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Chitinases degrade chitin, a linear homopolymer of b-1,4-linked N-acetyl-Dglucosamine (GlcNAc) residues found in the cell walls of fungi and the exoskeletons of arthropods. They are secreted by the roots into the rhizosphere, a complex and dynamic environment where intense nutrient exchange occurs between plants and microbes. Here we modeled, expressed, purified, and characterized Zea mays and Oryza sativa root chitinases, and the chitinase of a symbiotic bacterium, Chitinophaga oryzae 1303 for their activities with chitin, di-, tri-, and tetra-saccharides and Aspergillus niger, with the goal of determining their role(s) in the rhizosphere and better understanding the molecular mechanisms underlying plant-microbe interactions. We show that Zea mays basic endochitinase (ZmChi19A) and Oryza sativa chitinase (OsChi19A) are from the GH19 chitinase family. The Chitinophaga oryzae 1303 chitinase (CspCh18A) belongs to the GH18 family. The three enzymes have similar apparent KM values of (20-40 µM) for the substrate 4-MU-GlcNAc3. They vary in their pH and temperature optima with OsChi19A activity optimal between pH 5-7 and 30-40°C while ZmChi19A and CspCh18A activities were optimal at pH 7-9 and 50-60°C. Modeling and site-directed mutation of ZmChi19A identified the catalytic cleft and the active residues E147 and E169 strategically positioned at ~8.6Å from each other in the folded protein. Cleavage of 4-MU-GlcNAc3 was unaffected by the absence of the CBD but diminished in the absence of the flexible C-terminal domain. However, unlike for the soluble substrate, the CBD and the newly identified flexible C-terminal domain were vital for inhibiting Aspergillus niger growth. The results are consistent with the involvement of the plant chitinases in defense against pathogens like fungi that have chitin exoskeletons. In summary, we have characterized the functional features and structural domains necessary for the activity of two plant root chitinases that are believed to be involved in plant defense and a bacterial chitinase that, along with the plant chitinases, may participate in nutrient recycling in the rhizosphere. [ABSTRACT FROM AUTHOR]

Summary: Although most xyloglucans (XyGs) biosynthesis enzymes have been identified, the molecular mechanism that defines XyG branching patterns is unclear. Four out of five XyG xylosyltransferases (XXT1, XXT2, XXT4, and XXT5) are known to add the xylosyl residue from UDP‐xylose onto a glucan backbone chain; however, the function of XXT3 has yet to be demonstrated.Single xxt3 and triple xxt3xxt4xxt5 mutant Arabidopsis (Arabidopsis thaliana) plants were generated using CRISPR‐Cas9 technology to determine the specific function of XXT3.Combined biochemical, bioinformatic, and morphological data conclusively established for the first time that XXT3, together with XXT4 and XXT5, adds xylosyl residue specifically at the third glucose in the glucan chain to synthesize XXXG‐type XyGs. We propose that the specificity of XXT3, XXT4, and XXT5 is directed toward the prior synthesis of the acceptor substrate by the other two enzymes, XXT1 and XXT2. We also conclude that XXT5 plays a dominant role in the synthesis of XXXG‐type XyGs, while XXT3 and XXT4 complementarily contribute their activities in a tissue‐specific manner.The newly generated xxt3xxt4xxt5 mutant produces only XXGG‐type XyGs, which further helps to understand the impact of structurally deficient polysaccharides on plant cell wall organization, growth, and development. [ABSTRACT FROM AUTHOR]

A plant cell wall is a highly complex structure consisting of networks of polysaccharides, proteins, and polyphenols that dynamically change during growth and development in various tissues. The cell wall not only acts as a physical barrier but also dynamically responds to disturbances caused by biotic and abiotic stresses. Plants have well-established surveillance mechanisms to detect any cell wall perturbations. Specific immune signaling pathways are triggered to contrast biotic or abiotic forces, including cascades dedicated to reinforcing the cell wall structure. This review summarizes the recent developments in molecular mechanisms underlying maintenance of cell wall integrity in plant–pathogen and parasitic interactions. Subjects such as the effect of altered expression of endogenous plant cell-wall-related genes or apoplastic expression of microbial cell-wall-modifying enzymes on cell wall integrity are covered. Targeted genetic modifications as a tool to study the potential of cell wall elicitors, priming of signaling pathways, and the outcome of disease resistance phenotypes are also discussed. The prime importance of understanding the intricate details and complete picture of plant immunity emerges, ultimately to engineer new strategies to improve crop productivity and sustainability. [ABSTRACT FROM AUTHOR]

