Xyloglucosyl Transferase Research Articles

Proteomic Analysis to Understand the Promotive Effect of Ethanol on Soybean Growth Under Salt Stress

Finding solutions to mitigate the impact of salinity on crops is important for global food security because soil salinity significantly reduces plant growth and grain yield. Ethanol may play an important role in mitigating the negative salt-induced effects on crops. Soybean root growth was significantly reduced under salt stress; however, it was restored and comparable to control values by ethanol application even under stress. To study the positive mechanism of ethanol on soybean growth, a proteomic approach was carried out. The categories with the greatest changes in protein numbers were protein metabolism, transport, and cell organization in biological processes, nucleus and cytosol in cellular components, and nucleic acid binding activity in molecular functions. Proteomic data were confirmed using immunoblot analysis. Reactive oxygen species enzymes increased under salt stress; among them, mitochondrial ascorbate peroxidase was further accumulated by ethanol application. Among the cell wall and membrane-associated proteins, xyloglucan xyloglucosyl transferase and H+-ATPase increased and decreased, respectively, under salt stress; however, they were restored to control levels by ethanol application. These results suggest that soybeans were adversely affected by salt stress and recovered with ethanol application via the regulation of cell wall and membrane functions through the detoxification of reactive oxygen species.

Using transcriptome sequencing (RNA-Seq) to screen genes involved in β-glucan biosynthesis and accumulation during oat seed development.

Oat (Avena sativa L.) is an annual grass that has a high nutritional value and therapeutic benefits. β-glucan is one of the most important nutrients in oats. In this study, we investigated two oat varieties with significant differences in β-glucan content (high β-glucan oat varieties BY and low β-glucan content oat variety DY) during different filling stages. We also studied the transcriptome sequencing of seeds at different filling stages. β-glucan accumulation was highest at days 6-16 in the filling stage. Differentially expressed genes (DEGs) were selected from the dataset of transcriptome sequencing. Among them, three metabolic pathways were closely related to the biosynthesis of β-glucan by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, including xyloglucan:xyloglucosyl transferase activity, starch and sucrose metabolism, and photosynthesis. By analyzing the expression patterns of DEGs, we identified one CslF2 gene and 32 transcription factors. Five modules were thought to be positively correlated with β-glucan accumulation by weighted gene co-expression network analysis (WGCNA). Moreover, the expression levels of candidate genes obtained from the transcriptome sequencing were further validated by quantitative real-time PCR (RT-qPCR) analysis. Our study provides a novel way to identify the regulatory mechanism of β-glucan synthesis and accumulation in oat seeds and offers a possible pathway for the genetic engineering of oat breeding for higher-quality seeds.

Genome-Wide Analysis of the Xyloglucan Endotransglucosylase/Hydrolase (XTH) Gene Family: Expression Pattern during Magnesium Stress Treatment in the Mulberry Plant (Morus alba L.) Leaves.

Mulberry (Morus alba L.), a significant fruit tree crop, requires magnesium (Mg) for its optimal growth and productivity. Nonetheless, our understanding of the molecular basis underlying magnesium stress tolerance in mulberry plants remains unexplored. In our previous study, we identified several differential candidate genes associated with Mg homeostasis via transcriptome analysis, including the xyloglucan endotransglucosylase/hydrolase (XTH) gene family. The XTH gene family is crucial for plant cell wall reconstruction and stress responses. These genes have been identified and thoroughly investigated in various plant species. However, there is no research pertaining to XTH genes within the M. alba plant. This research systematically examined the M. alba XTH (MaXTH) gene family at the genomic level using a bioinformatic approach. In total, 22 MaXTH genes were discovered and contained the Glyco_hydro_16 and XET_C conserved domains. The MaXTHs were categorized into five distinct groups by their phylogenetic relationships. The gene structure possesses four exons and three introns. Furthermore, the MaXTH gene promoter analysis reveals a plethora of cis-regulatory elements, mainly stress responsiveness, phytohormone responsiveness, and growth and development. GO analysis indicated that MaXTHs encode proteins that exhibit xyloglucan xyloglucosyl transferase and hydrolase activities in addition to cell wall biogenesis as well as xyloglucan and carbohydrate metabolic processes. Moreover, a synteny analysis unveiled an evolutionary relationship between the XTH genes in M. alba and those in three other species: A. thaliana, P. trichocarpa, and Zea mays. Expression profiles from RNA-Seq data displayed distinct expression patterns of XTH genes in M. alba leaf tissue during Mg treatments. Real-time quantitative PCR analysis confirmed the expression of the MaXTH genes in Mg stress response. Overall, this research enhances our understanding of the characteristics of MaXTH gene family members and lays the foundation for future functional genomic study in M. alba.

