A., Zhong, X., Wu, Y., Zhang, J., van der Knaap, E., Olmstead, R., Qi, Y., & Ding, B. (2011). Function and evolution of a MicroRNA that regulates a Ca2þ-ATPase and triggers the formation of phased small interfering RNAs in tomato reproductive growth. The Plant Cell Online, 23, 3185e3203. Wang, H., Jiao, X., Kong, X., Hamera, S., Wu, Y., Chen, X., Fang, R., & Yan, Y. (2016). A signaling cascade from miR444 to RDR1 in rice antiviral RNA silencing pathway. Plant Physiology, 170, 2365e2377. Wang, B., Lin, Z., Li, X., Zhao, Y., Zhao, B., Wu, G., Ma, X., Wang, H., Xie, Y., & Li, Q. (2020). Genome-wide selection and genetic improvement during modern maize breeding. Nature Genetics, 52, 565e571. Wang, Q., Liu, N., Yang, X., Tu, L., & Zhang, X. (2016). Small RNA-mediated responses to low-and high-temperature stresses in cotton. Scientific Reports, 6, 1e14. Food crops improvement: comparative biotechnological approaches 503 Wang, T., Pan, H., Wang, J., Yang, W., Cheng, T., & Zhang, Q. (2014). Identification and profiling of novel and conserved microRNAs during the flower opening process in Prunus mume via deep sequencing. Molecular Genetics and Genomics, 289, 169e183. Wang, W., Shi, T., Ni, X., Xu, Y., Qu, S., & Gao, Z. (2018). The role of miR319a and its target gene TCP4 in the regulation of pistil development in Prunus mume. Genome, 61, 43e48. Wang, Y., Sun, F., Cao, H., Peng, H., Ni, Z., Sun, Q., & Yao, Y. (2012). TamiR159 directed wheat TaGAMYB cleavage and its involvement in anther development and heat response. PLoS One, 7, e48445. Wang, S.-t., Sun, X.-l., Hoshino, Y., Yu, Y., Jia, B., Sun, Z.-w., Sun, M.-z., Duan, X.-b., & Zhu, Y.-m. (2014). MicroRNA319 positively regulates cold tolerance by targeting OsPCF6 and OsTCP21 in rice (Oryza sativa L.). PLoS One, 9, e91357. Wang, L., Sun, S., Jin, J., Fu, D., Yang, X., Weng, X., Xu, C., Li, X., Xiao, J., & Zhang, Q. (2015). Coordinated regulation of vegetative and reproductive branching in rice. Proceedings of the National Academy of Sciences, 112, 15504e15509. Wang, T., Sun, M.-Y., Wang, X.-S., Li, W.-B., & Li, Y.-G. (2016). Over-expression of GmGIa-regulated soybean miR172a confers early flowering in transgenic Arabidopsis thaliana. International Journal of Molecular Sciences, 17, 645. Wang, M., Wang, Q., & Zhang, B. (2013). Response of miRNAs and their targets to salt and drought stresses in cotton (Gossypium hirsutum L.). Gene, 530, 26e32. Wang, S., Wu, K., Yuan, Q., Liu, X., Liu, Z., Lin, X., Zeng, R., Zhu, H., Dong, G., & Qian, Q. (2012). Control of grain size, shape and quality by OsSPL16 in rice. Nature Genetics, 44, 950e954. Wang, X., Xu, Y., Hu, Z., & Xu, C. (2018). Genomic selection methods for crop improvement: Current status and prospects. The Crop Journal, 6, 330e340. Wang, L., ZengJ, H. Q., Song, J., Feng, S. J., & Yang, Z. M. (2015). miRNA778 and SUVH6 are involved in phosphate homeostasis in Arabidopsis. Plant Science, 238, 273e285. Wang, Y., Zhang, J., Cui, W., Guan, C., Mao, W., & Zhang, Z. (2017). Improvement in fruit quality by overexpressing miR399a in woodland strawberry. Journal of Agricultural and Food Chemistry, 65, 7361e7370. Wei, S., Yu, B., Gruber, M. Y., Khachatourians, G. G., Hegedus, D. D., & Hannoufa, A. (2010). Enhanced seed carotenoid levels and branching in transgenic Brassica napus expressing the Arabidopsis miR156b gene. Journal of Agricultural and Food Chemistry, 58, 9572e9578. Wollmann, H., Mica, E., Todesco, M., Long, J. A., & Weigel, D. (2010). On reconciling the interactions between APETALA2, miR172 and AGAMOUS with the ABC model of flower development. Development, 137, 3633e3642. Xian, Z., Huang, W., Yang, Y., Tang, N., Zhang, C., Ren, M., & Li, Z. (2014). miR168 influences phase transition, leaf epinasty, and fruit development via SlAGO1s in tomato. Journal of Experimental Botany, 65, 6655e6666. Xia, K., Ou, X., Gao, C., Tang, H., Jia, Y., Deng, R., Xu, X., & Zhang, M. (2015). OsWS1 involved in cuticular wax biosynthesis is regulated by osa-miR1848. Plant, Cell and Environment, 38, 2662e2673. Xie, F., Wang, Q., Sun, R., & Zhang, B. (2015). Deep sequencing reveals important roles of microRNAs in response to drought and salinity stress in cotton. Journal of Experimental Botany, 66, 789e804. Xie, Y., Zhou, Q., Zhao, Y., Li, Q., Liu, Y., Ma, M., Wang, B., Shen, R., Zheng, Z., & Wang, H. (2020). FHY3 and FAR1 integrate light signal with the miR156-SPL module-mediated aging pathway to regulate Arabidopsis flowering. Molecular Plant, 483e498. 504 Plant Small RNA in Food Crops Xin, M., Wang, Y., Yao, Y., Xie, C., Peng, H., Ni, Z., & Sun, Q. (2010). Diverse set of microRNAs are responsive to powdery mildew infection and heat stress in wheat (Triticum aestivum L.). BMC Plant Biology, 10, 1e11. Xu, J., Hua, K., & Lang, Z. (2019). Genome editing for horticultural crop improvement. Horticulture research, 6, 1e16. Xu, M. Y., Zhang, L., Li, W. W., Hu, X. L., Wang, M.-B., Fan, Y. L., Zhang, C. Y., & Wang, L. (2014). Stress-induced early flowering is mediated by miR169 in Arabidopsis thaliana. Journal of Experimental Botany, 65, 89e101. Yang, C., Li, D., Mao, D., Liu, X., Ji, C., Li, X., Zhao, X., Cheng, Z., Chen, C., & Zhu, L. (2013). Overexpression of micro RNA 319 impacts leaf morphogenesis and leads to enhanced cold tolerance in rice (Oryza sativa L.). Plant, Cell and Environment, 36, 2207e2218. Ye, G., & Smith, K. F. (2010). Marker-assisted gene pyramiding in cultivar development. In Molecular plant breeding: Principle, method and application (pp. 321e342). Yi, M., Nwe, K. T., Vanavichit, A., Chai-arree, W., & Toojinda, T. (2009). Marker assisted backcross breeding to improve cooking quality traits in Myanmar rice cultivar Manawthukha. Field Crops Research, 113, 178e186. Yogindran, S., & Rajam, M. V. (2021). Host-derived artificial miRNA-mediated silencing of ecdysone receptor gene provides enhanced resistance to Helicoverpa armigera in tomato. Genomics, 113, 736e747. Yuan, N., Yuan, S., Li, Z., Li, D., Hu, Q., & Luo, H. (2016). Heterologous expression of a rice miR395 gene in Nicotiana tabacum impairs sulfate homeostasis. Scientific Reports, 6, 1e14. Zafar, S. A., Zaidi, S. S.-e.-A., Gaba, Y., Singla-Pareek, S. L., Dhankher, O. P., Li, X., Mansoor, S., & Pareek, A. (2020). Engineering abiotic stress tolerance via CRISPR/ Cas-mediated genome editing. Journal of Experimental Botany, 71, 470e479. Zhang, Z., Chen, J., Lin, S., Li, Z., Cheng, R., Fang, C., Chen, H., & Lin, W. (2012). Proteomic and phosphoproteomic determination of ABA’s effects on grain-filling of Oryza sativa L. inferior spikelets. Plant Science, 185, 259e273. Zhang, Y., Massel, K., Godwin, I. D., & Gao, C. (2018). Applications and potential of genome editing in crop improvement. Genome Biology, 19, 1e11. Zhang, B., & Wang, Q. (2015). MicroRNA-based biotechnology for plant improvement. Journal of Cellular Physiology, 230, 1e15. Zhang, X., Xia, F., Xu, C., & Han, Y. (2019). Stability analysis of near-wellbore reservoirs considering the damage of hydrate-bearing sediments. Journal of Marine Science and Engineering, 7, 102. Zhang, Y.-C., Yu, Y., Wang, C.-Y., Li, Z.-Y., Liu, Q., Xu, J., Liao, J.-Y., Wang, X.-J., Qu, L.-H., & Chen, F. (2013). Overexpression of microRNA OsmiR397 improves rice yield by increasing grain size and promoting panicle branching. Nature Biotechnology, 31, 848. Zhang, N., Zhang, D., Chen, S. L., Gong, B. Q., Guo, Y., Xu, L., Zhang, X. N., & Li, J. F. (2018). Engineering artificial microRNAs for multiplex gene silencing and simplified transgenic screen. Plant Physiology, 178, 989e1001. Zhang, H., Zhang, J., Yan, J., Gou, F., Mao, Y., Tang, G., Botella, J. R., & Zhu, J. K. (2017). Short tandem target mimic rice lines uncover functions of miRNAs in regulating important agronomic traits. Proceedings of the National Academy of Sciences of the United States of America, 114, 5277e5282. Zhang, T., Zhao, Y.-L., Zhao, J.-H., Wang, S., Jin, Y., Chen, Z.-Q., Fang, Y.-Y., Hua, C.L., Ding, S.-W., & Guo, H.-S. (2016). Cotton plants export microRNAs to inhibit virulence gene expression in a fungal pathogen. Nature plants, 2, 1e6. Food crops improvement: comparative biotechnological approaches 505 Zhang, X., Zou, Z., Gong, P., Zhang, J., Ziaf, K., Li, H., Xiao, F., & Ye, Z. (2011). Overexpression of microRNA169 confers enhanced drought tolerance to tomato. Biotechnology Letters, 33, 403e409. Zhang, X., Zou, Z., Zhang, J., Zhang, Y., Han, Q., Hu, T., Xu, X., Liu, H., Li, H., & Ye, Z. (2011). Over-expression of sly-miR156a in tomato results in multiple vegetative and reproductive trait alterations and partial phenocopy of the sft mutant. FEBS Letters, 585, 435e439. Zhao, L., Kim, Y. J., Dinh, T. T., & Chen, X. (2007). miR172 regulates stem cell fate and defines the inner boundary of APETALA3 and PISTILLATA expression domain in Arabidopsis floral meristems. The Plant Journal, 51, 840e849. Zhao, Y. F., Peng, T., Sun, H. Z., Teotia, S., Wen, H. L., Du, Y. X., Zhang, J., Li, J. Z., Tang, G. L., & Xue, H. W. (2019). miR1432-Os ACOT (Acyl-CoA thioesterase) module determines grain yield via enhancing grain filling rate in rice. Plant Biotechnology Journal, 17, 712e723. Zhao, J., Yuan, S., Zhou, M., Yuan, N., Li, Z., Hu, Q., Bethea, F. G., Jr., Liu, H., Li, S., & Luo, H. (2019). Transgenic creeping bentgrass overexpressing Osa-miR393a exhibits altered plant development and improved multiple stress tolerance. Plant Biotechnology Journal, 17, 233e251. Zhou, J., Deng, K., Cheng, Y., Zhong, Z., Tian, L., Tang, X., Tang, A., Zheng, X., Zhang, T., & Qi, Y. (2017). CRISPR-Cas9 based genome editing reveals new insights into microRNA function and regulation in rice. Frontiers of Plant Science, 8, 1598. Zhou, M., Li, D., Li, Z., Hu, Q., Yang, C., Zhu, L., & Luo, H. (2013). Constitutive expression of a miR319 gene alters plant development and enhances salt and drought tolerance in transgenic creeping bentgrass. Plant Physiology, 161, 1375e1391. Zhou, J., Li, D., Wang, G., Wang, F., Kunjal, M., Joldersma, D., & Liu, Z. (2020). Application and future perspective of CRISPR/Cas9 genome editing in fruit crops. Journal of Integrative Plant Biology, 62, 269e286. Zhou, J., Yuan, M., Zhao, Y., Quan, Q., Yu, D., Yang, H., Tang, X., Xin, X., Cai, G., & Qian, Q. (2021). Efficient deletion of multiple circle RNA loci by CRISPR-Cas9 reveals Os06circ02797 as a putative sponge for OsMIR408 in rice. Plant Biotechnology Journal, 19, 1240e1252. Zhou, J., Zhang, R., Jia, X., Tang, X., Guo, Y., Yang, H., Zheng, X., Qian, Q., Qi, Y., & Zhang, Y. (2022). CRISPR-Cas9 mediated OsMIR168a knockout reveals its pleiotropy in rice. Plant Biotechnology Journal, 20, 310e322. This page intentionally left blank CHAPTER 16 Small RNA transgenesis for abiotic stress tolerant food crops Jie Cui Guangdong Provincial Key Laboratory for Plant Epigenetics, Shenzhen University, College of Life Sciences and Oceanography, Shenzhen, Guangdong, China 1. Introduction Climate change brings about a higher incidence of extreme temperatures, droughts, and floods, resulting in a decreased yield of important crops. With a continuously growing world population, the declined production in rice, wheat, and corn will seriously affect global food safety (Mickelbart et al., 2015). The development of stress-resistant crops is the need of the hour to provide sustainable crop productivity for the ever-increasing population. Unlike animals, plants are sessile organisms that have evolved elaborate tolerance mechanisms to various abiotic stress conditions, enabling them to suitably respond to adverse environmental pressures. With the discovery and exploration of plant small RNAs (sRNAs) in the past few years, the role of sRNA in plant adaptation to environmental stresses has also been revealed. Complete understanding of these adaptive mechanisms will facilitate the development of recent breeding technology for genetically modified plants with enhanced abiotic stress tolerance. sRNA transgenesis provides valuable tools for abiotic stress gene investigation and theoretical basis for crop improvement applications. To know the molecular basis of stress response genes by functional genomics approaches is the first step to obtain climate-resilient crops for sustainable agriculture. Forward genetics is a traditional approach for studying a particular biological trait from phenotypes to defined genes. Researchers adopt irradiation or chemicals to randomly induce DNA lesions in the genome to obtain a mutant pool of desired plant species, followed by the observation of progeny to screen the expected phenotype. More work on backcrossing with parental lines and mapping will help identify candidate genes caused by mutations, thereby connecting genes with their biological functions. Using this approach, scientists have uncovered the bulk of regulatory genes involved in various aspects of plant Plant Small RNA in Food Crops ISBN 978-0-323-91722-3 https://doi.org/10.1016/B978-0-323-91722-3.00007-5 © 2023 Elsevier Inc. All rights reserved. 507 508 Plant Small RNA in Food Crops growth and stress adaptation. Forward genetic screening facilitates gene function studies, particularly in the discovery of factors that affect a certain pathway. However, both genetic screening and mapping are time consuming and laborious. Another problem is that isolating many types of mutants is not easy in most plants. For instance, a complex trait determined by multiple gene loci (random mutagenesis) will cause low frequency of phenotypic changes, making it difficult to determine the regulatory mechanisms of desired traits. Reverse genetics is another powerful tool used for gene function studies. It always starts from a specific gene sequence knocking down/out or overexpressing the target gene and then inferring its biological function, among which knocking down/out is the most commonly used strategy for loss of function studies. Two classic methods provide knockout gene mutation by insertional disruption at the DNA regiondtransferred DNA (T-DNA) (Krysan et al., 1999) and transposon tagging (Parinov et al., 1999; Speulman et al., 1999). However, there are also some limitations. First, similar to the random mutation library from artificial mutagenesis, the interruption of a certain gene by a T-DNA or transposon cannot be predicted. Second, addressing the function of duplicate genes required much hybridization and genotyping to obtain multiple gene mutants. Recently, the emergence of CRISPR-Cas9 technology allows targeted editing of the nucleotides of a specific gene (Komor et al., 2017). This technique can induce genetically stable knockout lines for a single gene or several genes simultaneously. However, an obvious pitfall of CRISPR-Cas9 is that it cannot retain genetic materials of some lethal genes. The sRNA-based gene silencing approach is a promising reverse genetics tool for functional genomic studies because of several reasons. One is ease of use. Further, transcripts of target genes may be attenuated to various levels in independent transgenic lines, which provide a convenient way of dissecting how a gene regulates the relative phenotype to obtain a knockdown mutant with lethal genetic factors. By the selection of different promoters, silencing may occur at any developmental stage or in tissues as desired without changing genome loci; thus, it can serve as a more elaborate investigation method for gene function studies. In addition to being a universal reverse genetics tool for genetic study, technologies based on the sRNA-directed regulatory mechanism are emerging as powerful tools for crop improvement, because of the advantages of speed, precision, and efficiency compared with conventional breeding strategies. In this chapter, we introduce various sRNA transgenic Small RNA transgenesis for abiotic stress tolerant food crops 509 technologies and their applications in the improvement of abiotic stress tolerance crops (Table 16.1). Further, we discuss the challenges and perspectives of the future potential of sRNA as an ideal tool for molecular plant breeding. 2. Introduction of sRNA transgenic technologies 2.1 RNAi technology RNA interference (RNAi) is a conserved regulatory system that silences specific target genes by sRNAs processed from double-stranded RNA (dsRNA). The phenomenon was first observed in plants when researchers attempted to overexpress the sense CHALCONE SYNTHASE (CHS) transcript (a critical gene related to anthocyanin biogenesis) to obtain a deep purple flower in petunia. Surprisingly, they obtained variegated flowers and even white petals, described as co-suppression or homology-dependent gene silencing, because the expression of both introduced transgene and its homolog endogenous gene was inhibited in vivo (Napoli et al., 1990). However, the steady-state levels of the mRNAs produced by endogenous and transgenic CHS genes were negatively correlated with the degree of flower bleaching. It was still blurred that whether the biogenesis of CHS mRNA or the degradation was responsible for this phenomenon. Further analysis revealed that this phenomenon occurs after transcription, implying that host genes and transgenes are usually transcribed in the nucleus, whereas the corresponding mRNAs do not accumulate in the cytosol. This phenomenon was designated as post-transcriptional gene silencing (PTGS) (Blokland et al., 1994; de Carvalho et al., 1992), as mRNAs undergo degradation after transcription. As early as 1987, the very first construct generated to obtain an RNAi plant was an antisense transcript overexpression of a nopaline synthase in tomato (Rothstein et al., 1987). Several other reports have also found that antisense transgenes could lead to the silencing of a homologous endogenous gene, although at a low frequency (Smith et al., 1990). Interestingly, a sense transgene can also lead to the silencing of a homologous endogenous gene at a low frequency (Napoli et al., 1990; Van Der Krol et al., 1990). Later research in Caenorhabditis elegans (C. elegans) revealed that not only single-stranded RNA (ssRNA), either sense or antisense, but also dsRNA could trigger RNA silencing, and the latter was much more effective (Fire et al., 1998). Effective dsRNA-mediated silencing by co-expression sense and antisense transcripts, crossing ssRNA expression alleles and using a 510 Plant Small RNA in Food Crops single transcript with self-complementarity, have also been demonstrated in plants (Waterhouse et al., 1998). Nowadays, the highly efficient intronspliced hairpin dsRNA technique is a well-established RNAi tool that is most commonly used (Smith et al., 2000). Since its discovery in the late 1980s, the molecular mechanism underlying RNA silencing intrigued the scientific community for almost a decade (Vaucheret et al., 1997). Until, small interfering RNA (siRNA) was then discovered as a critical molecule to trigger RNA degradation; it is a uniform length of about 25-nt antisense RNA that pairs with the mRNA target (Hamilton & Baulcombe, 1999). Subsequent biochemical and genetic analyses depicted a detailed process for RNAi or PTGS. Briefly, the mRNA transcript was used as a template and was converted to dsRNA by RNAdependent RNA polymerase (RDR), then Dicer-like (DCL) enzymes processed dsRNA into a 21e24-nt mature siRNA, which was incorporated into an Argonaute (AGO) protein to form an RNA-induced silencing complex (RISC). RISC downregulates target genes by cleavage of their mRNAs or methylates the promoter region in a sequence-specific manner, leading to PTGS or transcriptional gene silencing (TGS), respectively (Rogers & Chen, 2013). The well-established mechanism underlying siRNA-directed gene silencing promotes RNAi as a powerful approach for functional gene exploration and application. 2.2 Virus-induced gene silencing (VIGS) RNAi transgenic technology is primarily dependent on well-established transgenic approaches. However, the transgenic approach is not available for all kinds of crops, making it difficult to study functional genomics or silencing crucial genes to improve agricultural traits. Thus, the improvement of such crop species is hindered. The RNAi mechanism is the natural defense system plants use to protect themselves from invading viruses (Waterhouse et al., 1998). Virus infection triggered dsRNA-mediated PTGS to degrade viral RNAs in plants. The concept of VIGS was proposed by van Kammen to explain the mechanism of plant recovery from virus infection (van Kammen, 1997). Now VIGS has been developed as a technique for both reverse and forward genetics in gene function analysis. Genes inducing severe plant symptoms were removed from the viral genome, leaving the critical genes for virus survival, then the remaining fragments were cloned into binary vectors. A fragment of the desired target gene was selected and inserted into the VIGS vector to generate a transcript Small RNA transgenesis for abiotic stress tolerant food crops 511 that fused with virus genes. Soon after the vector was delivered into plants by either transformation or virus infection, the immune response started to suppress virus replication by the generation of siRNAs from virusetarget gene fusion, resulting in specific degradation of the target genes in plants. In addition, the target gene silencing phenotypes conferred by these siRNAs are observed in the whole plant within a short time due to the systemic movement property of siRNA. Because VIGS can rapidly generate phenotypes and does not require the development of stable transformants (or further improvement in viral vectors and inoculation methods), it has been adopted for high-throughput screening of a large number of genes involved in the regulation of multiple abiotic stress responses, saving both time and labor (Ramegowda et al., 2013, 2014). It was recently used to verify gene function in high-temperature pollen development and cadmium (heavy metal) tolerance in wheat (Li et al., 2022; Yang, Ye, et al., 2021). Some virus-based constructs were modified to carry endogenous or artificial miRNA (amiRNA), termed MIR-VIGS, which combined the specificity of amiRNA and versatility of VIGS (Tang et al., 2010). VIGS vectors with wide host range and mild symptoms of viral infection are ideal for gene function studies, now more and more virus vectors have also been developed for various crops research (Kumar et al., 2022; Singh et al., 2022; Singh & Mysore, 2022; Tiedge et al., 2022; Wang et al., 2022). 2.3 Drawbacks of RNAi The length of the target gene fragment for efficient RNAi is 300e800 bp (Watson et al., 2005), which means that hundreds of short siRNA fragments with various uncertain nucleotide composition are generated after splicing by DCL. These siRNAs are loaded into AGO proteins and downregulate target genes by PTGS or TGS according to their length and 50 nucleotide. However, the precise target is guaranteed by selecting a distinct region of target transcripts; thus, the length requirement makes it difficult to silence one specific member of a conserved gene family. The efficient silencing of RNAi requires activity of RDRs which involves the secondary siRNA generation to amplify suppression effects of targets, but the process also causes detrimental consequences in plants. The endogenous targets cleaved by the siRNAeAGO complex (primary siRNAs derived from the transgenic dsRNA fragment can be loaded into AGO proteins) may be captured by RDR. RDR-directed dsRNA synthesis occurs in the 50 e30 and 30 e50 directions along the non-target region, 512 Plant Small RNA in Food Crops leading to transitive secondary siRNA generation in untended areas. Transitive property leads to the phenomenon of RNA silencing spreading to the adjacent region along the mRNA sequence, which may cause decreased expression levels of genes without homologs to target genes (Baulcombe, 2007; García-Pérez et al., 2004). Efforts have been made to explore a more specific and less off-target gene silencing tool. It is better to generate a predictable and short sRNA fragment to knock down genes of interest. Moreover, short RNAs can be modified to carry 30 -end mismatches that minimize transitive silencing by blocking RDR activity (Baulcombe, 2007; Voinnet, 2008). miRNA, with a conserved and widespread regulatory mechanism, comes to mind. 2.4 Artificial miRNA (amiRNA) Besides siRNAs, miRNA is the other major class of crucial small noncoding RNAs, which offered a highly conserved gene regulatory mechanism in plants. Different from siRNAs, miRNA has been generated from the MIR gene distributed mostly in the intergenic region of the genome in plants. In brief, the MIR gene is transcribed by RNA polymerase II to primary miRNA, which is successively cleaved by DCL enzymes, and eventually turned into a mature 21-nt miRNA strand incorporated into AGO1. The miRNAeAGO1 complex forms RISC to exert transcript cleavage and/or translation inhibition of the mRNA that pairs with the mature miRNA sequence (Rogers & Chen, 2013). With the discovery of wide distribution and negative regulation effect of miRNAs, researchers on this topic realize that it could be a more specific way of silencing any target genes in vivo by ssRNAs pairing with the 21-nt region. For the short and highly complementary regions that miRNA require, amiRNA has less offtarget effects compared with dsRNA-mediated silencing (Zhang, 2014). Evidence indicates that mutations in 21-nt mature regions of the hairpin stem loop in miRNA precursor did not affect miRNA biogenesis (Parizotto et al., 2004; Schwab et al., 2006). Thus, by endogenous miRNA biogenesis of key enzymes, researchers replaced the mature region of an endogenous primary miRNA backbone with artificial sequences to obtain mature amiRNAs that target their base pair matched genes in plants. Few defined parameters are taken into account for the designation of amiRNA sequences, including no mismatches in the seed region (specifically at the 9e11 bp position), one or two mismatches may be allowed near the 18e21 position, and hybridization energy with its target sites (Schwab et al., 2006). In addition, it is worth emphasizing that the backbone from the same Small RNA transgenesis for abiotic stress tolerant food crops 513 species is preferred. WMDM3 (http://wmd3.weigelworld.org/cgi-bin/ webapp.cgi) is a popular and widely accepted online tool for amiRNA design. One can easily access the website and import the target gene sequence or entry number to obtain both amiRNA lists and primers of overlapping PCR for precursor replacement. Another user-friendly interface, such as P-SAMS, is also available for amiRNA design (Fahlgren et al., 2016). To explore strategies for the diploid barely improvement, recently, artificial miRNA has been used to investigate the properties of barley cysteine proteases for further modification and use in brewing or food industry (Gomez-Sanchez et al., 2021). However, in a study of silencing grain hardness genes, compared with siRNA, the effects of amiRNA were less stable when transferred to the next generation of transgenic wheat plants, which may hinder its application in polyploid cereals (Gasparis et al., 2017). Due to its specificity, researchers started to take poly-cistronic miRNA precursors to reinforce the silencing of one gene or targeting different genes at the same time. Poly-cistronic constructs carry multiple MIRNA hairpins under a single promoter on the same expression cassette. The backbone of hairpins could be either identical (Ai et al., 2011; Kung et al., 2012; Park et al., 2009) or different (Liang et al., 2012). To increase the production efficiency and processing precision of single miRNAs from a synthetic polycistronic MIR gene, the transfer RNA (tRNA) sequence was placed between each individual miRNA precursor (Zhang, Zhang, Chen, et al., 2018). Modifying plant endogenous polycistronic MIRNA genes is another way for expressing multiple amiRNAs on the same transgene, for example, the use of rice endogenous polycistronic MIR395ag locus (contains seven miRNA hairpins) in wheat (Fahim et al., 2012). 2.5 Artificial trans-acting siRNA (atasiRNA) Most miRNAs target mRNAs encoding transcription factors (TFs) (Mitsuda & Ohme-Takagi, 2009; Rhoades et al., 2002) or other kinds of protein-coding genes, such as hormone receptors (Navarro et al., 2006) or enzymes involved in catalyzing metabolic pathways (Fujii et al., 2005). In plants, almost all these miRNA targets experienced cleavage or translation repression leading to mRNA degradation. However, a few miRNA targets, including some non-coding genes and several families of protein-coding genes, remain after being cleaved by their miRNA trigger which are usually of 22-nt length. These RNA strands are converted to dsRNA by RDR. dsRNAs are subsequently spliced by DCL4 or DCL5 (mostly in monocots to generate 24-nt phasiRNAs) site by site to form a series of 514 Plant Small RNA in Food Crops arranged head-to-tail secondary siRNAs with a distinctive, phased configuration termed phased siRNA (phasiRNA) (Liu, Teng, et al., 2020). Similar to miRNA, some phasiRNAs loaded into AGO proteins negatively regulate their target genes in a homology-dependent manner. phasiRNAs generated from protein-coding genes usually target the loci from where they are derived because of the high degree of complementarity. Interestingly, one subclass of phasiRNA is trans-acting siRNA (tasiRNA), which is produced from the non-protein coding TAS locus and silences its mRNA target, which is transcribed from elsewhere (hence the term trans-acting) (Liu, Teng, et al., 2020). de la Luz et al. modified the TASlc locus in Arabidopsis to produce a set of artificial tasiRNAs to successfully obtain FAD2 gene mutants with comparable phenotype to the null allele, indicating that artificial tasiRNA (atasiRNA, also known as synthetic trans-acting, syn-tasiRNA) was a high efficacy silencing tool (de la Luz Gutiérrez-Nava et al., 2008). Later, atasiRNAs produced from other TAS loci (Felippes & Weigel, 2009; Montgomery, Howell, et al., 2008; Montgomery, Seong, et al., 2008) and gene fragment fusion with an upstream target site of the tasiRNA trigger miRNA173 (De Felippes et al., 2012) was found to cause knockdown of target genes. These studies not only illustrate the mechanism of tasiRNA biogenesis but also demonstrate its application potential. An advantage of this technology is that by integration in one atasiRNA cassette, it is easy to generate several 21-nt siRNAs to target either the same gene in different sites to enhance silencing efficiency or multiple genes in a complex pathway to simultaneously modify a specific trait (de la Luz Gutiérrez-Nava et al., 2008). Engineering a group of tasiRNAs into one cassette could preclude promoter homology-dependent gene silencing (Meyer & Saedler, 1996) when required to choose a promoter to construct multiple dsRNA cassettes to silence more than one target. atasiRNA has been used to generate multiple target sites for tomato virus in order to enhance viral immunity (Carbonell et al., 2019). However, a transiently transgenic test in tomato and Nicotiana benthamiana using several published methods for multiplexed gene silencing, such as poly-cistronic microRNA precursors and tasiRNAbased methods, did not show precisely and efficiently processing of small RNAs (Lunardon et al., 2021). This new finding also highlights the necessity of further improvement of artificial small RNA strategies which are aimed to silencing multiple genes simultaneously. Scientists can now precisely control gene expression by adjusting the precursor position where atasiRNA is expressed and modify the degree of base-pairing at the 30 end of atasiRNA to change the siRNA activity (López-Dolz et al., 2020). Table 16.1 Comparison of small RNA technologies. Technology Small RNA Precursor/ biogenesis Length Target complementarity Applied approach RNAi SiRNA Long double stranded RNA/selfcomplementary hairpin RNA 300e800 bp Perfect Transgene/ spray/ irrigation VIGS siRNA/ miRNA Modified virus genome 21e24 nt Perfect/ imperfect Nontransgene amiRNA MiRNA Endogenous miRNA precusor Mostly 21 nt Imperfect Transgene atasiRNA TasiRNA Endogenous tasiRNA precusor 21 nt Perfect Transgene Multiple target sites STTM Binding sites of miRNA 90e130 bp Imperfect Transgene/ VIGS Block the function of an entire miRNA family miRNA overexpression MiRNA Two non-cleavable miRNA binding sites separated by a spacer/the mechanism of the long non-coding RNA IPS1 Endogenous miRNA precusor Relying on the development of virus vectors Instability of silencing effect through generations Low processing efficiency in vivo Difficulty in construction due to multiple binding sites Mostly 21 nt Imperfect Transgene Gain of functional study of miRNAs Development defects caused by ectopic expression Advantage Disadvantage Down regulation of gene expression to various levels/at desired development stages Large scale screen, time saving High specificity Low specificity, off-target 516 Plant Small RNA in Food Crops 2.6 Short tandem target mimic (STTM) Artificial miRNA/tasiRNA is not only an alternative approach for siRNAbased gene silencing but also a powerful tool for overexpressing a certain miRNA or siRNA when the primary structure is driven under a strong promoter, thereby providing a method for gain of function studies of functional sRNAs. However, for loss of function studies, the exact functions of miRNA families could not be deciphered well because each miRNA family has multiple loci in the genome, and some of them are functionally redundant. Scientists have developed a method to inactivate miRNA, particularly miRNA families with multiple members whose mature sequences are highly conserved, according to the mechanism of the long non-coding RNA IPS1 (INDUCED BY PHOSPHATE STARVATION1). IPS1 serves as a miRNA sponge, which contains a motif with sequence complementarity to a phosphate (Pi) starvation induced miRNA, miR399 (Franco-Zorrilla et al., 2007). Functional blocking of miR399 by interruption with a mismatched loop (a CTA trinucleotide bulge) at the expected miRNA cleavage site of the pairing region was designated as target mimic (TM). TM has been used to sequester functions of several other miRNAs in Arabidopsis by taking advantage of the modified endogenous IPS1 (Franco-Zorrilla et al., 2007; Wu et al., 2009). Later, a report utilized IPS1-based TM to perform large-scale screening of miRNA knockdown mutants. Compared with plant overexpression of miRNAresistant target genes when blocking by TM, only a small proportion of miRNA mutants cause comparable obvious phenotypes (Todesco et al., 2010). A technology termed short tandem target mimic (STTM) was explored to solve this problem (Yan et al., 2012). STTM is more effective because it triggered degradation of targeted sRNAs by the SDN family of exonucleases rather than simply sequestering them in the IPS1 case (Franco-Zorrilla et al., 2007; Yan et al., 2012). Generally, STTM is a structure with two non-cleavable miRNA binding sites separated by a spacer (optimal length is 48e88 nt). With the availability of STTM, scientists have uncovered some conserved miRNA families that play a key role in crop development (Chu et al., 2021; Yang, Zhang, et al., 2021) and abiotic stress tolerance. One such example is miR166. Consistent with the discovery in Arabidopsis (Yan et al., 2016), inactivation of rice miR166 resulted in morphological changes that confer drought resistance (Zhang, Zhang, Srivastava, et al., 2018). In addition to drought toleracnce, knock down of the same miRNA in corn Small RNA transgenesis for abiotic stress tolerant food crops 517 led to enhanced resistance against abiotic stresses such as salt and high temperature (Li, Yang, et al., 2020). STTMs have also been successfully used to silence numerous miRNA families in model plants and various crops to improve agricultural traits (Chen et al., 2021; Peng et al., 2018; Zhang, Zhang, et al., 2017). STTMs may be introduced into plant cells by stable transformation (Yan et al., 2012) or Agrobacterium-mediated transient expression (Zhang, Li, et al., 2017). Based on VIGS vectors, several reports also expand the usage of STTM as a reliable tool independent of transformation (Jian et al., 2017; Liu, Liu, et al., 2019). Previous chapters have discussed the complex regulatory roles of sRNAs in various abiotic stress conditions. Hence, we will focus on the improvement in plant tolerance efficacy using sRNA transgenic technology (Fig. 16.1, Table 16.2). 