The plant's recalcitrant cell wall is composed of numerous polysaccharides, including cellulose, hemicellulose, and pectin. The most abundant hemicellulose in dicot cell walls is xyloglucan, which consists of a β-(1- > 4) glucan backbone with α-(1- > 6) xylosylation producing an XXGG or XXXG pattern. Xylose residues of xyloglucan are branched further with different patterns of arabinose, fucose, galactose, and acetylation that varies between species. Although xyloglucan research in other species lag behind Arabidopsis thaliana , significant advances have been made into the agriculturally relevant species Oryza sativa and Solanum lycopersicum , which can be considered model organisms for XXGG type xyloglucan. In this review, we will present what is currently known about xyloglucan biosynthesis in A. thaliana , O. sativa , and S. lycopersicum and discuss the recent advances in the characterization of the glycosyltransferases involved in this complex process and their organization in the Golgi. [ABSTRACT FROM AUTHOR]

Glycosyltransferases (GTs) are a large family of enzymes that add sugars to a broad range of acceptor substrates, including polysaccharides, proteins and lipids, by utilizing a wide variety of donor substrates in the form of activated sugars. Individual GTs have generally been considered to exhibit a high level of substrate specificity, but this has not been thoroughly investigated across the extremely large set of GTs. Here we investigate xyloglucan xylosyltransferase 1 (XXT1), a GT involved in the synthesis of the plant cell wall polysaccharide, xyloglucan. Xyloglucan has a glucan backbone, with initial side chain substitutions exclusively composed of xylose from uridine diphosphate (UDP)-xylose. While this conserved substitution pattern suggests a high substrate specificity for XXT1, our in vitro kinetic studies elucidate a more complex set of behavior. Kinetic studies demonstrate comparable k cat values for reactions with UDP-xylose and UDP-glucose, while reactions with UDP-arabinose and UDP-galactose are over 10-fold slower. Using k cat / K M as a measure of efficiency, UDP-xylose is 8-fold more efficient as a substrate than the next best alternative, UDP-glucose. To the best of our knowledge, we are the first to demonstrate that not all plant XXTs are highly substrate specific and some do show significant promiscuity in their in vitro reactions. Kinetic parameters alone likely do not explain the high substrate selectivity in planta, suggesting that there are additional control mechanisms operating during polysaccharide biosynthesis. Improved understanding of substrate specificity of the GTs will aid in protein engineering, development of diagnostic tools, and understanding of biological systems. [ABSTRACT FROM AUTHOR]

The primary plant cell wall is a complex matrix composed of interconnected polysaccharides including cellulose, hemicellulose, and pectin. Changes of this dynamic polysaccharide system play a critical role during plant cell development and differentiation. A better understanding of cell wall architectures can provide insight into the plant cell development. In this study, a Raman spectroscopic imaging approach was developed to visualize the distribution of plant cell wall polysaccharides. In this approach, Surface-enhanced Raman scattering (SERS through self-assembled silver nanoparticles) was combined with Raman labels (4-Aminothiophenol. 4ATP) and targeted enzymatic hydrolysis to improve the sensitivity, specificity, and throughput of the Raman imaging technique, and to reveal the distribution of pectin and its co-localization with xyloglucan inside onion epidermal cell (OEC) wall. This technique significantly decreased the required spectral acquisition time. The resulted Raman spectra showed a high Raman signal. The resulted Raman images successfully revealed and characterized the pectin distribution and its co-localization pattern with xyloglucan in OEC wall. [ABSTRACT FROM AUTHOR]

Glycosyltransferases (GTs) are enzymes that catalyze reactions attaching an activated sugar to an acceptor substrate, which may be a polysaccharide, peptide, lipid, or small molecule. In the past decade, notable progress has been made in revealing and cloning genes encoding polysaccharide-synthesizing GTs. However, the vast majority of GTs remain structurally and functionally uncharacterized. The mechanism by which they are organized in the Golgi membrane, where they synthesize complex, highly branched polysaccharide structures with high efficiency and fidelity, is also mostly unknown. This review will focus on current knowledge about plant polysaccharide-synthesizing GTs, specifically focusing on protein-protein interactions and the formation of multiprotein complexes. [ABSTRACT FROM AUTHOR]

