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Plant cell wall biosynthesis: making the bricks Monika S. Doblin, Claudia E. Vergara, Steve Read, Ed Newbigin and Antony Bade 6.1 Introduction 6.1. 1 Importance of polysaccharide synthesis Each cell in a plant is surrounded by an... more
Plant cell wall biosynthesis: making the bricks Monika S. Doblin, Claudia E. Vergara, Steve Read, Ed Newbigin and Antony Bade 6.1 Introduction 6.1. 1 Importance of polysaccharide synthesis Each cell in a plant is surrounded by an extracellular matrix called the cell wall that is ...
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Iben Sørensen, Filomena A. Pettolino, Sarah M. Wilson, Monika S. Doblin, Bo Johansen, Antony Bacic and William G. T. Willats Department of Biology, The University of Copenhagen, Ole Maaløes vej 5, Copenhagen DK-2200, Denmark, Plant Cell... more
Iben Sørensen, Filomena A. Pettolino, Sarah M. Wilson, Monika S. Doblin, Bo Johansen, Antony Bacic and William G. T. Willats Department of Biology, The University of Copenhagen, Ole Maaløes vej 5, Copenhagen DK-2200, Denmark, Plant Cell Biology Research Centre, School of Botany, University of Melbourne, Melbourne, Vic. 3010, Australia, Department of Biology, The University of Copenhagen, Østerfarimagsgade 2D, Copenhagen DK-1353, Denmark, and Australian Centre for Plant Functional Genomics, School of Botany, University of Melbourne, Melbourne, Vic. 3010, Australia
SummaryBarley (Hordeum vulgare L) grain is comparatively rich in (1,3;1,4)-β-glucan, a source of fermentable dietary fibre that protects against various human health conditions. However, low grain (1,3;1,4)-β-glucan content is preferred... more
SummaryBarley (Hordeum vulgare L) grain is comparatively rich in (1,3;1,4)-β-glucan, a source of fermentable dietary fibre that protects against various human health conditions. However, low grain (1,3;1,4)-β-glucan content is preferred for brewing and distilling. We took a reverse genetics approach, using CRISPR/Cas9 to generate mutations in members of the Cellulose synthase-like (Csl) gene superfamily that encode known (HvCslF6 and HvCslH1) and putative (HvCslF3 and HvCslF9) (1,3;1,4)-β-glucan synthases. Resultant mutations ranged from single amino acid (aa) substitutions to frameshift mutations causing premature stop codons, and led to specific differences in grain morphology, composition and (1,3;1,4)-β-glucan content. (1,3;1,4)-β-Glucan was absent in the grain of cslf6 knock-out lines whereas cslf9 knock-out lines had similar (1,3;1,4)-β-glucan content to WT. However, cslf9 mutants showed changes in the abundance of other cell wall-related monosaccharides compared to WT. Thousa...
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ABSTRACTThe mechanisms underlying rootzone-localised responses to salinity stress during early stage of barley development remains fragmentary and elusive. Here, we performed a comprehensive detection of the multi-root-omes... more
ABSTRACTThe mechanisms underlying rootzone-localised responses to salinity stress during early stage of barley development remains fragmentary and elusive. Here, we performed a comprehensive detection of the multi-root-omes (transcriptomes, metabolomes, lipidomes) of a domesticated barley cultivar (Clipper) and a landrace (Sahara) with seedling root growth maintained and restricted in response to salt stress, respectively. Novel generalized linear models were designed to determine differentially expressed genes (DEG) or abundant metabolites (DAM) specific to salt treatments, genotypes, or rootzones (meristematic Z1, elongation Z2, maturation Z3). Based on pathway over-representation of the DEG and DAM, phenylpropanoid biosynthesis is the most statistically over-represented biological pathways among all salinity responses observed. Together with the histological evidence, an intense salt-induced lignin impregnation was found only at the stelic cell wall of Clipper Z2, comparing to a ...