2.7 Drought Drought is a detrimental and widespread stress affecting crop productivity. The molecular mechanisms of how plants cope with drought stress have been intensively studied in the past few years including roles of both protein-coding genes and sRNAs. Producing the signal hormone abscisic acid (ABA), closing stomata, reprogramming gene expression, and adjusting osmotic pressure can lead to adaptive growth and development in plants. Using RNAi transgenesis to silence the negative regulators of these adaptation responses has successfully enhanced drought tolerance in various plants. Drought stress rapidly stimulates ABA accumulation to activate the ABA signaling pathway, which plays a central role in helping plants adapt to stress conditions (Cao et al., 2013). Farnesyltransferase is a key negative Figure 16.1 Applications to improve abiotic stress tolerance. Small RNAs-based strategies for development of abiotic stress tolerance plants. 518 Plant Small RNA in Food Crops Table 16.2 Utilization of small RNA technologies to improve abiotic stress tolerance of plants. Plant species Technologies Arabidopsis thaliana RNAi Canola (Brassica napus) RNAi Rice (Oryza sativa) Rice (Oryza sativa) RNAi Functional small RNA/ target gene Stress tolerance Drought [58] Rice (Oryza sativa) RNAi Rice (Oryza sativa) RNAi Rice (Oryza sativa) RNAi Potato (Solanum tuberosum) Artificial miRNA Rice (Oryza sativa) Maize (Zea mays) STTM FTB (FARNESYL TRANSFERASE BETA SUBUNIT) FTB (FARNESYL TRANSFERASE BETA SUBUNIT) SQS (SQUALENE SYNTHASE) RACK1 (RECEPTOR OF ACTIVATED CKINASE 1) OsDIS1 (DROUGHTINDUCED SINA PROTEIN 1) OsDSG1 (DELAYED SEED GERMINATION 1) OsGRXS17 (GLUTAREDOXINS 17) CBP80 (CAPBINDING PROTEIN 80) miR166 STTM miR166 Creeping bentgrass (Agrostis stolonifera) Arabidopsis thaliana Alfalfa (Medicago sativa) Rice (Oryza sativa) miRNA overexpression miR319 miRNA overexpression miRNA overexpression miRNA overexpression miR394 Drought [68] miR156 Drought [69] miR164 Drought [70] RNAi Drought [58] Drought [57] Drought [60] Drought [62] Drought and salinity [63] Drought [64] Drought [66] Drought [50] Drought, saltnity and heat [51] Drought and salinity [67] Small RNA transgenesis for abiotic stress tolerant food crops 519 Table 16.2 Utilization of small RNA technologies to improve abiotic stress tolerance of plants.dcont'd Functional small RNA/ target gene Stress tolerance miRNA overexpression miR396 miRNA overexpression miR2118 Drought, saltnity and cold [71] Drought [72] miRNA overexpression miRNA overexpression miRNA overexpression Artificial miRNA miRNA overexpression miRNA overexpression miRNA overexpression miRNA overexpression miRNA overexpression miR408 Drought [73] miR319 Cold [76,77] miR397 Cold [78] BCB (BLUE-COPPERBINDING PROTEIN) miR156 Freezing [79] Cold [80] miR156 Cold [80] miR528 Cold [81] miR528 Cold [81] miR402 Salinity, cold and drought [82] Salinity, cold and oxidative [83] Cold [85] Plant species Technologies Tobacco (Nicotiana tabacum) Tobacco (Nicotiana tabacum) Ryegrass (Lolium perenne) Rice (Oryza sativa) Arabidopsis thaliana Arabidopsis thaliana Pine (Pinus elliottii) Rice (Oryza sativa) Pine (Pinus elliottii) Rice (Oryza sativa) Arabidopsis thaliana Arabidopsis thaliana miRNA overexpression miR408 Arabidopsis thaliana Artificial miRNA Rice (Oryza sativa) Arabidopsis thaliana Alfalfa (Medicago sativa) Arabidopsis thaliana RNAi JAZ1 (JASMONATEZIM-DOMAIN PROTEIN 1) OsClo5 (CALEOSIN 5) miRNA overexpression miRNA overexpression RNAi Cold [86] miR156 Heat [87] miR156 Heat [88] AtProDH (PROLINE DEHYDROGENASE) Salinity and freezing [90] Continued 520 Plant Small RNA in Food Crops Table 16.2 Utilization of small RNA technologies to improve abiotic stress tolerance of plants.dcont'd Plant species Technologies Mulberry (Morus alba) RNAi Rice (Oryza sativa) RNAi Arabidopsis thaliana Tobacco (Nicotiana tabacum) Creeping bentgrass (Agrostis stolonifera) Tomato (Solanum Lycopersicon) Creeping bentgrass (Agrostis stolonifera) Barley (Hordeum vulgare) Potato (Solanum tuberosum) RNAi Functional small RNA/ target gene Stress tolerance MaRGS (REGULATOR OF GPROTEIN SIGNALING) OsRPK1 (RECEPTORLIKE PROTEIN KINASE 1) AtbZIP24 Salinity [91] Salinity [92] Salinity [93] miRNA overexpression miR408 Salinity and Pi starvation [94] miRNA overexpression miR528 RNAi SlHK2 (HISTIDINE KINASE 2) miRNA overexpression miR393 Salinity and nitrogen deficiency [95] Drought, heat and combined [110] Salinity and heat [111] miRNA overexpression Hhvu-miRX Drought [147] STTM miRSES Heat [148] regulator of ABA sensing in plants; thus, the manipulation of plant farnesyltransferase for improving plant drought tolerance is useful (Manavalan et al., 2012; Wang et al., 2005, 2009). As early as 2005, Wang et al. found that knock down of either the a- or b-subunit of farnesyltransferase enhances the response to ABA and drought tolerance in Arabidopsis (Wang et al., 2005). When the same strategy was applied to an important oilseed crop canola, three-year field collection data indicated a significantly higher productivity in transgenic RNAi crops than the controls under drought stress during flowering time (Wang et al., 2005). Likewise, inhibition of Small RNA transgenesis for abiotic stress tolerant food crops 521 SQS, a member of farnesyl-diphosphate farnesyltransferase, by RNAi also improved drought tolerance at both vegetative and reproductive stages in rice by reducing stomatal conductance (Manavalan et al., 2012). RECEPTOR OF ACTIVATED C-KINASE 1 (RACK1), a highly conserved scaffold protein with versatile functions, is involved in the abiotic stress response in rice and mediates negative regulation of the redox system under drought. RACK1 downregulation was achieved by RNAi resulting in the development of drought-tolerant rice plants (Li et al., 2009). The conserved ubiquitination system is a principal proteolytic mechanism for plants involved in plant response to abiotic stress (Smalle & Vierstra, 2004), in which ubiquitin ligase E3 is one of the three key enzymes. A C3HC4 RING finger E3 ligase OsDIS1 of rice negatively regulates drought response (Ning et al., 2011). Another RING finger E3 ligase OsDSG1, whose homolog negatively regulates ABA signaling, has also been studied in rice (Park et al., 2010). RNAi of both OsDISl and OsDSG1 genes produced drought-resistant rice without unfavorable effects on seed production or yield (Ning et al., 2011; Park et al., 2010). Repression of the other drought-associated genes using RNAi, such as OsGRXS17 (a glutaredoxin), which is involved in cellular redox homeostasis and redoxdependent signal pathway regulation, could also confer improved drought tolerance in rice (Hu, Wu, et al., 2017). By knock down of the gene critical for DNA modification, RNAi can be used to perform large-scale screening of candidate targets for crop improvement. RNAi of the chromatin remodeler to regulate DNA modification of poplar provided abundant information about its drought stress-response genes (Sow et al., 2021). amiRNA has also been used to target candidate genes to obtain drought-tolerant plants. In Arabidopsis, the CAP-BINDING PROTEIN 80 (CBP80, also known as Abscisic Acid Hypersensitive 1, ABH1) gene plays an important role in drought tolerance by regulating ABA transduction. Downregulation of CBP80 using amiRNA enhances drought tolerance of the cultivated tetraploid potato, which is sensitive to water shortage (Pieczynski et al., 2013). Manipulating miRNA expression levels can enhance plant adaptation to water-deficit conditions. miR166 knockdown lines of rice (STTM166) showed rolling leaves and higher drought resistance, which may be due to its regulation of polysaccharide biogenesis that contributes to cell wall formation and vascular development (Zhang, Zhang, Srivastava, et al., 2018). Overexpression of drought-inducible miRNAs could confer enhanced drought tolerance in various plants, 522 Plant Small RNA in Food Crops including miR319 in creeping bentgrass (Zhou et al., 2013), miR394a in Arabidopsis (Ni et al., 2012), and miR156, miR164, miR408, miR396 and miR2118 in crops such as alfalfa, rice, ryegrass and tobacco, respectively (Arshad et al., 2017; Chen et al., 2015; Fang et al., 2014; Hang et al., 2021; Wu et al., 2015). 2.8 Extreme temperatures Temperature-associated stress due to seasonal variations and climate changes is another inevitable environmental threat for plants, which limits their growth and productivity. sRNA-based gene silencing technology has been used to develop temperature-resilient plants (i.e., plants tolerant to cold or heat stresses). Lignin is a major component of the plant secondary cell wall that affects its stiffness and permeability. In several species, the amount of lignin was altered after cold treatment and conferred cold adaptation in plants (Ferrer et al., 2008; Shafi et al., 2014). Overexpressing OsmiR319 or downregulating its target genes of the OsPCF TF family members showed enhanced cold tolerance in transgenic rice seedlings (Wang et al., 2014; Yang et al., 2013). miR397a overexpression may allow transgenic Arabidopsis plants to endure low temperature stimuli by modulating lignification of its cell walls by the function of miR397 target genes (Dong & Pei, 2014). Similarly, a biosynthesis regulator of lignin, BLUE-COPPER-BINDING PROTEIN (BCB), which acts downstream of a specific cold-induced nuclear protein, TOLERANT TO CHILLING AND FREEZING 1 (TCF1), influenced cold acclimation in Arabidopsis (Ji et al., 2015). Knocked-down BCB expression by amiRNA reduced lignin accumulation under cold treatment, thereby resulting in increased freezing tolerance (Ji et al., 2015). Thus, inhibiting the negative regulatory genes of cold tolerance by overexpression of cold-responsive miRNA or amiRNA have successfully improved cold stress tolerance. Other examples include OsmiR528 and OsmiR156 in rice and pine (Tang & Thompson, 2019; Zhou & Tang, 2019), and miR402 and miR408 in Arabidopsis (Kim et al., 2010; Ma et al., 2015). The plant hormone jasmonate (JA) positively modulates freezing stress tolerance in various plants, and its biogenesis is activated under cold stress (Hu, Jiang, et al., 2017). Using amiRNA targeting JAZ1 gene transcripts to suppress the repressor of the JA pathway improved cold stress tolerance in Arabidopsis (Makhazen et al., 2021). A recent report on rice revealed a phenomenon consistent with that in the model plant. OsClo5, a universally Small RNA transgenesis for abiotic stress tolerant food crops 523 expressed gene localized in lipid droplets, inhibits JA biosynthesis and signal transduction. RNAi lines of OsClo5 showed higher survival than WT rice after recovery from cold treatment (Zeng et al., 2022). In addition to cold, high temperature has irreversible and detrimental effects on plants, leading to a sharp decrease in global yields of several crops. To obtain heat-tolerant crops, the manipulation of sRNA expression and alteration of sRNA-based gene targets present a valuable approach, especially in the background of global warming. Upregulation of miR156 has been shown to increase and sustain heat stress memory in Arabidopsis (Stief et al., 2014). Later, a study in the economic forage crop alfalfa also demonstrated that miR156 overexpression and RNAi knockdown of its target gene SPL13 showed increased tolerance to heat stress (Matthews et al., 2019). Foliar spray of exogenous spermidine (Spd) improved heat tolerance in cucumber. Interestingly, one research focused on the sRNA pattern changes underlying the application of the heat-resistance enhancer Spd, which provides insights into probing regulators of heat tolerance in plants that differs from information provided by conventional comparative studies of miRNAs (Wang et al., 2018). 2.9 Salinity Soil salinization seriously affects agricultural production worldwide because most important crops are sensitive to high salt concentration in the soil. Many plants accumulate soluble osmotic regulatory substances (e.g., proline) to achieve osmotolerance caused by salt stress. As early as 1999, Nanjo et al. used the antisense cDNA of AtProDH in Arabidopsis to successfully repress the expression of AtProDH, which encodes an enzyme that catalyzes proline degradation. These antisense transgenic plants showed enhanced accumulation of proline and more tolerance to high salinity and freezing than WT plants (Nanjo et al., 1999). Likewise, RNAi-silencing of MaRGS, a regulator of the G-protein signaling (RGS) protein, in mulberry plants showed higher proline content and salt stress tolerance than mulberry seedlings with MaRGS overexpression (Liu, Fan, et al., 2019). Similar to Gproteins, leucine-rich repeat receptor-like protein kinase (LRR-RLK) is another important signal transduction system that plays a fundamental role in plant response to environmental factors. OsRPK1-RNAi rice plants exhibited higher salt tolerance than WT plants, which again demonstrated the positive relevance between proline abundance and salt acclimation (Li, Chen, et al., 2020). Except for manipulating the metabolites to enhance salt 524 Plant Small RNA in Food Crops tolerance, engineering key TFs involved in salt response also worked well. Under normal growth conditions, the expression of stress-inducible genes involved in osmotic adjustment and ion homeostasis was activated in AtbZIP24-RNAi lines, indicating that this TF was a negative regulator of salt stress acclimation (Yang et al., 2009). When under salt stress, transgenic Arabidopsis with decreased AtbZIP24 expression exhibited higher tolerance with enhanced resistance to Naþ accumulation (Yang et al., 2009). The wheat miRNA TaemiR408 was identified as a stress gene in response to salt stress and Pi starvation, which is conserved in both monocot and dicot plants. Tobacco lines with overexpression of TaemiR408 showed improved growth phenotype, biomass, and photosynthesis compared with non-transgenic plants under both salt and Pi starvation treatments (Bai et al., 2018). Constitutive expression of rice OsmiR528, a conserved monocotspecific miRNA, showed improved salt stress and nitrogen-deficiency tolerance in the perennial creeping bentgrass (Yuan et al., 2015). These indicate the potential of manipulating conserved or specific miRNAs in improving plant abiotic stress resistance. Further studies should use genetic tools to validate in vivo function of more salt-induced sRNAs to promote molecular design of outstanding crop species. 3. Challenges: safety and specificity An important safety concern is the off-target effects across eukaryotic plants, especially when target regions are highly conserved protein domains. Theoretically, such a situation will cause undesired phenotypic pleiotropy of transgenic crops and may propagate to non-target crops exposed to the genetically modified plants. Thus, the ecological risk of sRNA transgenesis needs to be carefully evaluated. With the boom of next generation sequencing technology, enormous data (including genome, sRNA, and coding gene expression information) in more biological lineages can provide valuable resources when bioinformatics approaches are performed. Combined with the available and progressive theories and computational tools, it is feasible to achieve rigorous validation of sRNA targets in all concerned organisms. Various guidelines have been provided to remedy off-target effects using siRNA-based approaches, including effective sRNA design (Birmingham et al., 2007; Wesley et al., 2001), using tissue-specific or inducible promoters for producing sRNA (Chen et al., 2003; Li et al., 2015), selecting promoters with the lowest expression level under the premise of silencing efficiency (Li et al., 2015), or pooling multiple Small RNA transgenesis for abiotic stress tolerant food crops 525 synthetic siRNAs for the same gene to minimize the concentration of each piece of siRNA to reduce the contribution of off-target effects (Kittler et al., 2007; Neumeier et al., 2020). In plants, siRNA/miRNA-based gene silencing usually represses gene targets with full complementarity with nucleotides. However, in animals, sRNA-induced gene silencing does not need perfect pairing with targets. In miRNAs as short as 6e8 nt, full pairing occurring mostly at 30 -UTR (Untranslated Region) of a transcript was enough to trigger silencing (Rogers & Chen, 2013). When taken up by animal cells, there is a possibility that siRNAs may bind to undefined miRNA-like target sites in 30 UTRs of mRNAs by their seed sequences, which will lead to unwanted off-target effects. miRNAlike off-target silencing is another factor to consider when producing siRNAs, which are transferred from transgenic plants to higher trophic levels, such as people who eat the crop or hosts in the case of parasitoids. Owing to this, experimental procedures and strategies require to be well-designed before executing large-scale field studies of RNAi-based crops. Chemical modification of the specific position of siRNA with 20 -O-methyl ribosyl can avoid its targeting to unintended genes (Jackson et al., 2006). Later, a technology introducing a single nucleotide bulge placed in the antisense strand of a siRNA backbone was developed as “bulge-siRNA”. Bulge-siRNA occurring at the proper position was demonstrated as a superior alternative to chemical modifications to minimizing off-target silencing triggered by siRNAs structures, without loss in silencing of the intended targets (Dua et al., 2011). These modifications to avoid off-target effects are predominantly used in synthetic siRNAs for therapeutic applications. siRNAs produced in plants mainly depend on the endogenous system, which cannot generate these modified forms of sRNAs till date. Despite this situation, structural or chemical modification of siRNAs still provide new insights for further application in crop improvement, particularly for the use of spray-induced gene silencing (SIGS) to avoid cross-kingdom RNAi, which we will discuss in a subsequent section. With the advantages of predictable mature sequences and short length, much effort has been taken to make amiRNA a more manageable genetic and functional genomic technology. Strategies were developed to perform large-scale screening with various tag or reporter genes for the identification of more effective amiRNA sequences (Li et al., 2013, 2014; Zhang, Zhang, Chen, et al., 2018), even under conditions that target gene silencing generates no visible phenotype. Zhang et al. established a method to use endogenous transfer RNA (tRNA) processing to simultaneously produce 526 Plant Small RNA in Food Crops multiple amiRNAs for gene silencing, which provides an alternative tool for multiple gene silencing in addition to artificial tasiRNA (Zhang, Zhang, Chen, et al., 2018). miRNAs usually affect both stress response and plant growth. When choosing candidates for genetic engineering to improve abiotic stress tolerance in crops, it is critical to focus on transgenic plants without noticeable developmental or morphological changes and yield loss in field conditions. The opposite roles that miR169 plays in plant species under drought conditions suggest that even conserved miRNAs function in a species-specific manner (Li et al., 2008; Wang et al., 2011; Zhang et al., 2011). This should be carefully considered when tailoring genetic modification strategies for specific target species. Considering the diverse functions of one specific miRNA to explore more underlying mechanisms is a key point for utility. Besides avoiding unwanted traits when applying sRNA transgenesis in crop improvement, another factor that should be considered is multiple stress factors may simultaneously exist in the natural environment. By RNAi silencing a single gene or regulating the expression of a single miRNA, multiple stress tolerance and improvement of several agricultural traits can be obtained at the same time in crops. Downregulation of the expression of the cytokinin receptor gene SlHK2 by RNAi enhanced tolerance to either drought or heat, or both combined, in tomato (Mushtaq et al., 2022). Overexpression of rice miR393 in another grass species, creeping bentgrass, improved not only drought stress tolerance but also salt and heat stress tolerance by inducing several physical changes and gene expression (Zhao et al., 2019). miR168 is a key miRNA that targets AGO1, a major component of RISC, which mediates most miRNA/ siRNA-based gene silencing in plants. miR168 suppression by a TM affects several rice characters, including shortening flowering time, increasing grain yield, and enhancing immunity to pathogens (Wang et al., 2021). These studies shed light on the future exploration of using sRNA under combined stresses, which is closer to field conditions. 4. Perspectives: better strategies and efficiency 4.1 Cis-regulatory element editing By modifying the time or space specificity and gene expression level, cisregulatory variants of coding genes often showed subtle phenotypic changes and therefore could reduce undesired traits in crop improvement Small RNA transgenesis for abiotic stress tolerant food crops 527 (Hendelman et al., 2021; Wittkopp & Kalay, 2012). Quantitative trait variation in crops can be manipulated by CRISPR-Cas9 mutagenesis of promoters of the stem cell fate-decision genes CLAVATA and WUSCHEL (Rodriguez-Leal, 2017). Except for introducing functional sRNA constructs into plants to change the expression level of critical target proteins, cis-regulatory editing of important regulatory sRNAs or key genes in the pathway opens the door to a vast pool of candidate genes that could be exploited in stress resilience crops. 4.2 Spray method of sRNA In addition to the available sRNA transgenic technologies in crop improvement, public concerns about genetically modified crops make it an urge for scientists to look for alternative approaches. Spray-induced gene silencing (SIGS) stepped onto the stage. It is usually administered in the form of dsRNAs or sRNAs and is directly sprayed onto host plants instead of chemical pesticides (Wang & Jin, 2017; Wang, Thomas, & Jin, 2017). Compared with stable chemical molecules, RNA is much less stable when exposed to an RNase-rich environment. To obtain higher efficacy, researchers used layered double hydroxide nanoparticles (LDHs) as dsRNA carriers to extend the virus resistance of tobacco for up to 20 days (Mitter et al., 2017), very recently, the same group improved the delivery platform from MgAl-LDH to MgFe-LDH to decrease the toxicity of the delivery substance after repeated usage (Jain et al., 2022). Various types of nanomaterials have also been used in RNAi delivery systems aiming to protect RNA from degradation and improve its efficiency for entering into harmful insects (Yan et al., 2021). However, reducing the production cost of dsRNA or sRNA and standardizing the process for easy operation are still on the way. SIGS can also efficiently target endogenous plant genes and decrease their expression levels (Dalakouras et al., 2016; Dubrovina et al., 2019; Kiselev et al., 2021). Using SIGS to downregulate the negative effectors in abiotic stress tolerance in crops is another innovative way that deserves to be investigated soon. Furthermore, with chemical or structural modification of exogenous siRNA fragments to avoid miRNA-like off-targets in animals, SIGS will provide a more safety way for food crop application. 4.3 Great potential of non-canonical types of sRNAs sRNAs responding to abiotic stress have been detected in several comparative studies. Intensive studies on the functions and mechanisms of 528 Plant Small RNA in Food Crops miRNAs and siRNAs have been accomplished in model plants using established genetic transformation approaches. However, not all members of sRNAs have been well characterized in plants, including some nonconventional sRNAs, which are derived from housekeeping RNAs, such as transfer RNA (tRNA) and ribosomal RNA (rRNA). Except for their fundamental role in protein synthesis, tRNAs and rRNAs can also be cleaved to produce sRNAs, designated as tsRNAs and rsRNAs, respectively. In animals, tsRNAs are involved in diverse biological processes, such as stress response (Thompson et al., 2008) and tumorigenesis (Honda et al., 2015). Plant tsRNA was first detected in Arabidopsis tissues after oxidative stimuli treatment (Thompson et al., 2008). Subsequent studies revealed its widespread presence in rice (Chen et al., 2011), barley (Hackenberg et al., 2013), and other crop plant (Zhang et al., 2009). In some species, tsRNA was induced specifically under nutrient deficiency, ultraviolet (UV) exposure, and salt or drought stresses (Alves et al., 2017). Although the biological function of tsRNA is largely unknown, Gu et al. recently revealed that a 50 tsRNA negatively regulates anti-fungal defense in Arabidopsis after being loaded into AGO1 and directing the cleavage of the target gene Cytochrome P450 71A13 (Gu et al., 2022). This finding also sheds light on the biological significance of tsRNA in adverse environmental conditions. In the long term, rsRNAs were considered degradation fragments of mature rRNAs due to their abundance. Some studies have reported their existence in yeast and association with AGO1 (Bühler et al., 2008). In C. elegans, cold stress or UV irradiation elicited RNA processing disorders, triggering rsRNA biogenesis (Zhu et al., 2018), which resulted in nuclear RNAi-mediated gene silencing to inhibit rRNA precursor transcription elongation by RNA polymerase I (Liao et al., 2021). This system provides a quality control mechanism to maintain rRNA homeostasis and retain cell survival in specific situations. Our current knowledge of rsRNAs is mainly from the model plant Arabidopsis. In Arabidopsis, virus-infected sRNAs were produced from rRNA (Mengji Cao et al., 2014). A previous study also identified rsRNAs in mutants whose degradation of rRNA maturation byproducts were impaired (Lange et al., 2011). Recently, You et al. found that the abundant 21-nt rsRNAs in fry1 mutant could compete with miRNAs and form a putative functional RISC when loaded into AGO proteins (You et al., 2019). Remembering the accumulation of rsRNA under environmental stimuli in C. elegans, it prompts us to ask whether rsRNAs play some roles in plant stress response. Similar to tsRNAs, Small RNA transgenesis for abiotic stress tolerant food crops 529 rsRNAs can be loaded into AGO proteins, indicating that they may be functionally analogous to miRNAs (You et al., 2019). Although further deciphering the biogenesis, functional role, underlying mechanism, and association with stress is urgent, structural RNA derived non-coding RNAs as new regulating sRNAs provides a promising direction for crop yield improvement under stress conditions. New technologies of next generation sequencing combined with bioinformatics promote the discovery of circular RNAs (circRNAs) as another class of non-coding RNAs that may have regulatory roles in stress biology in the past few years. Different expression of circRNAs was observed in barley treated with iron and zinc (Darbani et al., 2016), tomato and soybean under extreme temperatures (Wang et al., 2020; Yang et al., 2020; Zuo et al., 2016), and wheat in drought conditions (Wang, Yang, et al., 2017). Take advantage of knockout or overexpression, several genetic evidence has illustrated that circRNA can be functional in enhancing abiotic stress tolerance of plants (Gao et al., 2019; Zhang et al., 2019; Zhou et al., 2021). The putative mechanisms of circRNAs in response to abiotic stress remain to be elucidated, expanding the repertoire of tools for crop biotech applications. 4.4 Exploring species-specific miRNAs Although there are conserved miRNA families across various crops, species-specific miRNAs still constitute a large portion. Numerous miRNAs, including both conserved and novel miRNAs, have been reported to mediate plants response against abiotic stress factors in a vast array of crops by next generation sequencing technologies (Noman et al., 2017). Unfortunately, genetic evidence for the functional species-specific miRNAs in abiotic stress is still scarce, hindering comprehensive understanding of miRNA and its further applications. According to a few available reports, species-specific miRNAs have huge potential. A strawberry miRNA, FanmiR73, was identified to target an important TF ABI5 in the ABA pathway. Under UV-B radiation and salt stress, the Fan-miR73 transcript level was decreased, indicating that this miRNA plays a crucial role in stress response (Li et al., 2016). In the cereal grain barley, Hackenberg et al. identified a group of Triticeae-specific miRNAs that show different spatiotemporal expression patterns under drought conditions, which may be regulated by drought-related TF DREB (Michael Hackenberg et al., 2015). Later, when further comparative analysis was performed using a 530 Plant Small RNA in Food Crops drought tolerant transgenic barley overexpression of wheat DREB TF, the same group characterized a new barley-specific miRNA that was highly expressed in transgenic plants, termed hvu-miRX. Transgenic barley with overexpression of hvu-miRX exhibited the drought tolerance phenotype, providing a candidate for designing drought tolerant cereals (Zhou et al., 2018). Tuber formation in potato is inhibited by heat, but Lehretz et al. found that a potato-specific 19-nt miRNA downregulated the mobile tuberization signal SP6A to block tuber initiation under high temperature (Lehretz et al., 2019). Emerging evidence reminds us that further studies should keep an eye on species-specific miRNAs to expand resources for transgenesis applications in crop improvement. Information on the adaptation of crop wild relatives to adverse environments could be more resourceful, as natural selection has already tested more options than humans ever will. The focus is on crops that survive well under extreme environmental conditions to explore specific stress tolerance mechanisms for further use. Researchers call for establishing a superpangenome, a gene bank with both cultivated gene pool and genetic stock from wild relatives, to accelerate crop improvement (Khan et al., 2020). Gladly, this effort has been made in soybeans recently (Liu, Du, et al., 2020). 5. Conclusion Under changing climates, a major challenge for the next few decades is the sustainable production of high-yield agricultural crops. The roles of various sRNAs have been well documented in plant adaptation to abiotic stress. Further studies need to focus on the application of sRNAs with known function and to explore mechanisms underlying other kinds of nonconventional sRNAs, such as tsRNAs, rsRNAs, and circRNAs. Wild crop species other than cultivars provide more useful information for developing abiotic stress-resilient plants. The sRNA-based gene silencing mechanism has emerged as an efficient and precise approach for functional genomic studies and crop improvement. In this chapter, we summarized several approaches based on the endogenous gene-silencing mechanism and their latest improvements and applications. This chapter also listed some published examples of sRNA transgenesis to improve tolerance to various environmental stress conditions, especially highlighting the perspectives for the improvement of technologies. Further studies should focus on (1) stress response miRNAs without adverse effects on crop growth and yield, and Small RNA transgenesis for abiotic stress tolerant food crops 531 (2) manipulation by a single miRNA to enhance tolerance to multiple stress factors simultaneously. Intensive studies have demonstrated the prospect for spraying sRNAs in biotic control, and adopting this environment-friendly way of silencing abiotic stress negative regulators should be carefully considered and designed to alleviate public concerns about the safety of transgenic crops. Acknowledgments This study was funded by the Shenzhen Grant Plan for Science and Technology (JCYJ20190808144415154). The author apologizes to colleagues whose works have not been cited or included because of space limitations. References Ai, T., Zhang, L., Gao, Z., Zhu, C. X., & Guo, X. (2011). Highly efficient virus resistance mediated by artificial microRNAs that target the suppressor of PVX and PVY in plants. Plant Biology, 13(2), 304e316. https://doi.org/10.1111/j.1438-8677.2010.00374.x Alves, C. S., Vicentini, R., Duarte, G. T., Pinoti, V. F., Vincentz, M., & Nogueira, F. T. S. (2017). Genome-wide identification and characterization of tRNA-derived RNA fragments in land plants. Plant Molecular Biology, 93(1e2), 35e48. https://doi.org/ 10.1007/s11103-016-0545-9 Arshad, M., Feyissa, B. A., Amyot, L., Aung, B., & Hannoufa, A. (2017). MicroRNA156 improves drought stress tolerance in alfalfa (Medicago sativa) by silencing SPL13. Plant Science, 258, 122e136. https://doi.org/10.1016/j.plantsci.2017.01.018 Bai, Q., Wang, X., Chen, X., Shi, G., Liu, Z., Guo, C., & Xiao, K. (2018). Wheat miRNA taemir408 acts as an essential mediator in plant tolerance to pi deprivation and salt stress via modulating stress-associated physiological processes. Frontiers of Plant Science, 9. https://doi.org/10.3389/fpls.2018.00499 Baulcombe, D. C. (2007). Amplified silencing. Science, 315(5809), 199e200. https:// doi.org/10.1126/science.1138030 Birmingham, A., Anderson, E., Sullivan, K., Reynolds, A., Boese, Q., Leake, D., Karpilow, J., & Khvorova, A. (2007). A protocol for designing siRNAs with high functionality and specificity. Nature Protocols, 2(9), 2068e2078. https://doi.org/ 10.1038/nprot.2007.278 Blokland, Van, Geest, Mol, J. N. M., & Kooter, J. M. (1994). Transgene-mediated suppression of chalcone synthase expression in Petunia hybrida results from an increase in RNA turnover. The Plant Journal, 6(6), 861e877. Bühler, M., Spies, N., Bartel, D. P., & Moazed, D. (2008). TRAMP-mediated RNA surveillance prevents spurious entry of RNAs into the Schizosaccharomyces pombe siRNA pathway. Nature Structural & Molecular Biology, 15(10), 1015e1023. https://doi.org/ 10.1038/nsmb.1481 Cao, Mengji, Du, P., Wang, X., Yu, Y.