The plant cell wall is a complex network of polysaccharides. A better understanding of the plant cell wall polysaccharide content, distribution, and interactions among them could be instrumental for our further understanding of plant cell wall in general. Confocal Raman microscopy (CRM) is a powerful tool that could reveal details about chemical landscape of the plant cell wall with micrometer resolution. However, the low signal‐to‐noise (S/N) ratio of Raman spectral signal led to low throughput of Raman imaging, which limited its application in plant cell wall study. In this study, the interaction between the cell wall polysaccharides and the pectin enzyme endo‐polygalacturonase (EPG) was characterized by analyzing Raman images collected before and after EPG treatment at high scanning speed. The obtained low S/N ratio Raman spectra were processed with principal component analysis (PCA) and hierarchical clustering analysis (HCA) to recover information‐rich signature changes in Raman signal dataset that reflected the changes in the polysaccharides caused specifically by the enzymatic hydrolysis. The PCA reconstruction technique significantly improved the S/N ratio of the spectra dataset while kept the Raman signal intact. Further analysis of principal components (PCs) revealed the pectin distribution and its interaction with the enzyme, which provided organizational details of pectin inside the onion plant cell wall. The technique could be used to reveal polysaccharide organization and distribution inside plant cell walls. [ABSTRACT FROM AUTHOR]

Summary: Plants mount defense responses during pathogen attacks, and robust host defense suppression by pathogen effector proteins is essential for infection success. 4E02 is an effector of the sugar beet cyst nematode Heterodera schachtii. Arabidopsis thaliana lines expressing the effector‐coding sequence showed altered expression levels of defense response genes, as well as higher susceptibility to both the biotroph H. schachtii and the necrotroph Botrytis cinerea, indicating a potential suppression of defenses by 4E02. Yeast two‐hybrid analyses showed that 4E02 targets A. thaliana vacuolar papain‐like cysteine protease (PLCP) 'Responsive to Dehydration 21A' (RD21A), which has been shown to function in the plant defense response. Activity‐based protein profiling analyses documented that the in planta presence of 4E02 does not impede enzymatic activity of RD21A. Instead, 4E02 mediates a re‐localization of this protease from the vacuole to the nucleus and cytoplasm, which is likely to prevent the protease from performing its defense function and at the same time, brings it in contact with novel substrates. Yeast two‐hybrid analyses showed that RD21A interacts with multiple host proteins including enzymes involved in defense responses as well as carbohydrate metabolism. In support of a role in carbohydrate metabolism of RD21A after its effector‐mediated re‐localization, we observed cell wall compositional changes in 4E02 expressing A. thaliana lines. Collectively, our study shows that 4E02 removes RD21A from its defense‐inducing pathway and repurposes this enzyme by targeting the active protease to different cell compartments. Significance Statement: The 4E02 effector of the sugar beet cyst nematode Heterodera schachtii targets Arabidopsis thaliana vacuolar PLCP RD21A, which functions in the plant defense response. 4E02 mediates a re‐localization of this protease to the nucleus and cytoplasm, which is accompanied by altered expression levels of defense response genes, higher susceptibility to H. schachtii and Botrytis cinerea, and cell wall compositional changes. These findings uncover mechanistic details in the parasitic strategy of a damaging pathogen, which could be exploited in future control strategies. [ABSTRACT FROM AUTHOR]

In dicotyledonous plants, xyloglucan (XyG) is the most abundant hemicellulose of the primary cell wall. The enzymes involved in XyG biosynthesis have been identified through reverse-genetics and activity was characterized by heterologous expression. Currently, there is no information on the atomic structures or amino acids involved in activity or substrate binding of any of the Golgilocalized XyG biosynthetic enzymes. A homology model of the xyloglucan xylosyltransferase 2 (XXT2) catalytic domain was built on the basis of the crystal structure of A64Rp. Molecular dynamics simulations revealed that the homology model retains the glycosyltransferase (GT)-A fold of the template structure used to build the homology model indicating that XXT2 likely has a GT-A fold. According to the XXT2 homology model, six amino acids (Phe204, Lys207, Asp228, Ser229, Asp230, His378) were selected and their contribution in catalytic activity was investigated. Sitedirected mutagenesis studies show that Asp228, Asp230 and His378 are critical for XXT2 activity and are predicted to be involved in coordination of manganese ion. Lys207 was also found to be critical for protein activity and the homology model indicates a critical role in substrate binding. Additionally, Phe204 mutants have less of an impact on XXT2 activity with the largest effect when replaced with a polar residue. This is the first study that investigates the amino acids involved in substrate binding of the XyG-synthesizing xylosyltransferases and contributes to the understanding of the mechanisms of polysaccharide-synthesizing GTs and XyG biosynthesis. [ABSTRACT FROM AUTHOR]

