The synthesis of xyloglucan, an abundant plant cell wall polysaccharide, requires CSLC function.

Xyloglucan (XyG) is an abundant component of the primary cell walls of most plants. While the structure of XyG has been well studied, much remains to be learned about its biosynthesis. Here we employed reverse genetics to investigate the role of Arabidopsis cellulose synthase like-C (CSLC) proteins in XyG biosynthesis. We found that single mutants containing a T-DNA in each of the five Arabidopsis CSLC genes had normal levels of XyG. However, higher-order cslc mutants had significantly reduced XyG levels, and a mutant with disruptions in all five CSLC genes had no detectable XyG. The higher-order mutants grew with mild tissue-specific phenotypes. Despite the apparent lack of XyG, the cslc quintuple mutant did not display significant alteration of gene expression at the whole-genome level, excluding transcriptional compensation. The quintuple mutant could be complemented by each of the five CSLC genes, supporting the conclusion that each of them encodes a XyG glucan synthase. Phylogenetic analyses indicated that the CSLC genes are widespread in the plant kingdom and evolved from an ancient family. These results establish the role of the CSLC genes in XyG biosynthesis, and the mutants described here provide valuable tools with which to study both the molecular details of XyG biosynthesis and the role of XyG in plant cell wall structure and function.

Polysaccharides Release in a Laboratory-Scale Batch Hydrothermal Pretreatment of Wheat Straw under Rigorous Isothermal Operation.

Hydrothermal pretreatment (HP) is an eco-friendly process for deconstructing lignocellulosic biomass (LCB) that plays a key role in ensuring the profitability of producing biofuels or bioproducts in a biorefinery. At the laboratory scale, HP is usually carried out under non-isothermal regimes with poor temperature control. In contrast, HP is usually carried out under isothermal conditions at the commercial scale. Consequently, significant discrepancies in the values of polysaccharide releases are found in the literature. Therefore, laboratory-scale HP data are not trustworthy if scale-up or retrofitting of HP at larger scales is required. This contribution presents the results of laboratory-scale batch HP for wheat straw in terms of xylan and glucan release that were obtained with rigorous temperature control under isothermal conditions during the reaction stage. The heating and cooling stages were carried out with fast rates (43 and -40 °C/min, respectively), minimizing non-isothermal reaction periods. Therefore, the polysaccharide release results can be associated exclusively with the isothermic reaction stage and can be considered as a reliable source of information for HP at commercial scales. The highest amount of xylan release was 4.8 g/L or 43% obtained at 180 °C and 20 min, while the glucan release exhibited a maximum of 1.2 g/L or 5.5%. at 160 °C/180 °C and 30 min.

Organization of Xylan Production in the Golgi During Secondary Cell Wall Biosynthesis.

Secondary cell wall (SCW) production during xylem development requires massive up-regulation of hemicellulose (e.g. glucuronoxylan) biosynthesis in the Golgi. Although mutant studies have revealed much of the xylan biosynthetic machinery, the precise arrangement of these proteins and their products in the Golgi apparatus is largely unknown. We used a fluorescently tagged xylan backbone biosynthetic protein (IRREGULAR XYLEM9; IRX9) as a marker of xylan production in the Golgi of developing protoxylem tracheary elements in Arabidopsis (Arabidopsis thaliana). Both live-cell confocal and transmission electron microscopy (TEM) revealed SCW deposition is accompanied by a significant proliferation of Golgi stacks. Furthermore, although Golgi stacks were randomly distributed, the organization of the cytoplasm ensured their close proximity to developing SCWs. Quantitative immuno-TEM revealed IRX9 is present in a specific subdomain of the Golgi stack and was most abundant in the ring of the inner margins of medial cisternae where fenestrations are abundant. Conversely, the xylan product accumulated in swollen trans cisternal margins and the Trans-Golgi network (TGN). The irx9 mutant lacked this expansion for both the cisternal margins and the TGN, whereas Golgi stack proliferation was unaffected. Golgi in irx9 also displayed dramatic changes in their structure, with increases in cisternal fenestration and tubulation. Our data support a new model where xylan biosynthesis and packaging into secretory vesicles are localized in distinct structural and functional domains of the Golgi. Rather than polysaccharide biosynthesis occurring in the center of the cisternae, IRX9 and the xylan product are arranged in successive concentric rings in Golgi cisternae.

Non-Starch Polysaccharides in Durum Wheat: A Review.