-Q., Qiu, Y.-H., Li, W., Gal-On, A., Zhou, C., Li, Y., & Ding, S.-W. (2014). Virus infection triggers widespread silencing of host genes by a distinct class of endogenous siRNAs in Arabidopsis. Proceedings of the National Academy of Sciences, 111(40), 14613e14618. https://doi.org/10.1073/pnas.1407131111 Cao, M., Liu, X., Zhang, Y., Xue, X., Zhou, X. E., Melcher, K., Gao, P., Wang, F., Zeng, L., Zhao, Y., Deng, P., Zhong, D., Zhu, J. K., Xu, H. E., & Xu, Y. (2013). An 532 Plant Small RNA in Food Crops ABA-mimicking ligand that reduces water loss and promotes drought resistance in plants. Cell Research, 23(8), 1043e1054. https://doi.org/10.1038/cr.2013.95 Carbonell, A., Lisón, P., & Daròs, J. A. (2019). Multi-targeting of viral RNAs with synthetic trans-acting small interfering RNAs enhances plant antiviral resistance. The Plant Journal, 100(4), 720e737. https://doi.org/10.1111/tpj.14466 de Carvalho, F., Gheysen, G., Kushnir, S., Van Montagu, M., Inzé, D., & Castresana, C. (1992). Suppression of beta-1,3-glucanase transgene expression in homozygous plants. The EMBO Journal, 11(7), 2595e2602. https://doi.org/10.1002/j.14602075.1992.tb05324.x Chen, S., Hofius, D., Sonnewald, U., & Börnke, F. (2003). Temporal and spatial control of gene silencing in transgenic plants by inducible expression of double-stranded RNA. The Plant Journal, 36(5), 731e740. https://doi.org/10.1046/j.1365-313X.2003.01914.x Chen, C. J., Liu, Q., Zhang, Y. C., Qu, L. H., Chen, Y. Q., & Gautheret, D. (2011). Genome-wide discovery and analysis of microRNAs and other small RNAs from rice embryogenic callus. RNA Biology, 8(3), 538e547. https://doi.org/10.4161/ rna.8.3.15199 Chen, L., Luan, Y., & Zhai, J. (2015). Sp-miR396a-5p acts as a stress-responsive genes regulator by conferring tolerance to abiotic stresses and susceptibility to Phytophthora nicotianae infection in transgenic tobacco. Plant Cell Reports, 34(12), 2013e2025. https://doi.org/10.1007/s00299-015-1847-0 Chen, J., Teotia, S., Lan, T., & Tang, G. (2021). MicroRNA techniques: Valuable tools for agronomic trait analyses and breeding in rice. Frontiers of Plant Science, 12. https:// doi.org/10.3389/fpls.2021.744357 Chu, L., He, X., Shu, W., Wang, L., & Tang, F. (2021). Knockdown of miR393 promotes the growth and biomass production in poplar. Frontiers of Plant Science, 12. https:// doi.org/10.3389/fpls.2021.714907 Dalakouras, A., Wassenegger, M., McMillan, J. N., Cardoza, V., Maegele, I., Dadami, E., Runne, M., Krczal, G., & Wassenegger, M. (2016). Induction of silencing in plants by high-pressure spraying of in vitro-synthesized small RNAs. Frontiers of Plant Science, 7. https://doi.org/10.3389/fpls.2016.01327 Darbani, B., Noeparvar, S., & Borg, S. (2016). Identification of circular RNAs from the parental genes involved in multiple aspects of cellular metabolism in barley. Frontiers of Plant Science, 7. https://doi.org/10.3389/fpls.2016.00776 De Felippes, F. F., Wang, J. W., & Weigel, D. (2012). MIGS: MiRNA-induced gene silencing. Plant Journal, 70(3), 541e547. https://doi.org/10.1111/j.1365313X.2011.04896.x Dong, C. H., & Pei, H. (2014). Over-expression of miR397 improves plant tolerance to cold stress in Arabidopsis thaliana. Journal of Plant Biology, 57(4), 209e217. https:// doi.org/10.1007/s12374-013-0490-y Dua, P., Yoo, J. W., Kim, S., & Lee, D. K. (2011). Modified siRNA structure with a single nucleotide bulge overcomes conventional siRNA-mediated off-target silencing. Molecular Therapy, 19(9), 1676e1687. https://doi.org/10.1038/mt.2011.109 Dubrovina, A. S., Aleynova, O. A., Kalachev, A. V., Suprun, A. R., Ogneva, Z. V., & Kiselev, K. V. (2019). Induction of transgene suppression in plants via external application of synthetic dsRNA. International Journal of Molecular Sciences, 20(7). https:// doi.org/10.3390/ijms20071585 Fahim, M., Millar, A. A., Wood, C. C., & Larkin, P. J. (2012). Resistance to wheat streak mosaic virus generated by expression of an artificial polycistronic microRNA in wheat. Plant Biotechnology Journal, 10(2), 150e163. https://doi.org/10.1111/j.14677652.2011.00647.x Small RNA transgenesis for abiotic stress tolerant food crops 533 Fahlgren, N., Hill, S. T., Carrington, J. C., & Carbonell, A. (2016). P-SAMS: A web site for plant artificial microRNA and synthetic trans-acting small interfering RNA design. Bioinformatics, 32(1), 157e158. https://doi.org/10.1093/bioinformatics/btv534 Fang, Y., Xie, K., & Xiong, L. (2014). Conserved miR164-targeted NAC genes negatively regulate drought resistance in rice. Journal of Experimental Botany, 65(8), 2119e2135. https://doi.org/10.1093/jxb/eru072 Felippes, F. F., & Weigel, D. (2009). Triggering the formation of tasiRNAs in Arabidopsis thaliana: The role of microRNA miR173. EMBO Reports, 10(3), 264e270. https:// doi.org/10.1038/embor.2008.247 Ferrer, J. L., Austin, M. B., Stewart, C., & Noel, J. P. (2008). Structure and function of enzymes involved in the biosynthesis of phenylpropanoids. Plant Physiology and Biochemistry, 46(3), 356e370. https://doi.org/10.1016/j.plaphy.2007.12.009 Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E., & Mello, C. C. (1998). Potent and specific genetic interference by double-stranded RNA in caenorhabditis elegans. Nature, 391(6669), 806e811. https://doi.org/10.1038/35888 Franco-Zorrilla, J. M., Valli, A., Todesco, M., Mateos, I., Puga, M. I., Rubio-Somoza, I., Leyva, A., Weigel, D., García, J. A., & Paz-Ares, J. (2007). Target mimicry provides a new mechanism for regulation of microRNA activity. Nature Genetics, 39(8), 1033e1037. https://doi.org/10.1038/ng2079 Fujii, H., Chiou, T. J., Lin, S. I., Aung, K., & Zhu, J. K. (2005). A miRNA involved in phosphate-starvation response in Arabidopsis. Current Biology, 15(22), 2038e2043. https://doi.org/10.1016/j.cub.2005.10.016 Gao, Z., Li, J., Luo, M., Li, H., Chen, Q., Wang, L., Song, S., Zhao, L., Xu, W., Zhang, C., Wang, S., & Ma, C. (2019). Characterization and cloning of grape circular RNAs identified the cold resistance-related vv-circATS1. Plant Physiology, 180(2), 966e985. https://doi.org/10.1104/pp.18.01331 García-Pérez, R. D., Houdt, H. V., & Depicker, A. (2004). Spreading of post-transcriptional gene silencing along the target gene promotes systemic silencing. The Plant Journal, 38(4), 594e602. https://doi.org/10.1111/j.1365-313x.2004.02067.x Gasparis, S., Kała, M., Przyborowski, M., Orczyk, W., & Nadolska-Orczyk, A. (2017). Artificial microRNA-based specific gene silencing of grain hardness genes in polyploid cereals appeared to be not stable over transgenic plant generations. Frontiers of Plant Science, 7. https://doi.org/10.3389/fpls.2016.02017 Gomez-Sanchez, A., Santamaria, M. E., Gonzalez-Melendi, P., Muszynska, A., Matthess, C., Martinez, M., & Diaz, I. (2021). Repression of barley cathepsins, HvPap19 and HvPap-1, differentially alters grain composition and delays germination. Journal of Experimental Botany, 72(9), 3474e3485. https://doi.org/10.1093/jxb/erab007 Gu, H, Lian, B, Yuan, Y, Kong, C, Li, Y, Liu, C, & Qi, Y (2022). A 5’ tRNA-Ala-derived small RNA regulates anti-fungal defense in plants. Science China Life Sciences, 65(1), 1e15. Hackenberg, Michael, Gustafson, P., Langridge, P., & Shi, B. (2015). Differential expression of micro RNA s and other small RNA s in barley between water and drought conditions. Plant Biotechnology Journal, 13(1), 2e13. https://doi.org/10.1111/pbi.12220 Hackenberg, M., Huang, P. J., Huang, C. Y., Shi, B. J., Gustafson, P., & Langridge, P. (2013). A comprehensive expression profile of microRNAs and other classes of noncoding small RNAs in barley under phosphorous-deficient and-sufficient conditions. DNA Research, 20(2), 109e125. https://doi.org/10.1093/dnares/dss037 Hamilton, A. J., & Baulcombe, D. C. (1999). A species of small antisense RNA in posttranscriptional gene silencing in plants. Science, 286(5441), 950e952. https://doi.org/ 10.1126/science.286.5441.950 534 Plant Small RNA in Food Crops Hang, N., Shi, T., Liu, Y., Ye, W., Taier, G., Sun, Y., Wang, K., & Zhang, W. (2021). Overexpression of Os-microRNA408 enhances drought tolerance in perennial ryegrass. Physiologia Plantarum, 172(2), 733e747. https://doi.org/10.1111/ppl.13276 Hendelman, A., Zebell, S., Rodriguez-Leal, D., Dukler, N., Robitaille, G., Wu, X., Kostyun, J., Tal, L., Wang, P., Bartlett, M. E., Eshed, Y., Efroni, I., & Lippman, Z. B. (2021). Conserved pleiotropy of an ancient plant homeobox gene uncovered by cisregulatory dissection. Cell, 184(7). https://doi.org/10.1016/j.cell.2021.02.001, 17241739.e16. Honda, S., Loher, P., Shigematsu, M., Palazzo, J. P., Suzuki, R., Imoto, I., Rigoutsos, I., & Kirino, Y. (2015). Sex hormone-dependent tRNA halves enhance cell proliferation in breast and prostate cancers. Proceedings of the National Academy of Sciences of the United States of America, 112(29), E3816eE3825. https://doi.org/10.1073/pnas.1510077112 Hu, Y., Jiang, Y., Han, X., Wang, H., Pan, J., & Yu, D. (2017). Jasmonate regulates leaf senescence and tolerance to cold stress: Crosstalk with other phytohormones. Journal of Experimental Botany, 68(6), 1361e1369. https://doi.org/10.1093/jxb/erx004 Hu, Y., Wu, Q., Peng, Z., Sprague, S. A., Wang, W., Park, J., Akhunov, E., Jagadish, K. S. V., Nakata, P. A., Cheng, N., Hirschi, K. D., White, F. F., & Park, S. (2017). Silencing of OsGRXS17 in rice improves drought stress tolerance by modulating ROS accumulation and stomatal closure. Scientific Reports, 7(1). https://doi.org/ 10.1038/s41598-017-16230-7 Jackson, A. L., Burchard, J., Leake, D., Reynolds, A., Schelter, J., Guo, J., Johnson, J. M., Lim, L., Karpilow, J., Nichols, K., Marshall, W., Khvorova, A., & Linsley, P. S. (2006). Position-specific chemical modification of siRNAs reduces “off-target” transcript silencing. RNA, 12(7), 1197e1205. https://doi.org/10.1261/rna.30706 Jain, R. G., Fletcher, S. J., Manzie, N., Robinson, K. E., Li, P., Lu, E., Brosnan, C. A., Xu, Z. P., & Mitter, N. (2022). Foliar application of clay-delivered RNA interference for whitefly control. Nature Plants, 8(5), 535e548. https://doi.org/10.1038/s41477022-01152-8 Ji, H., Wang, Y., Cloix, C., Li, K., Jenkins, G. I., Wang, S., Shang, Z., Shi, Y., Yang, S., Li, X., & Copenhaver, G. P. (2015). The arabidopsis RCC1 family protein TCF1 regulates freezing tolerance and cold acclimation through modulating lignin biosynthesis. PLoS Genetics, 11(9), e1005471. https://doi.org/10.1371/journal.pgen.1005471 Jian, C., Han, R., Chi, Q., Wang, S., Ma, M., Liu, X., & Zhao, H. (2017). Virus-based microRNA silencing and overexpressing in common wheat (Triticum aestivum L.). Frontiers of Plant Science, 8. https://doi.org/10.3389/fpls.2017.00500 van Kammen, A. (1997). Virus-induced gene silencing in infected and transgenic plants. Trends in Plant Science, 2(11), 409e411. https://doi.org/10.1016/s1360-1385(97) 01128-x Khan, A. W., Garg, V., Roorkiwal, M., Golicz, A. A., Edwards, D., & Varshney, R. K. (2020). Super-pangenome by integrating the wild side of a species for accelerated crop improvement. Trends in Plant Science, 25(2), 148e158. https://doi.org/10.1016/ j.tplants.2019.10.012 Kim, J. Y., Kwak, K. J., Jung, H. J., Lee, H. J., & Kang, H. (2010). MicroRNA402 affects seed germination of arabidopsis thaliana under stress conditions via targeting DEMETER-LIKE Protein3 mRNA. Plant and Cell Physiology, 51(6), 1079e1083. https://doi.org/10.1093/pcp/pcq072 Kiselev, K. V., Suprun, A. R., Aleynova, O. A., Ogneva, Z. V., Kalachev, A. V., & Dubrovina, A. S. (2021). External dsRNA downregulates anthocyanin biosynthesisrelated genes and affects anthocyanin accumulation in arabidopsis thaliana. International Journal of Molecular Sciences, 22(13). https://doi.org/10.3390/ijms22136749 Kittler, R., Surendranath, V., Heninger, A. K., Slabicki, M., Theis, M., Putz, G., Franke, K., Caldarelli, A., Grabner, H., Kozak, K., Wagner, J., Rees, E., Korn, B., Small RNA transgenesis for abiotic stress tolerant food crops 535 Frenzel, C., Sachse, C., Sönnichsen, B., Guo, J., Schelter, J., Burchard, J., … Buchholz, F. (2007). Genome-wide resources of endoribonuclease-prepared short interfering RNAs for specific loss-of-function studies. Nature Methods, 4(4), 337e344. https://doi.org/10.1038/nmeth1025 Komor, A. C., Badran, A. H., & Liu, D. R. (2017). CRISPR-based technologies for the manipulation of eukaryotic genomes. Cell, 168(1e2), 20e36. https://doi.org/10.1016/ j.cell.2016.10.044 Krysan, P. J., Young, J. C., & Sussman, M. R. (1999). T-DNA as an insertional mutagen in Arabidopsis. The Plant Cell Online, 11(12), 2283e2290. https://doi.org/10.1105/ tpc.11.12.2283 Kumar, G., Kumari, K., & Dasgupta, I. (2022). RTBV-based VIGS vector for functional genomics in rice: Methodology, advances, challenges, and future implications. In , Vol 2408. Methods in molecular biology (pp. 117e131). Humana Press Inc. https://doi.org/ 10.1007/978-1-0716-1875-2_8 Kung, Y. J., Lin, S. S., Huang, Y. L., Chen, T. C., Harish, S. S., Chua, N. H., & Yeh, S. D. (2012). Multiple artificial microRNAs targeting conserved motifs of the replicase gene confer robust transgenic resistance to negative-sense single-stranded RNA plant virus. Molecular Plant Pathology, 13(3), 303e317. https://doi.org/10.1111/j.13643703.2011.00747.x Lange, H., Sement, F. M., & Gagliardi, D. (2011). MTR4, a putative RNA helicase and exosome co-factor, is required for proper rRNA biogenesis and development in Arabidopsis thaliana. The Plant Journal, 68(1), 51e63. https://doi.org/10.1111/j.1365313X.2011.04675.x Lehretz, G. G., Sonnewald, S., Hornyik, C., Corral, J. M., & Sonnewald, U. (2019). Posttranscriptional regulation of FLOWERING LOCUS T modulates heat-dependent source-sink development in potato. Current Biology, 29(10). https://doi.org/10.1016/ j.cub.2019.04.027, 1614-1624.e3. Li, J. L., Chen, X. X., Shi, C. C., Liu, F. H., Sun, J., & Ge, R. C. (2020). Effects of OsRPK1 gene overexpression and RNAi on the salt-tolerance at seedling stage in rice. https://doi.org/10.3724/ Acta Agronomica Sinica, 46(8), 1217e1224. SP.J.1006.2020.92060 Li, J. F., Chung, H. S., Niu, Y., Bush, J., McCormack, M., & Sheen, J. (2013). Comprehensive protein-based artificial microRNA screens for effective gene silencing in plants. The Plant Cell Online, 25(5), 1507e1522. https://doi.org/10.1105/ tpc.113.112235 Li, H., Guan, R., Guo, H., & Miao, X. (2015). New insights into an RNAi approach for plant defence against piercing-sucking and stem-borer insect pests. Plant, Cell and Environment, 38(11), 2277e2285. https://doi.org/10.1111/pce.12546 Li, D.h., Liu, H., Yang, Y.l., Zhen, P.p., & Liang, J.s. (2009). Down-regulated expression of RACK1 gene by RNA interference enhances drought tolerance in rice. Rice Science, 16(1), 14e20. https://doi.org/10.1016/S1672-6308(08)60051-7 Li, D., Mou, W., Luo, Z., Li, L., Limwachiranon, J., Mao, L., & Ying, T. (2016). Developmental and stress regulation on expression of a novel miRNA, Fan-miR73, and its target ABI5 in strawberry. Scientific Reports, 6. https://doi.org/10.1038/srep28385 Li, W. X., Oono, Y., Zhu, J., He, X. J., Wu, J. M., Iida, K., Lu, X. Y., Cui, X., Jin, H., & Zhu, J. K. (2008). The Arabidopsis NFYA5 transcription factor is regulated transcriptionally and posttranscriptionally to promote drought resistance. The Plant Cell Online, 20(8), 2238e2251. https://doi.org/10.1105/tpc.108.059444 Li, N., Yang, T., Guo, Z., Wang, Q., Chai, M., Wu, M., Li, X., Li, W., Li, G., Tang, J., Tang, G., & Zhang, Z. (2020). Maize microRNA166 inactivation confers plant development and abiotic stress resistance. International Journal of Molecular Sciences, 21(24), 1e20. https://doi.org/10.3390/ijms21249506 536 Plant Small RNA in Food Crops Li, J. F., Zhang, D., & Sheen, J. (2014). Epitope-tagged protein-based artificial miRNA screens for optimized gene silencing in plants. Nature Protocols, 9(4), 939e949. https:// doi.org/10.1038/nprot.2014.061 Li, G. Z., Zheng, Y. X., Liu, H. T., Liu, J., & Kang, G. Z. (2022). WRKY74 regulates cadmium tolerance through glutathione-dependent pathway in wheat. Environmental Science and Pollution Research, 29(45), 68191e68201. https://doi.org/10.1007/s11356022-20672-6 Liang, G., He, H., Li, Y., & Yu, D. (2012). A new strategy for construction of artificial miRNA vectors in Arabidopsis. Planta, 235(6), 1421e1429. https://doi.org/10.1007/ s00425-012-1610-5 Liao, S., Chen, X., Xu, T., Jin, Q., Xu, Z., Xu, D., Zhou, X., Zhu, C., Guang, S., & Feng, X. (2021). Antisense ribosomal siRNAs inhibit RNA polymerase I-directed transcription in C. elegans. Nucleic Acids Research, 49(16), 9194e9210. https://doi.org/ 10.1093/nar/gkab662 Liu, Y., Du, H., Li, P., Shen, Y., Peng, H., Liu, S., Zhou, G. A., Zhang, H., Liu, Z., Shi, M., Huang, X., Li, Y., Zhang, M., Wang, Z., Zhu, B., Han, B., Liang, C., & Tian, Z. (2020). Pan-genome of wild and cultivated soybeans. Cell, 182(1), 162e176.e13. https://doi.org/10.1016/j.cell.2020.05.023 Liu, C., Fan, W., Zhu, P., Xia, Z., Hu, J., & Zhao, A. (2019). Mulberry RGS negatively regulates salt stress response and tolerance. Plant Signaling & Behavior, 14(12), 1672512. https://doi.org/10.1080/15592324.2019.1672512 Liu, X., Liu, S., Wang, R., Chen, X., Fan, Z., Wu, B., & Zhou, T. (2019). Analyses of MiRNA functions in maize using a newly developed ZMBJ-CMV-2bN81-STTM vector. Frontiers of Plant Science, 10. https://doi.org/10.3389/fpls.2019.01277 Liu, Y., Teng, C., Xia, R., & Meyers, B. C. (2020). PhasiRNAs in Plants: Their Biogenesis, genic sources, and roles in stress responses, development, and reproduction. The Plant Cell Online, 32(10), 3059e3080. https://doi.org/10.1105/tpc.20.00335 López-Dolz, L., Spada, M., Daròs, J.-A., & Carbonell, A. (2020). Fine-tune control of targeted RNAi efficacy by plant artificial small RNAs. Nucleic Acids Research, 48(11), 6234e6250. https://doi.org/10.1093/nar/gkaa343 Lunardon, A., Kariuki, S. M., & Axtell, M. J. (2021). Expression and processing of polycistronic artificial microRNAs and trans-acting siRNAs from transiently introduced transgenes in Solanum lycopersicum and Nicotiana benthamiana. The Plant Journal, 106(4), 1087e1104. https://doi.org/10.1111/tpj.15221 de la Luz Gutiérrez-Nava, M., Aukerman, M. J., Sakai, H., Tingey, S. V., & Williams, R. W. (2008). Artificial trans-acting siRNAs confer consistent and effective gene silencing. Plant Physiology, 147(2), 543e551. https://doi.org/10.1104/ pp.108.118307 Ma, C., Burd, S., & Lers, A. (2015). MiR408 is involved in abiotic stress responses in Arabidopsis. The Plant Journal, 84(1), 169e187. https://doi.org/10.1111/tpj.12999 Makhazen, D. S., Veremeichik, G. N., Shkryl, Y. N., Tchernoded, G. K., Grigorchuk, V. P., & Bulgakov, V. P. (2021). Inhibition of the JAZ1 gene causes activation of camalexin biosynthesis in Arabidopsis callus cultures. Journal of Biotechnology, 342, 102e113. https://doi.org/10.1016/j.jbiotec.2021.10.012 Manavalan, L. P., Chen, X., Clarke, J., Salmeron, J., & Nguyen, H. T. (2012). RNAimediated disruption of squalene synthase improves drought tolerance and yield in rice. Journal of Experimental Botany, 63(1), 163e175. https://doi.org/10.1093/jxb/ err258 Matthews, C., Arshad, M., & Hannoufa, A. (2019). Alfalfa response to heat stress is modulated by microRNA156. Physiologia Plantarum, 165(4), 830e842. https://doi.org/ 10.1111/ppl.12787 Small RNA transgenesis for abiotic stress tolerant food crops 537 Meyer, P., & Saedler, H. (1996). Homology-dependent gene silencing in plants. Annual Review of Plant Physiology and Plant Molecular Biology, 47(1), 23e48. https://doi.org/ 10.1146/annurev.arplant.47.1.23 Mickelbart, M. V., Hasegawa, P. M., & Bailey-Serres, J. (2015). Genetic mechanisms of abiotic stress tolerance that translate to crop yield stability. Nature Reviews Genetics, 16(4), 237e251. https://doi.org/10.1038/nrg3901 Mitsuda, N., & Ohme-Takagi, M. (2009). Functional analysis of transcription factors in Arabidopsis. Plant and Cell Physiology, 50(7), 1232e1248. https://doi.org/10.1093/pcp/ pcp075 Mitter, N., Worrall, E. A., Robinson, K. E., Li, P., Jain, R. G., Taochy, C., Fletcher, S. J., Carroll, B. J., Lu, G. Q., & Xu, Z. P. (2017). Clay nanosheets for topical delivery of RNAi for sustained protection against plant viruses. Nature Plants, 3. https://doi.org/ 10.1038/nplants.2016.207 Montgomery, T. A., Howell, M. D., Cuperus, J. T., Li, D., Hansen, J. E., Alexander, A. L., Chapman, E. J., Fahlgren, N., Allen, E., & Carrington, J. C. (2008). Specificity of ARGONAUTE7-miR390 interaction and dual functionality in TAS3 trans-acting siRNA formation. Cell, 133(1), 128e141. https://doi.org/10.1016/j.cell.2008.02.033 Montgomery, T. A., Seong, J. Y., Fahlgren, N., Gilbert, S. D., Howell, M. D., Sullivan, C. M., Alexander, A., Nguyen, G., Allen, E., Ji, H. A., & Carrington, J. C. (2008). AGO1-miR173 complex initiates phased siRNA formation in plants. Proceedings of the National Academy of Sciences of the United States of America, 105(51), 20055e20062. https://doi.org/10.1073/pnas.0810241105 Nanjo, T., Kobayashi, M., Yoshiba, Y., Kakubari, Y., Yamaguchi-Shinozaki, K., & Shinozaki, K. (1999). Antisense suppression of proline degradation improves tolerance to freezing and salinity in Arabidopsis thaliana. FEBS Letters, 461(3), 205e210. https:// doi.org/10.1016/s0014-5793(99)01451-9 Napoli, C., Lemieux, C., & Jorgensen, R. (1990). Introduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous genes in trans. The Plant Cell Online, 2(4), 279e289. https://doi.org/10.2307/3869076 Navarro, L., Dunoyer, P., Jay, F., Arnold, B., Dharmasiri, N., Estelle, M., Voinnet, O., & Jones, J. D. G. (2006). A plant miRNA contributes to antibacterial resistance by repressing auxin signaling. Science, 312(5772), 436e439. https://doi.org/10.1126/ science.1126088 Neumeier, J., Meister, G., & Specificity. (2020). RNAi mechanisms and strategies to reduce off-target effects. Frontiers of Plant Science, 11. Ni, Z., Hu, Z., Jiang, Q., & Zhang, H. (2012). Overexpression of gma-MIR394a confers tolerance to drought in transgenic Arabidopsis thaliana. Biochemical and Biophysical Research Communications, 427(2), 330e335. https://doi.org/10.1016/j.bbrc.2012.09.055 Ning, Y., Jantasuriyarat, C., Zhao, Q., Zhang, H., Chen, S., Liu, J., Liu, L., Tang, S., Park, C. H., Wang, X., Liu, X., Dai, L., Xie, Q., & Wang, G.-L. (2011). The SINA E3 ligase OsDIS1 negatively regulates drought response in rice. Plant Physiology, 157(1), 242e255. https://doi.org/10.1104/pp.111.180893 Noman, A., Fahad, S., Aqeel, M., Ali, U., Amanullah, Anwar, S., Baloch, S. K., & Zainab, M. (2017). miRNAs: Major modulators for crop growth and development under abiotic stresses. Biotechnology Letters, 39(5), 685e700. https://doi.org/10.1007/ s10529-017-2302-9 Parinov, S., Sevugan, M., Ye, D., Yang, W. C., Kumaran, M., & Sundaresan, V. (1999). Analysis of flanking sequences from dissociation insertion lines: A database for reverse genetics in Arabidopsis. The Plant Cell Online, 11(12), 2263e2270. https://doi.org/ 10.1105/tpc.11.12.2263 Parizotto, E. A., Dunoyer, P., Rahm, N., Himber, C., & Voinnet, O. (2004). In vivo investigation of the transcription, processing, endonucleolytic activity, and functional 538 Plant Small RNA in Food Crops relevance of the spatial distribution of a plant miRNA. Genes & Development, 18(18), 2237e2242. https://doi.org/10.1101/gad.307804 Park, G.-G., Park, J.-J., Yoon, J., Yu, S.-N., & An, G. (2010). A RING finger E3 ligase gene, Oryza sativa Delayed Seed Germination 1 (OsDSG1), controls seed germination and stress responses in rice. Plant Molecular Biology, 74(4e5), 467e478. https://doi.org/ 10.1007/s11103-010-9687-3 Park, W., Zhai, J., & Lee, J. Y. (2009). Highly efficient gene silencing using perfect complementary artificial miRNA targeting AP1 or heteromeric artificial miRNA targeting AP1 and CAL genes. Plant Cell Reports, 28(3), 469e480. https://doi.org/ 10.1007/s00299-008-0651-5 Peng, T., Qiao, M., Liu, H., Teotia, S., Zhang, Z., Zhao, Y., Wang, B., Zhao, D., Shi, L., Zhang, C., Le, B., Rogers, K., Gunasekara, C., Duan, H., Gu, Y., Tian, L., Nie, J., Qi, J., Meng, F., … Tang, G. (2018). A resource for inactivation of MicroRNAs using short tandem target mimic technology in model and crop plants. Molecular Plant, 11(11), 1400e1417. https://doi.org/10.1016/j.molp.2018.09.003 Pieczynski, M., Marczewski, W., Hennig, J., Dolata, J., Bielewicz, D., Piontek, P., Wyrzykowska, A., Krusiewicz, D., Strzelczyk-Zyta, D., Konopka-Postupolska, D., Krzeslowska, M., Jarmolowski, A., & Szweykowska-Kulinska, Z. (2013). Downregulation of CBP80 gene expression as a strategy to engineer a drought-tolerant potato. Plant Biotechnology Journal, 11(4), 459e469. https://doi.org/10.1111/pbi.12032 Ramegowda, V., Mysore, K. S., & Senthil-Kumar, M. (2014). Virus-induced gene silencing is a versatile tool for unraveling the functional relevance of multiple abiotic-stressresponsive genes in crop plants. Frontiers of Plant Science, 5. https://doi.org/10.3389/ fpls.2014.00323 Ramegowda, V., Senthil-kumar, M., Udayakumar, M., & Mysore, K. S. (2013). A highthroughput virus-induced gene silencing protocol identifies genes involved in multistress tolerance. BMC Plant Biology, 13(1). https://doi.org/10.1186/1471-2229-13-193 Rhoades, M. W., Reinhart, B. J., Lim, L. P., Burge, C. B., Bartel, B., & Bartel, D. P. (2002). Prediction of plant microRNA targets. Cell, 110(4), 513e520. https://doi.org/ 10.1016/S0092-8674(02)00863-2 Rodriguez-Leal, D. (2017). Engineering quantitative trait variation for crop improvement by genome editing. Cell, 171(2), 470e480. Rogers, K., & Chen, X. (2013). Biogenesis, turnover, and mode of action of plant microRNAs. The Plant Cell Online, 25(7), 2383e2399. https://doi.org/10.1105/ tpc.113.113159 Rothstein, S. J., DiMaio, J., Strand, M., & Rice, D. (1987). Stable and heritable inhibition of the expression of nopaline synthase in tobacco expressing antisense RNA. Proceedings of the National Academy of Sciences, 84(23), 8439e8443. https://doi.org/10.1073/ pnas.84.23.8439 Schwab, R., Ossowski, S., Riester, M., Warthmann, N., & Weigel, D. (2006). Highly specific gene silencing by artificial microRNAs in Arabidopsis. The Plant Cell Online, 18(5), 1121e1133. https://doi.org/10.1105/tpc.105.039834 Shafi, A., Dogra, V., Gill, T., Ahuja, P. S., Sreenivasulu, Y., & Pandey, G. K. (2014). Simultaneous over-expression of PaSOD and RaAPX in transgenic Arabidopsis thaliana confers cold stress tolerance through increase in vascular lignifications. PLoS One, 9(10), e110302. https://doi.org/10.1371/journal.pone.0110302 Singh, A. K., Ghosh, D., & Chakraborty, S. (2022). Optimization of tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) in tomato. In , 2408. Methods in Molecular Biology (pp. 133e145). Humana Press Inc. https://doi.org/10.1007/978-10716-1875-2_9 Small RNA transgenesis for abiotic stress tolerant food crops 539 Singh, D. K., & Mysore, K. S. (2022). Virus-induced gene silencing in sorghum using brome mosaic virus. In Methods in molecular biology (Vol 2408, pp. 109e115). Humana Press Inc. https://doi.org/10.1007/978-1-0716-1875-2_7 Smalle, J., & Vierstra, R. D. (2004). The ubiquitin 26S proteasome proteolytic pathway. Annual Review of Plant Biology, 55, 555e590. https://doi.org/10.1146/ annurev.arplant.55.031903.141801 Smith, N. A., Singh, S. P., Wang, M. B., Stoutjesdijk, P. A., Green, A. G., & Waterhouse, P. M. (2000). Total silencing by intronspliced hairpin RNAs. Nature, 407(6802), 319e320. https://doi.org/10.1038/35030305 Smith, C. J. S., Watson, C. F., Morris, P. C., Bird, C. R., Seymour, G. B., Gray, J. E., Arnold, C., Tucker, G. A., Schuch, W., Harding, S., & Grierson, D. (1990). Inheritance and effect on ripening of antisense polygalacturonase genes in transgenic tomatoes. Plant Molecular Biology, 14(3), 369e379. https://doi.org/10.1007/BF00028773 Sow, M. D., Le Gac, A. L., Fichot, R., Lanciano, S., Delaunay, A., Le Jan, I., LesageDescauses, M. C., Citerne, S., Caius, J., Brunaud, V., Soubigou-Taconnat, L., Cochard, H., Segura, V., Chaparro, C., Grunau, C., Daviaud, C., Tost, J., Brignolas, F., Strauss, S. H., … Maury, S. (2021). RNAi suppression of DNA methylation affects the drought stress response and genome integrity in transgenic poplar. New Phytologist, 232(1), 80e97. https://doi.org/10.1111/nph.17555 Speulman, E., Metz, P. L. J., Van Arkel, G., Hekkert, B. T. L., Stiekema, W. J., & Pereira, A. (1999). A two-component enhancer-inhibitor transposon mutagenesis system for functional analysis of the arabidopsis genome. The Plant Cell Online, 11(10), 1853e1866. https://doi.org/10.1105/tpc.11.10.1853 Stief, A., Altmann, S., Hoffmann, K., Pant, B. D., Scheible, W. R., & Bäurle, I. (2014). Arabidopsis miR156 regulates tolerance to recurring environmental stress through SPL transcription factors. The Plant Cell Online, 26(4), 1792e1807. https://doi.org/ 10.1105/tpc.114.123851 Tang, W., & Thompson, W. A. (2019). OsmiR528 enhances cold stress tolerance by repressing expression of stress response-related transcription factor genes in plant cells. Current Genomics, 20(2), 100e114. https://doi.org/10.2174/ 1389202920666190129145439 Tang, Y., Wang, F., Zhao, J., Xie, K., Hong, Y., & Liu, Y. (2010). Virus-based MicroRNA expression for gene functional analysis in plants. Plant Physiology, 153(2), 632e641. https://doi.org/10.1104/pp.110.155796 Thompson, D. M., Lu, C., Green, P. J., & Parker, R. (2008). tRNA cleavage is a conserved response to oxidative stress in eukaryotes. RNA, 14(10), 2095e2103. https://doi.org/ 10.1261/rna.1232808 Tiedge, K., Destremps, J., Solano-Sanchez, J., Arce-Rodriguez, M. L., & Zerbe, P. (2022). Foxtail mosaic virus-induced gene silencing (VIGS) in switchgrass (Panicum virgatum L.). Plant Methods, 18(1). https://doi.org/10.1186/s13007-022-00903-0 Todesco, M., Rubio-Somoza, I., Paz-Ares, J., Weigel, D., & Copenhaver, G. P. (2010). A collection of target mimics for comprehensive analysis of MicroRNA function in Arabidopsis thaliana. PLoS Genetics, 6(7), e1001031. https://doi.org/10.1371/ journal.pgen.1001031 Van Der Krol, A. R., Mur, L. A., Beld, M., Mol, J. N. M., & Stuitje, A. R. (1990). Flavonoid genes in petunia: Addition of a limited number of gene copies may lead to a suppression of gene expression. The Plant Cell Online, 2(4), 291e299. https://doi.org/ 10.1105/tpc.2.4.291 Vaucheret, H., Nussaume, L., Palauqui, J. C., Quilléré, I., & Elmayan, T. (1997). A transcriptionally active state is required for post-transcriptional silencing (cosuppression) of nitrate reductase host genes and transgenes. The Plant Cell Online, 9(8), 1495e1504. https://doi.org/10.1105/tpc.9.8.1495 540 Plant Small RNA in Food Crops Voinnet, O. (2008). Use, tolerance and avoidance of amplified RNA silencing by plants. Trends in Plant Science, 13(7), 317e328. https://doi.org/10.1016/j.tplants.2008.05.004 Wang, Y., Beaith, M., Chalifoux, M., Ying, J., Uchacz, T., Sarvas, C., Griffiths, R., Kuzma, M., Wan, J., & Huang, Y. (2009). Shoot-specific down-regulation of protein farnesyltransferase (a-subunit) for yield protection against drought in canola. Molecular Plant, 2(1), 191e200. https://doi.org/10.1093/mp/ssn088 Wang, X., Chang, X., Jing, Y., Zhao, J., Fang, Q., Sun, M., Zhang, Y., Li, W., & Li, Y. (2020). Identification and functional prediction of soybean CircRNAs involved in lowtemperature responses. Journal of Plant Physiology, 250. https://doi.org/10.1016/ j.jplph.2020.153188 Wang, T., Chen, L., Zhao, M., Tian, Q., & Zhang, W. H. (2011). Identification of drought-responsive microRNAs in Medicago truncatula by genome-wide highthroughput sequencing. BMC Genomics, 12. https://doi.org/10.1186/1471-2164-12367 Wang, Y., Guo, S., Wang, L., Wang, L., He, X., Shu, S., Sun, J., & Lu, N. (2018). Identification of microRNAs associated with the exogenous spermidine-mediated improvement of high-temperature tolerance in cucumber seedlings (Cucumis sativus L.). BMC Genomics, 19(1). https://doi.org/10.1186/s12864-018-4678-x Wang, M., & Jin, H. (2017). Spray-induced gene silencing: A powerful innovative strategy for crop protection. Trends in Microbiology, 25(1), 4e6. https://doi.org/10.1016/ j.tim.2016.11.011 Wang, H., Li, Y., Chern, M., Zhu, Y., Zhang, L. L., Lu, J. H., Li, X. P., Dang, W. Q., Ma, X. C., Yang, Z. R., Yao, S. Z., Zhao, Z. X., Fan, J., Huang, Y. Y., Zhang, J. W., Pu, M., Wang, J., He, M., Li, W. T., … Wang, W. M. (2021). Suppression of rice miR168 improves yield, flowering time and immunity. Nature Plants, 7(2), 129e136. https://doi.org/10.1038/s41477-021-00852-x Wang, S., Sun, X., Hoshino, Y., Yu, Y., Jia, B., Sun, Z., Sun, M., Duan, X., Zhu, Y., & Unver, T. (2014). MicroRNA319 positively regulates cold tolerance by targeting OsPCF6 and OsTCP21 in rice (Oryza sativa L.). PLoS One, 9(3), e91357. https:// doi.org/10.1371/journal.pone.0091357 Wang, M., Thomas, N., & Jin, H. (2017). Cross-kingdom RNA trafficking and environmental RNAi for powerful innovative pre- and post-harvest plant protection. Current Opinion in Plant Biology, 38, 133e141. https://doi.org/10.1016/j.pbi.2017.05.003 Wang, Z., Wang, X. Y., Martinho, C., & Baulcombe, D. C. (2022). Post-transcriptional gene silencing using virus-induced gene silencing to study plant gametogenesis in tomato. In , Vol 2484. Methods in molecular biology (pp. 201e212). Humana Press Inc. https://doi.org/10.1007/978-1-0716-2253-7_15 Wang, Y., Yang, M., Wei, S., Qin, F., Zhao, H., & Suo, B. (2017). Identification of circular RNAs and their targets in leaves of Triticum aestivum L. under dehydration stress. Frontiers of Plant Science, 7. https://doi.org/10.3389/fpls.2016.02024 Wang, Y., Ying, J., Kuzma, M., Chalifoux, M., Sample, A., McArthur, C., Uchacz, T., Sarvas, C., Wan, J., Dennis, D. T., McCourt, P., & Huang, Y. (2005). Molecular tailoring of farnesylation for plant drought tolerance and yield protection. The Plant Journal, 43(3), 413e424. https://doi.org/10.1111/j.1365-313X.2005.02463.x Waterhouse, P. M., Graham, M. W., & Wang, M. B. (1998). Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA. Proceedings of the National Academy of Sciences of the United States of America, 95(23), 13959e13964. https://doi.org/10.1073/pnas.95.23.13959 Watson, J. M., Fusaro, A. F., Wang, M. B., & Waterhouse, P. M. (2005). RNA silencing platforms in plants. FEBS Letters, 579(26), 5982e5987. https://doi.org/10.1016/ j.febslet.2005.08.014 Small RNA transgenesis for abiotic stress tolerant food crops 541 Wesley, S. V., Helliwell, C. A., Smith, N. A., Wang, M., Rouse, D. T., Liu, Q., Gooding, P. S., Singh, S. P., Abbott, D., Stoutjesdijk, P. A., Robinson, S. P., Gleave, A. P., Green, A. G., & Waterhouse, P. M. (2001). Construct design for efficient, effective and high-throughput gene silencing in plants. The Plant Journal, 27(6), 581e590. https://doi.org/10.1046/j.1365-313x.2001.01105.x Wittkopp, P. J., & Kalay, G. (2012). Cis-regulatory elements: Molecular mechanisms and evolutionary processes underlying divergence. Nature Reviews Genetics, 13(1), 59e69. https://doi.org/10.1038/nrg3095 Wu, B. F., Li, W. F., Xu, H. Y., Qi, L. W., & Han, S. Y. (2015). Role of cin-miR2118 in drought stress responses in Caragana intermedia and Tobacco. Gene, 574(1), 34e40. https://doi.org/10.1016/j.gene.2015.07.072 Wu, G., Park, M. Y., Conway, S. R., Wang, J. W., Weigel, D., & Poethig, R. S. (2009). The sequential action of miR156 and miR172 regulates developmental timing in arabidopsis. Cell, 138(4), 750e759. https://doi.org/10.1016/j.cell.2009.06.031 Yan, J., Gu, Y., Jia, X., Kang, W., Pan, S., Tang, X., Chen, X., & Tang, G. (2012). Effective small RNA destruction by the expression of a short tandem target mimic in Arabidopsis. The Plant Cell Online, 24(2), 415e427. https://doi.org/10.1105/tpc.111.094144 Yan, S., Ren, B. Y., & Shen, J. (2021). Nanoparticle-mediated double-stranded RNA delivery system: A promising approach for sustainable pest management. Insect Science, 28(1), 21e34. https://doi.org/10.1111/1744-7917.12822 Yan, Jun, Zhao, C., Zhou, J., Yang, Y., Wang, P., Zhu, X., Tang, G., Bressan, R. A., Zhu, J.-K., & Yu, H. (2016). The miR165/166 mediated regulatory module plays critical roles in ABA homeostasis and response in Arabidopsis thaliana. PLoS Genetics, 12(11), e1006416. https://doi.org/10.1371/journal.pgen.1006416 Yang, C., Li, D., Mao, D., Liu, X., Ji, C., Li, X., Zhao, X., Cheng, Z., Chen, C., & Zhu, L. (2013). Overexpression of microRNA319 impacts leaf morphogenesis and leads to enhanced cold tolerance in rice (Oryza sativa L.). Plant, Cell and Environment, 36(12), 2207e2218. https://doi.org/10.1111/pce.12130 Yang, X., Liu, Y., Zhang, H., Wang, J., Zinta, G., Xie, S., Zhu, W., & Nie, W. F. (2020). Genome-wide identification of circular RNAs in response to low-temperature stress in tomato leaves. Frontiers in Genetics, 11. https://doi.org/10.3389/fgene.2020.591806 Yang, O., Popova, O. V., Süthoff, U., Lüking, I., Dietz, K. J., & Golldack, D. (2009). The Arabidopsis basic leucine zipper transcription factor AtbZIP24 regulates complex transcriptional networks involved in abiotic stress resistance. Gene, 436(1e2), 45e55. https://doi.org/10.1016/j.gene.2009.02.010 Yang, X., Ye, J., Niu, F., Feng, Y., & Song, X. (2021). Identification and verification of genes related to pollen development and male sterility induced by high temperature in the thermo-sensitive genic male sterile wheat line. Planta, 253(4). https://doi.org/ 10.1007/s00425-021-03601-8 Yang, J., Zhang, N., Zhang, J., Jin, X., Zhu, X., Ma, R., Li, S., Lui, S., Yue, Y., & Si, H. (2021). Knockdown of MicroRNA160a/b by STTM leads to root architecture changes via auxin signaling in Solanum tuberosum. Plant Physiology and Biochemistry, 166, 939e949. https://doi.org/10.1016/j.plaphy.2021.06.051 You, C., He, W., Hang, R., Zhang, C., Cao, X., Guo, H., Chen, X., Cui, J., & Mo, B. (2019). FIERY1 promotes microRNA accumulation by suppressing rRNA-derived small interfering RNAs in Arabidopsis. Nature Communications, 10(1). https://doi.org/ 10.1038/s41467-019-12379-z Yuan, S., Li, Z., Li, D., Yuan, N., Hu, Q., & Luo, H. (2015). Constitutive expression of rice MicroRNA528 alters plant development and enhances tolerance to salinity stress and nitrogen starvation in creeping bentgrass. Plant Physiology, 169(1), 576e593. https:// doi.org/10.1104/pp.15.00899 542 Plant Small RNA in Food Crops Zeng, X., Jiang, J., Wang, F., Liu, W., Zhang, S., Du, J., & Yang, C. (2022). Rice OsClo5, a caleosin protein, negatively regulates cold tolerance through the jasmonate signalling pathway. Plant Biology, 24(1), 52e61. https://doi.org/10.1111/plb.13350 Zhang, Z. J. (2014). Artificial trans-acting small interfering RNA: A tool for plant biology study and crop improvements. Planta, 239(6), 1139e1146. https://doi.org/10.1007/ s00425-014-2054-x Zhang, P., Fan, Y., Sun, X., Chen, L., Terzaghi, W., Bucher, E., Li, L., & Dai, M. (2019). A large-scale circular RNA profiling reveals universal molecular mechanisms responsive to drought stress in maize and Arabidopsis. The Plant Journal, 98(4), 697e713. https:// doi.org/10.1111/tpj.14267 Zhang, Q., Li, Y., Zhang, Y., Wu, C., Wang, S., Hao, L., Wang, S., & Li, T. (2017). MdMiR156ab and Md-mir395 target WRKY transcription factors to influence apple resistance to leaf spot disease. Frontiers of Plant Science, 8. https://doi.org/10.3389/ fpls.2017.00526 Zhang, S., Sun, L., & Kragler, F. (2009). The phloem-delivered RNA pool contains small noncoding RNAs and interferes with translation1[W][OA]. Plant Physiology, 150(1), 378e387. https://doi.org/10.1104/pp.108.134767 Zhang, N., Zhang, D., Chen, S. L., Gong, B. Q., Guo, Y., Xu, L., Zhang, X. N., & Li, J. F. (2018). Engineering artificial microRNAs for multiplex gene silencing and simplified transgenic screen. Plant Physiology, 178(3), 989e1001. https://doi.org/10.1104/ pp.18.00828 Zhang, J., Zhang, H., Srivastava, A. K., Pan, Y., Bai, J., Fang, J., Shi, H., & Zhu, J. K. (2018). Knockdown of rice microRNA166 confers drought resistance by causing leaf rolling and altering stem xylem development. Plant Physiology, 176(3), 2082e2094. https://doi.org/10.1104/pp.17.01432 Zhang, H., Zhang, J., Yan, J., Gou, F., Mao, Y., Tang, G., Botella, J. R., & Zhu, J. K. (2017). Short tandem target mimic rice lines uncover functions of miRNAs in regulating important agronomic traits. Proceedings of the National Academy of Sciences of the United States of America, 114(20), 5277e5282. https://doi.org/10.1073/ pnas.1703752114 Zhang, X., Zou, Z., Gong, P., Zhang, J., Ziaf, K., Li, H., Xiao, F., & Ye, Z. (2011). Overexpression of microRNA169 confers enhanced drought tolerance to tomato. Biotechnology Letters, 33(2), 403e409. https://doi.org/10.1007/s10529-010-0436-0 Zhao, J., Yuan, S., Zhou, M., Yuan, N., Li, Z., Hu, Q., Bethea, F. G., Liu, H., Li, S., & Luo, H. (2019). Transgenic creeping bentgrass overexpressing Osa-miR393a exhibits altered plant development and improved multiple stress tolerance. Plant Biotechnology Journal, 17(1), 233e251. https://doi.org/10.1111/pbi.12960 Zhou, H., Hussain, S. S., Hackenberg, M., Bazanova, N., Eini, O., Li, J., Gustafson, P., & Shi, B. (2018). Identification and characterisation of a previously unknown drought tolerance-associated micro RNA in barley. The Plant Journal, 95(1), 138e149. https:// doi.org/10.1111/tpj.13938 Zhou, Man, Li, D., Li, Z., Hu, Q., Yang, C., Zhu, L., & Luo, H. (2013). Constitutive expression of a miR319 gene alters plant development and enhances salt and drought tolerance in transgenic creeping bentgrass. Plant Physiology, 161(3), 1375e1391. https:// doi.org/10.1104/pp.112.208702 Zhou, M., & Tang, W. (2019). MicroRNA156 amplifies transcription factor-associated cold stress tolerance in plant cells. Molecular Genetics and Genomics, 294(2), 379e393. https:// doi.org/10.1007/s00438-018-1516-4 Zhou, J., Yuan, M., Zhao, Y., Quan, Q., Yu, D., Yang, H., Tang, X., Xin, X., Cai, G., Qian, Q., Qi, Y., & Zhang, Y. (2021). Efficient deletion of multiple circle RNA loci by CRISPR-Cas9 reveals Os06circ02797 as a putative sponge for OsMIR408 in rice. Plant Biotechnology Journal, 19(6), 1240e1252. https://doi.org/10.1111/pbi.13544 Small RNA transgenesis for abiotic stress tolerant food crops 543 Zhu, C., Yan, Q., Weng, C., Hou, X., Mao, H., Liu, D., Feng, X., & Guang, S. (2018). Erroneous ribosomal RNAs promote the generation of antisense ribosomal siRNA. Proceedings of the National Academy of Sciences of the United States of America, 115(40), 10082e10087. https://doi.org/10.1073/pnas.1800974115 Zuo, J., Wang, Q., Zhu, B., Luo, Y., & Gao, L. (2016). Deciphering the roles of circRNAs on chilling injury in tomato. Biochemical and Biophysical Research Communications, 479(2), 132e138. https://doi.org/10.1016/j.bbrc.2016.07.032 Mushtaq N, Wang Y, Fan J, Li Y, Ding J. (2022). Down-regulation of cytokinin receptor gene SlHK2 improves plant tolerance to drought, heat, and combined stresses in tomato. Plants (Basel).11(2):154.doi: 10.3390/plants11020154. This page intentionally left blank CHAPTER 17 Scope of small RNA technology to develop biotic stress tolerant food crops Urvashi Mittala, Vijay Kumara, Sarvjeet Kukrejab, Baljeet Singha, c and Umesh Goutama a School of Bioengineering and Biosciences, Lovely Professional University, Phagwara, Punjab, India; School of Agriculture, Lovely Professional University, Phagwara, Punjab, India; cICAR-Central Potato Research Institute, Shimla, Himachal Pradesh, India b 1. Introduction The growing world population and livestock necessitates a significant increase in food and fodder output. According to the UN Population Division, the world’s population will reach 8.3 billion by 2030 (Chaudhary et al., 2021). The scientific community’s critical mission in the 21st century is to supply higher quality food and feed to the ever-growing population. Moreover, climatological circumstances have a negative impact on agricultural output all over the world and are the leading source of abiotic and biotic stressors, resulting in a global crop output loss of more than 50% each year (Singhal et al., 2016). Biotic factors whether bacteria, viruses, nematodes, oomycetes, or any other pathogen, they all pose a serious threat to crop production and transportation, putting food security at risk. For hundreds of years, traditional crop breeding has been used to establish highyielding crop varieties, and great progress has been achieved in using genetic variants found in germplasm resources to build crops with desired agronomical qualities. Although, traditional breeding procedures are timeconsuming and inconvenient due to the extended generation period and self-crossing of crops. Genetic engineering is another approach that is now being used throughout the world to increase crop yields by developing stress- and disease-resistant crop types. But, the so-called pleiotropic effect, which occurs when a single characteristic is regulated by many genes or vice versa, makes agronomical features genetically complicated. As a result, enhancing one characteristic quality through genetic engineering can often have unintended consequences for other key qualities. Therefore, genetic modulators that function precisely and target in a specific manner are Plant Small RNA in Food Crops ISBN 978-0-323-91722-3 https://doi.org/10.1016/B978-0-323-91722-3.00008-7 © 2023 Elsevier Inc. All rights reserved. 545 546 Plant Small RNA in Food Crops needed to manipulate agronomical features to boost crop yield (Roberts & Mattoo, 2018). Plants do have some of their own decent mechanisms to tolerate stress caused by biotic stressors. They have the ability to recognize pathogenassociated molecular patterns (PAMPs) and host danger-associated molecular patterns (DAMPs). Both PAMPs and DAMPs induce PAMP-triggered immunity (PTI) which involves the expression of pathogenesis-related (PR) genes, deposition of callose, reactive oxygen species (ROS) production and accumulation of salicylic acid (SA) (Jwa & Hwang, 2017; Withers & Dong, 2017). But pathogens also have evolved mechanisms to suppress the PTI and climaxing by triggering effector-triggered susceptibility (ETS) (de Wit, 2016; Schuebel et al., 2016; Gouveia et al., 2017). Plants initiate effector-triggered immunity (ETI), also known as their secondary immune response, counteracting pathogen ETS. Plants also have certain novel and more effective proteins that, if nothing else works, lead to the disruption of the infected cell by limiting pathogen growth. These novel proteins are known as R-proteins or Hypersensitive Response (HR) proteins, and they boost the plant defense system (Bashir et al., 2013; Kushalappa et al., 2016). All the products of HR proteins are produced via domains such as: serine/ threonine kinase domain, Toll/interleukin-1 receptor-like domains, nucleotide-binding domains/sites, and motifs like, leucine-rich repeats, leucine zippers. Upon catching a signal, receptors of R-gene are able to trigger a signal transduction cascade, that includes protein phosphorylation, ion flux control, and ROS, among other signaling activities. Plant immune responses are strictly regulated by immunity-associated factors; like transcription factors and small RNA (sRNA). In the recent past, sRNA technology have emerged as a novel solution to develop biotic stress resistant food crops. sRNA are non-coding 20e30 nucleotide (nt) long molecules that are commonly present in eukaryotic organisms and are involved in gene silencing (Holoch & Moazed, 2015; Itaya et al., 2008) (Fig. 17.1). MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) are the two main types of plant sRNAs. Most miRNAs have a length of 21e24 nt and are made up of RNAs with poorly base-paired hairpin structures, whereas siRNAs are made up of precisely complementary long dsRNAs (Chen, 2009; Xie et al., 2004). Post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS) are considered to be the primary signaling pathways involved in the production of small-RNAs that shield plants from the variety of stressors. Long siRNAs (lsiRNAs), heterochromatic siRNAs (hc-siRNAs), trans-acting siRNAs (ta-siRNAs), 547 Scope of small RNA technology to develop biotic stress tolerant food crops If partially matching mRNA then inhibition at transcription level. If perfectly matching mRNA then inhibition at a genetic level. D C L1 + H Y L1 Cytosol Nucleu s Loaded onto AGO protein and form RISC complex ss-miRNA pre-miRNA 3 Dicer (DCL) 3 Figure 17.1 Showing a general mechanism of gene silencing. natural antisense transcript-derived siRNAs (nat-siRNAs), cis-acting siRNAs (cis-siRNAs) and coding derived sRNAs are plant associated siRNAs (Fig. 17.2). Dicer like proteins (DCLs) are endonucleases which are one of the major proteins to be involved in biogenesis of sRNA. DCLs have been found to be responsible for the generation of serrate (SE), hyponastic leaves (HYL1), sRNAs and last Hua enhancer protein1 (HEN1) (Holoch & Moazed, 2015; Katiyar-Agarwal & Jin, 2010; Rogers and Chen, 2013). Other proteins like RDRs and SGS3 are required for the Doublestranded RNA (dsRNA) HAIRPINRNA (hpRNA) Precursors of Plant small RNAs Natural antisense transcript siRNAs Heterochrom atin derived siRNAs MicroRNAs Transacting siRNAs Cisacting siRNAs Exon/codin g transcript derived siRNAs Figure 17.2 Classification of plant-siRNAs. Microisoforms RNAs (isomiRs) 548 Plant Small RNA in Food Crops amplification of certain siRNAs (Sijen et al., 2007). Regulation of gene expression of plants under several stressors like: water, bacterial, salt, viral, temperature, nematode infections includes TGS and PTGS as a response pathway. Afterward the generation of pre-miRNA/sRNA and amplification, pre-miRNA/sRNA are put onto Argonaute proteins and then elimination of passenger strand occurs. In mammals, the passenger strand is eliminated by an energy-dependent process by utilizing ATP (Liu and Paroo 2010), however, in plants, no such process is known yet (Huang et al., 2016). The guide strands of mature-RNA-induced silencing complexes (RISCs) anneal to their complementary sequence and regulate gene expression at the transcriptional and post-transcriptional level via DNA methylation (Covarrubias and Reyes, 2010), chromatin remodeling (de Alba et al., 2013), mRNA slicing (Huang et al., 2016), mRNA destruction or translational inhibition (Ghildiyal & Zamore, 2009; Liu & Paroo, 2010; Zhang et al., 2011). 2. Effect of biotic stress on food crops Biotic stress is a circumstance in which the natural development of plant is hampered by harmful pathogens. These pathogens typically grow on or even inside plant tissues, causing symptoms such as lesions, stunting, rotting, and chlorosis (Sapre et al., 2021). Plants face a variety of biotic stressors like fungus, bacterium, phytoplasma, virus, viroid, and nematode. As per the reports of Wang et al. (2013), biotic stressors cause major loss in the yield of cotton, maize, rice, wheat, potatoes, and soybeans crops by approximately, 28.8%, 31.2%, 37.4%, 28.2%, 40.3% and 26.3%, respectively. Different pathogens have different effects on food crops (Table 17.1), such as Tomato mosaic virus (ToMV), Tomato yellow leaf curl virus (TYLCV) both can cause devastating yield losses in tomato. TYLCV is more disastrous to tomatoes, which is spread by Bemisia tabaci (white fly) and is responsible for causing stunted growth, yellowing, reduction and curling of leaves (Yang et al., 2021). Ralstonia solanacearum causes bacterial wilt in the food crops belonging to Solanaceous category such as potatoes, pepper, eggplant, and tomatoes. The disease caused by this bacterium is called as bacterial wilt or green wilt as the leaves of the plant appears green when the wilt symptoms start to appear (Munns & Tester, 2008). Cucumber mosaic virus (CMV) belonging to the Bromoviridae family of plant viruses and has wide range of host affecting more than 1200 species worldwide including peppers, tomatoes, celery, potatoes, etc. Symptoms associated with CMV Table 17.1 Effect of different pathogens on food crops. Biotic stressors Plant Effect 1. Snap beans 2. Cucumber Mosaic Virus (CMVs) CMV 3. CMV Tomato 4. Tomato Mosaic Virus (ToMVs) CMV Tomato Lettuce Mosaic and blistering of leaves Pods are deformed Vein clearing (loss of normal green coloration of plant veins) Shoe-string (leaf lamina and other tissues become narrow) Leaf suffers from filiformity and attains fern like shape Stunted growth Leaves are curved and deformed Can take filiform shape in winters Yellow green mottling of leaves and fruits. 6. Tobamoviruses (Tobacco and Tomato mosaic virus) Tomato and Tobacco 7. Tomato Chlorosis Virus (ToCVs) Tomato 8. Melon necrotic spot virus (MNSVs) Melon, cucumber, watermelon 5. Peppers Tomato- affected leaves shows mottling, older leaves give fern-like appearance and younger ones are twisted. Fruits are distorted and may have necrotic spots. Yellow Leaf Disorder Yellowing of the leaf occurs the size and number of the fruits reduces Local or large necrotic spots on leaves. Necrosis on stems and leaves References Li et al. (2020) Jiao & Peng (2018) Yang et al. (2021) Huseynova et al. (2014), Lin et al. (2003) Huseynova et al. (2014) Huseynova et al. (2014), Wintermantel et al. (2005) Huseynova et al. (2014), Sela et al. (2013) 549 Continued Scope of small RNA technology to develop biotic stress tolerant food crops S.No. Plant Effect References 9. Tomato yellow leaf curl virus (TYLCVs e geminivirus) Tomato (Lycopersicon esculentum). Huseynova et al. (2014), Sade et al. (2012) 10. Faba Bean Necrotic Yellow Virus (FBNYVs) Xanthomonas axonopodis pv. Manihotis XAM (Bacterial Blight) Xanthomonas oryzae pv. Oryzae (bacterial blight) Faba-bean (Vicia faba) Cassava (Manihot esculenta) Rice At Early stage- Growth is stunted, shoots are erect, leaves are abnormally short. Plant will not produce salable fruits. At Later stage- No symptoms of flowers as such but falling of flowers is observed. Early infection can lead to necrosis, yellowing of leaves and plant death. Leaf wilting and angular spots on leaves Leads to reduction in productivity. 13. Pseudomonas plantarii (Seedling blight) Rice 14. Pseudomonas avenae and P. syringae pv. Panici (bacterial brown stripe) Sarocladium oryzae, Pseudomonas fuscovaginae and Fusarium fujikuroi (Rice sheath rot) Rice 11. 12. 15. Rice Huseynova et al. (2014) Li et al. (2018) Most common Leaves starts to appear yellow-grey in color and curved, overall entire plants starts to degenerate 50% of rice yield is affected annually. Basal chlorosis is observed and seedlings appear red-brown and severe infection can retard the root growth Seed germination is hindered Mohapatra et al. (2019) Sheath discoloration and rotting Sterility is seen in infected grains Bigirimana et al. (2015) Saha et al. (2015) Saha et al. (2015) Plant Small RNA in Food Crops Biotic stressors 550 Table 17.1 Effect of different pathogens on food crops.dcont'd S.No. 16. Wheat 18. Blumeria graminis sp. Tritici (Powdery mildew) Wheat 19. Ustilago tritici (Loose smut) Wheat 20. Verticillium dahliae causes Verticillium wilt Rhizoctonia solani Cotton 22. Cotton aphid caused by Aphis gossypii Cotton 23. Helicoverpa armigera (Cotton bollworm) Cotton 17. 21. Wheat Cotton Severely affects the wheat ears (tip of the grain-bearing plant) Causes orange to yellow pustules or uredia on leaves, necks, leaf sheaths and glumes. Infested plants mature uredia matures into telia and changes red to black color. Symptoms are visible on leaves and leaf sheaths Leaves appear pale yellow. Seen as loose mass of brown spores which replaces the tissues of plant with spike. Infected plants produce sterile kernel containing seed coat. Reduction in growth rate, affects vascular tissuses Affects the seedlings and result in seed decay, seedling root rot and girdling of seedlings. Early season- cause crinkling and cupping of leaves, stunting of seedling Mid-season- stunts the plant growth Late season- is most sensitive as the cotton lint is exposed. Damages cotton bolls Miao et al. (2021) Kayim et al. (2022) Kayim et al. (2022) Kayim et al. (2022) Erdogan (2010) Erdogan (2010) Erdogan (2010) Erdogan (2010) 551 Continued Scope of small RNA technology to develop biotic stress tolerant food crops Fusarium graminearum Fusarium head blight (FHB) Puccinia striiformis f. Sp. Tritici (Stripe rust, Stem rust, 552 Table 17.1 Effect of different pathogens on food crops.dcont'd Biotic stressors Plant Effect References 24. Mylabris pustulata (Blister bettle) Chickpea Dry root rot and collar rot of chickpea 25. Maruca vitrata (Spotted pod borer) Pigeon pea Phytophthora stem blight of pigeon pea ICAR, Status paper e Crop Protection. Division of Crops Science, ICAR, Krishi Bhawan, New Delhi, 2018 (ICAR, 2018). ICAR, Status paper e Crop Protection. Division of Crops Science, ICAR, Krishi Bhawan, New Delhi, 2018 (ICAR, 2018). Plant Small RNA in Food Crops S.No. Scope of small RNA technology to develop biotic stress tolerant food crops 553 are dependent on the age of plant as young plants are more susceptible as compared to old plants. Leaves of young plants are pale green, wrinkled and develops a chlorotic mosaic pattern while fruits show necrotic spots (Li et al., 2020). Rice seed rot and rice sheath rot is caused by a variety of Fusarium, Pythium, and other water mold species. Blights in rice seedlings are caused by fungi such as Fusarium spp., Curvularia spp., and Rhizoctonia solani. These infections are usually transmitted by seeds and have an impact on the seed germination and development. Wheat is another crop which is considered to be most vital among food crops worldwide and is affected by various soil-borne bacteria and fungus pathogens. These pathogens cause great deal of reduction in crop productivity and yield annually. Loose smut, seed rust, stem rust, powdery mildew, fusarium head blight are the most severe diseases caused by Ustilago tritici, Puccinia striiformis, Blumeria graminis, and Fusarium graminearum, respectively. Loose smut is recognized as loose mass of brown spores and the infected plants produce sterile kernel. While seed rust and stem rust produces almost same symptoms such as orange to yellow uredia (or pustules) which may change to telia (black) on plant maturity. Powdery mildew is an endemic disease-causing yield losses up to 22.5%, 35%, 62% in Egypt, Russia and Brazil, respectively. This pathogen attacks wheat at vegetative phase decreasing photosynthesis, appearance of gray colonies leaves, alteration in leaf assimilation index and increased respiration rate that ultimately affects the grain quality (Kayim et al., 2022). Overall, pathogens cause heavy reduction in the yield of food crops and threatens the world food security. Hence, solutions needs to be identified which can protect the quality, yield and productivity of plants without harming the ecosystem. One of the best solution which recently has become popular is the use of sRNA in the field of agriculture biotechnology. sRNA can be exogenous of endogenous to plant in the form of siRNA and miRNA that can targets specific mRNA, breaking it into small pieces, hence silencing the targeted gene. 3. Role of small RNA in eliminating various biotic stress 3.1 Bacterial stress Several proteins participate to initiate an effective defense response against pathogens through sRNA synthesis and activities. Endoribonucleases DICER or DICER-like (DCL) proteins play a role in the formation of sRNAs, argonautes (AGOs) play a role in sRNA-directed gene suppression, and 554 Plant Small RNA in Food Crops RDRs forms the precursors of dsDNA. miRNA393 is broadly engaged in inducing resistance to plants against various infections (Table 17.2) and stressors including salinity and water stress. At the transcriptional level, hcsiRNA directs DNA methylation and histone modification to silence transposons, palindromic repeats, and genes. Immunity modulation is also influenced by the RNA-directed DNA methylation (RdDM) pathway. RdDM-created mutants display phenotypic illness changes in response to bacterial and fungal infections. The fact that a triple mutant of the noncytosine (CG loci) methyltransferases (drm1-2/drm2-2/cmt3-11) with POL-IV subunit showed great resistance to Pseudomonas syringae pathogenicity supports this hypothesis (Dowen et al., 2012; Matzke & Mosher, 2014; López Sánchez et al., 2016). The levels of miRNA160 and miRNA167 (Table 17.2) are elevated when Pst DC3000 hrcC strain of P. syringae infects the plants. They target the ARF (Auxin response factor) family to promote the antibacterial plant response. Flg22 is a flagellin protein having highly conserved 22 amino acids from the bacterial flagellin protein, first identified in mutants of Arabidopsis. The flg 22 therapy is the treatment in which flg 22 binds with flagellin sensing 2 receptor kinase (FLS2) and induces the ROS and ethylene production in tomatoes. Additionally, other receptor kinases were also identified in tomatoes such as FLS3 which binds with another epitope of flagellin, such as flgII-28, and leads to the production of ROS, opens up Ca2þ channels, which in turn, open upon several anion channels (aquaporin) and finally leads to stomatal closure (Czekus et al., 2021). Further research revealed that flg22 therapy induces miRNA160a and fifteen additional miRNAs. miRNA398b, miRNA773, and 9 additional miRNAs, on the other hand, are not induced via flg22 therapy (Li et al., 2010). High-expression of miRNA398b and miRNA773 reduces PTI via suppressing the accumulation of callose induced by bacteria or by inhibiting the flg22 protein, indicating that miRNAs have key role in resistance against several diseases. Although the over-expression of miRNA160 leads to enhancement in deposition of callose induced by PAMP, but it showed no effect on plant defense against Pst DC3000 bacteria, which indicates a complex network of regulation of miRNAs in disease response pathway of plants (Navarro et al., 2006). The complementary strand of miRNA393, miRNA393b, is also uploaded onto AGO2 which helps in downregulating the MEMB12. Downregulation of MEMB12, which is a SNARE protein found in Golgi apparatus, leading to enhanced exocytosis of PR-1, improves plant tolerance. Therefore, two sRNAs derived from Table 17.2 Several sRNAs and their role in plant defense in food crops. Small RNA Plant Pathogen Role References 1. miRNA393 Oryza sativa Pseudomonas syringae 2. miRNA408 Triticum aestivum Bian et al. (2012), Xia et al. (2012) Feng et al. (2013) 3. miRNA7695, 169a, 172a, and 398b miRNA167, 171, 444, 408 and 1138 miRNA5300 Oryza sativa Puccinia striiformis f.sp. Tritici (Wheat stripe rust) M. oryzae Suppress auxin signaling Promote Salicylic acid consumption Promote resistance toward bacteria. Targets plantacyanin-related proteins Negatively mediates TaCLP1 Li et al. (2014b) 4. 5. 6. miRNA159 and miR166 7. miRNA1023 8. miRNA1510, 393, 1507, and 2109 Triticum aestivum Blumeria graminis Overexpression decreases fungal growth Enhances hydrogen peroxide accumulation and genes associated with defense. Involved in PTI Solanum lycopersicum Gossypium hirsutum F. oxysporum Fungi decreases the expression of the miRNA Verticillium dahlia Silences both virulence genes HiC-15 (isotrichodermin C-15 hydroxylase) and Clp-1 (Ca2þ dependent cysteine protease) Suppress the Alpha/Beta Hydrolase gene (FGSG_03101) Mediates defenses responses in the plant Enhances the Glyma.16G135500 gene associated with plant diseases resistant pathway. Triticum aestivum Glycine max Fusarium graminearum Phytophthora sojae Gupta et al. (2012) Ouyang et al. (2014) Zhang et al. (2016) Jiao & Peng (2018) Wong et al. (2014) 555 Continued Scope of small RNA technology to develop biotic stress tolerant food crops S. No. 556 Table 17.2 Several sRNAs and their role in plant defense in food crops.dcont'd Small RNA Plant Pathogen Role References 9. miRNA160 Phytophthora. Infestans Plays role in systemic acquired resistance (SAR) pathway Natarajan et al. (2018) 10. miRNA477 Verticillium dahlia Increase the resistance against the fungi. 11. miRNA6019 and miRNA6020 Solanum chacoense and Solanum tuberosum Gossypium hirsutum Tobacco Tobacco Mosaic Virus Provide resistance Hu et al. (2020) Li et al. (2012b) Plant Small RNA in Food Crops S. No. Scope of small RNA technology to develop biotic stress tolerant food crops 557 the similar s RNA duplex, miRNA393 binds with AGO1 and miRNA393b binds with AGO2, to boost up the immunity of plants (Navarro et al., 2006; Zhang et al., 2011). Other noteworthy result concerning miRNA in bacterial defense is that one miRNA is enough to nullify both positive and negative regulators of immunity, although, it depends upon the time and intensity of defense responses. During early infection, miRNA863e3p promotes plant defense by inhibiting a typical receptorlike pseudokinase1, ARLPK1 and ARLPK2, but later infection adversely impacts defense by silencing SE (serrate) gene (Niu et al., 2016). Pathogens releases effectors into the plants to counter plant mediated PTI, however plants activates ETI in response to pathogenic effectors. The ETI reaction is tightly controlled by siRNAs and miRNAs because it is sturdy and generally induces a hypersensitivity response (HR). Pst DC3000 effector protein AvrRpt2-induced siRNA nat-siRNAATGB2 inhibits the production of the pentatricopeptide repeats (PPR) protein-like gene (PPRL) and prevents the bad influence of PPRL’s on the plant pathway of resistant which is controlled via RPS2, which recognizes effector AvrRpt2 (Katiyar-Agarwal et al., 2006). AtlsiRNA-1, produced by AvrRpt2, promotes resistance against disease by reducing the activity of AtRAP, a down-regulator of plant disease resistance. After a deep sequencing, it has been found out that there are several miRNAs (>20 microRNAs) and nat-siRNAs that accumulated upon the ETI mechanism. And these miRNAs target the hormones which are responsible for biogenesis pathways and signaling pathways responsible for plant resistance. R-proteins are yet another proteins which are utilized by plants, such as: RPP4 and SNC1, localized in the RPP5 locus, to shield themselves from infections caused by bacteria and fungus (Baldrich et al., 2014). There was a report which also showed that the sRNA negatively regulated the R proteins, SNC1 gene was upregulated in mutants dcl4 and ago1. This happens because sometimes pathogen interferes with the normal functioning of sRNA mediated R-proteins activation/inhibition and pathogen itself only activates R-proteins. However, it has also been reported that sRNAs complementary to SNC1 gene were not amplified suggesting that there are other sRNA which are contributing in the enhancement of SNC1 gene. This was proved when infection with Pst DC3000 caused decrease in miRNA842 accumulation, however, this was not the same case when host was infected with Pst DC3000 hrcC- strain (Shivaprasad et al., 2012; Yi & Richards, 2007). Other studies indicated that miRNA482 targets the mRNAs of CC-NBS-LRR (58 coiled coil-nucleotide binding site-leucine 558 Plant Small RNA in Food Crops rich repeats). Meanwhile, siRNA synthesis has been stimulated via an RDR6-processing pathway, which might be used to attack different mRNAs of proteins involved in the plant defense mechanism. Therefore, when bacteria or virus infects, miRNA482 accumulation decrease to inhibit miRNA482-controlled pathway and consequently upregulate the level of mRNAs associated with defense mechanism (Shivaprasad et al., 2012). Studies conducted on cotton described that miRNA482 can attack 10% genes of NBS-LRR and also activates the secondary siRNAs. Verticillium dahliae, a fungi infecting cotton, decreases accumulation of miRNA482 and upregulates expression of NBS-LRR (Zhu et al., 2013). Elicitors are nothing but the compounds which are responsible for activating a signaling cascade in response to some pathogen attack. These compounds are very diverse with little to no similarity with each other, except they all trigger hypersensitive responses in plants (Patel et al., 2020). Elicitors have ability to down-regulate miRNAs, and the same was observed in rice infected with Magnaportbe oryzae. The expression of miRNA529/1879 was lowered but the genes associated with oxidative stress were up-regulated (Baldrich et al., 2015). Furthermore, elicitor treatment negatively altered the accumulation of miRNA393b/ miRNA156 in rice infected with M. oryzae (Campo et al., 2013). OsamiRNA1871 plays a key role in plant resistance as its over-expression leads to reduced resistance against M. oryzae and decreases the yield. A mutant MIM1871 that blocked the Osa-miRNA1871 unexpectedly enhanced the resistance against M. oryzae, increased the panicle number and enhanced the yield in mutants (Holoch & Moazed, 2015; Li et al., 2021; Xin et al., 2010). Hence, blocking miRNA1871 increases the PTI. From all of these studies, it can be clearly stated that plant sRNAs are clearly important in controlling the roles of genes involved in plant defense pathway (Li et al., 2012a; Zhai et al., 2011). Nevertheless, each and every sRNA have their own role in plant defense and immunity pathways (Shivaprasad et al., 2012) and their functions and expressions are dependent upon the pathogens (Liu et al., 2014; Zhu et al., 2013). As a result, more sRNAs’ functions must be explored in order to determine the systematic involvement of RNA silencing in plant resistance (Fei et al., 2015). 