Engineering of substrate specificity in a plant cell-wall modifying enzyme through alterations of carboxyl-terminal amino acid residues.

Structural determinants of substrate recognition remain inadequately defined in broad specific cell-wall modifying enzymes, termed xyloglucan xyloglucosyl transferases (XETs). Here, we investigate the Tropaeolum majus seed TmXET6.3 isoform, a member of the GH16_20 subfamily of the GH16 network. This enzyme recognises xyloglucan (XG)-derived donors and acceptors, and a wide spectrum of other chiefly saccharide substrates, although it lacks the activity with homogalacturonan (pectin) fragments. We focus on defining the functionality of carboxyl-terminal residues in TmXET6.3, which extend acceptor binding regions in the GH16_20 subfamily but are absent in the related GH16_21 subfamily. Site-directed mutagenesis using double to quintuple mutants in the carboxyl-terminal region - substitutions emulated on barley XETs recognising the XG/penta-galacturonide acceptor substrate pair - demonstrated that this activity could be gained in TmXET6.3. We demonstrate the roles of semi-conserved Arg238 and Lys237 residues, introducing a net positive charge in the carboxyl-terminal region (which complements a negative charge of the acidic penta-galacturonide) for the transfer of xyloglucan fragments. Experimental data, supported by molecular modelling of TmXET6.3 with the XG oligosaccharide donor and penta-galacturonide acceptor substrates, indicated that they could be accommodated in the active site. Our findings support the conclusion on the significance of positively charged residues at the carboxyl terminus of TmXET6.3 and suggest that a broad specificity could be engineered via modifications of an acceptor binding site. The definition of substrate specificity in XETs should prove invaluable for defining the structure, dynamics, and function of plant cell walls, and their metabolism; these data could be applicable in various biotechnologies.

Hormone-regulated expansins: Expression, localization, and cell wall biomechanics in Arabidopsis root growth.

Expansins facilitate cell expansion by mediating pH-dependent cell wall (CW) loosening. However, the role of expansins in controlling CW biomechanical properties in specific tissues and organs remains elusive. We monitored hormonal responsiveness and spatial specificity of expression and localization of expansins predicted to be the direct targets of cytokinin signaling in Arabidopsis (Arabidopsis thaliana). We found EXPANSIN1 (EXPA1) homogenously distributed throughout the CW of columella/lateral root cap, while EXPA10 and EXPA14 localized predominantly at 3-cell boundaries in the epidermis/cortex in various root zones. EXPA15 revealed cell-type-specific combination of homogenous vs. 3-cell boundaries localization. By comparing Brillouin frequency shift and AFM-measured Young's modulus, we demonstrated Brillouin light scattering (BLS) as a tool suitable for non-invasive in vivo quantitative assessment of CW viscoelasticity. Using both BLS and AFM, we showed that EXPA1 overexpression upregulated CW stiffness in the root transition zone (TZ). The dexamethasone-controlled EXPA1 overexpression induced fast changes in the transcription of numerous CW-associated genes, including several EXPAs and XYLOGLUCAN:XYLOGLUCOSYL TRANSFERASEs (XTHs), and associated with rapid pectin methylesterification determined by in situ Fourier-transform infrared spectroscopy in the root TZ. The EXPA1-induced CW remodeling is associated with the shortening of the root apical meristem, leading to root growth arrest. Based on our results, we propose that expansins control root growth by a delicate orchestration of CW biomechanical properties, possibly regulating both CW loosening and CW remodeling.

Transcriptome Analysis Reveals the Defense Mechanism of Cotton against Verticillium dahliae Induced by Hypovirulent Fungus Gibellulopsis nigrescens CEF08111.