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One of the most important interactions within the paracrystalline matrix of the plant cell wall occurs between cellulose microfibrils to allow for the formation of larger diameter macrofibrils. Here, we have used computational techniques... more
One of the most important interactions within the paracrystalline matrix of the plant cell wall occurs between cellulose microfibrils to allow for the formation of larger diameter macrofibrils. Here, we have used computational techniques to investigate how different microfibril surfaces might adsorb onto one another. Molecular dynamics simulations show that limited direct adsorption occurs between non-polar surfaces and free energy of desorption calculations suggest this is due to a high energy barrier for the removal of a single layer of water between these surfaces. Further, it is predicted that when microfibril aggregation occurs, significant conformational changes take place at the surfaces of interaction involving O2 dihedral angles, exocyclic C6 conformation, and microfibril chain tilt. It is more likely that direct interactions initially take place between polar (110) surfaces, and that surface interactions occur between the same types of surface, such as 110 to 110, 1–10 to 1–10 or 200 to 100, where hydrogen bonds can be formed, to stabilise the aggregate. Additionally, we have identified that for the exocyclic group of a glucose residue to change conformation in origin layers, the O2 dihedral in residues before and adjacent to the glucose must rotate to a more cis-like conformation, compared to the trans-like conformation observed in crystalline cellulose. This change in exocyclic conformation occurs due to a slight shift in adjacent chains that preferentially stabilises the exocyclic conformation change in a specific glucose residue of each cellobiose repeat.
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Oat (Avena sativa) is a cereal crop whose grains are rich in (1,3; 1,4)-β-D-glucan (mixed linkage glucan or MLG), a soluble dietary fiber. In our study, we analyzed oat endosperm development in two Canadian varieties with differing MLG... more
Oat (Avena sativa) is a cereal crop whose grains are rich in (1,3; 1,4)-β-D-glucan (mixed linkage glucan or MLG), a soluble dietary fiber. In our study, we analyzed oat endosperm development in two Canadian varieties with differing MLG content and nutritional value. We confirmed that oat undergoes a nuclear type of endosperm development but with a shorter cellularisation phase than barley (Hordeum vulgare). Callose and cellulose were the first polysaccharides to be detected in the early anticlinal cell walls at 11 days post-emergence (DPE) of the panicle. Other polysaccharides such as heteromannan and homogalacturonan were deposited early in cellularisation around 12 DPE after the first periclinal walls are laid down. In contrast to barley, heteroxylan deposition coincided with completion of cellularisation and was detected from 14 DPE but was only detectable after demasking. Notably, MLG was the last polysaccharide to be laid down at 18 DPE within the differentiation phase, rather ...
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Arabinogalactan-proteins (AGPs) are members of the hydroxyproline-rich glycoprotein (HRGP) superfamily, a group of highly diverse proteoglycans that are present in the cell wall, plasma membrane as well as secretions of almost all plants,... more
Arabinogalactan-proteins (AGPs) are members of the hydroxyproline-rich glycoprotein (HRGP) superfamily, a group of highly diverse proteoglycans that are present in the cell wall, plasma membrane as well as secretions of almost all plants, with important roles in many developmental processes. The role of GALT8 (At1g22015), a Glycosyltransferase-31 (GT31) family member of the Carbohydrate-Active Enzyme database (CAZy), was examined by biochemical characterization and phenotypic analysis of a galt8 mutant line. To characterize its catalytic function, GALT8 was heterologously expressed in tobacco leaves and its enzymatic activity tested. GALT8 was shown to be a β-(1,3)-galactosyltransferase (GalT) that catalyzes the synthesis of a β-(1,3)-galactan, similar to the in vitro activity of KNS4/UPEX1 (At1g33430), a homologous GT31 member previously shown to have this activity. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) confirmed the products were of 2-6 degree of polymerisation...
Secondary cell walls (SCW) in stem xylem cells provide mechanical strength and structural support for growth. SCW thickening is light- regulated and varies under different light growth conditions. Our previous study revealed that blue... more
Secondary cell walls (SCW) in stem xylem cells provide mechanical strength and structural support for growth. SCW thickening is light- regulated and varies under different light growth conditions. Our previous study revealed that blue light enhances SCW thickening through the activity of MYC2 directed by CRYPTOCHROME1 (CRY1) signaling in stem xylary fiber cells. In this study, we demonstrate that the low ratio of red: far-red light (R:FR) of the shaded light condition inhibits SCW thickening in the inflorescence stem of Arabidopsis. Phytochrome B (PHYB) plays a dominant role in perceiving the R:FR balance. Under white and red-light conditions, phyB mutants display thinner SCWs in xylary fibers, but thicker SCWs are deposited in the PHYTOCHROME INTERACTING FACTORS (PIFs) quadruple mutant pif1pif3pif4pif5 (pifq), suggesting involvement of the PHYB-PIFs signaling module in regulating SCW thickening. Interaction of PIF4 with MYC2 affects MYC2 localization in nuclei and inhibits its tran...