4. Viral stress Viral pathogens invade plant cells and use the host machinery to replicate their DNA/RNA. PTGS was firstly observed in transgenic processing and Scope of small RNA technology to develop biotic stress tolerant food crops 559 infection with the Potato virus X (PVX). The identification of complimentary sRNAs toward transcript of sense transgene and the PVX positive strand shows that sRNAs are involved in viral defense and PTGS transgene silencing (Hamilton & Baulcombe, 1999). More studies suggested that the dsRNAs are produced during the replication of viruses and viroids, as well as the folding of their RNA genomes and transcripts, which attracts gene-silencing pathway (Ding, 2009). Viruses have doublestranded DNA (dsDNA), double-stranded RNA (dsRNA), singlestranded DNA (ssDNA), and single-stranded RNA (ssRNA) (Ding & Voinnet, 2007). There are many viruses such as CuYV (Cucumber yellow virus), TuMV (turnip mosaic virus), CMV (cytomegalovirus), WMV (watermelon mosaic virus), PVX (potato virus X), TYLCV (tomato yellow leaf curl virus) belonging to positive ssRNA family (Yoo et al., 2004) which can yield almost equal numbers of positive and negative strand vsiRNAs with no positional bias (Ho et al., 2006). At the time of replication of viral genome single stranded RNA, a complementary strand of RNA is generated, resulting in lengthy double stranded RNA containing the original strand of viral genome (Donaire et al., 2009). Host RNA silencing machines can target the dsRNA replicative intermediate forms of ssRNA viruses as well as the dsRNA genomes of dsRNA viruses. Host RDRs boost both anti-viral and anti-viroid signals, for instance, RDR1 is induced in tobacco and Arabidopsis following the SA and TMV infection (Simon-Loriere & Holmes, 2011), and, RDR6 mutants are vulnerable to both ssRNA and ssDNA viruses (Willmann et al., 2011). To prove this point, rice was infected with RSV and RDV (ssRNA and dsDNA) and as expected, RDR6 expression was lost. Further, it was observed that the antisense transformation of OsRDR6 made it highly susceptible to RDV (Hong et al., 2015). Apple stem grooving virus (ASGV), is yet another devastating virus which was studied by using short RNA sequencing, and it was shown that vsiRNAs are generated during infection. ASGV infections modify expression of sRNAs produced from tRNA, while microRNAs, natural-antisense transcript siRNA, phasedsiRNA, and repeat-associated siRNA levels remain unaltered (Viser et al., 2014). In Piper nigrum L., recognition and cloning of tRNA-derived short RNAs as a response to a fast wild disease (Phytophthora capsici) was also accomplished (Asha & Soniya, 2016). Their findings revealed the existence of short RNAs which are derived from t-RNA in P. capsici during infections, as well as their regulatory functions. Under pathogen 560 Plant Small RNA in Food Crops stress, sRNAs produced from mature tRNAs (50 tRFs) 50 -end are abundantly expressed. 50 AlaCGCtRFs and 50 MetCATtRF-mRNAs were discovered because they target mRNAs involved in defensive response, such as NPR1 and ubiquitin ligase. These mRNAs are important for pathogenesis-related protein activation signaling processes, which are important in plant pathogen interactions. Rice stripe virus (RSV) infection causes miRNA-444 to be produced, which reduces the repressive effects of MADS box genes on RDR1 transcription, hence activating the RDR1-dependent antiviral silencing mechanism (Diao et al., 2019). Furthermore, rice plants which are transgenic bearing OsRDR6 knockdown gene have found to be more susceptible to RSV, with more severe complications, demonstrating that R-genes are required for plant survival. Another sRNA, miRNA-403a, controls the expression of the AGO2 gene, which is involved in Nicotiana benthamiana for defense against the tomato mosaic virus (ToMV) (Wang et al., 2016). The RNA silencing suppressor (RSS) of the lettuce necrotic yellow virus (LNYV), phospo-P protein, interacts with the proteins associated with gene silencing AGO1, AGO2, AGO4, RDR6, and SGS3. RDR6/SGS3-dependent amplification of silencing, translational repression, and miRNA-guided AGO1 cleavage are all suppressed by LNYV-P (lettuce necrotic yellow virusphosphoprotein) (Mann et al., 2016). To prevent cleavage, tomato chlorosis virus P22 RNA silencing suppressors prefer to bind long dsRNAs for silencing suppression (Landeo-Rios et al., 2016). Silva et al. (2011) published the first report on the sRNA profiling from plants infected with a virus from the Luteoviridae family, which have not been explored much. Dicer-like (DCL) ribonucleases generated viral DNA after infection. sRNAs derived from genomes of viruses are utilized like guides for the purpose of silencing its genome (Silva et al., 2011). The cotton leafroll dwarf virus (CLRDV) agent, was transferred to plants by Aphis gossypii (aphid vector). By utilizing an in-silico method, it was hypothesized that cotton leaf curl Allahabad virus (CLCuAV) genes can be attacked by the miRNAs present in cotton, allowing host repression and virus proliferation. Cotton miRNA-2950 could attack viral genome, whilst miRNA408 aims were interwoven transcripts of the AC1 and AC2 genes in CLCuAV, that code for replication-associated protein (Rep) and transcriptional activator protein, respectively. There was also the discovery of a collection of miRNAs (miRNA-394, 395a, and 395d) having complementary sites on the transcripts of the AC1 and AC4 genes (Shweta, 2014). Romanel and his group became the first to investigate Scope of small RNA technology to develop biotic stress tolerant food crops 561 regional modifications of sRNAs in virus-infected cotton plants using sRNAs in cotton leaves infected with Cotton leafroll dwarf virus (CLRDV). Their analysis found that expression of 60 potential conserved miRNA were altered on viral infection, out of which 19 belonged to novel miRNA families. These alterations in miRNA expression might be responsible for the viral induced pathogenicity in plants. They also discovered that 24 nucleotide long heterochromatin-associated siRNAs in the infected plant were altered quantitatively and qualitatively, resulting in the reactivation of at least one cotton transposable element (Romanel et al., 2012). Pathogen effectors-triggered immunity is a kind of hypersensitive disease resistance induced by plant R proteins that detect viral proteins (ETI) (Moffett, 2009; Sanseverino & Raffaella Ercolano, 2012). R genes have low expression levels in the absence of viral infection for appropriate development and basal immunity. Brassica miRNA-1885, also known as bramiR1885, is the first plant miRNA to target a R gene for cleavage. Since then, other miRNAs have been discovered or projected to target R genes that give viral resistance (Holoch & Moazed, 2015). The cleavage of the R genes that give resistance to tobacco mosaic virus is guided by two tobacco miRNAs, nta-miRNA6019 (22 nt) and nta-miRNA-6020 (21 nt) (TMV) (Table 17.2) (Li et al., 2012a). The synthesis of 21-nt phased secondary siRNAs is triggered by cleavage of the R mRNA targeted by nta-miRNA6019 (phasiRNAs). Another collection of miRNAs, miRNA1507a, miRNA-1507c, and miRNA-482a, is elevated by SMV infection in the susceptible soybean, and is anticipated to influence the expression of multiple disease resistance genes in the NBSLRR family (Bao et al., 2018). Methylation of chromatin is important for preventing viral replication. The lack of methylation in host plant genomes renders the plant more vulnerable to infections by viruses such as ss-DNA geminiviruses. Although geminivirus, like most other viruses, is susceptible to RNA silencing, it creates inhibitory proteins such as AL2 and L2 to evade this protection. These proteins interact with adenosine kinase (ADK), an essential co-factor of methyl-transferase. The viral genome is targeted by sRNA-directed methylation. In plants with Cytosin or histone H3 lysin 9 (H3K9) methyltransferase defects, RNA-mediated methylation pathway components or ADK-encoding genes show increased susceptibility to geminivirus (Raja et al., 2008). For sRNA production, DCLs collaborate with DRBs. For methylation-mediated antiviral defense, the DRB3 protein collaborates with DCL3 and AGO4. (Raja et al., 2014). 562 Plant Small RNA in Food Crops 5. Fungal stress Plant immunity is a complex and tightly controlled mechanism. Plant immunity sRNAs are better known for detecting the genes involved in fungal pathogen response, thanks to improved novel techniques for plant disease tolerance. Plant sRNA has been more well-known in the last decade as a potential target for fungal diseases (Baldrich et al., 2014; Chen, 2009; Hussain et al., 2018; Song et al., 2018). Especially methods like microarray and sequencing aimed at identifying sRNAs whose expression is influenced by fungal elicitors became more popular (Kohli et al., 2014; Shen et al., 2014). In the fungus Neurospora crassa, both DCL-independent and DCLdependent small interfering-RNA synthesis processes have been discovered. Furthermore, according to Lee et al. (2010), the manufacture of micro-RNA-like short RNAs (milRNAs) in N. crassa has been found to include union of several proteins, involving an RNAse III domaincontaining protein MRPL3, the exonuclease QIP, QDE-2, and Dicers. The powdery mildew fungus Puccinia graminis f. sp. Tritici (Bgt) causes catastrophic diseases in wheat, barley, and other plants. miRNA159,164,167,171,444,408,1129, and 1138 (Table 17.2), which control three separate defensive response mechanisms, are all considerably elevated during the early stage of Bgt infection, but not at the late stage. As a result, these miRNAs might play an important part in HR during the onset of illness (Gupta et al., 2012). The sRNA role in plant immune response have been shown in B. graminis f. sp. Hordei (Bgh) (powdery mildew fundus) (Liu et al., 2014). The miRNA family miRNA-9863 targets the Mildew resistance locus a (Mla), which encodes the set of CC-NBS-LRR proteins which react towards Bgh. In N. benthamiana expression system, miRNA9863 was observed to perform the division of MLA-1 transcripts and to suppress the aggregation of MLA-1 protein. Furthermore, miRNA9863 promote the synthesis of 21 long nucleotide phased siRNAs (phasiRNAs) and suppress Mla-1 expression. Mla1-mediated cell death and disease resistance are reduced when miRNA9863 is overexpressed. miRNAs with varied expression patterns play significant roles against various fungal infections, according to computational analyses based on EST sequence information (Hussain et al., 2018). They looked at the role of microRNAs in Cajanus cajan’s (pigeon pea) defensive responses against Fusarium wilt. Using EST data from C. cajan, they discovered five new miRNAs and their targets from the miRNA169i-3p, Scope of small RNA technology to develop biotic stress tolerant food crops 563 1214, 3695, 9666b-3p, and 8182 families. These miRNAs have been shown to have different patterns of expression toward inoculation of Fusarium as a biotic-stress response. Botrytis and Verticillium spp. are aggressive fungal diseases that cause major crop losses all over the world. According to latest findings, Botrytis cinerea transports short RNAs (Bc-sRNAs) into plant cells to quiet host immunity genes. Dicer-like protein 1 (Bc-DCL1) and Bc-DCL2 of B. cinerea are the most common sRNA effectors. When sRNAs targeting Bc-DCL1 and Bc-DCL2 are expressed in tomato, Bc-DCL genes are silenced, and fungal pathogenicity and growth are reduced, suggesting bilateral cross-kingdom of RNAi and sRNA transport across fungal organisms and plants. This method can be used to combat numerous fungal infections at the same time. External sRNAs and dsRNAs can also be absorbed via Botrytis (dsRNAs). It was reported that incidence of gray mold disease is greatly reduced when the surface of vegetables, flowers and fruits were applied with ssRNAs or dsRNAs which targets the DCL1 and DCL2 genes of Botrytis (Wang et al., 2016; Weiberg et al., 2013). Sclerotinia sclerotiorum infests about 600 species of plants, yet there is no to little information about the genes involved in infecting the host plant. sRNAs generated by S. sclerotiorum were sequenced in culture as well as during invasion of two host species, Arabidopsis thaliana and Phaseolus vulgaris. During infection, S. sclerotiorum creates at least 374 different highabundance sRNAs, the majority of which originate from repeat-rich plastic genomic regions. The soil-borne fungus disease Verticillium wilt (Verticillium dahliae Kleb) causes wilting, yellowing and finally mortality in cotton. Yin and his colleagues were the first to notice differences among G. barbadense L., Hai-7124 (Verticillium resistant) and G. hirsutum L., Yi11 (susceptible) in their miRNA expression levels. They discovered 215 miRNA groups having altered patterns of expression between verticillium susceptible and resistant libraries of sRNA. Afterward of Verticillium inoculation, they discovered two trans-acting siRNAs and hundreds of endogenous siRNA candidates. They also discovered that numerous siRNAs matched exactly with retrotransposons, suggesting, retrotransposons might have a part in synthesis of endogenous plant siRNAs (Yin & et al., 2007). Another study that looked for miRNAs and their target genes found 140 recognized and 58 new miRNAs in the roots of cotton plants infested by Verticillium dahlia (Zhang et al., 2015). Another group recently found 37 new mi-RNAs by analyzing complementaryDNA libraries of two sRNA derived by upland variety KV-1 (resistant 564 Plant Small RNA in Food Crops to Verticillium) following inoculation with this disease in order to better understand the modulation of genes related to resistance (Holoch & Moazed, 2015). 24 new mi-RNAs could target 49 mRNAs/genes responsible for the production of secondary metabolite, mitogenactivated protein kinase (MAPK) pathway, and plant-pathogen interactions. Few new miRNAs and selected genes discovered in this work have been linked to the promotion of Verticillium wilt resistance (Holoch & Moazed, 2015). 6. Conclusion This complex “arms race” is never be going to stop between plants and pathogens, which is, obviously, an integral part of evolution. Studies have confirmed the role of various regulatory proteins and sRNAs but it won’t be too long when pathogens find new and improved ways to counteract plant strategies. New studies will help us to find out more about the working of gene silencing and also about the ‘how’ to manipulate these sRNAs against several types of stressors which can help researchers and breeders to develop stress-tolerant crops. After all, the one who can fight in the more improvised way will survive more as it is already been said it is indeed the “Survival of the fittest”. References de Alba, A. E. M., Elvira-Matelot, E., & Vaucheret, H. (2013). Gene silencing in plants: A diversity of pathways. Biochimica et Biophysica Acta (BBA)-Gene Regulatory Mechanisms, 1829(12), 1300e1308. Asha, S., & Soniya, E. V. (2016). Transfer RNA derived small RNAs targeting defense responsive genes are induced during Phytophthora capsici infection in black pepper (Piper nigrum L.). Frontiers of Plant Science, 7, 767. Baldrich, P., et al. (2015). MicroRNA-mediated regulation of gene expression in the response of rice plants to fungal elicitors. RNA Biology, 12(8), 847e863. Baldrich, P., Kakar, K., Siré, C., Moreno, A. B., Berger, A., García-Chapa, M., & San Segundo, B. (2014). Small RNA profiling reveals regulation of Arabidopsis miR168 and heterochromatic siRNA415 in response to fungal elicitors. BMC genomics, 15(1), 1e16. Bao, D., et al. (2018). Down-regulation of genes coding for core RNAi components and disease resistance proteins via corresponding microRNAs might be correlated with successful Soybean mosaic virus infection in soybean. Molecular Plant Pathology, 19(4), 948e960. Bashir, Z., et al. (2013). Hypersensitive responseda biophysical phenomenon of producers. European Journal of Microbiology and Immunology, 3(2), 105e110. Bian, H., et al. (2012). Distinctive expression patterns and roles of the miRNA393/TIR1 homolog module in regulating flag leaf inclination and primary and crown root growth in rice (Oryza sativa). New Phytologist, 196(1), 149e161. Scope of small RNA technology to develop biotic stress tolerant food crops 565 Bigirimana, V. D. P., Hua, G. K., Nyamangyoku, O. I., & Höfte, M. (2015). Rice sheath rot: an emerging ubiquitous destructive disease complex. Frontiers in Plant Science, 6, 1066. Campo, S., Peris-Peris, C., Siré, C., Moreno, A. B., Donaire, L., Zytnicki, M., & San Segundo, B. (2013). Identification of a novel micro RNA (mi RNA) from rice that targets an alternatively spliced transcript of the N ramp6 (Natural resistance-associated macrophage protein 6) gene involved in pathogen resistance. New Phytologist, 199(1), 212e227. Chaudhary, S., Grover, A., & Sharma, P. C. (2021). MicroRNAs: Potential targets for developing stress-tolerant crops. Life, 11(4), 289. Chen, X. (2009). Small RNAs and their roles in plant development. Annual Review of Cell and Developmental Biology, 25, 21e44. Chen, L., Luan, Y., & Zhai, J. (2015). Sp-miR396a-5p acts as a stress-responsive genes regulator by conferring tolerance to abiotic stresses and susceptibility to Phytophthora nicotianae infection in transgenic tobacco. Plant Cell Reports, 34(12), 2013e2025. Covarrubias, A. A., & Reyes, J. L. (2010). Post-transcriptional gene regulation of salinity and drought responses by plant microRNAs. Plant, Cell and Environment, 33(4), 481e489. Czékus, Z., et al. (2021). Activation of local and systemic defence responses by Flg22 is dependent on daytime and ethylene in intact tomato plants. International Journal of Molecular Sciences, 22(15), 8354. Diao, P., et al. (2019). miR403a and SA are involved in NbAGO2 mediated antiviral defenses against TMV infection in Nicotiana benthamiana. Genes, 10(7), 526. Ding, B. (2009). The biology of viroid-host interactions. Annual Review of Phytopathology, 47, 105e131. Ding, S. W., & Voinnet, O. (2007). Antiviral immunity directed by small RNAs. Cell, 130(3), 413e426. Donaire, L., et al. (2009). Deep-sequencing of plant viral small RNAs reveals effective and widespread targeting of viral genomes. Virology, 392(2), 203e214. Dowen, R. H., et al. (2012). Widespread dynamic DNA methylation in response to biotic stress. Proceedings of the National Academy of Sciences, 109(32), E2183eE2191. Erdo
gan, O. (2010). Cotton diseases, insects and control. International Workshop on Sustainable Cotton Production. Fei, Q., et al. (2015). Secondary si RNA s from Medicago NB-LRR s modulated via mi RNAetarget interactions and their abundances. The Plant Journal, 83(3), 451e465. Feng, H., et al. (2013). Target of tae-miR408, a chemocyanin-like protein gene (TaCLP1), plays positive roles in wheat response to high-salinity, heavy cupric stress and stripe rust. Plant Molecular Biology, 83(4), 433e443. Ghildiyal, M., & Zamore, P. D. (2009). Small silencing RNAs: An expanding universe. Nature Reviews Genetics, 10(2), 94e108. Gouveia, B. C., et al. (2017). Immune receptors and co-receptors in antiviral innate immunity in plants. Frontiers in Microbiology, 7, 2139. Gupta, O. P., et al. (2012). MicroRNA regulated defense responses in Triticum aestivum L. during Puccinia graminis f. sp. tritici infection. Molecular Biology Reports, 39(2), 817e824. Hamilton, A. J., & Baulcombe, D. C. (1999). A species of small antisense RNA in posttranscriptional gene silencing in plants. Science, 286(5441), 950e952. Ho, T., et al. (2006). A simplified method for cloning of short interfering RNAs from Brassica juncea infected with Turnip mosaic potyvirus and Turnip crinkle carmovirus. Journal of Virological Methods, 136(1e2), 217e223. Holoch, D., & Moazed, D. (2015). RNA-mediated epigenetic regulation of gene expression. Nature Reviews Genetics, 16(2), 71e84. 566 Plant Small RNA in Food Crops Hong, W., et al. (2015). OsRDR6 plays role in host defense against double-stranded RNA virus, Rice Dwarf Phytoreovirus. Scientific Reports, 5(1), 1e11. Huang, J., et al. (2016). Diverse functions of small RNAs in different plantepathogen communications. Frontiers in Microbiology, 7, 1552. Huang, J., Yang, M., & Zhang, X. (2016). The function of small RNAs in plant biotic stress response. Journal of Integrative Plant Biology, 58(4), 312e327. Hu, G., et al. (2020). The cotton miR477-CBP60A module participates in plant defense against Verticillium dahlia. Molecular Plant-Microbe Interactions, 33(4), 624e636. Huseynova, I., et al. (2014). Biotic stress and crop improvement. In Improvement of crops in the era of climatic changes (pp. 91e120). New York, NY: Springer. Hussain, K., et al. (2018). Identification, characterization and expression analysis of pigeonpea miRNAs in response to Fusarium wilt. Gene, 653, 57e64. ICAR. (2018). Status paper e crop protection. Krishi Bhawan, New Delhi: Division of Crops Science, ICAR. Itaya, A., et al. (2008). Small RNAs in tomato fruit and leaf development. Biochimica et Biophysica Acta (BBA)-Gene Regulatory Mechanisms, 1779(2), 99e107. Jiao, J., & Peng, D. (2018). Wheat microRNA1023 suppresses invasion of Fusarium graminearum via targeting and silencing FGSG_03101. Journal of Plant Interactions, 13(1), 514e521. Jwa, N. S., & Hwang, B. K. (2017). Convergent evolution of pathogen effectors toward reactive oxygen species signaling networks in plants. Frontiers of Plant Science, 8, 1687. Katiyar-Agarwal, S., et al. (2006). A pathogen-inducible endogenous siRNA in plant immunity. Proceedings of the National Academy of Sciences, 103(47), 18002e18007. Katiyar-Agarwal, S., & Jin, H. (2010). Role of small RNAs in host-microbe interactions. Annual Review of Phytopathology, 48, 225e246. Kayim, M., Nawaz, H., & Alsalmo, A. (2022). Fungal Diseases of Wheat. Dr. Mahmood-urRahman Ansari. https://doi.org/10.5772/intechopen.102661 Kohli, D., Joshi, G., Deokar, A. A., Bhardwaj, A. R., Agarwal, M., Katiyar-Agarwal, S., … Jain, P. K. (2014). Identification and characterization of wilt and salt stress-responsive microRNAs in chickpea through high-throughput sequencing. PloS one, 9(10). Article e108851. Kushalappa, A. C., Yogendra, K. N., & Karre, S. (2016). Plant innate immune response: Qualitative and quantitative resistance. Critical Reviews in Plant Sciences, 35(1), 38e55. Landeo-Ríos, Y., et al. (2016). The p22 RNA silencing suppressor of the crinivirus Tomato chlorosis virus preferentially binds long dsRNAs preventing them from cleavage. Virology, 488, 129e136. Lee, H. C., et al. (2010). Diverse pathways generate microRNA-like RNAs and Dicerindependent small interfering RNAs in fungi. Molecular Cell, 38(6), 803e814. Li, Y., et al. (2010). Identification of microRNAs involved in pathogen-associated molecular pattern-triggered plant innate immunity. Plant Physiology, 152(4), 2222e2231. Li, Y., et al. (2014). Multiple rice microRNAs are involved in immunity against the blast fungus Magnaporthe oryzae. Plant Physiology, 164(2), 1077e1092. Li, X., et al. (2018). Identification and functional analysis of cassava DELLA proteins in plant disease resistance against cassava bacterial blight. Plant Physiology and Biochemistry, 124, 70e76. Li, G. M., et al. (2012a). Pandemic H1N1 influenza vaccine induces a recall response in humans that favors broadly cross-reactive memory B cells. Proceedings of the National Academy of Sciences, 109(23), 9047e9052. Li, Y., et al. (2021a). Blocking miR530 improves rice resistance, yield, and maturity. Frontiers of Plant Science, 12, 729560. Li, X., et al. (2012b). Identification of soybean microRNAs involved in soybean cyst nematode infection by deep sequencing. PLoS One, 7(6), e39650. Scope of small RNA technology to develop biotic stress tolerant food crops 567 Li, Y., et al. (2021b). Blocking Osa-miR1871 enhances rice resistance against Magnaporthe oryzae and yield. Plant Biotechnology Journal, 20, 646e659. Li, N., Yu, C., Yin, Y., Gao, S., Wang, F., Jiao, C., & Yao, M. (2020). Pepper crop improvement against cucumber mosaic virus (CMV): A review. Frontiers in Plant Science, 11, 598798. Lin, H. X., et al. (2003). Genetic diversity and biological variation among California isolates of Cucumber mosaic virus. Journal of General Virology, 84(1), 249e258. Liu, J., et al. (2014). The miR9863 family regulates distinct Mla alleles in barley to attenuate NLR receptor-triggered disease resistance and cell-death signaling. PLoS Genetics, 10(12), e1004755. Liu, Q., & Paroo, Z. (2010). Biochemical principles of small RNA pathways. Annual Review of Biochemistry, 79, 295e319. López Sánchez, A., et al. (2016). The role of DNA (de) methylation in immune responsiveness of Arabidopsis. The Plant Journal, 88(3), 361e374. Mann, K. S., et al. (2016). Cytorhabdovirus P protein suppresses RISC-mediated cleavage and RNA silencing amplification in planta. Virology, 490, 27e40. Matzke, M. A., & Mosher, R. A. (2014). RNA-directed DNA methylation: An epigenetic pathway of increasing complexity. Nature Reviews Genetics, 15(6), 394e408. Miao, J., et al. (2021). The orange wheat blossom midge promotes fusarium head blight disease, posing a risk to wheat production in northern China. Acta Ecologica Sinica. https://doi.org/10.1016/j.chnaes.2021.10.010 Moffett, P. (2009). Mechanisms of recognition in dominant R gene mediated resistance. Advances in Virus Research, 75, 1e229. Mohapatra, D., et al. (2019). Priming with salicylic acid induces defense against bacterial blight disease by modulating rice plant photosystem II and antioxidant enzymes activity. Physiological and Molecular Plant Pathology, 108, 101427. Munns, R., & Tester, M. (2008). Mechanisms of salinity tolerance. Annual Review of Plant Biology, 59, 651e681. Natarajan, B., et al. (2018). MiRNA160 is associated with local defense and systemic acquired resistance against Phytophthora infestans infection in potato. Journal of Experimental Botany, 69(8), 2023e2036. Navarro, L., et al. (2006). A plant miRNA contributes to antibacterial resistance by repressing auxin signaling. Science, 312(5772), 436e439. Niu, D., Lii, Y. E., Chellappan, P., Lei, L., Peralta, K., Jiang, C., & Jin, H. (2016). miRNA863-3p sequentially targets negative immune regulator ARLPKs and positive regulator SERRATE upon bacterial infection. Nature Communications, 7(1), 1e13. Ouyang, S., et al. (2014). MicroRNAs suppress NB domain genes in tomato that confer resistance to Fusarium oxysporum. PLoS Pathogens, 10(10), e1004464. Patel, Z. M., Mahapatra, R., & Jampala, S. S. M. (2020). Role of fungal elicitors in plant defense mechanism. In Molecular aspects of plant beneficial microbes in agriculture (pp. 143e158). Academic Press. Raja, P., et al. (2008). Viral genome methylation as an epigenetic defense against geminiviruses. Journal of Virology, 82(18), 8997e9007. Raja, P., et al. (2014). Arabidopsis double-stranded RNA binding protein DRB3 participates in methylation-mediated defense against geminiviruses. Journal of Virology, 88(5), 2611e2622. Roberts, D. P., & Mattoo, A. K. (2018). Sustainable agriculturedenhancing environmental benefits, food nutritional quality and building crop resilience to abiotic and biotic stresses. Agriculture, 8(1), 8. Rogers, K., & Chen, X. (2013). Biogenesis, turnover, and mode of action of plant microRNAs. The Plant Cell Online, 25(7), 2383e2399. Romanel, E., Silva, T. F., Corrêa, R. L., Farinelli, L., Hawkins, J. S., Schrago, C. E., & Vaslin, M. F. (2012). Global alteration of microRNAs and transposon-derived small 568 Plant Small RNA in Food Crops RNAs in cotton (Gossypium hirsutum) during Cotton leafroll dwarf polerovirus (CLRDV) infection. Plant Molecular Biology, 80(4), 443e460. Sade, D., et al. (2012). A developmentally regulated lipocalin-like gene is overexpressed in Tomato yellow leaf curl virus-resistant tomato plants upon virus inoculation, and its silencing abolishes resistance. Plant Molecular Biology, 80(3), 273e287. Saha, S., Garg, R., Biswas, A., & Rai, A. B. (2015). Bacterial diseases of rice: an overview. Journal of Pure and Applied Microbiology, 9(1), 725e736. Sanseverino, W., & Raffaella Ercolano, M. (2012). In silico approach to predict candidate R proteins and to define their domain architecture. BMC Research Notes, 5(1), 1e11. Sapre, S., et al. (2021). Molecular techniques used in plant disease diagnosis. In Food security and plant disease management (pp. 405e421). Woodhead Publishing. Schuebel, F., et al. (2016). 