Verticillium wilt is a kind of plant vascular disease caused by the soilborne fungus Verticillium dahliae, which severely limits cotton production. Our previous studies showed that the endophytic fungus Gibellulopsis nigrescens CEF08111 can effectively control Verticillium wilt and induce a defense response in cotton plants. However, the comprehensive molecular mechanism governing this response is not yet clear. To study the signaling mechanism induced by strain CEF08111, the transcriptome of cotton seedlings pretreated with CEF08111 was sequenced. The results revealed 249, 3559 and 33 differentially expressed genes (DEGs) at 3, 12 and 48 h post inoculation with CEF08111, respectively. At 12 h post inoculation with CEF08111, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that the DEGs were enriched mainly in the plant-pathogen interaction, mitogen-activated protein kinase (MAPK) signaling pathway-plant, and plant hormone signal transduction pathways. Gene ontology (GO) analysis revealed that these DEGs were enriched mainly in the following terms: response to external stimulus, systemic acquired resistance, kinase activity, phosphotransferase activity, xyloglucan: xyloglucosyl transferase activity, xyloglucan metabolic process, cell wall polysaccharide metabolic process and hemicellulose metabolic process. Moreover, many genes, such as calcium-dependent protein kinase (CDPK), flagellin-sensing 2 (FLS2), resistance to Pseudomonas syringae pv. maculicola 1(RPM1) and myelocytomatosis protein 2 (MYC2), that regulate crucial points in defense-related pathways were identified and may contribute to V. dahliae resistance in cotton. Seven DEGs of the pathway phenylpropanoid biosynthesis were identified by weighted gene co-expression network analysis (WGCNA), and these genes are related to lignin synthesis. The above genes were compared and analyzed, a total of 710 candidate genes that may be related to the resistance of cotton to Verticillium wilt were identified. These results provide a basis for understanding the molecular mechanism by which the biocontrol fungus CEF08111 increases the resistance of cotton to Verticillium wilt.

Integrated Physiochemical, Hormonal, and Transcriptomic Analysis Revealed the Underlying Mechanisms for Granulation in Huyou (Citrus changshanensis) Fruit.

Juice sac granulation is a common internal physiological disorder of citrus fruit. In the present study, we compared the physiochemical characteristics and transcriptome profiles of juice sacs in different granulation levels from Huyou fruit (Citrus changshanensis). The accumulation of cell wall components, including the water-soluble pectin, protopectin, cellulose, and lignin, were significantly correlated with the granulation process, resulting in the firmness increase of the juice sac. The in situ labeling of the cell wall components indicated the early accumulation of cellulose and high-methylesterified pectin in the outer layer cells, as well as the late accumulation of lignin in the inner layer cells of the juice sac. Several phytohormones, including auxins, abscisic acids, cytokinins, jasmonic acid, salicylic acid, and/or their metabolites, were positively correlated to the granulation level, indicating an active and complex phytohormones metabolism in the granulation process. Combining the trend analysis by the Mfuzz method and the module-trait correlation analysis by the Weighted Gene Co-expression Network Analysis method, a total of 2940 differentially expressed genes (DEGs) were found to be positively correlated with the granulation level. Gene Ontology (GO) enrichment indicated that the selected DEGs were mainly involved in the cell wall organization and biogenesis, cell wall macromolecule metabolic process, carbohydrate metabolic process, and polysaccharide metabolic process. Among these selected genes, those encoding β-1,4-xylosyltransferase IRX9, cellulose synthase, xyloglucan: xyloglucosyl transferase, xyloglucan galactosyltransferase MUR3, α-1,4-galacturonosyltransferase, expansin, polygalacturonase, pectinesterase, β-glucosidase, β-galactosidase, endo-1,3(4)-β-glucanase, endoglucanase and pectate lyase that required for the biosynthesis or structural modification of cell wall were identified. In addition, NAC, MYB, bHLH, and MADS were the top abundant transcription factors (TFs) families positively correlated with the granulation level, while the LOB was the top abundant TFs family negatively correlated with the granulation level.

Broad Specific Xyloglucan:Xyloglucosyl Transferases Are Formidable Players in the Re-Modelling of Plant Cell Wall Structures.