Cell walls are essential components of plant cells which perform a variety of important functions for the different cell types, tissues and organs of a plant. Besides mechanical function providing cell shape, cell walls participate in intercellular communication, defense during plant-microbe interactions, and plant growth. The plant cell wall consists predominantly of polysaccharides with the addition of structural glycoproteins, phenolic esters, minerals, lignin, and associated enzymes. Alterations in the cell wall composition created through either changes in biosynthesis of specific constituents or their post-synthetic modifications in the apoplast compromise cell wall integrity and frequently induce plant compensatory responses as a result of these alterations. Here we report that post-synthetic removal of fucose residues specifically from arabinogalactan proteins in the Arabidopsis plant cell wall induces differential expression of fucosyltransferases and leads to the root and hypocotyl elongation changes. These results demonstrate that the post-synthetic modification of cell wall components presents a valuable approach to investigate the potential signaling pathways induced during plant responses to such modifications that usually occur during plant development and stress responses. [ABSTRACT FROM AUTHOR]

The complexity of cell wall composition and structure determines the strength, flexibility, and function of the primary cell wall in plants. However, the contribution of the various components to cell wall integrity (CWI) and function remains unclear. Modifications of cell wall composition can induce plant responses known as CWI control. In this study, we used transgenic expression of the fungal feruloyl esterase AnFAE to examine the effect of post-synthetic modification of Arabidopsis and Brachypodium cell walls. Transgenic Arabidopsis plants expressing AnFAE showed a significant reduction of monomeric ferulic acid, decreased amounts of wall-associated extensins, and increased susceptibility to Botrytis cinerea, compared with wild type. Transgenic Brachypodium showed reductions in monomeric and dimeric ferulic acids and increased susceptibility to Bipolaris sorokiniana. Upon infection, transgenic Arabidopsis and Brachypodium plants also showed increased expression of several defense-related genes compared with wild type. These results demonstrate a role, in both monocot and dicot plants, of polysaccharide feruloylation in plant CWI, which contributes to plant resistance to necrotrophic pathogens. [ABSTRACT FROM AUTHOR]

The immutans (im) variegation mutation of Arabidopsis has green- and white- sectored leaves due to action of a nuclear recessive gene. IM codes for PTOX, a plastoquinol oxidase in plastid membranes. Previous studies have revealed that the green and white sectors develop into sources (green tissues) and sinks (white tissues) early in leaf development. In this report we focus on white sectors, and show that their transformation into effective sinks involves a sharp reduction in plastid number and size. Despite these reductions, cells in the white sectors have near-normal amounts of plastid RNA and protein, and surprisingly, a marked amplification of chloroplast DNA. The maintenance of protein synthesis capacity in the white sectors might poise plastids for their development into other plastid types. The green and white im sectors have different cell wall compositions: whereas cell walls in the green sectors resemble those in wild type, cell walls in the white sectors have reduced lignin and cellulose microfibrils, as well as alterations in galactomannans and the decoration of xyloglucan. These changes promote susceptibility to the pathogen Pseudomonas syringae. Enhanced susceptibility can also be explained by repressed expression of some, but not all, defense genes. We suggest that differences in morphology, physiology and biochemistry between the green and white sectors is caused by a reprogramming of leaf development that is coordinated, in part, by mechanisms of retrograde (plastid-to-nucleus) signaling, perhaps mediated by ROS. We conclude that variegation mutants offer a novel system to study leaf developmental programming, cell wall metabolism and host-pathogen interactions. [ABSTRACT FROM AUTHOR]