Durum wheat is one of most important cereal crops that serves as a staple dietary component for humans and domestic animals. It provides antioxidants, proteins, minerals and dietary fibre, which have beneficial properties for humans, especially as related to the health of gut microbiota. Dietary fibre is defined as carbohydrate polymers that are non-digestible in the small intestine. However, this dietary component can be digested by microorganisms in the large intestine and imparts physiological benefits at daily intake levels of 30-35 g. Dietary fibre in cereal grains largely comprises cell wall polymers and includes insoluble (cellulose, part of the hemicellulose component and lignin) and soluble (arabinoxylans and (1,3;1,4)-ß-glucans) fibre. More specifically, certain components provide immunomodulatory and cholesterol lowering activity, faecal bulking effects, enhanced absorption of certain minerals, prebiotic effects and, through these effects, reduce the risk of type II diabetes, cardiovascular disease and colorectal cancer. Thus, dietary fibre is attracting increasing interest from cereal processors, producers and consumers. Compared with other components of the durum wheat grain, fibre components have not been studied extensively. Here, we have summarised the current status of knowledge on the genetic control of arabinoxylan and (1,3;1,4)-ß-glucan synthesis and accumulation in durum wheat grain. Indeed, the recent results obtained in durum wheat open the way for the improvement of these important cereal quality parameters.

Expression profiles of cell-wall related genes vary broadly between two common maize inbreds during stem development.

BACKGROUND: The cellular machinery for cell wall synthesis and metabolism is encoded by members of large multi-gene families. Maize is both a genetic model for grass species and a potential source of lignocellulosic biomass from crop residues. Genetic improvement of maize for its utility as a bioenergy feedstock depends on identification of the specific gene family members expressed during secondary wall development in stems. RESULTS: High-throughput sequencing of transcripts expressed in developing rind tissues of stem internodes provided a comprehensive inventory of cell wall-related genes in maize (Zea mays, cultivar B73). Of 1239 of these genes, 854 were expressed among the internodes at ≥95 reads per 20 M, and 693 of them at ≥500 reads per 20 M. Grasses have cell wall compositions distinct from non-commelinid species; only one-quarter of maize cell wall-related genes expressed in stems were putatively orthologous with those of the eudicot Arabidopsis. Using a slope-metric algorithm, five distinct patterns for sub-sets of co-expressed genes were defined across a time course of stem development. For the subset of genes associated with secondary wall formation, fifteen sequence motifs were found in promoter regions. The same members of gene families were often expressed in two maize inbreds, B73 and Mo17, but levels of gene expression between them varied, with 30% of all genes exhibiting at least a 5-fold difference at any stage. Although presence-absence and copy-number variation might account for much of these differences, fold-changes of expression of a CADa and a FLA11 gene were attributed to polymorphisms in promoter response elements. CONCLUSIONS: Large genetic variation in maize as a species precludes the extrapolation of cell wall-related gene expression networks even from one common inbred line to another. Elucidation of genotype-specific expression patterns and their regulatory controls will be needed for association panels of inbreds and landraces to fully exploit genetic variation in maize and other bioenergy grass species.

Downregulating aspen xylan biosynthetic GT43 genes in developing wood stimulates growth via reprograming of the transcriptome.

Xylan is one of the main compounds determining wood properties in hardwood species. The xylan backbone is thought to be synthesized by a synthase complex comprising two members of the GT43 family. We downregulated all GT43 genes in hybrid aspen (Populus tremula × tremuloides) to understand their involvement in xylan biosynthesis. All three clades of the GT43 family were targeted for downregulation using RNA interference individually or in different combinations, either constitutively or specifically in developing wood. Simultaneous downregulation in developing wood of the B (IRX9) and C (IRX14) clades resulted in reduced xylan Xyl content relative to reducing end sequence, supporting their role in xylan backbone biosynthesis. This was accompanied by a higher lignocellulose saccharification efficiency. Unexpectedly, GT43 suppression in developing wood led to an overall growth stimulation, xylem cell wall thinning and a shift in cellulose orientation. Transcriptome profiling of these transgenic lines indicated that cell cycling was stimulated and secondary wall biosynthesis was repressed. We suggest that the reduced xylan elongation is sensed by the cell wall integrity surveying mechanism in developing wood. Our results show that wood-specific suppression of xylan-biosynthetic GT43 genes activates signaling responses, leading to increased growth and improved lignocellulose saccharification.

A glycosyl transferase family 43 protein involved in xylan biosynthesis is associated with straw digestibility in Brachypodium distachyon.