Plant xyloglucan:xyloglucosyl transferases, known as xyloglucan endo-transglycosylases (XETs) are the key players that underlie plant cell wall dynamics and mechanics. These fundamental roles are central for the assembly and modifications of cell walls during embryogenesis, vegetative and reproductive growth, and adaptations to living environments under biotic and abiotic (environmental) stresses. XET enzymes (EC 2.4.1.207) have the β-sandwich architecture and the β-jelly-roll topology, and are classified in the glycoside hydrolase family 16 based on their evolutionary history. XET enzymes catalyse transglycosylation reactions with xyloglucan (XG)-derived and other than XG-derived donors and acceptors, and this poly-specificity originates from the structural plasticity and evolutionary diversification that has evolved through expansion and duplication. In phyletic groups, XETs form the gene families that are differentially expressed in organs and tissues in time- and space-dependent manners, and in response to environmental conditions. Here, we examine higher plant XET enzymes and dissect how their exclusively carbohydrate-linked transglycosylation catalytic function inter-connects complex plant cell wall components. Further, we discuss progress in technologies that advance the knowledge of plant cell walls and how this knowledge defines the roles of XETs. We construe that the broad specificity of the plant XETs underscores their roles in continuous cell wall restructuring and re-modelling.

Plant Xyloglucan Xyloglucosyl Transferases and the Cell Wall Structure: Subtle but Significant.

Plant xyloglucan xyloglucosyl transferases or xyloglucan endo-transglycosylases (XET; EC 2.4.1.207) catalogued in the glycoside hydrolase family 16 constitute cell wall-modifying enzymes that play a fundamental role in the cell wall expansion and re-modelling. Over the past thirty years, it has been established that XET enzymes catalyse homo-transglycosylation reactions with xyloglucan (XG)-derived substrates and hetero-transglycosylation reactions with neutral and charged donor and acceptor substrates other than XG-derived. This broad specificity in XET isoforms is credited to a high degree of structural and catalytic plasticity that has evolved ubiquitously in algal, moss, fern, basic Angiosperm, monocot, and eudicot enzymes. These XET isoforms constitute gene families that are differentially expressed in tissues in time- and space-dependent manners during plant growth and development, and in response to biotic and abiotic stresses. Here, we discuss the current state of knowledge of broad specific plant XET enzymes and how their inherently carbohydrate-based transglycosylation reactions tightly link with structural diversity that underlies the complexity of plant cell walls and their mechanics. Based on this knowledge, we conclude that multi- or poly-specific XET enzymes are widespread in plants to allow for modifications of the cell wall structure in muro, a feature that implements the multifaceted roles in plant cells.

Another building block in the plant cell wall: Barley xyloglucan xyloglucosyl transferases link covalently xyloglucan and anionic oligosaccharides derived from pectin.

We report on the homo- and hetero-transglycosylation activities of the HvXET3 and HvXET4 xyloglucan xyloglucosyl transferases (XET; EC 2.4.1.207) from barley (Hordeum vulgare L.), and the visualisation of these activities in young barley roots using Alexa Fluor 488-labelled oligosaccharides. We discover that these isozymes catalyse the transglycosylation reactions with the chemically defined donor and acceptor substrates, specifically with the xyloglucan donor and the penta-galacturonide [α(1-4)GalAp]5 acceptor - the homogalacturonan (pectin) fragment. This activity is supported by 3D molecular models of HvXET3 and HvXET4 with the docked XXXG donor and [α(1-4)GalAp]5 acceptor substrates at the -4 to +5 subsites in the active sites. Comparative sequence analyses of barley isoforms and seed-localised TmXET6.3 from nasturtium (Tropaeolum majus L.) permitted the engineering of mutants of TmXET6.3 that could catalyse the hetero-transglycosylation reaction with the xyloglucan/[α(1-4)GalAp]5 substrate pair, while wild-type TmXET6.3 lacked this activity. Expression data obtained by real-time quantitative polymerase chain reaction of HvXET transcripts and a clustered heatmap of expression profiles of the gene family revealed that HvXET3 and HvXET6 co-expressed but did not share the monophyletic origin. Conversely, HvXET3 and HvXET4 shared this relationship, when we examined the evolutionary history of 419 glycoside hydrolase 16 family members, spanning monocots, eudicots and a basal Angiosperm. The discovered hetero-transglycosylation activity in HvXET3 and HvXET4 with the xyloglucan/[α(1-4)GalAp]5 substrate pair is discussed against the background of roles of xyloglucan-pectin heteropolymers and how they may participate in spatial patterns of cell wall formation and re-modelling, and affect the structural features of walls.