30 -NADP and 30 -NAADP, two metabolites formed by the bacterial type III effector AvrRxo1*A. Journal of Biological Chemistry, 291(44), 22868e22880. Sela, N., et al. (2013). A new cryptic virus belonging to the family Partitiviridae was found in watermelon co-infected with Melon necrotic spot virus. Virus Genes, 47(2), 382e384. Shen, D., et al. (2014). Identification and characterization of micro RNA s in oilseed rape (Brassica napus) responsive to infection with the pathogenic fungus Verticillium longisporum using Brassica AA (Brassica rapa) and CC (Brassica oleracea) as reference genomes. New Phytologist, 204(3), 577e594. Shivaprasad, P. V., et al. (2012). A microRNA superfamily regulates nucleotide binding siteeleucine-rich repeats and other mRNAs. The Plant Cell Online, 24(3), 859e874. Shweta, J. A. K. (2014). In silico prediction of cotton (Gossypium hirsutum) encoded microRNAs targets in the genome of Cotton leaf curl Allahabad virus. Bioinformation, 10(5), 251. Sijen, T., et al. (2007). Secondary siRNAs result from unprimed RNA synthesis and form a distinct class. Science, 315(5809), 244e247. Silva, T. F., Romanel, E. A., Andrade, R. R., Farinelli, L., Østerås, M., Deluen, C., … Vaslin, M. F. (2011). Profile of small interfering RNAs from cotton plants infected with the polerovirus Cotton leafroll dwarf virus. BMC molecular biology, 12(1), 1e12. Simon-Loriere, E., & Holmes, E. C. (2011). Why do RNA viruses recombine? Nature Reviews Microbiology, 9(8), 617e626. Singhal, P., Jan, A. T., Azam, M., & Haq, Q. M. R. (2016). Plant abiotic stress: A prospective strategy of exploiting promoters as alternative to overcome the escalating burden. Frontiers in Life Science, 9(1), 52e63. Song, F., et al. (2018). Small RNA profiling reveals involvement of microRNA-mediated gene regulation in response to mycorrhizal symbiosis in Poncirus trifoliata L. Raf. Tree Genetics & Genomes, 14(3), 1e14. Visser, M., et al. (2014). High-throughput sequencing reveals small RNAs involved in ASGV infection. BMC Genomics, 15(1), 1e10. Wang, M., et al. (2013). The critical role of potassium in plant stress response. International Journal of Molecular Sciences, 14(4), 7370e7390. Wang, H., Jiao, X., Kong, X., Hamera, S., Wu, Y., Chen, X., … Yan, Y. (2016). A signaling cascade from miR444 to RDR1 in rice antiviral RNA silencing pathway. Plant Physiology, 170(4), 2365e2377. Wang, Y., Li, H., Si, Y., Zhang, H., Guo, H., & Miao, X. (2012). Microarray analysis of broad-spectrum resistance derived from an indica cultivar Rathu Heenati. Planta, 235(4), 829e840. Weiberg, A., et al. (2013). Fungal small RNAs suppress plant immunity by hijacking host RNA interference pathways. Science, 342(6154), 118e123. Scope of small RNA technology to develop biotic stress tolerant food crops 569 Willmann, M. R., et al. (2011). The functions of RNA-dependent RNA polymerases in Arabidopsis. The Arabidopsis Book/American Society of Plant Biologists, 9. Wintermantel, W. M., et al. (2005). The complete nucleotide sequence and genome organization of Tomato chlorosis virus. Archives of Virology, 150(11), 2287e2298. de Wit, P. J. (2016). Cladosporium fulvum effectors: Weapons in the arms race with tomato. Annual Review of Phytopathology, 54, 1e23. Withers, J., & Dong, X. (2017). Post-translational regulation of plant immunity. Current Opinion in Plant Biology, 38, 124e132. Wong, J., et al. (2014). Roles of small RNA s in soybean defense against P hytophthora sojae infection. The Plant Journal, 79(6), 928e940. Xia, K., et al. (2012). OsTIR1 and OsAFB2 downregulation via OsmiR393 overexpression leads to more tillers, early flowering and less tolerance to salt and drought in rice. PLoS One, 7(1), e30039. Xie, Z., et al. (2004). Genetic and functional diversification of small RNA pathways in plants. PLoS Biology, 2(5), e104. Xin, M., et al. (2010). Diverse set of microRNAs are responsive to powdery mildew infection and heat stress in wheat (Triticum aestivum L.). BMC Plant Biology, 10(1), 1e11. Yin, L., et al. (2007). Continued spread of HIV among injecting drug users in southern Sichuan Province, China. Harm Reduction Journal, 4(1), 1e7. Yin, Z., Li, Y., Han, X., & Shen, F. (2012). Genome-wide profiling of miRNAs and other small non-coding RNAs in the Verticillium dahliaeeinoculated cotton roots. PLoS One, 7(4), e35765. Yang, Z., Yang, C., Yang, Z., & Wu, Y. (2021). Long noncoding RNAs in tomato: Roles in development and stress response. In Long Noncoding RNAs in Plants (pp. 177e187). Academic Press. Yi, H., & Richards, E. J. (2007). A cluster of disease resistance genes in Arabidopsis is coordinately regulated by transcriptional activation and RNA silencing. The Plant Cell Online, 19(9), 2929e2939. Yoo, B. C., et al. (2004). A systemic small RNA signaling system in plants. The Plant Cell Online, 16(8), 1979e2000. Zhai, J., et al. (2011). MicroRNAs as master regulators of the plant NB-LRR defense gene family via the production of phased, trans-acting siRNAs. Genes & Development, 25(23), 2540e2553. Zhang, W., et al. (2011). Bacteria-responsive microRNAs regulate plant innate immunity by modulating plant hormone networks. Plant Molecular Biology, 75(1), 93e105. Zhang, X., et al. (2011). Expression of artificial microRNAs in tomato confers efficient and stable virus resistance in a cell-autonomous manner. Transgenic Research, 20(3), 569e581. Zhang, C., et al. (2016a). Suppression of jasmonic acid-mediated defense by viral-inducible microRNA319 facilitates virus infection in rice. Molecular Plant, 9(9), 1302e1314. Zhang, T., et al. (2016b). Cotton plants export microRNAs to inhibit virulence gene expression in a fungal pathogen. Nature plants, 2(10), 1e6. Zhang, Y., Wang, W., Chen, J., Liu, J., Xia, M., & Shen, F. (2015). Identification of miRNAs and their targets in cotton inoculated with Verticillium dahliae by highthroughput sequencing and degradome analysis. International Journal of Molecular Sciences, 16(7), 14749e14768. Zhang, Y., Wang, Y., Xie, F., Li, C., Zhang, B., Nichols, R. L., & Pan, X. (2016c). Identification and characterization of microRNAs in the plant parasitic root-knot nematode Meloidogyne incognita using deep sequencing. Functional & Integrative Genomics, 16(2), 127e142. Zhu, Q. H., et al. (2013). miR482 regulation of NBS-LRR defense genes during fungal pathogen infection in cotton. PLoS One, 8(12), e84390. This page intentionally left blank CHAPTER 18 Future prospective of small RNA molecules: food crop improvement and agricultural sustainability Jafar K. Lonea, Muntazir Mushtaqa, Om Prakash Guptab and a Gayacharan a ICAR-National Bureau of Plant Genetic Resources, Division of Germplasm Evaluation, Hyderabad, Telangana, India; bICAR-Indian Institute of Wheat and Barley Research, Karnal, Haryana, India 1. Introduction Plants are an essential component of the environment and may be utilized by humans for shelter, medicine and food. Global warming has a detrimental influence on plants, including dramatic temperature fluctuations, changes in rainfall patterns, hunger or drought situations, and pest and disease outbreaks. These, in turn, have an impact on crop productivity, decreasing both the quality and quantity of agricultural produce (Munaweera et al., 2022). Climate change and rapid population expansion of 10 billion by 2050 have significantly increased the global food demands (Cushman et al., 2022; Zandalinas et al., 2022; Zsogon et al. 2022). As a result, achieving the objective of food security for current and future generations is critical (Munaweera et al., 2022). Biotechnology facilitates the creation of radical changes in crops to endure stress, which is difficult to do using traditional breeding methods. It is a reliable approach for increasing agricultural yield. The advancement of biotechnological technologies such as RNA-mediated gene silencing has created new possibilities for more precise and rapid plant genetic modifications (Borges & Martienssen, 2015). Such intense efforts are presently ongoing to develop desired crop cultivars in order to fulfill food demand while also supporting sustainable agriculture in the face of climate change. Small RNAs (sRNAs) are RNA molecules present in bacteria, fungi, plants, and animals, which act as negative regulators through base-pairing to regulate specific target genes. In the past decade, much of the research interest has been focused on the functions of sRNA mostly described in Plant Small RNA in Food Crops ISBN 978-0-323-91722-3 https://doi.org/10.1016/B978-0-323-91722-3.00014-2 © 2023 Elsevier Inc. All rights reserved. 571 572 Plant Small RNA in Food Crops plants in various stages of the life cycle along with their development and responses to various abiotic and biotic stresses (Khraiwesh et al., 2012). Plant sRNAs are 20e24 nucleotides (nt) long molecules, dimeric, transacting RNA molecules that block the mRNA in a site-specific manner. RNA interference (RNAi), which is based on small interfering RNA (siRNA), is a commonly used reverse genetics technology that aids in the discovery of gene functions in various species. This technique has been widely used to manipulate gene expression in plants in order to obtain desirable traits. RNAi has been used to improve crop performance and productivity by modifying the gene involved in biomass, grain yield, and fruit and vegetable shelf life. It has also been used to develop tolerance to biotic and abiotic stresses. Crop nutrition has also been improved by supplementing crops with vital amino acids, fatty acids, antioxidants, and other nutrients helpful to human health, or by lowering allergens or pesticides (Kamthan et al., 2015). miRNAs are significant regulators of biologically important activities such as growth, development, and stress response in crop plants. Despite their comparable size (20e24 nt), miRNA and siRNA differ in precursor structures, biogenesis pathways, and mechanisms of action. This review also focuses on miRNA-based genetic modification technologies, in which diverse miRNAs/artificial miRNAs and their targets may be used to improve a variety of desired plant traits. Because of their selectivity and lack of undesired off-target effects, microRNA-based RNAi approaches are far more effective than siRNA-based RNAi strategies. The prime significance of miRNAs can be determined by the fact that research on miRNA has allured an immense curiosity from researchers all across the world (Shriram et al., 2016). MicroRNAs have been widely characterized and revealed as crucial for regulation of numerous developmental and physiological processes (Tyagi et al., 2019), and therefore their miss-expression causes different imperfections in plants. Various studies have reported a number of miRNAs in different crops that govern various traits such as flowering, root, seed and leaf morphology besides abiotic stress, biotic stress and hormonal responses (Ganie et al., 2016; Shriram et al., 2016; Tyagi et al., 2019). Here, we tried to assess the recent developments in the role of small RNA in crop improvement and agricultural sustainability. 2. Classification of sRNA In the past decade, sRNA has been considered the hotspot regulator of genetic expression in plant growth, development, and physiology Future prospective of small RNA molecules 573 (Chen, 2005; Jones-Rhoades et al., 2006; Mallory & Vaucheret, 2006). Broadly, sRNA has been classified into two groups in plants viz: microRNAs (miRNAs) and small-interfering RNAs (siRNAs). Both types of small RNAs are known to regulate plant growth and development (Khraiwesh et al., 2012). The classification of miRNA in the plant kingdom is shown in Fig. 18.1 sRNAs are broadly classified into two main categories viz, hairpin RNA (hpRNA) and small interfering RNA (siRNA). The precursor for hpRNA which is further classified into two major categories viz. miRNA and other hpRNA (Axtell, 2013). miRNA families may be divided into two classes based on their conservation and diversity during plant kingdom evolution. The ancient miRNAs are typically widely expressed and evolutionarily conserved, while the young miRNAs are expressed at comparably low levels or are only induced under certain circumstances, and typically occur only in a few species, making them evolutionarily non-conserved (Djami-Tchatchou et al., 2017; Qin et al., 2014). Previously, evidence of frequent birth and death of MIRNA genes in Arabidopsis was described (Fahlgren et al., 2007). The results suggested that novel small RNA-generating loci might develop into MIRNA genes by aberrant replication/recombination or transposition events from expressed gene sequences. Furthermore, it was shown that Figure 18.1 Categorization of sRNA in plants. cis-nat. siRNA, cis-natural siRNA; hcsiRNA, heterochromatin siRNA; hpRNA, hairpin RNA; miRNA, micro RNA; siRNA, small interfering RNA; siRNA, small interfering RNA; trans-nat. siRNA, trans-natural siRNA. Other hpRNA includes PIWI-interacting RNA (piRNA). 574 Plant Small RNA in Food Crops several miRNAs are commonly lost throughout evolution (Fahlgren et al., 2007). The precursor for siRNA is dsRNA, classified into three secondary classes viz, secondary siRNA, heterochromatin siRNA, and nat-siRNA. The secondary siRNA synthesized from dsRNA depends on the upstream small RNA trigger and subsequent RDR activity. This is further subclassified into phased siRNA and trans-acting siRNA. The heterochromatin-siRNA is mainly produced from the intergenic and repetitive regions, 23e24 nucleotides long. The nat-siRNA is produced from dsRNA owing to hybridization of completely and independently transcribed RNAs. This is further sub-classified into cis-nat-siRNA, which is transcribed from overlapping genes in opposite polarities and trans-natsiRNA produced from the non-overlapping genes whose miRNA have a complementary nature. Plant-based sRNAs are derived from the cleavage of long dsRNA precursors by DICER-like proteins (DCLs). Nevertheless, siRNAs and miRNAs are generated from different precursor molecules (Voinnet, 2009). The DCL cleaves dsRNA into 20-24-base-pair long RNA duplexes (Vazquez, 2006). Double-stranded sRNA after production is stabilized by 3ʹ-OH methylation (Bove et al., 2006). One of the strands of this duplex is attached to RNA induced silencing complex (RISC), which contains one ARGONAUTE (AGO) protein, for mediating RNAi activity via basepairing with its target (Mallory & Vaucheret, 2010). 3. Micro RNA (miRNA) miRNAs are 20e24-nt RNA molecules produced by the dicer-like complex from incorrectly folded hairpin-like precursors (Ramachandran & Chen, 2008). For miRNA discovery, three fundamental approaches are used: forward genetics, direct cloning, and bioinformatics prediction, followed by experimental validation. The direct method, which involves isolating and cloning short RNAs from biological sources, is the simplest and cheapest method, and various researchers have used it to find plantbased small RNAs. In the case of plants, these molecules regulate gene expression by cleaving or repressing mRNAs. They are engaged in a variety of activities, including floral organ identification, leaf morphogenesis, and root development (Sunkar et al., 2007). They are also engaged in the transacting siRNA synthesis as well as the feedback control of sRNA pathways (Allen et al., 2005). They have been investigated in a variety of stress Future prospective of small RNA molecules 575 situations, including drought stress, salt stress, extreme cold, and high temperature stress (Fujii et al., 2005; Yang et al., 2007; Zhang, Pan, Cannon, et al., 2006). When such molecules are involved, multi-complex gene network pathways are activated. Although there have been more studies on the tolerance mechanisms mediated by miRNA, there is still a scarcity of information on their functional roles (Zhang, Pan, Cobb, & Anderson, 2006). Such research is required to completely understand how plants develop in difficult conditions and to aid crop improvement and agricultural sustainability. In this race, Next Generation Sequencing (NGS) platform has significantly accelerated the discovery and characterization of plant-based miRNAs in a wide range of diverse species (Li et al., 2011). 4. Short-interfering RNAs (siRNA) siRNAs are short 50 -phosphorylated dsRNA with two nucleotide overhangs at the 30 end fragments, acting as gene silencers (Elbashir et al., 2001). The gene silencing can be initiated by homologous sequence of the target gene, which needs to be silenced by either short-hairpin RNA (shRNA) or long dsRNA precursors (Fire, 1999; Tuschl, 2001). The siRNAs are known for acting as co-transcriptional silencers of gene expression through regulation of chromatin (Burkhart et al., 2011). The mechanism of action is triggered by the entry of long dsRNA for example a rogue genetic element or a viral intruder and through an introduced transgene inside the cytosol by recruiting the dicer enzyme (Bernstein et al., 2001). This gene silencing activity occurs by the activation of several DNA and histone-modifying proteins, including the cytosine methyltransferase and Chromomethylase3 (CMT3). 5. Biogenesis and mode of action of sRNA in plants In the plant kingdom, the genome encodes hundreds to several thousands of sRNA genes existing in families (Budak & Akpinar, 2015). The biogenesis of miRNA and siRNA molecules is almost the same. Here we have explained about the biogenesis of miRNAs (Fig. 18.2). The miRNA genes are found to be on both introns and exons. In plants most of the miRNA genes are found in intergenic regions. On transcriptional processing, plant miRNAs are folded to from hairpin structure with more variations in the arms than animal miRNA. Transcription of miRNA genes in plants by RNA Pol II results in the formation of primary miRNAs (pri- 576 Plant Small RNA in Food Crops Figure 18.2 Biogenesis and regulation of miRNA mediated gene silencing in plants. miRNA, microRNA; Pol II, DNA dependent RNA polymerase II; pre-miRNA, precursor miRNA; pri-miRNA, primary miRNA. miRNA) with 50 cap and polyadenylation, and this process further requires RNA-binding protein Hyponastic leaves 1 (HYL1) and a C2H2-zinc finger protein SERRATE in the nucleus for converting pri-miRNA into premiRNA (Fang & Spector, 2007; Kurihara et al., 2006). Specialized RNA molecules are transcribed and self-folded into the hairpin loop like structure called primary miRNA (pri-miRNA). The hairpin structure of long primiRNA is locally cropped by Drosha, a nuclear RNase III, and precursor miRNA (pre-miRNA is formed (Rogers & Chen, 2013). The hairpin structure consists of a terminal loop, an upper stem, the miRNA/miRNA* duplex region, a lower stem, and two arms that show more variations. The mature miRNA duplexes stabilized by a methyl transferase supported by S-adenosyl methionine dependent Hua Enhancer 1 (HEN1) for methylation. This methylation occurs at the 30 region of the miRNA duplex to prevent degradation and 30 uridylation (Li et al., 2005; Yang et al., 2006). This nascent miRNA/miRNA* duplex exhibits 2-nt 30 overhangs structure at both strands, and each strand possesses a 5ʹ end phosphate and two 3ʹ end hydroxyl groups (2ʹ OH and 3ʹ OH), where only the 2ʹ eOH position is methylated by the small RNA methyltransferase HUA Enhancer-1 (HEN1) (Yang et al., 2006; Yu et al., 2005). After processing, mature miRNA duplex is transported into the cytoplasm through a specialized exportin-5 homolog (HASTY) in plants, but that Future prospective of small RNA molecules 577 actual exportation is still entirely unknown (Park et al., 2005). The stemloops of pri-miRNAs in plants are highly variable in length have more complex structures than their w70 nt animal counterparts (Bologna & Voinnet, 2014). This results in the processing of miRNA in plants from either the loop-proximal site to the loop-distal site or vice versa (Bologna et al., 2013). From the mature duplex miRNAs (miRNA/miRNA*), the guide strand, miRNA is assembled into the AGO protein, while the passenger strand, miRNA* is degraded. The guide strand is then loaded to the RISC (binding complex) for target cleavage or translational inhibition of target mRNAs. RISC, a multiprotein complex, is an RNA-induced gene silencing complex that serve as the template to recognize complementary mRNA sites. RISC is linked with ARGONAUTE, which cleaves the mRNA. In Arabidopsis thaliana, 10 Argonaute protein paralogs have been reported (Vaucheret, 2008; Zhang et al., 2015). AGO1 regulates miRNAmediated gene regulation either by translational repression (Brodersen et al., 2008) or by cutting miRNA target mRNAs (Baumberger & Baulcombe, 2005). AGO1 consists of 2 domains, the PAZ domain containing a cleft that binds mRNA and a PIWI domain with ribonuclease activity. miRNAs guide the RISC complex to target particular genes through base pairing, which mediates gene silencing by target specific cleavage and/or inhibition of translation. Recently, it has been reported that RISC/AGO1 complex seems too prevalent in translational inhibition, as compared to target cleavage, which is essential for plant development at the post-germination stage (Carbonell et al., 2012; Yang et al., 2019). 6. Comparison between miRNA and siRNA Both miRNA and siRNA are gene expression regulators at posttranscriptional and translational stages (Vazquez, 2006). Although they show similarities in size (20e24 nt) and biogenesis pathway and modes of action, they differ in many other structural and functional parameters (Axtell, 2013, Table 18.1). 7. Role of miRNA in food crop improvement The responses to different abiotic stresses in plants at the molecular level entail miRNA-based regulation of various interactions and cross talks with various molecular pathways (Bej & Basak, 2014). Therefore, it is essential to 578 Plant Small RNA in Food Crops Table 18.1 Contrasting properties of miRNA and siRNA. Properties miRNA siRNA Length Structure 19e25 nt Double stranded Organisms Higher plants, animals and other mammals Endogenous mRNA 21e23 nt Single stranded with hairpin Lower plants and animals Exogenous mRNA Site of action Mode of action Targeting range Required Argonaute protein Stability Gene regulation mechanism Biomarker Significance Repression of translation, degradation of mRNA miRNA is capable of targeting more than 100 types of mRNA simultaneously AGO1, AGO10 miRNAs are extremely stable and resistant to RNase activity, extreme pH as well as freeze-thaw cycles Only post-transcriptional Used as molecular markers in plant breeding Defense against viruses and genome stability mRNA degradation, modification of histones, DNA methylation siRNA targets only one type of mRNA AGO1, AGO4, AGO6, AGO7 Less stable than miRNA Both transcriptional and post-transcriptional Not serve as a molecular marker Regulates expression of endogenous genes breed new crop plant cultivars by targeting their miRNA genes associated with abiotic stress to increase yield as well as quality of crop plants. The miRNAs are considered critical gene regulators for controlling growth and development as well as response to various stressors in plants. So, miRNAbased gene editing platform is the most promising technologies that could be a game changer for both agricultural productivity and reproducibility in order to develop elite crop cultivars. miRNA-based regulation of gene expression can be manipulated by transgenic approaches by overexpressing the target-specific miRNA-resistant genes and creating novel artificial targets (Gupta, 2015). This strategy has been widely used for producing stress-tolerant plants by targeting miRNAs-based negative stress regulators Future prospective of small RNA molecules 579 (Franco-Zorrilla et al., 2007). Further various research groups tried to suppress a gene expression of a protein-coding miRNA by utilizing the artificial miRNA (amiRNA) technology. This novel post-transcriptional gene silencing strategy has been efficiently used in rice and numerous plant species from moss to dicots (Khraiwesh et al., 2008; Sharma et al., 2015). The over-expression of the miR319 gene in the morphogenesis of rice (Oryza sativa) leaves resulted into cold tolerant rice plant on chilling treatment (Yang et al., 2013). In the model plant Arabidopsis, the functional analyses of miRNAs have been reported in various studies (Zhang & Wang, 2015). In Medicago truncatula, the overexpression of miR160 resulted defects in root growth and development, which causes a reduction in the nitrogen-fixing nodule formation (Bustos-Sanmamed et al., 2013). Therefore, the miRNAs can be anticipated to play critical role during plant response to different stresses and can significantly improve crop. 8. Role of siRNA in crop improvement Plant genome architecture can be edited and manipulated to achieve high crop yields like in rice and other plants (Jiao et al., 2010; Miura et al., 2010; Springer, 2010; Wang et al., 2012). In rice, suppressing OsGA20ox2 gene produces semi-dwarf variety from QX1 (taller variety). The transgenic plants produced through RNA interference (RNAi) revealed a significant increase in seeds number per panicle and panicle length and higher test rate (1000 grain) weight (Qiao et al., 2007). In rice, the over-expression of the OsSPL14 gene causes increased grain yield and decrease in tiller number, resulting, which could modify the basic plant architecture (Wang et al., 2012). It has been reported that the overexpression of OsSPL16 positively regulates the cell proliferation and results in increased gain width and higher crop yield in rice plants (Wang et al., 2012). Drought stress or Water deficit is the primary abiotic stress that limits the agricultural production. RNAi machinery has the unprecedented potential engineer crops tolerant to drought stress. In canola, farnesyl transferase was down regulated using RNAi exhibited more tolerance to seed abortion under drought condition (Wang et al., 2009). Li et al. (2009) developed transgenic drought-tolerant plants by silencing the receptor for activated C-kinase 1 (RACK1). The RNAi targets the ubiquitin ligase gene to enhance drought tolerance in rice. RNAi approaches specific to seeds has been successfully used to produce dominant high lysine corn by repressing the expression of 22-kDa storage proteins (zein) in maize (Segal et al., 2003|). Starch content can be increased 580 Plant Small RNA in Food Crops through RNAi technology by manipulating the phosphate metabolism genes in Arabidopsis and maize (Weise et al., 2012). In rice, the glutenin content has been reduced by the gene silencing mediated RNAi approach which remains undigested by kidney patients. Kusaba et al. (2003) employed this tool to develop LGC-1 (low glutenin content1), a rice variety by constructing GluB hairpin RNA in RNAi-technology. Further, this tool was utilized by Gil-Humanes et al. (2008) to reduce the g-gliadins levels in diverse wheat lines. Therefore, this technology can be utilized to produce climate-tolerant crops with enhanced crop yield and quality. 9. siRNA for nutritional enhancement in food crops Crop nutritional value has been improved using RNA interference (RNAi) technology. Crops have unique composition of metabolites; some of them are allergies for humans, pollutants for the environment, and allergens for various crops. However, there are plants that have very important nutritional values and are consumed by humans or animals. RNAi technology provides a publicly acceptable option for developing biofortified foods. Transgenic plants with desired RNAi constructs targeting specific genes involved in a metabolic pathway helps in increasing accumulation of beneficial metabolites or also can lower the anti-nutritional compounds. For example, essential fatty acids and their composition play a vital part in keeping a good heart health in humans, but the desired combinations and compositions of fatty acids are lacking in most of the oil sources. Therefore, in a study RNAi machinery has been utilized effectively to alter the fatty acid composition of oil (Kamthan et al., 2015). In another study to improve the oil quality of cotton seeds, which is generally considered as unhealthy for human consumption, two fatty acid desaturase genes were targeted using RNAi machinery Liu et al. (2002). Oil flavor and stability in soybean was improved, to avoid the hydrogenation process by decreasing alphalinolenic acid to prolong storage quality. There are other studies used RANi to improve the oil quality such as, Flores et al. (2008) employed RNAi approach to down-regulate omega-3 fatty acid. A considerable decrease in alpha-linoleic acid content (1e3%) of RNAi targeted transgenic soybean was reported in contrast to non-transgenic soybean seed (7e10%). Furthermore, seed-specific RNAi strategies have been successfully applied to produce corn with high lysine content through suppression of zein storage proteins (Segal et al., 2003). In another study, RNAi was successfully applied to down-regulate the starch-branching enzyme, thus Future prospective of small RNA molecules 581 wheat with high-amylose possess great potential in improving human health (Regina et al., 2006) used RNAi machinery to generate wheat with high amylose content through down-regulation of the starch-branching enzyme. RNAi technology has been employed to increase the leaf starch content. Starch’s phosphorylation and dephosphorylation processes are central steps of starch degradation in leaves. On the basis of this fact, Weise et al. (2012) enhanced starch content in Zea mays and Arabidopsis by manipulating genes involved in phosphate metabolism (Weise et al., 2012). RNAi has been employed to decrease glutenin content in rice that kidney patients digest. Kusaba et al. (2003) used GluB hairpin RNA to generate a low glutenin rice variety called LGC-1s. At low temperatures (4 C), potato tubers undergo unfavorable phenomena of “cold sweetening” that results in the conversion of starch into glucose and fructose. To deal with this challenge of cold-induced sweetening in potatoes, Chen et al. (2008) targeted the tobacco gene, NtSPP2, for RNAi-mediated repression of SPP in potato tubers. The transgenic potato tubers exhibited increased sucrose-6 phosphate (Suc6P) levels when stored at low temperatures. Further, conversion of sucrose into hexose was decreased in potato tubers due to the blocked cold-induced expression of vacuolar invertase (VI). Furthermore, Gil-Humanes et al. (2012) observed a slight upsurge in the protein levels of transgenic plants because of the compensatory effect created as a result of g-gliadin down-regulation on the rest of gluten proteins. The glutenin levels were enhanced although no change was experienced in the content of total gliadins. Nonetheless, experiments have been conducted to explore the development of seedless fruits in fructiferous plants by GmMIPS1 silencing approach to inhibit seed development. RNAi reduction resulted in increased amylose type starch synthesis in four wheat lines (Regina et al., 2006); similarly, barley lines with low expression of SBE IIa or SBE IIb, as well as low expression of both isoforms, were developed using RNA-mediated silencing technology (Regina et al., 2010). RNAi has also been utilised to down-regulate the starch branching enzyme, resulting in high-amylose wheat with incredible health benefits (Zhou & Luo, 2013). RNAi has been used to generate corn with higher essential amino acids (Hasan & Rima, 2021), improved soybean oil quality (Yang et al., 2018), and cotton with improved fatty acid composition. Overexpression of GmPDAT genes increased seed size and oil content, but RNAi strains lowered seed size and oil content of soybeans 582 Plant Small RNA in Food Crops (Gao et al., 2020; Liu et al., 2020). Rice carotenoid concentration increases when three carotenoid-cleavage dioxygenase genes, OsCCD1, 4a, and 4b, are suppressed (Ko et al., 2018). The RNAi technique has also been utilized in apples to improve fruit quality by increasing self-life and decreasing the amount of a key apple allergy, metabolites, and sugar accumulation in the fruits via sorbitol production, which affects fruit starch accumulation (Romer et al., 2020). High lysine maize for the production of Zein proteins and silencing of carotenoid b-hydroxylases to boost the b-carotene content of maize are two examples adding plants through RNAi technology (Berman et al., 2017; Choudhary et al., 2021; Li & Song, 2020) respectively. 10. sRNA in developing climate-resilient food crop plants Climate change and abiotic stress have a considerable consequence on total agricultural productivity and demand growing crops that can endure various climate changes and environmental stresses. Plant miRNAs (miRNAs) have become crucial post-transcriptional and translational geneexpression regulators for stress modulation. Recent research establishes their significant function in epigenetics of stress/adaptive responses and provides genome-stability in plants. Several miRNAs have been identified from diverse crop plants under different stresses, and miRNA-driven RNAinterference (RNAi) technology has become the alternative for crop trait improvement and offers phenotypic plasticity in harsh environmental conditions. Recently, sRNAs have been reported to play role in heat stress responses and plant thermotolerance (Zuo et al., 2021). Here in Table 18.2, we present the investigation of miRNAs as potential targets for production of crops that can endure multi-stress environments via loss-/gain-offunction approaches. 11. sRNA-based approaches to develop biotic stress tolerance in crop plants In crop plants under biotic stress, small RNAs are recognized to play an important function. Plants use RNA silencing technology to activate pathogen-associated molecular pattern-triggered immunity and effectortriggered immunity in order to resist pathogens and defend themselves against insect herbivores. Pathogens, on the other hand, create sRNAs and Table 18.2 Role of miRNAs in abiotic stress tolerance in transgenic plants. Strategy miR156 miR166 [ [ miR319 [ miR165 miR398 [ [ miR156 AthmiR399f AthmiR408 AthmiR408 [ [ GmmiR172a GmmiR172c [ [ [ [ Transgenic plant Sesame Oryza sativa Medicago sativa Arabidopsis Triticum aestivum Zea mays Arabidopsis thaliana Cicer arietinum Arabidopsis thaliana Glycine max Arabidopsis thaliana Target genes Improved characters References SPL TF HD-ZipIII Salinity stress tolerance Salt stress tolerance Zhang et al. (2020) Parmer et al. (2020) TCP TF Response to salt stress Ma et al. (2019) HD-ZipIII CSD Drought stress tolerance Salt stress tolerance Yang et al. (2019) Qiu et al. (2018) SPL TF CSP41b, ABF3 Salt stress tolerance Enhanced salinity stress tolerance Enhanced drought stress tolerance Enhanced salt and cold stress tolerance oxidative stress tolerance Salt tolerance Fu et al. (2017) Baek et al. (2016) Salt tolerance, drought tolerance and ABA sensitivity Li, Xue, and Yi (2016) and Li, Wang, et al. (2016) DREB1/2A, Rd17/29A PLANTACYANINS, CUPREDOXIN, UCLACYANIN LAC3SSAC1 Glyma01g39520 Hajyzadeh et al. (2015) Ma et al. (2015) Pan et al. (2016) 583 Continued Future prospective of small RNA molecules miRNA 584 Table 18.2 Role of miRNAs in abiotic stress tolerance in transgenic plants.dcont'd Strategy HvmiR827 OsmiR397a OsmiR166 OsmiR319 OsmiR319b OsmiR393a Os-miR 528 OsmiR529a PtrmiR396b [ [ Y [ [ [ [ [ [ Transgenic plant Hordeum vulgare Arabidopsis thaliana Oryza sativa Agrostis stolonifera Oryza sativa Agrostis Stolonifera Agrostis stolonifera Oryza sativa Citrus limon Target genes Improved characters References SPX Drought tolerance Ferdous et al. (2017) CBF, COR Cold tolerance Dong and Pei (2014) OsHB4 Drought resistance Zhang et al. (2018) AsPCF5/6/8, AsTCP14 and AsNAC60 OsPCF6, OsTCP21 Salt tolerance Zhou and Luo (2014) Cold tolerance Wang et al. (2014) Salt tolerance, drought tolerance and heat tolerance Enhanced salt tolerance Zhang et al. (2018) Enhanced oxidative stress tolerance Cold tolerance Yue et al. (2017) AsAFB2, AsTIR 1 AsAAO, AsCBP1 OsSPL2, OsSPL14 ACO Yuan et al. (2015) Zhang et al. (2016) Plant Small RNA in Food Crops miRNA Future prospective of small RNA molecules 585 effectors in order to combat plant immunity. The development of sRNAs, RNAi machinery, and pathogen effectors has resulted from the arms race between host plants and pathogens/insect herbivores. Several research have been conducted to investigate the role of sRNAs in plant defense and protection (Fig. 18.3). Plants are evolved with complex defense mechanisms in order to combat different pathogens, in turn pathogens also generate diverse effector or suppressor molecules as counter defenses. The result of interactions between plant and the pathogen relies on the comparative contribution of resistance and the susceptibility factors. The role of sRNAs against different plant pathogen stressors is given in Table 18.3. Plant bacteria are exceptionally complex to manage owing to their short generation period. Dunoyer et al. (2006) demonstrated the repression of genes of Agrobacterium tumefaciens (iaaMandipt) could considerably decrease the tumor production in Arabidopsis using RNAi approach. This approach has been utilized in other plants as well. Navarro et al. (2006) reported the improved resistance against bacteria due to over-expression of miRNA393 in transgenic Arabidopsis, although with some developmental changes. In two different studies, miR398 and miR825 (Jagadeeswaran et al., 2009) were demonstrated to be downregulated upon bacterial infectivity. RNA silencing has been used to target fatty acid metabolism genes to increase disease resistance in crop plants and is a fundamental approach for generating disease resistance. Jiang et al. (2009) reported the improved resistance against blast fungus and leaf blight by suppressing OsSSI2 gene in rice using RNAi technology. In addition, Yara et al. (2007) reported the increased resistance to rice blast fungus by suppressing OsFAD7 and OsFAD8 genes. Peltier et al. (2009) demonstrated the increased tolerance to Sclerotinia sclerotiorum in soybean by targeting lignin production genes using RNAi owing to reduced lignin content. In 2010, Xin et al. (2010) showed the response of 24 miRNAs in wheat against Blumeria graminis f. sp. tritici (Bgt), a responsible causal agent of powdery mildew. In another study conducted by Campo et al. (2013), enhanced resistance against Magnaporthe oryzae was accomplished by over-expressing Osa-miR 7696 in rice (Campo et al., 2013). Antiviral responses based on PAMP Triggered Immunity (PTI) are probably elicited by DAMPs (Zvereva & Pooggin, 2012). Proteins encoded by viruses are recognized by R protein and the RNAi can also elicit defense responses to combat viruses (Moffett, 