Background: Fusarium graminearum, one of the causal agents of Fusarium Head Blight (FHB, scab), leads to severe losses in grain yield and quality due to the production of mycotoxins which are harmful to human and livestock. Different traits for FHB resistance in wheat were identified for common wheat (Triticum aestivum L.) while the sources of FHB resistance in durum wheat (Triticum turgidum ssp. Durum), one of the cereals most susceptible to F. graminearum infection, have not been found. New lines of evidence indicate that content and composition of cell wall polymers affect the susceptibility of the wall to degrading enzymes produced by pathogens during infection and can play a role in the outcome of host-pathogen interactions. The objective of our research is to identify potential cell wall biochemical traits linked to Fusariosis resistance to be transferred from a resistant common wheat to a susceptible durum wheat line. Results: A detailed analysis of cell wall composition in spikes isolated from a highly resistant common wheat accession “02-5B-318”, a breeding line derived from the FHB-resistant Chinese cv. Sumai-3 and a high susceptible durum wheat cv. Saragolla was performed. Significant differences in lignin monolignols composition, arabinoxylan (AX) substitutions and pectin methylesterification were found between resistant and susceptible plants. We isolated and characterized a pectin methylesterase gene WheatPME1, which we found being down regulated in the FHB-resistant line and induced by fungal infection in the susceptible wheat. Conclusions: Our results indicate cell wall traits differing between the FHB sensitive and resistant wheat genotypes, possibly related to FHB-resistance, and identify the line 02-5B-318R as a potential resource of such traits. Evidence suggests that WheatPME1 is involved in wheat response to F. graminearum. [ABSTRACT FROM AUTHOR]

All living cells generate structurally complex and compositionally diverse spectra of glycans and glycoconjugates, critical for organismal evolution, development, functioning, defense, and survival. Glycosyltransferases (GTs) catalyze the glycosylation reaction between activated sugar and acceptor substrate to synthesize a wide variety of glycans. GTs are distributed among more than 130 gene families and are involved in metabolic processes, signal pathways, cell wall polysaccharide biosynthesis, cell development, and growth. Glycosylation mainly takes place in the endoplasmic reticulum (ER) and Golgi, where GTs and glycosidases involved in this process are distributed to different locations of these compartments and sequentially add or cleave various sugars to synthesize the final products of glycosylation. Therefore, delivery of these enzymes to the proper locations, the glycosylation sites, in the cell is essential and involves numerous secretory pathway components. This review presents the current state of knowledge about the mechanisms of protein trafficking between ER and Golgi. It describes what is known about the primary components of protein sorting machinery and trafficking, which are recognition sites on the proteins that are important for their interaction with the critical components of this machinery. [ABSTRACT FROM AUTHOR]

The primary cell wall of higher plants consists of a mixture of polysaccharides whose spatial proximities and interactions with each other are not well understood. We recently obtained the first two-dimensional (2D) and three-dimensional high-resolution magic-angle-spinning 13C solid-state nuclear magnetic resonance spectra of the uniformly 13C-labeled primary cell wall of Arabidopsis thaliana, which allowed us to assign the majority of 13C resonances of the three major classes of polysaccharides: cellulose, hemicellulose, and pectins. In this work, we measured the intensity buildup of 13C-13C cross-peaks in a series of 2D 13C correlation spectra to obtain semiquantitative information about the spatial proximities between different polysaccharides. Comparison of 2D spectra measured at different spin diffusion mixing times identified intermolecular pectin-cellulose cross-peaks as well as interior cellulose-surface cellulose cross-peaks. The intensity buildup time constants are only modestly longer for cellulose-pectin cross-peaks than for interior cellulose-surface cellulose cross-peaks, indicating that pectins come into direct contact with the cellulose microfibrils. Approximately 25-50% of the cellulose chains exhibit close contact with pectins. The 13C magnetization of the wall polysaccharides is not fully equilibrated by 1.5 s, indicating that pectins and cellulose are not homogeneously mixed on the molecular level. We also assigned the 13C signals of cell wall proteins, identifying common residues such as Pro, Hyp, Tyr, and Ala. The chemical shifts indicate significant coil and sheet conformations in these structural proteins. Interestingly, few cross- peaks were observed between the proteins and the polysaccharides. Taken together, these data indicate that the three major types of polysaccharides in the primary wall of Arabidopsis form a single cohesive network, while structural proteins form a relatively separate domain. [ABSTRACT FROM AUTHOR]