Characterization of the transcriptional profiles in common buckwheat (Fagopyrum esculentum) under PEG-mediated drought stress

Background: Common buckwheat (Fagopyrum esculentum) is an important staple food crop in southwest China, where drought stress is one of the largest limiting factors that lead to decreased crop production. To reveal the molecular mechanism of common buckwheat in response to drought stress, we performed a comprehensive transcriptomics study to evaluate gene expression profiles of common buckwheat during PEG-mediated drought treatment. Results: In total, 45 million clean reads were assembled into 53,404 unigenes with an average length of 749 bp and N50 length of 1296 bp. A total of 1329 differentially expressed genes (DEGs) were identified by comparing wellwatered and drought-treated plants, out of which 666 were upregulated and 663 were downregulated. Furthermore, we defined the functional characteristics of DEGs using GO and KEGG classifications. GO enrichment analysis showed that the DEGs were significantly overrepresented in four categories, namely, oxidoreductase activity, oxidation-reduction process, xyloglucan:xyloglucosyl transferase activity, and apoplast. Using KEGG pathway analysis, a large number of annotated genes were overrepresented in terms such as plant hormone signal transduction, phenylpropanoid biosynthesis, photosynthesis, and carbon metabolism. Conclusions: These results can be further exploited to investigate the molecular mechanism of common buckwheat in response to drought treatment and could supply with valuable molecular sources for abiotic-tolerant elite breeding programs in the future.

Physiological and Growth Response of Pepper (Capsicum annum L.) Seedlings to Supplementary Red/Blue Light Revealed through Transcriptomic Analysis

Red and blue light have great effects on physiological processes and growth of plants. In this experiment, we investigated the physiological and growth response of pepper (Capsicum annuum L.) to supplementary red:blue (4:1) light for 1 h (T1), 3 h (T2), and 5 h (T3), and the full-spectrum light-emitting diodes, LEDs, as control (CK). Thirty-day-old seedlings were grown under these treatments for 20 days in a climate-controlled room before data measurement. The results showed that the light treatments significantly (p < 0.05) affected the photosynthesis and growth indexes as well as gene expression in the pepper seedlings. Plants under T2 generally had better performance in terms of seedling growth. A total of 124, 1283, and 1091 differentially expressed genes were found in CK vs. T1, CK vs. T2, and CK vs. T3, respectively. Among the treatments, T2 in comparison with CK had 705 upregulated and 578 downregulated differentially expressed genes (DEGs). We also found that CPRF2, Paggis, HLIPS, GIGANTEA, LSH1, and FTSH genes were expressed differently under the various light treatments. Based on GeneOntology (GO) enrichment analysis, DEGs were significantly enriched on 15 GO terms of which xyloglucan:xyloglucosyl transferase activity and apoplastic, cellular polysaccharide metabolic, and cellular carbohydrate metabolic processes were closely related to light responses. A total of 96 genes that are related to plant–pathogen interaction, zeatin biosynthesis, plant hormone signal transduction, and wax/cutin/suberine biosynthesis which are involved in the pathway of light reaction in plants were significantly enriched in T2 plants compared with plants under CK. The application of red:blue light at 4:1 for 3 h improved the growth of pepper seedlings better than the other treatments and this can be tested under the Chinese Solar Greenhouse condition.

Engineering the acceptor substrate specificity in the xyloglucan endotransglycosylase TmXET6.3 from nasturtium seeds (Tropaeolum majus L.).