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Plant proteins that are secreted without a classical signal peptide leader sequence are termed leaderless secretory proteins (LSPs) and are implicated in both plant development and (a)biotic stress responses. In plant proteomics... more
Plant proteins that are secreted without a classical signal peptide leader sequence are termed leaderless secretory proteins (LSPs) and are implicated in both plant development and (a)biotic stress responses. In plant proteomics experimental workflows, identification of LSPs is hindered by the possibility of contamination from other subcellar compartments upon purification of the secretome. Applying machine learning algorithms to predict LSPs in plants is also challenging due to the rarity of experimentally validated examples for training purposes. This work attempts to address this issue by establishing criteria for identifying potential plant LSPs based on experimental observations and training random forest classifiers on the putative datasets. The resultant plant protein database LSPDB and bioinformatic prediction tools LSPpred and SPLpred are available at lsppred.lspdb.org. The LSPpred and SPLpred modules are internally validated on the training dataset, with false positives co...
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A polysaccharide-rich mucilage is released from the seed coat epidermis of numerous plant species and has been intensively studied in the model plant Arabidopsis. This has led to the identification of a large number of genes involved in... more
A polysaccharide-rich mucilage is released from the seed coat epidermis of numerous plant species and has been intensively studied in the model plant Arabidopsis. This has led to the identification of a large number of genes involved in the synthesis, secretion and modification of cell wall polysaccharides such as pectin, hemicellulose and cellulose being identified. These genes include a small network of transcription factors (TFs) and transcriptional co-regulators, that not only regulate mucilage production, but epidermal cell differentiation and in some cases flavonoid biosynthesis in the internal endothelial layer of the seed coat. Here we focus on the function of these regulators and propose a simplified model where they are assigned to a hierarchical gene network with three regulatory levels (tiers) as a means of assisting in the interpretation of the complexity. We discuss limitations of current methodologies and highlight some of the problems associated with defining the fun...
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Defective Kernel1 (DEK1) is a plant-specific calpain involved in epidermis specification and maintenance. DEK1 regulation of the epidermal cell wall is proposed to be key to ensure tissue integrity and coordinated growth. Changes in the... more
Defective Kernel1 (DEK1) is a plant-specific calpain involved in epidermis specification and maintenance. DEK1 regulation of the epidermal cell wall is proposed to be key to ensure tissue integrity and coordinated growth. Changes in the expression of DEK1 are correlated with changes in the expression of cell wall-related genes. For example, we have found that Lipid transfer protein 3 (LTP3), EXPANSIN 11 (EXP11), and an AP2 transcription factor (AP2TF) are misexpressed in plants with constitutively altered levels of DEK1 activity. RT-qPCR studies show that LTP3 and AP2TF may respond to a DEK1-generated signal whereas EXP11 is not altered immediately after dexamethasone induction of CALPAIN suggesting it is not in the direct signalling pathway downstream of DEK1. Our data suggest these genes are regulated by a feedback mechanism in response to DEK1-induced changes in the cell wall, and contribute to the phenotypes seen in plants with altered DEK1 expression.
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Intrinsically disordered proteins (IDPs) are functional proteins that lack a well-defined three dimensional structure. The study of IDPs is a rapidly growing area as the crucial biological functions of more of these proteins are... more
Intrinsically disordered proteins (IDPs) are functional proteins that lack a well-defined three dimensional structure. The study of IDPs is a rapidly growing area as the crucial biological functions of more of these proteins are uncovered. In plants, IDPs are implicated in plant stress responses, signalling and regulatory processes. A superfamily of cell wall proteins, the hydroxyproline-rich glycoproteins (HRGPs), have characteristic features of IDPs. Their protein backbones are rich in the disordering amino acid proline, they contain repeated sequence motifs and extensive post-translational modifications (glycosylation) and have been implicated in many biological functions. HRGPs are evolutionarily ancient, having been isolated from the protein-rich walls of chlorophyte algae to the cellulose-rich walls of embryophytes. Examination of HRGPs in a range of plant species should provide valuable insights into how they have evolved. Commonly divided into the arabinogalactan-proteins (A...
