PLANT SMALL RNA
IN FOOD CROPS
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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
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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
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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.
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SECTION 1
Basics
1
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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
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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
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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/.
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our traditional culture: Worldwide. Journal of Medicinal Plants Studies, 6, 116e122.
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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
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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.
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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).
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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.
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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
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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.
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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
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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
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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
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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
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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.
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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
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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
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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.
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SECTION 2
Small RNA from food
crops: mechanism
and regulation
107
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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
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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.
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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
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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
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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.
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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.
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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
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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
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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
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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).
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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
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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.,
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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
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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
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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).
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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
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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).
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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).
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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
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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
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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.
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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
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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) (Secic &
Kogel, 2021) (Fig. 7.1).
Figure 7.1 Schematic representation of the possible role of RNAi in crop development.
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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
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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
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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.
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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
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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
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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
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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).
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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.
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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).
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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).
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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
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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.
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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.
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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)
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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.
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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
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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).
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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
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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
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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
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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 (Kriznik 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
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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.
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microRNA-like RNAs in a plant pathogenic fungus Sclerotinia sclerotiorum by highthroughput sequencing. Molecular Genetics and Genomics, 287(4), 275e282.
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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
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(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
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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
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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).
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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
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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
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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
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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.
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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).
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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
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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).
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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
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All rights reserved.
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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,
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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).
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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
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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
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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
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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).
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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
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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
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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
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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
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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
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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).
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& 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,
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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.
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(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
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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.)
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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.
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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.
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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
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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
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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)
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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
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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.
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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).
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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.
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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
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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
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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
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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
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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
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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
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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
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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
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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,
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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
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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.)
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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.
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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),
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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
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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
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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
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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
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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
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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)?
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SECTION 3
Applications and
future scope
469
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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
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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
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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.
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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
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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.
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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
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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
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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,
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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
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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
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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,
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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
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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
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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.
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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
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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).
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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
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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”.
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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
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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).
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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-
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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
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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
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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.
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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