The knowledge of substrate specificity of XET enzymes is important for the general understanding of metabolic pathways to challenge the established notion that these enzymes operate uniquely on cellulose-xyloglucan networks. Xyloglucan xyloglucosyl transferases (XETs) (EC 2.4.1.207) play a central role in loosening and re-arranging the cellulose-xyloglucan network, which is assumed to be the primary load-bearing structural component of plant cell walls. The sequence of mature TmXET6.3 from Tropaeolum majus (280 residues) was deduced by the nucleotide sequence analysis of complete cDNA by Rapid Amplification of cDNA Ends, based on tryptic and chymotryptic peptide sequences. Partly purified TmXET6.3, expressed in Pichia occurred in N-glycosylated and unglycosylated forms. The quantification of hetero-transglycosylation activities of TmXET6.3 revealed that (1,3;1,4)-, (1,6)- and (1,4)-β-D-glucooligosaccharides were the preferred acceptor substrates, while (1,4)-β-D-xylooligosaccharides, and arabinoxylo- and glucomanno-oligosaccharides were less preferred. The 3D model of TmXET6.3, and bioinformatics analyses of identified and putative plant xyloglucan endotransglycosylases (XETs)/hydrolases (XEHs) of the GH16 family revealed that H94, A104, Q108, K234 and K237 were the key residues that underpinned the acceptor substrate specificity of TmXET6.3. Compared to the wild-type enzyme, the single Q108R and K237T, and double-K234T/K237T and triple-H94Q/A104D/Q108R variants exhibited enhanced hetero-transglycosylation activities with xyloglucan and (1,4)-β-D-glucooligosaccharides, while those with (1,3;1,4)- and (1,6)-β-D-glucooligosaccharides were suppressed; the incorporation of xyloglucan to (1,4)-β-D-glucooligosaccharides by the H94Q variant was influenced most extensively. Structural and biochemical data of non-specific TmXET6.3 presented here extend the classic XET reaction mechanism by which these enzymes operate in plant cell walls. The evaluations of TmXET6.3 transglycosylation activities and the incidence of investigated residues in other members of the GH16 family suggest that a broad acceptor substrate specificity in plant XET enzymes could be more widespread than previously anticipated.

Spatiotemporal Changes in Xylan-1/Xyloglucan and Xyloglucan Xyloglucosyl Transferase (XTH-Xet5) as a Step-In of Ultrastructural Cell Wall Remodelling in Potato⁻Potato Virus Y (PVYNTN) Hypersensitive and Susceptible Reaction.

One type of monitoring system in a plant cell is the cell wall, which intensively changes its structure during interaction with pathogen-stress factors. The wall plays a role as a dynamic and controlled structure, although it is not fully understood how relevant these modifications are to the molecular mechanisms during plant–virus interactions. In this work we localise the non-cellulosic polysaccharides such as xyloglucan, xylan (xylan-1) and xyloglucosyl transferase (XTH-Xet5), the enzyme that participates in the metabolism of xyloglucan. This provided us with information about the in situ distribution of the components of the hemicellulotic cell wall matrix in hypersensitive and susceptible potato–PVYNTN interactions. The loosening of the cell wall was accompanied by an increase in xylan depositions during susceptible interactions, whereas, during the hypersensitive response, when the cell wall was reinforced, the xylan content decreased. Moreover, the PVY inoculation significantly redirected XTH-Xet5 depositions, regardless of types of interactions, compared to mock-inoculated tissues. Furthermore, the immunogold localisation clearly revealed the domination of Xet5 in the cell wall and in vesicles in the susceptible host. In contrast, in the resistant host increased levels of Xet5 were observed in cytoplasm, in the cell wall and in the trans-Golgi network. These findings show that the hypersensitive reaction activated XTH-Xet5 in the areas of xyloglucan endo-transglycosylase (XET) synthesis, which was then actively transported to cytoplasm, cell wall and to vacuoles. Our results provide novel insight into cell wall reorganisation during PVYNTN infection as a response to biotic stress factors. These novel findings help us to understand the mechanisms of defence responses that are incorporated into the cell wall signalling network.

Physiological performance and differential expression profiling of genes associated with drought tolerance in root tissue of four contrasting varieties of two Gossypium species.