Xyloglucan is an important hemicellulosic polysaccharide in dicot primary cell wails. Most of the enzymes involved in xyloglucan synthesis have been identified. However, many important details of its synthesis in vivo remain unknown. The roles of three genes encoding xylosyltransferases participating in xyloglucan biosynthesis in Arabidopsis (Arabidopsis thaliana) were further investigated using reverse genetic, biochemical, and immunological approaches. New double mutants (xxtl xxt5 and xxt2 xxt5) and a triple mutant (xxtl xxt2 xxt5) were generated, characterized, and compared with three single mutants and the xxtl xxt2 double mutant that had been isolated previously. Antibody-based glycome profiling was applied in combination with chemical and immunohistochemical analyses for these characterizations. From the combined data, we conclude that XXT1 and XXT2 are responsible for the bulk of the xylosylation of the glucan backbone, and at least one of these proteins must be present and active for xyloglucan to be made. XXT5 plays a significant but as yet uncharacterized role in this process. The glycome profiling data demonstrate that the lack of detectable xyloglucan does not cause significant compensatory changes in other polysaccharides, although changes in nonxyloglucan polysaccharide amounts cannot be ruled out. Structural rearrangements of the polysaccharide network appear responsible for maintaining wall integrity in the absence of xyloglucan, thereby allowing nearly normal plant growth in plants lacking xyloglucan. Finally, results from immunohistochemical studies, combined with known information about expression patterns of the three genes, suggest that different combinations of xylosyltransferases contribute differently to xyloglucan biosynthesis in the various cell types found in stems, roots, and hypocotyls. [ABSTRACT FROM AUTHOR]

Plant cell wall (CW) polysaccharides are responsible for the mechanical strength and growth of plant cells; however, the high-resolution structure and dynamics of the CW polysaccharides are still poorly understood because of the insoluble nature of these molecules. Here, we use 2D and 3D magic-angle-spinning (MAS) solid-state NMR (SSNMR) to investigate the structural role of pectins in the plant CW. Intact and partially depectinated primary CWs of Arabidopsis thaliana were uniformly labeled with 13C and their NMR spectra were compared. Recent 13C resonance assignment of the major polysaccharides in Arabidopsis thaliana CWs allowed us to determine the effects of depectination on the intermolecular packing and dynamics of the remaining wall polysaccharides. 2D and 3D correlation spectra show the suppression of pectin signals, confirming partial pectin removal by chelating agents and sodium carbonate. Importantly, higher cross peaks are observed in 2D and 3D 13C spectra of the depectinated CW, suggesting higher rigidity and denser packing of the remaining wall polysaccharides compared with the intact CW. 13C spin-lattice relaxation times and 1H rotating-frame spin-lattice relaxation times indicate that the polysaccharides are more rigid on both the nanosecond and microsecond timescales in the depectinated CW. Taken together, these results indicate that pectic polysaccharides are highly dynamic and endow the polysaccharide network of the primary CW with mobility and flexibility, which may be important for pectin functions. This study demonstrates the capability of multidimensional SSNMR to determine the intermolecular interactions and dynamic structures of complex plant materials under near-native conditions. Copyright © 2012 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Xyloglucan is an important hemicellulosic polysaccharide in dicot primary cell wails. Most of the enzymes involved in xyloglucan synthesis have been identified. However, many important details of its synthesis in vivo remain unknown. The roles of three genes encoding xylosyltransferases participating in xyloglucan biosynthesis in Arabidopsis (Arabidopsis thaliana) were further investigated using reverse genetic, biochemical, and immunological approaches. New double mutants (xxtl xxt5 and xxt2 xxt5) and a triple mutant (xxtl xxt2 xxt5) were generated, characterized, and compared with three single mutants and the xxtl xxt2 double mutant that had been isolated previously. Antibody-based glycome profiling was applied in combination with chemical and immunohistochemical analyses for these characterizations. From the combined data, we conclude that XXT1 and XXT2 are responsible for the bulk of the xylosylation of the glucan backbone, and at least one of these proteins must be present and active for xyloglucan to be made. XXT5 plays a significant but as yet uncharacterized role in this process. The glycome profiling data demonstrate that the lack of detectable xyloglucan does not cause significant compensatory changes in other polysaccharides, although changes in nonxyloglucan polysaccharide amounts cannot be ruled out. Structural rearrangements of the polysaccharide network appear responsible for maintaining wall integrity in the absence of xyloglucan, thereby allowing nearly normal plant growth in plants lacking xyloglucan. Finally, results from immunohistochemical studies, combined with known information about expression patterns of the three genes, suggest that different combinations of xylosyltransferases contribute differently to xyloglucan biosynthesis in the various cell types found in stems, roots, and hypocotyls. [ABSTRACT FROM AUTHOR]