2009). Antiviral immunity elicits the Figure 18.3 sRNAs as key players in plant defense against different pathogens (A) Function of sRNAs in plant defense response. (B) RNAi as a silencing tool in plant defense against viral pathogens (DNA/RNA) Here both the miRNAs and vsiRNAs (virus derived- siRNAs) are involved. (C) In response to insect herbivore attack, plants produce sRNAs which are involved in the generation of phytochemicals that are toxic to insect herbivores or increase plant resistance response. Table 18.3 Role of sRNAs in plant-pathogen interactions. Target genes Gene expression upon infection Function during plantpathogen interaction Arabidopsis/P. syringae PPRL Upregulation KatiyarAgarwal et al. (2006) Plant Arabidopsis/P. syringae Upregulation miR 393 Plant Arabidopsis/ P. syringae Remorin, zinc finger homeobox family, frataxinrelated TIR1, AFB2, and AFB3 Disease resistant lines were obtained repressing a negative regulator of the RPS2 pathway ? Fahlgren et al. (2007) AtlsiRNA-1 Plant Arabidopsis/P. syringae AtRAP Upregulation Regulate auxin signaling and enhance plant immunity Enhances plant defense miR 1885 Plant TIR-NBSLRR Upregulation Repress effectortriggered immunity TMV vsiRNA Pathogen Brassica napus/ Turnip mosaic virus Arabidopsis/ Tobacco mosaic virus CPSF30,TRAPa ? ? sRNA Source NatsiRNAATGB2 Plant miR825 Host/pathogen interaction Fahlgren et al. (2007) KatiyarAgarwal et al. (2007) Wroblewski et al. (2007) Qi et al. (2009) 587 Continued Future prospective of small RNA molecules Upregulation References 588 Table 18.3 Role of sRNAs in plant-pathogen interactions.dcont'd Gene expression upon infection Function during plantpathogen interaction XOX5b.1, CSD1 and CSD2 Downregulation Jagadeeswaran et al. (2009), Li et al. (2010) ARF10, ARF16, and ARF17 MET2 Upregulation Callose deposition is negatively regulated and suppress auxin signaling and detoxification of ROS Enhance callose deposition Li et al. (2010) Arabidopsis/ P. syringe COX5B.1, CSD1 and CSD2 Downregulation Arabidopsis/ Bacteria ARF8, ARF6 Upregulation Plays role in negative regulation of callose deposition and disease resistance to bacteria Callose deposition is negatively regulated and suppresses the auxin signaling and induces ROS detoxification Regulation of auxin and enhances plant immunity Source miR398 Plant Arabidopsis/ P. Syringae miR160 Plant Arabidopsis/ P. syringae MiR 773 Plant Arabidopsis/ P. syringae miR398 Plant miR 167 Plant Target genes Downregulation References Li et al. (2010) Jagadeeswaran et al. (2009), Li et al. (2010) Zhang et al. (2011) Plant Small RNA in Food Crops Host/pathogen interaction sRNA Plant miS 482 Plant miR408 Plant miR398 Plant miR472 Plant NBS-LRR Downregulation Expression of miR482 is downregulated on virus and bacteria infection and R protein expression is induced Shivprasad et al. (2012) NBS-LRR Downregulation Zhu et al. (2013) Wheat/fungus Puccinia striiformis f. sp. tritici O. sativa/ M. oryzae TaCLP1, a type of plantacyanin protein Upregulation/ downregulation miR482 expression is downregulated and R protein expression is induced due to fungal infection Negatively regulation of stripe rust in wheat SOD2 Upregulation Li, Lu et al. (2014) CC-NBS-LRR ? Increased accumulation of H2O2 and defense-related genes and decreased fungal growth due to over-expression of miR398 Over-expressing miR472 increases plant susceptibility to bacteria Arabidopsis/ P. syringe Feng et al. (2013) Boccara et al. (2014) 589 S. lycopersicum/ tobacco mosaic virus, cauliflower mosaic virus and tobacco rattle virus G. raimondii/ V. dahliae Future prospective of small RNA molecules miR482 590 Plant Small RNA in Food Crops generation of vsiRNAs to target and destroy the infecting RNA virus (Fig. 18.3B). vsiRNAs were first revealed in tobacco plant (Hamilton & Baulcombe, 1999). Various reports have revealed the vsiRNA-based antiviral immunity in different plant species (Duan et al., 2012; Parent et al., 2015). In order to respond to different viruses, plants exploit several RNA silencing pathways. Viruses with dsRNA are targeted by DCL enzymes that convert dsRNA into siRNAs, while ssRNA viruses need RDRs to form dsRNAs, eventually recognized by DCLs (Huang et al., 2016) (Fig. 18.3B). Besides, dsRNA formed during the ssRNA replication could be also targeted by DCLs to form vsiRNAs. In addition to RDR1 and RDR6, DCL2 and DCL4 are reported to play role in plant immunity. The mutants viz., dcl2, dcl4, rdr1, and rdr6 showed considerable decrease of vsiRNAs, suggesting their role in biosynthesis of vsiRNA (Qi et al., 2009). The miRNAs have also been shown to profoundly involve in plant defense against viral pathogens. The miRNA accumulation is appreciably influenced due to viruses. The miRNA expression in roots in addition to leaves altered considerably following virus infection (Sun et al., 2015). Pradhan et al. (2015) studied the expression of 53 miRNAs in response to infection due to ToLCNDV virus (Pradhan et al., 2015). In addition, several miRNAs are known to target R genes in response to viral infection. Shiva prasad et al. (2012) demonstrated the enhanced miR482-targeted mRNA expression in tomato plants in response to Cucumber mosaic virus (CMV), Tobacco rattle virus (TRV), and Turnip crinkle virus (TCV) by suppression of miR482-mediated silencing cascade. In another study conducted by Li et al. (2012), miR 6019 and miR 6020 were shown to confer resistance in tobacco via NSB-LRRs regulation and the generation of secondary 21-nt siRNAs. In order to combat RSV and RDV, in one of the studies, enhanced expression of miR 168 was observed to repress the AGO1 gene in rice resulting in broad-spectrum resistance against virus (Wu et al., 2015). Viral resistance is accomplished due to inhibition of replication of viruses inside the cell, thus limiting movement of viruses between cells. In case of higher plants, sRNA-mediated viral immunity is not restricted to the infected cells only however, also silences viral RNAs in distant tissues (Palauqui et al., 1997). The role of siRNAs mainly relies on the RNAi machinery instead of siRNA mobility (Sarkies & Miska, 2014). Plants use sRNA as a defense approach to combat viruses. In turn, viruses use viral suppressors of RNA silencing (specific proteins) to repress RNAi Future prospective of small RNA molecules 591 (Csorba et al., 2015). Viral miRNAs are known to target genes and pathways in host plants to increase their infectivity (Zhuo et al., 2013). Though, only a few investigations have reported miRNAs encoded by viruses in plants, but their role is still unidentified (Zhuo et al., 2013). Betasatellites are known to be responsible for eliciting disease symptoms in begomoviruses (Qazi et al., 2007). Beta-satellites are known to produce bC1 protein that impedes DNA methylation (Yang et al., 2011). PTGS is suppressed by bC1 through upregulation of NbrgsCaM in N. benthamiana (Li, Huang, et al., 2014). 12. Pest and nematode resistance RNAi is effective against some insects that belong to the order Coleoptera. However, Lepidoptera and Hemiptera pests emerge to be mostly recalcitrant in their response to environmental RNAi, signifying biological barriers limiting the use of RNAi in managing such insect pests (Zotti et al., 2017). Unraveling these disadvantages could most possibly allocate this machinery to be integrated into IPM approaches as an exceptional and new mode of action. Baum et al. demonstrated a dsRNA construct in a genetically altered maize plant could cause mortality in Diabrotica virgifera larvae This discovery alerted scientists to the possibility of dsRNA as a revolutionary insect-pest control approach employing transgenic plants. The “SmartStax PRO,” a novel maize event MON87411 that expresses three Cry genes and a dsRNA carrying a 240 bp fragment of the D. virgifera Snf7 gene (DvSnf7), was certified for sale and release by the Canadian Food Inspection Agency (CFIA) in September 2016. In June 2017, the United States Environmental Protection Agency (US-EPA) gave its approval for commercialization of this event. The DvSnf7 gene codes for a protein that is required for vacuolar sorting, but no pesticide has been developed to target it. However, due to the mechanism of RNAi, the Snf7 dsRNA alone takes a long time to kill WCR effectively. Thus, the event MON87411 was produced in amalgamation with the Cry genes from Bacillus thuringiensis to facilitate targeting of both lepidopteran pests and CRWin addition to Diabrotica spp. Complex. The key idea of combining those mechanisms (i.e., Bt and RNAi) is also to surmount the incidence of resistance in insect pests to Bt technology. In this fashion, Snf7 dsRNA expressing genetically engineered maize has been confirmed to defend maize roots against WCR larvae. 592 Plant Small RNA in Food Crops 13. Conclusion Plant originated Small RNA (sRNA) molecules play important roles in regulation of several biological processes. These molecules move to their target sites through extracellular vehicles and modulate the target gene expression following either of the specific mechanism viz. DNA methylation, RNA interference (RNAi), and translational repression. Among these, the RNAi mechanism has been prominently used by scientists to decipher and alter gene function. The technology is successfully used to create plants with desired changes in a trait expressions and phenotype development. It is a powerful tool for understanding the impact of individual genes, and it helps molecular breeders to design better cultivars. In plants, the technology is primarily used in conferring stress tolerance in crop plants via transgenics with desired changes in target gene expression based on whether known miRNAs are positive or negative regulators. The application of the sRNAs in the crop improvement is unlimited, however, further research is needed to for enhancing its precision and efficiency. References Allen, E., Xie, Z., Gustafson, A. M., & Carrington, J. C. (2005). microRNA-directed phasing during trans-acting siRNA biogenesis in plants. Cell, 121(2), 207e221. Axtell, M. J. (2013). Classification and comparison of small RNAs from plants. Annual Review of Plant Biology, 64, 137e159. Baek, D., Chun, H. J., Kang, S., Shin, G., Park, S. J., Hong, H., Kim, C., Kim, D. H., Lee, S. Y., Kim, M. C., & Yun, D. J. (2016). A role for Arabidopsis miR399f in salt, drought, and ABA signaling. Molecules and cells, 39(2), 111. Baumberger, N., & Baulcombe, D. C. (2005). Arabidopsis ARGONAUTE1 is an RNA Slicer that selectively recruits microRNAs and short interfering RNAs. Proceedings of the National Academy of Sciences, 102(33), 11928e11933. Bej, S., & Basak, J. (2014). MicroRNAs: The potential biomarkers in plant stress response. American Journal of Plant Sciences, 5, 748e759. Berman, J., Zorrilla-López, U., Sandmann, G., Capell, T., Christou, P., & Zhu, C. (2017). The silencing of carotenoid b-hydroxylases by RNA interference in different maize genetic backgrounds increases the b-carotene content of the endosperm. International Journal of Molecular Sciences, 18(12), 2515. Bernstein, E., Caudy, A. A., Hammond, S. M., & Hannon, G. J. (2001). Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature, 409(6818), 363e366. Boccara, M., Sarazin, A., Thiebeauld, O., Jay, F., Voinnet, O., Navarro, L., & Colot, V. (2014). The Arabidopsis miR472-RDR6 silencing pathway modulates PAMP-and effector-triggered immunity through the post-transcriptional control of disease resistance genes. PLoS Pathogens, 10(1), e1003883. Bologna, N. G., Schapire, A. L., & Palatnik, J. F. (2013). Processing of plant microRNA precursors. Briefings in Functional Genomics, 12(1), 37e45. Future prospective of small RNA molecules 593 Bologna, N. G., & Voinnet, O. (2014). The diversity, biogenesis, and activities of endogenous silencing small RNAs in Arabidopsis. Annual Review of Plant Biology, 65, 473e503. Borges, F., & Martienssen, R. A. (2015). The expanding world of small RNAs in plants. Nature Reviews Molecular Cell Biology, 16(12), 727e741. Bove, J., Hord, C. L., & Mullen, M. A. (2006). The blossoming of RNA biology: Novel insights from plant systems. RNA, 12, 2035e2046. Brodersen, P., Sakvarelidze-Achard, L., Bruun-Rasmussen, M., Dunoyer, P., Yamamoto, Y. Y., Sieburth, L., & Voinnet, O. (2008). Widespread translational inhibition by plant miRNAs and siRNAs. Science, 320(5880), 1185e1190. Budak, H., & Akpinar, B. A. (2015). Plant miRNAs: Biogenesis, organization and origins. Functional & Integrative Genomics, 15(5), 523e531. Burkhart, K. B., Guang, S., Buckley, B. A., Wong, L., Bochner, A. F., & Kennedy, S. (2011). A Pre-mRNAeassociating factor links endogenous siRNAs to chromatin regulation. PLoS Genetics, 7(8), e1002249. Bustos-Sanmamed, P., Mao, G., Deng, Y., Elouet, M., Khan, G. A., Bazin, J., … LelandaisBrière, C. (2013). Overexpression of miR160 affects root growth and nitrogen-fixing nodule number in Medicago truncatula. Functional Plant Biology, 40(12), 1208e1220. Campo, S., Peris-Peris, C., Siré, C., Moreno, A. B., Donaire, L., Zytnicki, M., … San Segundo, B. (2013). Identification of a novel microRNA (mi RNA) from rice that targets an alternatively spliced transcript of the N ramp 6 (N natural resistance-associated macrophage protein 6) gene involved in pathogen resistance. New Phytologist, 199(1), 212e227. Carbonell, A., Fahlgren, N., Garcia-Ruiz, H., Gilbert, K. B., Montgomery, T. A., Nguyen, T., … Carrington, J. C. (2012). Functional analysis of three Arabidopsis ARGONAUTES using slicer-defective mutants. The Plant Cell Online, 24(9), 3613e3629. Chen, X. (2005). MicroRNA biogenesis and function in plants. FEBS Letters, 579(26), 5923e5931. Chen, S., Hajirezaei, M. R., ZANOR, M. I., Hornyik, C., Debast, S., Lacomme, C., … Boernke, F. (2008). RNA interference-mediated repression of sucrose-phosphatase in transgenic potato tubers (Solanum tuberosum) strongly affects the hexose-to-sucrose ratio upon cold storage with only minor effects on total soluble carbohydrate accumulation. Plant, Cell and Environment, 31(1), 165e176. Choudhary, M., Grover, K., & Singh, M. (2021). Maize’s significance in the Indian food situation to mitigate malnutrition. Cereal Chemistry, 98(2), 212e221. Csorba, T., Kontra, L., & Burgyan, J. (2015). Viral silencing suppressors: Tools forged to fine-tune host-pathogen coexistence. Virology, 479, 85e103. Cushman, J. C., Denby, K., & Mittler, R. (2022). Plant responses and adaptations to a changing climate. The Plant Journal: For Cell and Molecular Biology, 109(2), 319e322. Djami-Tchatchou, A. T., Sanan-Mishra, N., Ntushelo, K., & Dubery, I. A. (2017). Functional roles of microRNAs in agronomically important plantsdpotential as targets for crop improvement and protection. Frontiers of Plant Science, 8, 378. Dong, C. H., & Pei, H. (2014). Over-expression of miR 397 improves plant tolerance to cold stress in Arabidopsis thaliana. Journal of Plant Biology, 57(4), 209e217. Duan, C. G., Wang, C. H., & Guo, H. S. (2012). Application of RNA silencing to plant disease resistance. Silence, 3(1), 1e8. Dunoyer, P., Himber, C., & Voinnet, O. (2006). Induction, suppression and requirement of RNA silencing pathways in virulent Agrobacterium tumefaciens infections. Nature Genetics, 38(2), 258e263. 594 Plant Small RNA in Food Crops Eady, C. C., Kamoi, T., Kato, M., Porter, N. G., Davis, S., Shaw, M., … Imai, S. (2008). Silencing onion lachrymatory factor synthase causes a significant change in the sulfur secondary metabolite profile. Plant Physiology, 147(4), 2096e2106. Elbashir, S. M., Lendeckel, W., & Tuschl, T. (2001). RNA interference is mediated by 21and 22-nucleotide RNAs. Genes & Development, 15(2), 188e200. Fahlgren, N., Howell, M. D., Kasschau, K. D., Chapman, E. J., Sullivan, C. M., Cumbie, J. S., … Carrington, J. C. (2007). High-throughput sequencing of Arabidopsis microRNAs: Evidence for frequent birth and death of MIRNA genes. PLoS One, 2(2), e219. Fang, Y., & Spector, D. L. (2007). Identification of nuclear dicing bodies containing proteins for microRNA biogenesis in living Arabidopsis plants. Current Biology, 17(9), 818e823. Feng, H., Zhang, Q., Wang, Q., Wang, X., Liu, J., Li, M., … Kang, Z. (2013). Target of tae-miR408, a chemocyanin-like protein gene (TaCLP1), plays positive roles in wheat response to high-salinity, heavy cupric stress and stripe rust. Plant Molecular Biology, 83(4e5), 433e443. Ferdous, J., Whitford, R., Nguyen, M., Brien, C., Langridge, P., & Tricker, P. J. (2017). Drought-inducible expression of Hv-miR827 enhances drought tolerance in transgenic barley. Functional & Integrative Genomics, 17(2e3), 279e292. Fire, A. (1999). RNA-triggered gene silencing. Trends in Genetics, 15(9), 358e363. Flores, T., Karpova, O., Su, X., Zeng, P., Bilyeu, K., Sleper, D. A., … Zhang, Z. J. (2008). Silencing of Gm FAD3 gene by siRNA leads to low a-linolenic acids (18: 3) of fad3mutant phenotype in soybean [Glycine max (Merr.)]. Transgenic Research, 17(5), 839e850. Franco-Zorrilla, J. M., Valli, A., Todesco, M., Mateos, I., Puga, M. I., Rubio-Somoza, I., … Paz-Ares, J. (2007). Target mimicry provides a new mechanism for regulation of microRNA activity. Nature Genetics, 39(8), 1033e1037. Fujii, H., Chiou, T. J., Lin, S. I., Aung, K., & Zhu, J. K. (2005). A miRNA involved in phosphate-starvation response in Arabidopsis. Current Biology, 15(22), 2038e2043. Fu, R., Zhang, M., Zhao, Y., He, X., Ding, C., Wang, S., Feng, Y., Song, X., Li, P., & Wang, B. (2017). Identification of salt tolerance-related microRNAs and their targets in maize (Zea mays L.) using high-throughput sequencing and degradome analysis. Frontiers of Plant Science, 8, 864. https://doi.org/10.3389/fpls.2017.00864 Ganie, S. A., Dey, N., & Mondal, T. K. (2016). Promoter methylation regulates the abundance of osa-miR393a in contrasting rice genotypes under salinity stress. Functional & Integrative Genomics, 16, 1e11. Gao, L., Luo, J., Ding, X., Wang, T., Hu, T., Song, P., Zhai, R., Zhang, H., Zhang, K., Li, K., & Zhi, H. (2020). Soybean RNA interference lines silenced for eIF4E show broad potyvirus resistance. Molecular Plant Pathology, 21(3), 303e317. Gil-Humanes, J., Pistón, F., Hernando, A., Alvarez, J. B., Shewry, P. R., & Barro, F. (2008). Silencing of g-gliadins by RNA interference (RNAi) in bread wheat. Journal of Cereal Science, 48(3), 565e568. Gil-Humanes, J., Pistón, F., Rosell, C. M., & Barro, F. (2012). Significant down-regulation of g-gliadins has minor effect on gluten and starch properties of bread wheat. Journal of Cereal Science, 56(2), 161e170. Guo, W., Chen, W., Zhang, Z., Guo, N., Liu, L., Ma, Y., & Dai, H. (2020). The hawthorn CpLRR-RLK1 gene targeted by ACLSV-derived vsiRNA positively regulate resistance to bacteria disease. Plant Science, 300, 110641. Gupta, P. K. (2015). MicroRNAs and target mimics for crop improvement. Current Science, 1624e1633. Hajyzadeh, M., Turktas, M., Khawar, K. M., & Unver, T. (2015). miR408 overexpression causes increased drought tolerance in chickpea. Gene, 555(2), 186e193. Future prospective of small RNA molecules 595 Hamilton, A. J., & Baulcombe, D. C. (1999). A species of small antisense RNA in posttranscriptional gene silencing in plants. Science, 286(5441), 950e952. Hasan, M., & Rima, R. (2021). Genetic engineering to improve essential and conditionally essential amino acids in maize: Transporter engineering as a reference. Transgenic Research, 30(2), 207e220. Huang, J., Yang, M., & Zhang, X. (2016). The function of small RNAs in plant biotic stress response. Journal of Integrative Plant Biology, 58(4), 312e327. Jagadeeswaran, G., Saini, A., & Sunkar, R. (2009). Biotic and abiotic stress down-regulate miR398 expression in Arabidopsis. Planta, 229(4), 1009e1014. Jiang, C. J., Shimono, M., Maeda, S., Inoue, H., Mori, M., Hasegawa, M., Shoji, S., & Takatsuji, H. (2009). Suppression of the rice fatty-acid desaturase gene OsSSI2 enhances resistance to blast and leaf blight diseases in rice. Molecular Plant-Microbe Interactions, 22(7), 820e829. Jiao, Y., Wang, Y., Xue, D., Wang, J., Yan, M., Liu, G., … Li, J. (2010). Regulation of OsSPL14 by OsmiR156 defines ideal plant architecture in rice. Nature Genetics, 42(6), 541e544. Jones-Rhoades, M. W., Bartel, D. P., & Bartel, B. (2006). MicroRNAs and their regulatory roles in plants. Annual Review of Plant Biology, 57(1), 19e53. Kamthan, A., Chaudhuri, A., Kamthan, M., & Datta, A. (2015). Small RNAs in plants: Recent development and application for crop improvement. Frontiers of Plant Science, 6, 208. Katiyar-Agarwal, S., Gao, S., Vivian-Smith, A., & Jin, H. (2007). A novel class of bacteriainduced small RNAs in Arabidopsis. Genes & Development, 21(23), 3123e3134. Katiyar-Agarwal, S., Morgan, R., Dahlbeck, D., Borsani, O., Villegas, A., Zhu, J. K., … Jin, H. (2006). A pathogen-inducible endogenous siRNA in plant immunity. Proceedings of the National Academy of Sciences, 103(47), 18002e18007. Khraiwesh, B., Ossowski, S., Weigel, D., Reski, R., & Frank, W. (2008). Specific gene silencing by artificial MicroRNAs in physcomitrella patens: An alternative to targeted gene knock-outs. Plant Physiology, 148(2), 684e693. Khraiwesh, B., Zhu, J. K., & Zhu, J. (2012). Role of miRNAs and siRNAs in biotic and abiotic stress responses of plants. Biochimica et BiophysicaActa (BBA)-Gene Regulatory Mechanisms, 1819(2), 137e148. Ko, M. R., Song, M. H., Kim, J. K., Baek, S. A., You, M. K., Lim, S. H., & Ha, S. H. (2018). RNAi-mediated suppression of three carotenoid-cleavage dioxygenase genes, OsCCD1, 4a, and 4b, increases carotenoid content in rice. Journal of Experimental Botany, 69(21), 5105e5116. Kurihara, Y., Takashi, Y., & Watanabe, Y. (2006). The interaction between DCL1 and HYL1 is important for efficient and precise processing of pri-miRNA in plant microRNA biogenesis. Rna, 12(2), 206e212. Kusaba, M., Miyahara, K., Iida, S., Fukuoka, H., Takano, T., Sassa, H., … Nishio, T. (2003). Low glutelin content1: A dominant mutation that suppresses the glutelin multigene family via RNA silencing in rice. The Plant Cell Online, 15(6), 1455e1467. Li, H., Dong, Y., Yin, H., Wang, N., Yang, J., Liu, X., … Li, X. (2011). Characterization of the stress associated microRNAs in Glycine max by deep sequencing. BMC Plant Biology, 11(1), 1e12. Li, F., Huang, C., Li, Z., & Zhou, X. (2014). Suppression of RNA silencing by a plant DNA virus satellite requires a host calmodulin-like protein to repress RDR6 expression. PLoS Pathogens, 10(2), e1003921. Li, D. H., Hui, L. I. U., Yang, Y. L., Zhen, P. P., & Liang, J. S. (2009). Down-regulated expression of RACK1 gene by RNA interference enhances drought tolerance in rice. Rice Science, 16(1), 14e20. 596 Plant Small RNA in Food Crops Li, Y., Lu, Y. G., Shi, Y., Wu, L., Xu, Y. J., Huang, F., … Wang, W. M. (2014). Multiple rice microRNAs are involved in immunity against the blast fungus Magnaporthe oryzae. Plant Physiology, 164(2), 1077e1092. Li, F., Pignatta, D., Bendix, C., Brunkard, J. O., Cohn, M. M., Tung, J., … Baker, B. (2012). MicroRNA regulation of plant innate immune receptors. Proceedings of the National Academy of Sciences, 109(5), 1790e1795. Li, C., & Song, R. (2020). The regulation of zein biosynthesis in maize endosperm. Theoretical and Applied Genetics, 133(5), 1443e1453. Liu, Q., Singh, S. P., & Green, A. G. (2002). High-stearic and high-oleic cottonseed oils produced by hairpin RNA-mediated post-transcriptional gene silencing. Plant Physiology, 129(4), 1732e1743. Liu, J. Y., Zhang, Y. W., Han, X., Zuo, J. F., Zhang, Z., Shang, H., … Zhang, Y. M. (2020). An evolutionary population structure model reveals pleiotropic effects of GmPDAT for traits related to seed size and oil content in soybean. Journal of Experimental Botany, 71(22), 6988e7002. Li, W., Wang, T., Zhang, Y., & Li, Y. (2016b). Overexpression of soybean miR172c confers tolerance to water deficit and salt stress, but increases ABA sensitivity in transgenic Arabidopsis thaliana. Journal of Experimental Botany, 67(1), 175e194. Li, L., Xue, M., & Yi, H. (2016a). Uncovering microRNA-mediated response to SO2 stress in Arabidopsis thaliana by deep sequencing. Journal of Hazardous Materials, 316, 178e185. Li, J., Yang, Z., Yu, B., Liu, J., & Chen, X. (2005). Methylation protects miRNAs and siRNAs from a 30 -end uridylation activity in Arabidopsis. Current Biology, 15(16), 1501e1507. Li, Y., Zhang, Q., Zhang, J., Wu, L., Qi, Y., & Zhou, J. M. (2010). Identification of microRNAs involved in pathogen-associated molecular pattern-triggered plant innate immunity. Plant Physiology, 152(4), 2222e2231. Ma, C., Burd, S., & Lers, A. (2015). Mi R 408 is involved in abiotic stress responses in Arabidopsis. The Plant Journal, 84(1), 169e187. Mallory, A. C., & Vaucheret, H. (2006). Functions of microRNAs and related small RNAs in plants. Nature Genetics, 38(6), S31eS36. Mallory, A., & Vaucheret, H. (2010). Form, function, and regulation of ARGONAUTE proteins. The Plant Cell Online, 22(12), 3879e3889. Ma, J., Wang, Y., & Li, J. (2019). Global identification and analysis of microRNAs involved in salt stress responses in two alfalfa (Medicago sativa ‘Millennium’) lines. Canadian Journal of Plant Science, 100(4), 445e455. https://doi.org/10.1139/cjps-2018-0327 Miura, K., Ikeda, M., Matsubara, A., Song, X. J., Ito, M., Asano, K., … Ashikari, M. (2010). OsSPL14 promotes panicle branching and higher grain productivity in rice. Nature Genetics, 42(6), 545e549. Moffett, P. (2009). Mechanisms of recognition in dominant R gene mediated resistance. Advances in Virus Research, 75, 1e229. Munaweera, T. I. K., Jayawardana, N. U., Rajaratnam, R., & Dissanayake, N. (2022). Modern plant biotechnology as a strategy in addressing climate change and attaining food security. Agriculture & Food Security, 11(1), 1e28. Navarro, L., Dunoyer, P., Jay, F., Arnold, B., Dharmasiri, N., Estelle, M., Voinnet, O., & Jones, J. D. (2006). A plant miRNA contributes to antibacterial resistance by repressing auxin signaling. Science, 312(5772), 436e439. Palauqui, J. C., Elmayan, T., Pollien, J. M., & Vaucheret, H. (1997). Systemic acquired silencing: Transgene-specific post-transcriptional silencing is transmitted by grafting from silenced stocks to non-silenced scions. The EMBO Journal, 16(15), 4738e4745. Pan, W. J., Tao, J. J., Cheng, T., Bian, X. H., Wei, W., Zhang, W. K., Ma, B., Chen, S. Y., & Zhang, J. S. (2016). Soybean miR172a improves salt tolerance and can function as a Future prospective of small RNA molecules 597 long-distance signal. Molecular Plant, 9, 1337e1340. https://doi.org/10.1016/ j.molp.2016.05.010 Parent, J. S., Bouteiller, N., Elmayan, T., & Vaucheret, H. (2015). Respective contributions of Arabidopsis DCL 2 and DCL 4 to RNA silencing. The Plant Journal, 81(2), 223e232. Park, M. Y., Wu, G., Gonzalez-Sulser, A., Vaucheret, H., & Poethig, R. S. (2005). Nuclear processing and export of microRNAs in Arabidopsis. Proceedings of the National Academy of Sciences, 102(10), 3691e3696. Parmar, S., Gharat, S. A., Tagirasa, R., Chandra, T., Behera, L., Dash, S. K., & Shaw, B. P. (2020). Identification and expression analysis of miRNAs and elucidation of their role in salt tolerance in rice varieties susceptible and tolerant to salinity. PLoS One, 15(4), e0230958. https://doi.org/10.1371/journal.pone.0230958 Peltier, A. J., Hatfield, R. D., & Grau, C. R. (2009). Soybean stem lignin concentration relates to resistance to Sclerotinia sclerotiorum. Plant Disease, 93(2), 149e154. Pradhan, B., Naqvi, A. R., Saraf, S., Mukherjee, S. K., & Dey, N. (2015). Prediction and characterization of Tomato leaf curl New Delhi virus (ToLCNDV) responsive novel microRNAs in Solanum lycopersicum. Virus Research, 195, 183e195. Qazi, J., Amin, I., Mansoor, S., Iqbal, M. J., & Briddon, R. W. (2007). Contribution of the satellite encoded gene bC1 to cotton leaf curl disease symptoms. Virus Research, 128(1e2), 135e139. Qi, X., Bao, F. S., & Xie, Z. (2009). Small RNA deep sequencing reveals role for Arabidopsis thaliana RNA-dependent RNA polymerases in viral siRNA biogenesis. PloS One, 4(3), Article e4971. Qiao, F., Yang, Q., Wang, C. L., Fan, Y. L., Wu, X. F., & Zhao, K. J. (2007). Modification of plant height via RNAi suppression of OsGA20ox2 gene in rice. Euphytica, 158(1), 35e45. Qin, Z., Li, C., Mao, L., & Wu, L. (2014). Novel insights from non-conserved microRNAs in plants. Frontiers in Plant Science, 5, 586. Qiu, Z., He, Y., Zhang, Y., Guo, J., & Wang, L. (2018). Characterization of miRNAs and their target genes in He-Ne laser pretreated wheat seedlings exposed to drought stress. Ecotoxicology and Environmental Safety, 164, 611e617. https://doi.org/10.1016/ j.ecoenv.2018.08.077 Ramachandran, V., & Chen, X. (2008). Small RNA metabolism in Arabidopsis. Trends in Plant Science, 13(7), 368e374. Regina, A., Bird, A., Topping, D., Bowden, S., Freeman, J., Barsby, T., … Morell, M. (2006). High-amylose wheat generated by RNA interference improves indices of largebowel health in rats. Proceedings of the National Academy of Sciences, 103(10), 3546e3551. Regina, A., Kosar-Hashemi, B., Ling, S., Li, Z., Rahman, S., & Morell, M. (2010). Control of starch branching in barley defined through differential RNAi suppression of starch branching enzyme IIa and IIb. Journal of Experimental Botany, 61(5), 1469e1482. Rogers, K., & Chen, X. (2013). Biogenesis, turnover, and mode of action of plant microRNAs. The Plant Cell Online, 25(7), 2383e2399. Romer, E., Chebib, S., Bergmann, K. C., Plate, K., Becker, S., Ludwig, C., … Schwab, W. (2020). Tiered approach for the identification of Mal d 1 reduced, well tolerated apple genotypes. Scientific Reports, 10(1), 1e13. Sarkies, P., & Miska, E. A. (2014). Small RNAs break out: The molecular cell biology of mobile small RNAs. Nature Reviews Molecular Cell Biology, 15(8), 525e535. Segal, G., Song, R., & Messing, J. (2003). A new opaque variant of maize by a single dominant RNA-interference-inducing transgene. Genetics, 165(1), 387e397. Sharma, N., Tripathi, A., & Sanan-Mishra, N. (2015). Profiling the expression domains of a rice-specific microRNA under stress. Frontiers in Plant Science, 6, 333. Shivaprasad, P. V., Chen, H. M., Patel, K., Bond, D. M., Santos, B. A., & Baulcombe, D. C. (2012). A microRNA superfamily regulates nucleotide binding siteeleucine-rich repeats and other mRNAs. The Plant Cell Online, 24(3), 859e874. 598 Plant Small RNA in Food Crops Shriram, V., Kumar, V., Devarumath, R. M., Khare, T. S., & Wani, S. H. (2016). MicroRNAs as potential targets for abiotic stress tolerance in plants. Frontiers of Plant Science, 7, 817. Springer, N. (2010). Shaping a better rice plant. Nature Genetics, 42(6), 475e476. Sun, Z., He, Y., Li, J., Wang, X., & Chen, J. (2015). Genome-wide characterization of rice black streaked dwarf virus-responsive microRNAs in rice leaves and roots by small RNA and degradome sequencing. Plant and Cell Physiology, 56(4), 688e699. Sunkar, R., Chinnusamy, V., Zhu, J., & Zhu, J. K. (2007). Small RNAs as big players in plant abiotic stress responses and nutrient deprivation. Trends in Plant Science, 12(7), 301e309. Tuschl, T. (2001). RNA interference and small interfering RNAs. Chembiochem, 2(4), 239e245. Tyagi, S., Sharma, S., Ganie, S. A., Tahir, M., Mir, R. R., & Pandey, R. (2019). Plant microRNAs: Biogenesis, gene silencing, web-based analysis tools and their use as molecular markers. 3 Biotech, 9(11), 1e12. Vaucheret, H. (2008). Plant argonautes. Trends in Plant Science, 13(7), 350e358. Vazquez, F. (2006). Arabidopsis endogenous small RNAs: Highways and byways. Trends in Plant Science, 11(9), 460e468. Voinnet, O. (2009). Origin, biogenesis, and activity of plant microRNAs. Cell, 136(4), 669e687. Wang, Q. (2015). MicroRNA-based biotechnology for plant improvement. Journal of Cellular Physiology, 230(1), 1e15. Wang, Y., Beaith, M., Chalifoux, M., Ying, J., Uchacz, T., Sarvas, C., … Huang, Y. (2009). Shoot-specific down-regulation of protein farnesyltransferase (a-subunit) for yield protection against drought in canola. Molecular Plant, 2(1), 191e200. Wang, T., Chen, L., Zhao, M., Tian, Q., & Zhang, W. H. (2011a). Identification of drought-responsive microRNAs in Medicago truncatula by genome-wide highthroughput sequencing. BMC Genomics, 12(1), 1e11. Wang, X. B., Jovel, J., Udomporn, P., Wang, Y., Wu, Q., Li, W. X., … Ding, S. W. (2011b). The 21-nucleotide, but not 22-nucleotide, viral secondary small interfering RNAs direct potent antiviral defense by two cooperative argonautes in Arabidopsis thaliana. The Plant Cell Online, 23(4), 1625e1638. Wang, S. T., Sun, X. L., Hoshino, Y., Yu, Y., Jia, B., Sun, Z. W., … Zhu, Y. M. (2014). MicroRNA319 positively regulates cold tolerance by targeting OsPCF6 and OsTCP21 in rice (Oryza sativa L.). PLoS One, 9(3), e91357. Wang, S., Wu, K., Yuan, Q., Liu, X., Liu, Z., Lin, X., Zeng, R., Zhu, H., Dong, G., Qian, Q., & Fu, X. (2012). Control of grain size, shape and quality by OsSPL16 in rice. Nature Genetics, 44(8), 950e954. Weise, S. E., Aung, K., Jarou, Z. J., Mehrshahi, P., Li, Z., Hardy, A. C., … Sharkey, T. D. (2012). Engineering starch accumulation by manipulation of phosphate metabolism of starch. Plant Biotechnology Journal, 10(5), 545e554. Wroblewski, T., Piskurewicz, U., Tomczak, A., Ochoa, O., & Michelmore, R. W. (2007). Silencing of the major family of NBSeLRR-encoding genes in lettuce results in the loss of multiple resistance specificities. The Plant Journal, 51(5), 803e818. Wu, J., Yang, Z., Wang, Y., Zheng, L., Ye, R., Ji, Y., … Li, Y. (2015). Viral-inducible Argonaute 18 confers broad-spectrum virus resistance in rice by sequestering a host microRNA. Elife, 4, e05733. Xin, M., Wang, Y., Yao, Y., Xie, C., Peng, H., Ni, Z., & Sun, Q. (2010). Diverse set of microRNAs are responsive to powdery mildew infection and heat stress in wheat (Triticumaestivum L.). BMC Plant Biology, 10(1), 1e11. Future prospective of small RNA molecules 599 Yang, Z., Ebright, Y. W., Yu, B., & Chen, X. (2006). HEN1 recognizes 21e24 nt small RNA duplexes and deposits a methyl group onto the 20 OH of the 30 terminal nucleotide. Nucleic Acids Research, 34(2), 667e675. Yang, C., Li, D., Mao, D., Liu, X. U. E., Ji, C., Li, X., … Zhu, L. (2013). Overexpression of micro RNA 319 impacts leaf morphogenesis and leads to enhanced cold tolerance in rice (Oryza sativa L.). Plant, Cell and Environment, 36(12), 2207e2218. Yang, G., Li, Y., Wu, B., Zhang, K., Gao, L., & Zheng, C. (2019a). MicroRNAs transcriptionally regulate promoter activity in Arabidopsis thaliana. Journal of Integrative Plant Biology, 61(11), 1128e1133. Yang, Z., Wang, Z., Yang, C., Yang, Z., Li, H., & Wu, Y. (2019b). Physiological responses and small RNAs changes in maize under nitrogen deficiency and resupply. Genes Genomics, 41(10), 1183e1194. https://doi.org/10.1007/s13258-019-00848-0 Yang, X., Xie, Y., Raja, P., Li, S., Wolf, J. N., Shen, Q., … Zhou, X. (2011). Suppression of methylation-mediated transcriptional gene silencing by bC1-SAHH protein interaction during geminivirus-betasatellite infection. PLoS Pathogens, 7(10), e1002329. Yang, J., Xing, G., Niu, L., He, H., Guo, D., Du, Q., … Yang, X. (2018). Improved oil quality in transgenic soybean seeds by RNAi-mediated knockdown of GmFAD2-1B. Transgenic Research, 27(2), 155e166. Yang, Q., Zhang, R., Horikawa, I., Fujita, K., Afshar, Y., Kokko, A., … Harris, C. C. (2007). Functional diversity of human protection of telomeres 1 isoforms in telomere protection and cellular senescence. Cancer Research, 67(24), 11677e11686. Yara, A., Yaeno, T., Hasegawa, M., Seto, H., Montillet, J. L., Kusumi, K., … Iba, K. (2007). Disease resistance against Magnaporthe grisea is enhanced in transgenic rice with suppression of u-3 fatty acid desaturases. Plant and Cell Physiology, 48(9), 1263e1274. Yuan, S., Li, Z., Li, D., Yuan, N., Hu, Q., & Luo, H. (2015). Constitutive expression of rice microRNA528 alters plant development and enhances tolerance to salinity stress and nitrogen starvation in creeping bentgrass. Plant Physiology, 169(1), 576e593. Yue, E., Liu, Z., Li, C., Li, Y., Liu, Q., & Xu, J. H. (2017). Overexpression of miR529a confers enhanced resistance to oxidative stress in rice (Oryza sativa L.). Plant Cell Reports, 36(7), 1171e1182. Yu, B., Yang, Z., Li, J., Minakhina, S., Yang, M., Padgett, R. W., … Chen, X. (2005). Methylation as a crucial step in plant microRNA biogenesis. Science, 307(5711), 932e935. Zandalinas, S. I., & Mittler, R. (2022). Plant responses to multifactorial stress combination. New Phytologist, 234(4), 1161e1167. Zhang, W., Gao, S., Zhou, X., Chellappan, P., Chen, Z., Zhou, X., … Jin, H. (2011). Bacteria-responsive microRNAs regulate plant innate immunity by modulating plant hormone networks. Plant Molecular Biology, 75(1), 93e105. Zhang, S., Liu, Y., & Yu, B. (2015a). New insights into pri-miRNA processing and accumulation in plants. Wiley Interdisciplinary Reviews: RNA, 6(5), 533e545. Zhang, B., Pan, X., Cannon, C. H., Cobb, G. P., & Anderson, T. A. (2006). Conservation and divergence of plant microRNA genes. The Plant Journal, 46(2), 243e259. Zhang, B., Pan, X., Cobb, G. P., & Anderson, T. A. (2006). Plant microRNA: A small regulatory molecule with big impact. Developmental Biology, 289(1), 3e16. Zhang, S., Shi, W., Chen, Y., Xu, Z., Zhu, J., Zhang, T., … Zhang, X. (2015b). Overexpression of SYF2 correlates with enhanced cell growth and poor prognosis in human hepatocellular carcinoma. Molecular and Cellular Biochemistry, 410(1), 1e9. Zhang, X., Wang, W., Wang, M., Zhang, H. Y., & Liu, J. H. (2016a). The miR396b of Poncirus trifoliata functions in cold tolerance by regulating ACC oxidase gene expression and modulating ethyleneepolyamine homeostasis. Plant and Cell Physiology, 57(9), 1865e1878. 