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The carbohydrate-rich cell walls of land plants and algae have been the focus of much interest given the value of cell wall based products to our current and future economies. Hydroxyproline-rich glycoproteins (HRGPs), a major group of... more
The carbohydrate-rich cell walls of land plants and algae have been the focus of much interest given the value of cell wall based products to our current and future economies. Hydroxyproline-rich glycoproteins (HRGPs), a major group of wall glycoproteins, play important roles in plant growth and development, yet little is known about how they have evolved in parallel with the polysaccharide components of walls. We investigate the origins and evolution of the HRGP superfamily, which is commonly divided into three major multigene families: the arabinogalactan-proteins (AGPs), extensins (EXTs) and proline-rich proteins (PRPs). Using MAAB, a newly developed bioinformatics pipeline, we identified HRGPs in sequences from the 1000 plants (1KP) transcriptome project (www.onekp.com). Our analyses provide new insights into the evolution of HRGPs across major evolutionary milestones, including the transition to land and early radiation of angiosperms. Significantly, data mining reveals the ori...
Research Interests: Botany, Molecular Evolution, Biology, Medicine, Transcriptome, and 13 moreBiological Sciences, Phylogeny, Plant Physiology, P-glycoprotein, Plants, Molecular Phylogenetics and Evolution, Glycoproteins, Time Factors, Hydroxyproline, Amino Acid Sequence, Cell Wall, PLANT PROTEINS, and Likelihood Functions
Pollen exine is essential for protection from the environment of the male gametes of seed-producing plants, but its assembly and composition remain poorly understood. We previously characterized Arabidopsis (Arabidopsis thaliana) mutants... more
Pollen exine is essential for protection from the environment of the male gametes of seed-producing plants, but its assembly and composition remain poorly understood. We previously characterized Arabidopsis (Arabidopsis thaliana) mutants with abnormal pollen exine structure and morphology that we named kaonashi (kns). Here we describe the identification of the causal gene of kns4 that was found to be a member of the CAZy glycosyltransferase 31 gene family, identical to UNEVEN PATTERN OF EXINE1, and the biochemical characterization of the encoded protein. The characteristic exine phenotype in the kns4 mutant is related to an abnormality of the primexine matrix laid on the surface of developing microspores. Using light microscopy with a combination of type II arabinogalactan (AG) antibodies and staining with the arabinogalactan-protein (AGP)-specific β-Glc Yariv reagent, we show that the levels of AGPs in the kns4 microspore primexine are considerably diminished, and their location di...
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In proteomic analyses of the plant secretome, the presence of putative leaderless secretory proteins (LSPs) is difficult to confirm due to the possibility of contamination from other sub-cellular compartments. In the absence of a... more
In proteomic analyses of the plant secretome, the presence of putative leaderless secretory proteins (LSPs) is difficult to confirm due to the possibility of contamination from other sub-cellular compartments. In the absence of a plant-specific tool for predicting LSPs, the mammalian-trained SecretomeP has been applied to plant proteins in multiple studies to identify the most likely LSPs. This study investigates the effectiveness of using SecretomeP on plant proteins, identifies its limitations and provides a benchmark for its use. In the absence of experimentally verified LSPs we exploit the common-feature hypothesis behind SecretomeP and use known classically secreted proteins (CSPs) of plants as a proxy to evaluate its accuracy. We show that, contrary to the common-feature hypothesis, plant CSPs are a poor proxy for evaluating LSP detection due to variation in the SecretomeP prediction scores when the signal peptide (SP) is modified. Removing the SP region from CSPs and comparin...
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Cell walls and cellular turgor pressure shape and suspend the bodies of all vascular plants. In response to attack by fungal and oomycete pathogens, which usually breach their host's cell walls by mechanical force or by secreting... more
Cell walls and cellular turgor pressure shape and suspend the bodies of all vascular plants. In response to attack by fungal and oomycete pathogens, which usually breach their host's cell walls by mechanical force or by secreting lytic enzymes, plants often form local cell wall appositions (papillae) as an important first line of defence. The involvement of cell wall biosynthetic enzymes in the formation of these papillae is still poorly understood, especially in cereal crops. To investigate the role in plant defence of a candidate gene from barley (Hordeum vulgare) encoding cellulose synthase-like D2 (HvCslD2), we generated transgenic barley plants in which HvCslD2 was silenced through RNA interference (RNAi). The transgenic plants showed no growth defects but their papillae were more successfully penetrated by host-adapted, virulent as well as avirulent nonhost isolates of the powdery mildew fungus Blumeria graminis. Papilla penetration was associated with lower contents of ce...