Xyloglucan is the major hemicellulosic polysaccharide in the primary cell walls of most vascular dicotyledonous plants and has important structural and physiological functions in plant growth and development. In Arabidopsis (Ambidopsis thaliana), the 1,4-β-glucan synthase, Cellulose Synthase-Like C4 (CSLC4), and three xylosyltransferases, XXT1, XXT2, and XXT5, act in the Golgi to form the xylosylated glucan backbone during xyloglucan biosynthesis. However, the functional organization of these enzymes in the Golgi membrane is currently unknown. In this study, we used bimolecular fluorescence complementation and in vitro pull-down assays to investigate the supramolecular organization of the CSLC4, XXT1, XXT2, and XXT5 proteins in Arabidopsis protoplasts. Quantification of bimolecular fluorescence complementation fluorescence by flow cytometry allowed us to perform competition assays that demonstrated the high probability of protein-protein complex formation in vivo and revealed differences in the abilities of these proteins to form multiprotein complexes. Results of in vitro pull-down assays using recombinant proteins confirmed that the physical interactions among XXTs occur through their catalytic domains. Additionally, coimmunoprecipitation of XXT2YFP and XXT5HA proteins from Arabidopsis protoplasts indicated that while the formation of the XXT2-XXT2 homocomplex involves disulfide bonds, the formation of the XXT2-XXT5 heterocomplex does not involve covalent interactions. The combined data allow us to propose that the proteins involved in xyloglucan biosynthesis function in a multiprotein complex composed of at least two homocomplexes, CSLC4-CSLC4 and XXT2-XXT2, and three heterocomplexes, XXT2-XXT5, XXT1-XXT2, and XXT5-CSLC4. [ABSTRACT FROM AUTHOR]

The hemicellulosic polysaccharide xyloglucan (XyG), found in the primary cell walls of most plant tissues, is important for structural organization of the cell wall and regulation of growth and development. Significant recent progress in structural characterization of XyGs from different plant species has shed light on the diversification of XyG during plant evolution. Also, identification of XyG biosynthetic enzymes and examination of their interactions suggests the involvement of a multiprotein complex in XyG biosynthesis. This mini-review presents an updated overview of the diversity of XyG structures in plant taxa and recent findings on XyG biosynthesis. [ABSTRACT FROM AUTHOR]

Chemical genetics as a part of chemical genomics is a powerful and fast developing approach to dissect biological processes that may be difficult to characterize using conventional genetics because of gene redundancy or lethality and, in the case of polysaccharide biosynthesis, plant flexibility. Polysaccharide synthetic enzymes are located in two main compartments—the Golgi apparatus and plasma membrane—and can be studied in vitro using membrane fractions. Here, we first developed a high-throughput assay that allowed the screening of a library of chemicals with a potential effect on glycosyltransferase activities. Out of the 4800 chemicals screened for their effect on Golgi glucosyltransferases, 66 compounds from the primary screen had an effect on carbohydrate biosynthesis. Ten of these compounds were confirmed to inhibit glucose incorporation after a second screen. One compound exhibiting a strong inhibition effect (ID 6240780 named chemical A) was selected and further studied. It reversibly inhibits the transfer of glucose from UDP-glucose by Golgi membranes, but activates the plasma membrane-bound callose synthase. The inhibition effect is dependent on the chemical structure of the compound, which does not affect endomembrane morphology of the plant cells, but causes changes in cell wall composition. Chemical A represents a novel drug with a great potential for the study of the mechanisms of Golgi and plasma membrane-bound glucosyltransferases. [ABSTRACT FROM PUBLISHER]