Root growth in drying soil is generally limited by a combination of mechanical impedance and water stress. As the major function of root tissue is water and nutrient uptake, so it imparts an important role in plant growth and stress management. Previously, we have studied physiological performance and expression profiling of gene associated with drought tolerance in leaf tissue of four cotton varieties. Here, we have further continued our studies with the root tissue of these varieties. The Gossypium hirsutum species JKC-770 is drought-tolerant and KC-2 is drought-sensitive, while Gossypium herbaceum species JKC-717 is drought-tolerant and RAHS-187 is drought-sensitive. JKC-770 and JKC-717 the drought-tolerant varieties showed a comparatively high glutathione-S-transferase, superoxide dismutase, proline along with their gene expression, and low malondialdehyde content indicating low membrane damage and better antioxidative defense under drought condition. The expression levels of cellulose synthase, xyloglucan:xyloglucosyl transferase, and glycosyl hydrolases suggest modulation in cell wall structure and partitioning of sugars towards osmoprotectants instead of cell wall biosynthesis in tolerant varieties. Heat shock proteins and serine/threonine protein phosphotases show upregulation under drought condition, which are responsible for temperature tolerance and protein phosphorylation, respectively. These effects many metabolic processes and may be playing a key role in drought tolerance and adaptability of JKC-770 towards drought tolerance. The long-term water use efficiency (WUE) estimated in terms of carbon isotope discrimination (∆(13)C) in the root tissues showed maximum depletion in the ∆(13)C values in JKC-770 variety, while minimum in RAHS-187 under drought stress with reference to their respective control, suggesting a high WUE in JKC-770 variety.

Rice Os9BGlu31 Is a Transglucosidase with the Capacity to Equilibrate Phenylpropanoid, Flavonoid, and Phytohormone Glycoconjugates

Glycosylation is an important mechanism of controlling the reactivities and bioactivities of plant secondary metabolites and phytohormones. Rice (Oryza sativa) Os9BGlu31 is a glycoside hydrolase family GH1 transglycosidase that acts to transfer glucose between phenolic acids, phytohormones, and flavonoids. The highest activity was observed with the donors feruloyl-glucose, 4-coumaroyl-glucose, and sinapoyl-glucose, which are known to serve as donors in acyl and glucosyl transfer reactions in the vacuole, where Os9BGlu31 is localized. The free acids of these compounds also served as the best acceptors, suggesting that Os9BGlu31 may equilibrate the levels of phenolic acids and carboxylated phytohormones and their glucoconjugates. The Os9BGlu31 gene is most highly expressed in senescing flag leaf and developing seed and is induced in rice seedlings in response to drought stress and treatment with phytohormones, including abscisic acid, ethephon, methyljasmonate, 2,4-dichlorophenoxyacetic acid, and kinetin. Although site-directed mutagenesis of Os9BGlu31 indicated a function for the putative catalytic acid/base (Glu(169)), catalytic nucleophile residues (Glu(387)), and His(386), the wild type enzyme displays an unusual lack of inhibition by mechanism-based inhibitors of GH1 β-glucosidases that utilize a double displacement retaining mechanism.

Incorporation of β-(1,6)-linked glucooligosaccharides (pustulooligosaccharides) into plant cell wall structures

AbstractProtein extract of germinating nasturtium (Tropaeolum majus) seeds containing xyloglucan endotransglycosylase (xyloglucan xyloglucosyl transferase, EC 2.4.1.207, abbreviated XET) exhibited the heterotransglycosylating activity with donor/acceptor substrate pair xyloglucan/sulphorhodamine labelled pustulooligosaccharides (XG/PUOS-SR) in a dot blot assay. The heterotransglycosylating activity was confirmed by the substrate-product changes during transglycosylation by HPLC size-exclusion chromatography. Another donor substrate capable of being coupled with PUOS-SR was cellulose, probably owing to its structural similarity to xyloglucan. Surprisingly, microscopic comparison of the incorporation of the labelled xyloglucan nonasaccharide XGO9-SR (specific substrate for XET) and PUOS-SR into the cell wall structures clearly showed differences in their binding to specific cell structures: the primary cell wall and the plasma membrane. These findings indicate the existence in nasturtium of XETs with different localisation, substrate specificity and, probably, function.