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As the most abundant biopolymer on Earth, cellulose is a key structural component of the plant cell wall. Cellulose is produced at the plasma membrane by cellulose synthase (CesA) complexes (CSCs), which are assembled in the endomembrane... more
As the most abundant biopolymer on Earth, cellulose is a key structural component of the plant cell wall. Cellulose is produced at the plasma membrane by cellulose synthase (CesA) complexes (CSCs), which are assembled in the endomembrane system and trafficked to the plasma membrane. While several proteins that affect CesA activity have been identified, components that regulate CSC assembly and trafficking remain unknown. Here we show that STELLO1 and 2 are Golgi-localized proteins that can interact with CesAs and control cellulose quantity. In the absence of STELLO function, the spatial distribution within the Golgi, secretion and activity of the CSCs are impaired indicating a central role of the STELLO proteins in CSC assembly. Point mutations in the predicted catalytic domains of the STELLO proteins indicate that they are glycosyltransferases facing the Golgi lumen. Hence, we have uncovered proteins that regulate CSC assembly in the plant Golgi apparatus.
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UDP-xylose (UDP-Xyl) is the Xyl donor used in the synthesis of major plant cell wall polysaccharides such as xylan (as a backbone-chain monosaccharide) and xyloglucan (as a branching monosaccharide). The biosynthesis of UDP-Xyl from... more
UDP-xylose (UDP-Xyl) is the Xyl donor used in the synthesis of major plant cell wall polysaccharides such as xylan (as a backbone-chain monosaccharide) and xyloglucan (as a branching monosaccharide). The biosynthesis of UDP-Xyl from UDP-glucuronic acid (UDP-GlcA) is irreversibly catalyzed by UDP-glucuronic acid decarboxylase (UXS). Until now, little is known about the physiological roles of AtUXS in plants. Here, we report that AtUXS1, AtUXS2 and AtUXS4 are located in the Golgi apparatus whereas AtUXS3, AtUXS5 and AtUXS6 are located in the cytosol. Although all six single AtUXS T-DNA mutants and the uxs1usx2uxs4 triple mutant show no obvious phenotype, the uxs3uxs5uxs6 triple mutant has an irregular xylem phenotype. Monosaccharide analysis showed that Xyl levels decreased in the uxs3uxs5uxs6 mutant and linkage analysis confirmed that the content of xylan in the uxs3xus5uxs6 mutant declined, indicating that UDP-Xyl from cytosol AtUXS participates in xylan synthesis. Gel permeation ch...
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Nanoindentation experiments are performed using an atomic force microscope (AFM) to quantify the spatial distribution of mechanical properties of plant cell walls at nanometre length scales. At any specific location on the cell wall, a... more
Nanoindentation experiments are performed using an atomic force microscope (AFM) to quantify the spatial distribution of mechanical properties of plant cell walls at nanometre length scales. At any specific location on the cell wall, a complex (non-linear) force-indentation response occurs that can be deconvoluted using a unique multiregime analysis (MRA). This allows an unambiguous evaluation of the local transverse elastic modulus of the wall. Nanomechanical measurements on suspension-cultured cells (SCCs), derived from Italian ryegrass (Lolium multiflorum) starchy endosperm, show three characteristic modes of deformation and a spatial distribution of elastic moduli across the surface. 'Soft' and 'hard' domains are found across length scales between 0.1 µm and 3 µm, which is well above a typical pore size of the polysaccharide mesh. The generality and wider applicability of this mechanical heterogeneity is verified through in planta characterization on leaf epiderm...
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Sucrose non-fermenting-1 (SNF1)-related kinase 1 (SnRK1) lies at the heart of metabolic homeostasis in plants and is crucial for normal development and response to stress. Evolutionarily related to SNF1 in yeast and AMP-activated kinase... more
Sucrose non-fermenting-1 (SNF1)-related kinase 1 (SnRK1) lies at the heart of metabolic homeostasis in plants and is crucial for normal development and response to stress. Evolutionarily related to SNF1 in yeast and AMP-activated kinase (AMPK) in mammals, SnRK1 acts protectively to maintain homeostasis in the face of fluctuations in energy status. Despite a conserved function, the structure and regulation of the plant kinase differ considerably from its relatively well-understood opisthokont orthologues. In this review, we highlight the known plant-specific modes of regulation involving SnRK1 together with new insights based on a 3D molecular model of the kinase. We also summarise how these differences from other orthologues may be specific adaptations to plant metabolism, and offer insights into possible avenues of future inquiry into this enigmatic enzyme.