Simple Summary: In the present study, we created transgenic Arabidopsis plants overexpressing two fungal acetylesterases and a fungal feruloylesterase that acts on cell wall polysaccharides and studied their possible complementary additive effects on host defense reactions against the fungal pathogen, Botrytis cinerea. Our results showed that the Arabidopsis plants overexpressing two acetylesterases together contributed significantly higher resistance to B. cinerea in comparison with single protein expression. Conversely, coexpression of either of the acetyl esterases together with feruloylesterase compensates the latter's negative impact on plant resistance. The results also provided evidence that combinatorial coexpression of some cell wall polysaccharide-modifying enzymes might exert an additive effect on plant immune response by constitutively priming plant defense pathways even before pathogen invasion. These findings have potential uses in protecting valuable crops against pathogens. The plant cell wall (CW) is an outer cell skeleton that plays an important role in plant growth and protection against both biotic and abiotic stresses. Signals and molecules produced during host–pathogen interactions have been proven to be involved in plant stress responses initiating signal pathways. Based on our previous research findings, the present study explored the possibility of additively or synergistically increasing plant stress resistance by stacking beneficial genes. In order to prove our hypothesis, we generated transgenic Arabidopsis plants constitutively overexpressing three different Aspergillus nidulans CW-modifying enzymes: a xylan acetylesterase, a rhamnogalacturonan acetylesterase and a feruloylesterase. The two acetylesterases were expressed either together or in combination with the feruloylesterase to study the effect of CW polysaccharide deacetylation and deferuloylation on Arabidopsis defense reactions against a fungal pathogen, Botrytis cinerea. The transgenic Arabidopsis plants expressing two acetylesterases together showed higher CW deacetylation and increased resistance to B. cinerea in comparison to wild-type (WT) Col-0 and plants expressing single acetylesterases. While the expression of feruloylesterase alone compromised plant resistance, coexpression of feruloylesterase together with either one of the two acetylesterases restored plant resistance to the pathogen. These CW modifications induced several defense-related genes in uninfected healthy plants, confirming their impact on plant resistance. These results demonstrated that coexpression of complementary CW-modifying enzymes in different combinations have an additive effect on plant stress response by constitutively priming the plant defense pathways. These findings might be useful for generating valuable crops with higher protections against biotic stresses. [ABSTRACT FROM AUTHOR]

The acquisition of freeze tolerance in winter plants involves, among other cellular responses, activated catabolism of cell wall polysaccharides, thereby liberating oligosaccharides. One of these was identified as an oligosaccharin (physiologically active fragment) that most likely originates from hemicelluloses. Treatment of winter wheat seedlings with the oligosaccharin at 2°C increased their freeze tolerance by ~30%. Results obtained to date suggest that the oligosaccharin acts as an endogenous and systemic signaling molecule during cold adaptation. [ABSTRACT FROM AUTHOR]

Pectins are a highly complex family of cell wall polysaccharides comprised of homogalacturonan (HGA), rhamnogalacturonan I and rhamnogalacturonan II. We have specifically modified HGA in both tobacco (Nicotiana tabacum) and Arabidopsis by expressing the endopolygalacturonase II of Aspergillus niger (AnPGII). Cell walls of transgenic tobacco plants showed a 25% reduction in GalUA content as compared with the wild type and a reduced content of deesterified HGA as detected by antibody labeling. Neutral sugars remained unchanged apart from a slight increase of Rha, Ara, and Gal. Both transgenic tobacco and Arabidopsis were dwarfed, indicating that unesterified HGA is a critical factor for plant cell growth. The dwarf phenotypes were associated with AnPGII activity as demonstrated by the observation that the mutant phenotype of tobacco was completely reverted by crossing the dwarfed plants with plants expressing PGIP2, a strong inhibitor of AnPGII. The mutant phenotype in Arabidopsis did not appear when transformation was performed with a gene encoding AnPGII inactivated by site directed mutagenesis. [ABSTRACT FROM AUTHOR]

Keywords: polysaccharide biosynthesis; glycosyltransferases; protein complexes; plant Golgi; structural organization EN polysaccharide biosynthesis glycosyltransferases protein complexes plant Golgi structural organization 1 1 1 07/09/21 20210706 NES 210706 The name of the second author "Ning Zhang" was incorrectly spelled as "Ning Zang" in the article when published originally. Polysaccharide biosynthesis, glycosyltransferases, protein complexes, plant Golgi, structural organization. [Extracted from the article]