600 Plant Small RNA in Food Crops Zhang, H., Xia, R., Meyers, B. C., & Walbot, V. (2015). Evolution, functions, and mysteries of plant ARGONAUTE proteins. Current Opinion in Plant Biology, 27, 84e90. Zhang, B., You, C., Zhang, Y., Zeng, L., Hu, J., Zhao, M., & Chen, X. (2020). Linking key steps of microRNA biogenesis by TREX-2 and the nuclear pore complex in Arabidopsis. Native Plants, 6(8), 957e969. https://doi.org/10.1038/s41477-020-0726-z Zhang, J., Zhang, H., Srivastava, A. K., Pan, Y., Bai, J., Fang, J., … Zhu, J. K. (2018). Knockdown of rice microRNA166 confers drought resistance by causing leaf rolling and altering stem xylem development. Plant Physiology, 176(3), 2082e2094. Zhang, X., Zhu, Y., Wu, H., & Guo, H. (2016b). Post-transcriptional gene silencing in plants: A double-edged sword. Science China Life Sciences, 59(3), 271e276. Zhao, J., Yuan, S., Zhou, M., Yuan, N., Li, Z., Hu, Q., … Luo, H. (2019). Transgenic creeping bentgrass overexpressing Osa-miR393a exhibits altered plant development and improved multiple stress tolerance. Plant biotechnology journal, 17(1), 233e251. Zhou, M., & Luo, H. (2013). MicroRNA-mediated gene regulation: Potential applications for plant genetic engineering. Plant Molecular Biology, 83(1), 59e75. Zhou, M., & Luo, H. (2014). Role of microRNA319 in creeping bentgrass salinity and drought stress response. Plant Signaling & Behavior, 9(4), 1375e1391. Zhu, Q. H., Fan, L., Liu, Y., Xu, H., Llewellyn, D., & Wilson, I. (2013). miR482 regulation of NBS-LRR defense genes during fungal pathogen infection in cotton. PLoS One, 8(12), e84390. Zhuo, Y., Gao, G., an Shi, J., Zhou, X., & Wang, X. (2013). miRNAs: biogenesis, origin and evolution, functions on virus-host interaction. Cellular Physiology and Biochemistry, 32(3), 499e510. Zotti, M., Dos Santos, Cagliari, D., Christiaens, O., Taning, C. N. T., & Smagghe, G. (2017). RNA interference technology in crop protection against arthropod pests, pathogens and nematodes. Pest Management Science, 74(6), 1239e1250. Zsögön, A., Peres, L. E., Xiao, Y., Yan, J., & Fernie, A. R. (2022). Enhancing crop diversity for food security in the face of climate uncertainty. The Plant Journal, 109(2), 402e414. Zuo, Z. F., He, W., Li, J., Mo, B., & Liu, L. (2021). Small RNAs: The essential regulators in plant thermotolerance. Frontiers of Plant Science, 12, 726762. Zvereva, A. S., & Pooggin, M. M. (2012). Silencing and innate immunity in plant defense against viral and non-viral pathogens. Viruses, 4(11), 2578e2597. Index ‘Note: Page numbers followed by “f ” indicate figures and “t” indicate tables.’ A ABA responsive element (ABRE) binding protein 2 (ABP2), 50 Abiotic stresses climate change molecular and biotechnological approaches, 57e58, 59te60t plant-hormones (PGRs), 56e57, 57f environmental pollution, 45e46 eukaryotes, 51e52 extremes of temperatures, 46e47 heavy metal stress (HMs), 47 molecular mechanism of, 48f, 50e51 in plants biogenesis, 52e53, 54f micro-RNA (miRNA), 53e56, 55f post transcriptional process, 51e52 salt concentrations, 45e46 signal transduction, 48e50, 48f Abiotic stress resistance Cis-regulatory element editing, 526e527 drought stress, 191 functional genomics approaches, 507e508 heat and cold stress, 191e192 heavy metal, 187e191 non-canonical sRNAs types, 527e529 phosphate deficiency, 192 salinity stress, 192e193, 194t species-specific miRNAs, 529e530 spray-induced gene silencing (SIGS), 527 transcription factors (TFs), 373t ABA-responsive element binding proteins (AREBs), 370e371 cis-elements interaction, 370e371 dehydration responsive element (DRE), 371e372 DREB proteins, 371e372 ethylene-responsive element-binding factor (ERF) proteins, 371e372 RD29A/COR78/LT178 gene, 370e371 transgenic technologies artificial miRNA (amiRNA), 512e513 artificial trans-acting siRNA (atasiRNA), 513e514 drought, 517e522 RNA interference (RNAi), 509e512 safety and specificity, 524e526 salinity, 523e524 short tandem target mimic (STTM), 516e517 temperature-associated stress, 522e523 virus-induced gene silencing (VIGS), 510e511 Abscisic acid (ABA), drought stress tolerance ABA-importing carrier (AIT1), 369 AtDTX50, 369 cell-to-cell transfer, 369 leaves, 370f roots, 367e368, 369f stomata, 369 30 Adapter ligation, 116 50 Adapter ligation, 117 Advanced genomics tools biogenesis, 52e53, 54f micro-RNA (miRNA), 53e56, 55f molecular and biotechnological approaches, 57e58, 59te60t plant-hormones (PGRs), 56e57, 57f AGO protein, 186e187 Agricultural traits biogenesis, classification, and modes of action, 70e72 development and establishment, 69 601 602 Index Agricultural traits (Continued) future perspectives, 86e95 high-throughput deep sequencing, 70 miRNAs, 72, 72f phase transition and flowering time, 79e81 QTLs, 69 reproductive organ development and fertility, 82e84 root architecture establishment, 74e76 seed dormancy and germination, 72e74 shoot architecture, 76e79 size and yield of seeds/tubers, 84e86 targets, 86e95, 87te94t Agriculture. See also Food production climate change, 37e38, 40e41 food patterns, 27 in income-group countries, 28 soil, 35e36 water, 36 Apple stem grooving virus (ASGV) infection, 559e561 Arabidopsis, 81, 218e219, 222 Arachis duranensis, 151 ARGONAUTE 1 (AGO1), 349e350 Artificial manipulation of microRNA (AmiRNAs), 258 Artificial microRNAs (amiRNA) strategy, 512e513 disadvantage, 478e479 endogenous target mimicry (eTM), 479 food crop improvement, 478t pre-amiRNA treatment, 478e479 primary target specificity, 478e479 short tandem target mimic (STTM) strategy, 479 viral satellite DNA vectors, 480 Artificial trans-acting siRNA (atasiRNA) miRNA targets, 513e514 phasiRNAs, 513e514 TASlc locus modification, 514 tomato and Nicotiana benthamiana, 514 Auxin, 221e222 Auxin response factors (ARFs), 132e133 , 325, 405e406 Auxin signaling, 75e76 Azospirillum brasilense, 285 B Bacillus thuringiensis (Bt) crops, 39e40 Bacterial pathogens, 198 Banana (Musa acuminata), salt-responsive small RNAs, 319 Barley (Hordeum vulgare), 144e146, 243, 244te245t deep sequencing and computational analysis, 314 differentially expressed miRNAs, 314e315 Barley leaf stripe (BLS), 145 Barley yellow dwarf virus (BYDV), 145 Beta-N-oxalyl-ami-noalanine-L-alanine (BOAA), 202e203 Biogenesis, 130e132 Hua Enhancer 1 (HEN1), 576e577 mature duplex miRNAs (miRNA/miRNA), 577 miRNA mediated gene silencing, 575e576, 576f transcriptional processing, 575e576 Biotechnology-assisted crop improvement artificial microRNAs (amiRNA) strategy, 478e480 genome editing technology, 480e481 miRNAs agronomic traits, 493e494 crop architecture, 483e486 crop fertility, 487e488 crop flowering, 486e487 crop hybrid breeding, 493e494 crop seed/fruit development, 488e489 crop stress resistance traits, 490e492 RNA interference (RNAi), 477f gene silencing technique, 476 MdSE RNAi transgenic plants, 476e478 tomato fruit ripening, 476e478 transgenes and food crop improvement, 474te476t cisgenesis, 472e473 intragenesis, 472e473 tobacco lines, 473e476 transgenesis, 472e473 transgenic melon plants, 473 Index Biotic stress bacterial pathogens, 198 bacterial stress AtlsiRNA-1, 554e557 elicitors, 558 endoribonucleases DICER or DICER-like (DCL) proteins, 553e554 flg22 therapy, 554e557 immunity modulation, 553e554 miRNA160 and miRNA167, 554e557 R-proteins, 557e558 sRNAs, plant defense, 555te556t circRNA animal and plants, 253 cis sequence and trans factor, 252e253, 254f definition, 252e253 regulatory roles, 255e256, 255t cucumber mosaic virus (CMV), 548e553 fungal resistance, 198, 199t fungal stress, 562e564 insect and nematode resistance, 195e196 loose smut, 548e553 microRNAs Barley, 243, 244te245t biogenesis of, 235e236, 237f maize, 239 mechanism of action, 243e246 miR164, 238 miR168, 238 miR169, 238 miR393, 237e238 miR398, 239, 240te242t rice, 239 soyabean, 243 wheat, 239e243 parasitic weeds, 195 pathogens, 548, 549te552t plant disease control amiRNAs, 258 CRISPR-Cas9, 259 hairpin RNA, 259e260 603 HIGS, 257e258, 259f plant immunity, 234e235 plantepathogen interactions, 256e257, 258f powdery mildew, 548e553 productivity, 233 PTGS, 193 regulation bacteria, 251e252 fungi, 252, 253f viruses and viroids, 249e251, 251f small interfering RNA mRNA, 246 regulatory roles, 248e249, 250t RNAi, 246e247 types of, 247e248, 247f tolerance defense mechanisms, 585 PAMP triggered immunity (PTI), 585e590 plant-pathogen interactions, 587te589t RNA silencing, 585 RNA silencing pathways, 590e591 tolerant food crops biotic factors, 545e546 danger-associated molecular patterns (DAMPs), 546 Dicer like proteins (DCLs), 546e548 effector-triggered immunity (ETI), 546 gene silencing mechanism, 547f genetic engineering, 545e546 pathogen-associated molecular patterns (PAMPs), 546 plant immune responses, 546e548 pleiotropic effect, 545e546 pre-miRNA/sRNA and amplification, 546e548 traditional breeding, 545e546 tomato yellow leaf curl virus (TYLCV), 548e553 viral stress, 558e561 virus resistance, 196e197 Broccoli (Brassica oleracea), salt-responsive small RNAs, 322 604 Index C Capsicum (Capsicum annuum), miRNA networks, 414 Carbon sequestration, 35e36 Cassava Brown Streak Disease (CBSD), 196e197 Chickpea (Cicer arietinum), salt-responsive small RNAs, 316e317 Circular intronic RNAs (circincRNAs), 374 Circular lncRNAs (circncRNAs), 374 Circular RNA (circRNA), 529 animal and plants, 253 cis sequence and trans factor, 252e253, 254f definition, 252e253 regulatory roles, 255e256, 255t Cisgenesis, 472e473 Cis-regulatory element editing, 526e527 Climate change, 507 abiotic stresses molecular and biotechnological approaches, 57e58, 59te60t plant-hormones (PGRs), 56e57, 57f crop growth and yield, 38 greenhouse gases emission, 37e38 pest pressure and, 40e41 sustainable agriculture, 5e7 weather events, 38 Climate-resilient food crop plants, 582, 583te584t Clustered regularly interspaced short palindromic repeat-associated protein-9 nuclease (CRISPR/Cas9), 259 exogenous DNA sequences, 482 single guide RNA (sgRNA), 481e482 soybean miR1514 and miR1509, 482 types, 481e482 Cold deacclimation, 145 Cold stress, 191e192 Conservation agriculture, 38e39 Copper/zinc superoxide dismutase (CSD), 191e192 Cotton (Gossypium hirsutum L.), 153e154 Cotton leaf curl Allahabad virus (CLCuAV) genes, 559e561 Cover cropping, 38e39 Cover crops, 9e10 Cowpea (Vigna unguiculata L.), 150, 318 Crop production, 10e11 Cross-kingdom transfer, 156e164 Cucumber mosaic virus (CMV), 548e553 Cytochrome P450 enzyme, 202e203 Cytokinins, 222 D Danger-associated molecular patterns (DAMPs), 546 Date palm (Phoenix dactylifera), saltresponsive small RNAs, 319 Deforestation, 36e37 Degradation, 26, 37 Dehydration responsive element (DRE), 371e372 Diabrotica virgifera Snf7 gene (DvSnf7), 591 Dicer-like (DCL) proteins, 130e131, 186e187, 546e548 Disease resistance, 222e223 Double-stranded breaks (DSBs), 481 Drought stress, 45e46, 191 abscisic acid (ABA), 517e521 amiRNA, 521e522 miR166 knockdown, 521e522 Drought stress tolerance abscisic acid (ABA) biosynthesis, 366e367, 368f C-15 ABA skeleton, 366e367 transport, 367e369 defense mechanisms, 365e366 non-coding RNAs (ncRNA) long non-coding RNAs (lncRNAs), 373e374 micro RNA (miRNA), 375e377 small interference RNA (siRNA), 377e378 small non-coding RNAs (sncRNAs), 374e375 regulatory gene products, 365e366 restraining factors, 365e366 RNAi technology, 378e379 Index E Effector-triggered immunity (ETI), 234, 272e273, 546 Endogenous target mimicry (eTM), 479 Environmental stress, 48e52, 55f Environmental sustainability, 7e8 Ethylene-responsive element-binding factor (ERF) proteins, 371e372 Expressed sequence tags (ESTs), 152 Extracellular vesicles (EVs), 271 Extra-exon circular ncRNAs (ee-circncRNAs), 374 F Faba bean (Vicia faba), salt-responsive small RNAs, 318 Fatty acids, 223 Fertilizers efficacy, 11e13 mismanagement of, 5e7 Field crops barley, 144e146 cotton, 153e154 cowpea, 150 functional role of, 134te141t maize, 142e143 peanut, 150e152 rice, 133e142 sorghum, 146e147 soybean, 149 sugarcane, 147e149 sunflower, 152 tobacco plant, 154e155 Finger millet (Eleusine coracana), salt-responsive small RNAs, 315e316 Flowering locus T (FT), 79e80 Flowering time, 79e81 Food allergy, 202e203 Food and agriculture organization (FAO), 7, 13e15 Food demand aging, 31e32, 33t migration, 32e34, 34t population growth, 28e31, 31t Food patterns, 27 Food production, 25t aging, 31e32, 33t 605 biodiversity erosion, 26 changes in land cover/use, 36e37 climate change, 37e38, 40e41 economic growth, 28 food patterns, 27 food supply, 26e27, 27t food wastage, 27e28 fresh/underground water depletion, 26 migration, 32e34, 34t natural resources, 35e36 pesticide resistance, 38e40 pest pressure, 38e41 policy-making, 41 population growth, 28e31, 31t regional disparity, 28, 29te30t Food security, 5e9, 15e16 Food supply, 26e27, 27t Food wastage, 27e28 Forward genetics, 507e508 Functional genomics approaches, 507e508 Fungal resistance, 198, 199t Fungal stress Botrytis and Verticillium spp, 563e564 Neurospora crassa, 562e563 plant immunity, 562 powdery mildew fungus Puccinia graminis, 562e563 Sclerotinia sclerotiorum infection, 563e564 Verticillium dahlia, 563e564 G Gamma irradiation, 13e15 Gamma rays, 11e13 Gene Ontology (GO), 255e256 Gene silencing, 256e257 Genetically modified crops, 13 Genome editing technology clustered regularly interspaced short palindromic repeat-associated endonucleases (CRISPR/Cas), 481e482 oligonucleotide-directed mutagenesis (ODM), 480e481 sequence-specific nuclease (SSN), 480e481 synthetic oligonucleotides, 480e481 606 Index Gibberellic acid (GA), 72e73 Global warming, 571 Glutathione peroxidase (GPx), 313e314 Grain development and crop productivity Arabidopsis thaliana genome, 387e389 asterids capsicum (Capsicum annuum), 414 Oleaceae, Sesame, and Actinidiaceae, 414e415 potato (Solanum tuberosum L.), 412e414 tomato (Solanum lycopersicum), 403e411 Brassica, 397e403 citrus agronomic importance, 392e393 boron (B) toxicity, 394 csi-miRNA-435, 393e394 greening disease, 396e397 juice sac granulation, 392e393 miR156 isomiR, 393e394 miRNAome and degradome transcripts, 395e396 fruit development, 390e392 microRNA database (miRBase22), 385e386 orphan miRNAs, 389f phylogenomic clustering, 389e390 rosids, 415e424 translational improvements, 392 Green foxtail (Setaria viridis), salt-responsive small RNAs, 315 Gross Domestic Product (GDP), 25e26, 30e31 H Hairpin RNA (hpRNA), 259e260, 572e574 Heat stress, 191e192 Heavy metal stress (HMs), 47 Heavy metal tolerance, 187e191 Herbaspirillum seropedicae, 285 Heterochromatic small interfering RNA (hc-siRNA), 71, 378 High-throughput next-generation sequencing (HT-NGS) computational analysis, 118e119, 119f conserved/known sRNA identification, 121 differential expression, 123 emergence of, 110 future perspectives, 123e124 novel sRNA identification, 121e123 prediction, 123 pre-processing, 120e121 sRNA library preparation, 113e118, 117f 30 adapter ligation, 116 50 adapter ligation, 117 cDNA strand synthesis, 118 30 HD adapter removal, 116 PCR amplification and size selection, 118 sRNAs extraction isolation, 113 recovery of, 111e113, 112f strategy, 111e113 High-throughput phenotyping (HTP) platforms, 57e58 Horticultural breeding, 11e13 Horticultural crops, 155e156, 157te162t Host induced gene silencing (HIGS), 156e164, 257e258, 259f Host-microbe interaction crop improvement, 285e286, 287t extracellular vesicles, 271 microRNA (miRNA) characteristic of, 274e275 Lin-4, 274 next-generation sequencing, 278e281 plant-bacteria pathogenic interaction, 275e276, 277t plant-fungus pathogenic interaction, 276e278, 279t plant-virus pathogenic interaction, 278, 280t pt-mil-RNAs, 281 plant-microbe beneficial interactions, 283e285 Index small interfering RNA (siRNA) definition, 281e282 plant-bacterial pathogenic interaction, 282e283 plant-viral pathogenic interaction, 283 stress responsive small RNA, 272, 272f Huanglongbing (HLB), 396e397 Hydrological fluctuation, 347 I Indian mustard (Brassica juncea), salt-responsive small RNAs, 322e323 Industrial crops, 3 Innovative approaches, 15e16 Insecticide resistance, 195e196 Insects, 252 Intragenesis, 472e473 Intron derived lncRNAs (incRNAs), 374 Ionizing radiation, 13e15 Ion toxicity, salt stress, 296e297 J Jasmonate (JA), 522e523 L LAGLIDADG homing endonucleases (LHEs), 204e206 Land cover/use, 36e37 Lettuce necrotic yellow virus (LNYV), 559e561 Leucine-rich repeat receptor-like protein kinase (LRR-RLK), 523e524 Lin-4, 274 Long intergenic ncRNAs (lincRNAs), 373e374 Long non-coding RNAs (lncRNAs) drought stress circular lncRNAs (circncRNAs), 374 intron derived lncRNAs (incRNAs), 374 long intergenic ncRNAs (lincRNAs), 373e374 natural antisense transcripts (NATs), 374 607 transposable element derived lncRNAs (TE-derived lncRNAs), 374 monocots, 433 salt tolerance, 356te358t M Magnaporthe grisea, 198 Maize (Zea mays), 142e143, 239, 313e314 Male sterility and fertility, 200e201 Malnutrition, 15e16, 26e27 Microbe-associated molecular pattern (MAMP), 133, 272e273 MicroRNA (miRNA), 53e56, 55f, 70e71, 73e74, 82e84, 574e575 agronomic traits, 493e494 biogenesis, 349e350 biotic stress Barley, 243, 244te245t biogenesis of, 235e236, 237f maize, 239 mechanism of action, 243e246 miR164, 238 miR168, 238 miR169, 238 miR393, 237e238 miR398, 239, 240te242t rice, 239 soyabean, 243 wheat, 239e243 crop architecture, 483e486, 484te485t branching patterns, 485e486 miR164, 486 miR156 and miR529, 485e486 miR393 and miR444, 486 MiR398 and miR172, 486 regulatory networks interaction, 483e485 crop fertility, 487e488 crop flowering, 486e487 crop hybrid breeding, 493e494 crop seed/fruit development miR156, 488e489 miR159 and MiR164, 488 miR397 overexpression, 488 608 Index MicroRNA (miRNA) (Continued) tomato, 489 crop stress resistance traits, 491t abiotic and biotic stress, 490 drought and salinity stress, 491e492 NAC transcription factors, 490e491 temperature stress, 490 drought stress tolerance direct stress response, 375e376 genome-wide analyses, 376e377 miR166 and miR474, 375e376 miR393 and miR159, 375e376 miR397 and miR408, 375e376 reactive oxygen species (ROS), 376e377 superoxide dismutase (SOD), 376e377 transcription factors, 375e376 field crops barley, 144e146 cotton, 153e154 cowpea, 150 functional role of, 134te141t maize, 142e143 peanut, 150e152 rice, 133e142 sorghum, 146e147 soybean, 149 sugarcane, 147e149 sunflower, 152 tobacco plant, 154e155 food crop improvement, 577e579 genes, 572e574 grain development and crop productivity asterids (Solanaceae), 403e415 Brassica, 397e403 citrus, 392e397 crop yield, 435te449t monocots, 424e433 rosids, 415e424 host-microbe interaction characteristic of, 274e275 Lin-4, 274 next-generation sequencing, 278e281 plant-bacteria pathogenic interaction, 275e276, 277t plant-fungus pathogenic interaction, 276e278, 279t plant-virus pathogenic interaction, 278, 280t pt-mil-RNAs, 281 morphogenesis, 215e216 stress resistance traits, 492 MID domain, 186e187 Migration, 32e34, 34t MIR genes, 215e216 Mitochondrial stress 70 protein precursor (MSP), 195e196 Molecular-assisted breeding technologies, 471 Monocots agronomic traits, 424e425 barley embryos, 427 copper homeostasis, 431 epigenetic modification, 433 long noncoding RNAs (lncRNA), 433 miR168, 428e429 miR169, 429 miR172, 426e427 miR390, 429e430 miR396, 430 miR397, 430e431 miR398, 431e432 miR156 expression, 425e426 miR156-SPL module, 424e425 rice grain, miR530, 432 wheat grains, 428 Morphogenesis AGO proteins, 216 disease resistance and stress tolerance, 222e223 fruit development and quality improvement, 221 future prospects, 223e224 hormone signaling, 221e222 maize, 219 miRNAs, 215e216 modes of transport, 217 rice, Arabidopsis, 218e219 RNAi, 217 vegetative growth, 217, 219 Index Mungbean yellow mosaic Indian virus (MYMIV), 149 N Natural antisense transcripts (NATs), 374 Natural antisense transcript small interfering RNAs (NAT-siRNAs), 71e72, 84 Natural resources soil, 35e36 water, 36 Nematode resistance, 195e196 Next Generation Sequencing (NGS), 574e575 Non-coding RNAs (ncRNAs), 129 Non-homologous end joining (NHEJ), 481 Nuclear binding site (NBS) family genes, 255e256 Nuclear pore complexes (NPCs), 186e187 Nuclear technology, 11e13 Nutrient imbalances, 35e36 Nutritional security, 164e165 O Oil crops, 3 Oligonucleotide-directed mutagenesis (ODM), 480e481 Ornithine decarboxylase (ODC) gene, 259e260 Oxidative stress, salt stress, 296e297 P PAMP triggered immunity (PTI), 234, 272e273, 546 Parasitic weeds, 195 Pathogen-associated molecular pattern (PAMP), 234, 272e273, 546 Pattern recognition receptors (PRRs), 234, 272e273 Pattern-triggered immunity, 193 PAZ domain, 186e187 Peanut (Arachis hypogaea L.), 150e152 Pearl millet (Pennisetum glaucum), saltresponsive small RNAs, 316 Pest and nematode resistance, 591 609 Pesticide resistance, 38e40 Pest pressure, 38e41 Phased small interfering RNAs (phasiRNAs), 71 Phase transition, 79e81 Phenylalanine ammonia-lyase (PAL) enzyme, 11e13 Phosphate deficiency, 192 Photoperiod-sensitive male sterility (PSMS), 83e84 Phytohormones, 199e200 PIWI domains, 186e187 Plant architecture, 471e472 Plant-bacteria pathogenic interaction, 275e276, 277t Plant cell receptors (PRR), 234 Plant defense, 234e235, 235f Plant-fungus pathogenic interaction, 276e278, 279t Plant growth-promoting microorganisms (PGPMs), 10e11 Plant homeodomain (PHD), 133e142 Plant-hormones (PGRs), 56e57, 57f Plant immunity, 234e235 Plant-virus pathogenic interaction, 278, 280t Policy-making, 41 Population growth, 28e31, 31t Postharvest loss, 13e15 Post-transcriptional gene silencing (PTGS), 546e548 Post-translational gene silencing (PTGS), 193 Potassium deficiency, 154 Potato (Solanum tuberosum L.), tuberization, 412e414 Potato virus X (PVX), 558e559 Potato virus Y (PVY), 155 Primary miRNA transcripts (priemiRNAs), 70e71 Puccinia triticina mil-RNAs (pt-mil-RNAs), 281 Pumpkin (Cucurbita), salt-responsive small RNAs, 322 Pyrroline-5-carboxylate synthetase (P5CS), 313e314 610 Index Q Quantitative trait loci (QTL), 57e58, 69 R Radish (Raphanus sativus), salt-responsive small RNAs, 320e321 Ralstonia solanacearum, 151 Rapeseed (Brassica napus) auxin response factor (ARF) effector, 399 carpel patterning, 401f deep sequencing, 398e399 DEMETER-like target mRNAs, 401e402 miR159, 398 miR5801, 401e402 miRNA regulation networks, 400f pre-MIR394 transgene, 397e398 seed maturation, 397e401 Reactive oxygen species (ROSs), 49e50 Receptor for the activated C kinase 1 (RACK1) gene, 379 Regulatory role, plants miR164, 238 miR168, 238 miR169, 238 miR393, 237e238 miR398, 239, 240te242t Repeat associated RNA (ra-siRNA), 378 Reproductive organ development and fertility, 82e84 Resource use efficiency, 8e9 Reverse genetics, 508 Rice (Oryza sativa) miR397 overexpression, 133, 239 miR7695 overexpression, 309 plant homeodomain (PHD) finger proteins, 309 salt-responsive small RNAs comparative miRNA profiling, 307 deep sequencing, 308 genes coding regulator and effector proteins, 297 ion homeostasis, 308 microRNAs (miRNAs), 297e307 multicomponent signaling pathways, 308 Rice stripe virus (RSV) infection, 559e561 RNA-dependent RNA polymerases (RdRs), 130e131 RNA-directed DNA methylation (RdDM) pathway, 131e132, 553e554 RNA-induced silencing complex (RISC), 70e71, 131e132, 246e247 RNA interference (RNAi), 109e110, 509e512, 571e572 abiotic stress tolerance drought stress, 191 heat and cold stress, 191e192 heavy metal, 187e191 phosphate deficiency, 192 salinity stress, 192e193, 194t biotic stress, 246e247 bacterial pathogens, 198 fungal resistance, 198, 199t insect and nematode resistance, 195e196 parasitic weeds, 195 PTGS, 193 virus resistance, 196e197 crop development, 183e185, 185f allergen and toxin elimination, 202e203 biofortification, 201e202 male sterility and fertility, 200e201 phenotype change and altered architecture, 203e204 seedless fruit, 199e200 shelf life, 200 crop nutritional value, 580 drought stress tolerance functional genomics analysis, 379 gene detection techniques, 379 genetic makeup manipulation, 379 post-transcriptional regulation, 378e379 receptor for the activated C kinase 1 (RACK1) gene, 379 food crop improvement, 477f Index gene silencing technique, 476 MdSE RNAi transgenic plants, 476e478 tomato fruit ripening, 476e478 future prospective, 204e206 growth, 183e185, 185f mechanism and biogenesis, 186e187, 188f, 189te190t miRNAs, 183e185 morphogenesis, 217 Root architecture establishment, 74e76 Rosids anthocyanins, 421e422 fruit development, 416 miR156, 423 miR159, 419e420 miR169, 418 miR172, 416e417, 422e423, 422f miR319, 420e421 miR395, 421 miR397, 418 miR2950, 417e418 miR4414, 417 miR398 expression, 418e419 miR396-GRF, 417 pear miRNAome, 424 Rosaceae model plants, 415e416 S Salinity stress, 192e193, 194t. See also Salt stress agricultural production, 523e524 leucine-rich repeat receptor-like protein kinase (LRR-RLK), 523e524 wheat miRNA TaemiR408, 524 Salt-responsive small RNAs in food crops, 298te306t banana (Musa acuminata), 319 barley (Hordeum vulgare), 314e315 broccoli (Brassica oleracea), 322 chickpea (Cicer arietinum), 316e317 cowpea (Vigna unguiculata), 318 date palm (Phoenix dactylifera), 319 faba bean (Vicia faba), 318 finger millet (Eleusine coracana), 315e316 green foxtail (Setaria viridis), 315 611 Indian mustard (Brassica juncea), 322e323 maize (Zea mays), 313e314 pearl millet (Pennisetum glaucum), 316 pumpkin (Cucurbita), 322 radish (Raphanus sativus), 320e321 rice (Oryza sativa), 297e308 sesame (Sesamum indicum), 320 soybean (Glycine max), 318 sweet potato (Ipomoea batatas), 321 wheat (Triticum aestivum), 309e312 yuzu (Citrus junos), 319e320 Salt stress adverse effects, 295 common salt-responsive small RNAs Ca+2 mediated signaling proteins, 325 food crops, 323, 324f MiR169, 325e326 miR396 and miR167, 323e324 miR172/IDS1 regulatory module, 325 phytohormone signaling, 325 salt stress treatments, 323 crops growth and production restriction ion toxicity, 296e297 microRNAs (miRNAs), 295e296 non-coding RNAs (ncRNAs), 295e296 oxidative stress, 296e297 reprogramming, 295 signal transduction pathways, plants, 295 tolerance improvement, small RNAs manipulation miR156, 335 miR172, 334e335 miR396, 336 miR528, 333e334 miR172a, 335 miR164 family, 326 miR396 family members, 333 miR393 members, 333 miR156/SPL module, 335e336 TaemiR408, 334 target genes, 327te332t Seed dormancy, 72e74 612 Index Seed germination, 72e74 Seedless fruit development, 199e200 Sequence-specific nuclease (SSN), 481 Sesame (Sesamum indicum), salt-responsive small RNAs, 320 Shoot architecture, 76e79 Short interfering RNAs (siRNAs), 575 definition, 281e282 plant-bacterial pathogenic interaction, 282e283 plant-viral pathogenic interaction, 283 Short tandem target mimic (STTM), 78, 164, 479, 516e517 Signal transduction, 48e50, 48f SlARF7 repression, 199e200 Small interfering RNA (siRNA), 53, 130, 571e572 crop improvement, 579e580 drought stress tolerance, 377e378 heterochromatic siRNA (hc-siRNA), 378 repeat associated RNA (ra-siRNA), 378 trans-acting siRNAs (ta-siRNAs), 377e378 viral siRNA (v-siRNA), 378 vs. micro RNA (miRNA), 577, 578t mRNA, 246 nutritional enhancement, 580e582 regulatory roles, 248e249, 250t RNAi, 246e247 secondary, 574 synthesis, 377 types of, 247e248, 247f Small non-coding RNAs (sncRNAs), drought stress tolerance, 374e375 Social protection, 32 Soil amelioration, 10e11 Soil erosion, 35e36 Soil health, 8e10 Soil organic carbon change, 35e36 Sorghum (Sorghum bicolor (L.), 146e147 Soybean (Glycine max), 149, 243, 318 Spermidine (Spd), 523 Spray-induced gene silencing (SIGS), 286, 527 sRNA-based gene silencing approach, 508 Stakeholders, 7e8 Stress, 132e133 Stress tolerance, 222e223 Sucrose phosphatase (SPP), 201e202 Sucrose-phosphate synthase (SPS), 201e202 Sugarcane (Saccharum sp.), 147e149 Sugarcane bacilliform virus (SCBV), 148e149 Sunflower (Helianthus annuus), 152 Sustainable agriculture challenges, 5e8 consumers and retailers, 8e9 cover crops, 9e10 efficiency and output ability, 16e17 food security, 8e9 fruits, 4 genetically modified crops, 13 industrial crops, 3 innovation, 15e16 nuclear technology, 11e13 oil crops, 3 postharvest loss, 13e15 seeds and nuts, 4e5 for soil amelioration, and sustainable crop production, 10e11 vegetables crops, 4 Sustainable development goal (SDG), 26, 35e36 Sweet potato (Ipomoea batatas), salt-responsive small RNAs, 321 Syncytium, 195e196 T Temperature-associated stress, 522e523 Tobacco (Nicotiana tabacum), 154e155 Tobacco Streak Virus (TSVs), 196e197 Tomato (Solanum lycopersicum) ABC model, 408e409 BLIND/BL and FISTULATA/FIS genes, 408e409 lncRNA1459 and lncRNA2155 p, 411 miR160, 405e406 MIR164, 406e407 miR171, 409e410 Index miR396, 410 miR1917, 411 miR172 isoforms, 410 miR166-SlHB15A/PF1 module, 407e408, 408f miR156-SPL module, 403e404 miR156/157 targets SPL family, 404 ovule development, 404e405, 405f Tomato yellow leaf curl virus (TYLCV), 548e553 Trans-acting siRNAs (ta-siRNAs), small interference RNA (siRNA), 377e378 Trans-acting siRNA synthesis, 574e575 Transcription activator-like effector nucleases (TALENs), 204e206, 481 Transcriptional gene silencing (PTGS), 546e548 Transcription Coupled Export 2 (TREX2), 186e187 Transcription factors (TFs), 132e133 Transferred DNA (T-DNA), 508 Transposable element derived lncRNAs (TE-derived lncRNAs), 374 tRNA derived fragments (tRFs), 256e257 Tyrosine phosphatase (TP), 195e196 U Ultra-low gossypol levels (ULGCS), 202e203 Urbanization, 32e34, 37 V Vegetables crops, 4 Verticillium dahliae, 281 Verticillium longisporum, 276e278 Viral siRNA (v-siRNA), drought stress tolerance, 378 Viral stress AGO2 gene control, 559e561 apple stem grooving virus (ASGV) infection, 559e561 cotton leaf curl Allahabad virus (CLCuAV) genes, 559e561 613 cotton leafroll dwarf virus (CLRDV) agent, 559e561 lettuce necrotic yellow virus (LNYV), 559e561 pathogen effectors-triggered immunity, 561 Potato virus X (PVX), 558e559 rice stripe virus (RSV) infection, 559e561 Virus-activated siRNAs (vasiRNAs), 283 Virus-derived small-interfering RNAs (vsiRNAs), 278 Virus induced gene silencing (VIGS), 156e164, 249e251, 257, 309e310, 391e392, 510e511 Virus resistance, 196e197 W Waterlogging stress abiotic and biotic stresses, 348 agronomic traits, 347e348 genetic engineering, 347 miRNAs and morphological adaptations arabidopsis roots, 353 auxin response factor (ARF) expression, 353 auxin signaling, 352e353 cis-regulatory elements, 350e351 environmental signals, 350e351 flooding-responsive miRNAs, 350e351 gene expression, 347e348 maize roots, 351 miR393, 354 miR159 and miR319 families, 355 MiR390 and tasiRNA3, 353 miR156 expression, 354 miR167 targets, 353 target mRNAs, 350e351 TIR1 transcripts, 355 transcription factors (TFs), 348 plant growth and development flooding, 349 hypoxia, 349 submergence, 349 toxic metabolite accumulation, 349 pleiotropic effect, 347 yield loss, 347 614 Index Water use efficiency, 36 Wheat (Triticum aestivum), 239e243 salt-responsive small RNAs bioinformatics tools, 311 bread wheat cultivar (JN177), 309e310 KEGG analysis, 309e310 miRNA profiles, root, 309 NGS technologies, 309 real-time PCR and northern blot analysis, 311e312 salt stressed SR3 library (SR3S), 309e310 Tae-miR319 and tae-miR9666b-3p, 310e311 virus-induced gene silencing (VIGS) method, 309e310 salt tolerant cultivars, 309 Y Yellow dragon disease, 396e397 Yellow mosaic virus (YMV), 149 Yuzu (Citrus junos), salt-responsive small RNAs, 319e320 Z Zero tillage, 38e39 Zinc finger nucleases (ZFNs), 204e206, 481