Xyloglucan xyloglucosyl transferases from barley ( Hordeum vulgare L.) bind oligomeric and polymeric xyloglucan molecules in their acceptor binding sites

Background Xyloglucan xyloglucosyl transferases (EC 2.4.1.207), known as xyloglucan endotransglycosylases (XETs) use a disproportionation reaction mechanism and modulate molecular masses of xyloglucans. However, it is not known precisely how these size modulations and transfer reactions occur with polymeric acceptor substrates. Methods cDNAs encoding three barley HvXETs were expressed in Pichia pastoris and reaction mechanism and molecular properties of HvXETs were investigated. Results Significant differences in catalytic efficiencies ( k cat· K m − 1) were observed and these values were 0.01, 0.02 and 0.2 s − 1·mg − 1·ml for HvXET3, HvXET4 and HvXET6, respectively, using tamarind xyloglucan as a donor substrate. HPLC analyses of the reaction mixtures showed that HvXET6 followed a stochastic reaction mechanism with fluorescently or radioactively labelled tamarind xyloglucans and xyloglucan-derived oligosaccharides. The analyses from two successive reaction cycles revealed that HvXET6 could increase or decrease molecular masses of xyloglucans. In the first reaction cycle equilibrium was reached under limiting donor substrate concentrations, while xyloglucan mass modulations occurred during the second reaction cycle and depended on the molecular masses of incoming acceptors. Deglycosylation experiments indicated that occupancy of a singular N-glycosylation site was required for activity of HvXET6. Experiments with organic solvents demonstrated that HvXET6 tolerated DMSO, glycerol, methanol and 1,4-butanediol in 20% (v/v) concentrations. Conclusions The two-phase experiments demonstrated that large xyloglucan molecules can bind in the acceptor sites of HvXETs. General significance The results characterise donor and acceptor binding sites in plant XET, report that HvXETs act on xyloglucan donor substrates adsorbed onto nanocrystals and that HvXETs tolerate the presence of organic solvents.

Barley xyloglucan xyloglucosyl transferases bind xyloglucan-derived oligosaccharides in their acceptor-binding regions in multiple conformational states

Three barley xyloglucan endotransglycosylases (HvXETs), known as xyloglucan xyloglucosyl transferases (EC 2.4.1.207), were subjected to kinetic and computational docking studies. The k cat· K m −1 values with the reduced [ 3H]-labelled XXXG, XXLG/XLXG and XLLG acceptor substrates were 0.02 × 10 −2, 0.1 × 10 −2 and 3.2 × 10 −2 s −1 μM −1, while the K m constants were 10.6, 8.6 and 5.3 mM, obtained for HvXET3, HvXET4 and HvXET6, respectively. Docking of XLLG in acceptor-binding regions revealed that at least two conformational states were likely to participate in all isoforms. The assessments of kinetic and computational data indicated that the disposition of aromatic residues at the entrance to the active sites and the flexibility of proximal COOH-terminal loops could orient acceptors more or less favourably during binding, thus leading to tighter or weaker K m constants. The data suggested that binding of acceptors in HvXETs is guided by contributions from the conserved residues in the active sites and by the of neighbouring loops.

Analysis of functional divergence within two structurally related glycoside hydrolase families

Two glycoside hydrolase (GH) families were analyzed to detect the presence of functional divergence using the program DIVERGE. These two families, GH7 and GH16, each contain members related by amino acid sequence similarity, retaining hydrolytic mechanisms, and catalytic residue identity. GH7 and GH16 comprise GH Clan B, with a shared beta-jelly roll topology and mechanism. GH7 contains fungal cellobiohydrolases and endoglucanases and is divided into five main subfamilies, four of the former and one of the latter. Cluster comparisons between three of the cellobiohydrolase subfamilies and the endoglucanase subfamily identified specific amino acid residues that play a role in the functional divergence between the two enzyme types. GH16 contains subfamilies of bacterial agarases, xyloglucosyl transferases, 1,3-beta-D-glucanases, lichenases, and other enzymes with various substrate specificities and product profiles. Four cluster comparisons between these four main subfamilies again have identified amino acid residues involved in functional divergence between the subfamilies. (c) 2009 Wiley Periodicals, Inc. Biopolymers 91: 478-495, 2009.