The recalcitrance of secondary plant cell walls to digestion constrains biomass use for the production of sustainable bioproducts and for animal feed. We screened a population of Brachypodium recombinant inbred lines (RILs) for cell wall digestibility using commercial cellulases and detected a quantitative trait locus (QTL) associated with this trait. Examination of the chromosomal region associated with this QTL revealed a candidate gene that encodes a putative glycosyl transferase family (GT) 43 protein, orthologue of IRX14 in Arabidopsis, and hence predicted to be involved in the biosynthesis of xylan. Arabinoxylans form the major matrix polysaccharides in cell walls of grasses, such as Brachypodium. The parental lines of the RIL population carry alternative nonsynonymous polymorphisms in the BdGT43A gene, which were inherited in the RIL progeny in a manner compatible with a causative role in the variation in straw digestibility. In order to validate the implied role of our candidate gene in affecting straw digestibility, we used RNA interference to lower the expression levels of the BdGT43A gene in Brachypodium. The biomass of the silenced lines showed higher digestibility supporting a causative role of the BdGT43A gene, suggesting that it might form a good target for improving straw digestibility in crops.

Three UDP-xylose transporters participate in xylan biosynthesis by conveying cytosolic UDP-xylose into the Golgi lumen in Arabidopsis.

UDP-xylose (UDP-Xyl) is synthesized by UDP-glucuronic acid decarboxylases, also termed UDP-Xyl synthases (UXSs). The Arabidopsis genome encodes six UXSs, which fall into two groups based upon their subcellular location: the Golgi lumen and the cytosol. The latter group appears to play an important role in xylan biosynthesis. Cytosolic UDP-Xyl is transported into the Golgi lumen by three UDP-Xyl transporters (UXT1, 2, and 3). However, while single mutants affected in the UDP-Xyl transporter 1 (UXT1) showed a substantial reduction in cell wall xylose content, a double mutant affected in UXT2 and UXT3 had no obvious effect on cell wall xylose deposition. This prompted us to further investigate redundancy among the members of the UXT family. Multiple uxt mutants were generated, including a triple mutant, which exhibited collapsed vessels and reduced cell wall thickness in interfascicular fiber cells. Monosaccharide composition, molecular weight, nuclear magnetic resonance, and immunolabeling studies demonstrated that both xylan biosynthesis (content) and fine structure were significantly affected in the uxt triple mutant, leading to phenotypes resembling those of the irx mutants. Pollination was also impaired in the uxt triple mutant, likely due to reduced filament growth and anther dehiscence caused by alterations in the composition of the cell walls. Moreover, analysis of the nucleotide sugar composition of the uxt mutants indicated that nucleotide sugar interconversion is influenced by the cytosolic UDP-Xyl pool within the cell. Taken together, our results underpin the physiological roles of the UXT family in xylan biosynthesis and provide novel insights into the nucleotide sugar metabolism and trafficking in plants.

KNAT7 positively regulates xylan biosynthesis by directly activating IRX9 expression in Arabidopsis.

Xylan is the major plant hemicellulosic polysaccharide in the secondary cell wall. The transcription factor KNOTTED-LIKE HOMEOBOX OF ARABIDOPSIS THALIANA 7 (KNAT7) regulates secondary cell wall biosynthesis, but its exact role in regulating xylan biosynthesis remains unclear. Using transactivation analyses, we demonstrate that KNAT7 activates the promoters of the xylan biosynthetic genes, IRREGULAR XYLEM 9 (IRX9), IRX10, IRREGULAR XYLEM 14-LIKE (IRX14L), and FRAGILE FIBER 8 (FRA8). The knat7 T-DNA insertion mutants have thinner vessel element walls and xylary fibers, and thicker interfascicular fiber walls in inflorescence stems, relative to wild-type (WT). KNAT7 overexpression plants exhibited opposite effects. Glycosyl linkage and sugar composition analyses revealed lower xylan levels in knat7 inflorescence stems, relative to WT; a finding supported by labeling of inflorescence walls with xylan-specific antibodies. The knat7 loss-of-function mutants had lower transcript levels of the xylan biosynthetic genes IRX9, IRX10, and FRA8, whereas KNAT7 overexpression plants had higher mRNA levels for IRX9, IRX10, IRX14L, and FRA8. Electrophoretic mobility shift assays indicated that KNAT7 binds to the IRX9 promoter. These results support the hypothesis that KNAT7 positively regulates xylan biosynthesis.

Cytosol-Localized UDP-Xylose Synthases Provide the Major Source of UDP-Xylose for the Biosynthesis of Xylan and Xyloglucan.

Xylan and xyloglucan are the two major cell wall hemicelluloses in plants, and their biosynthesis requires a steady supply of the sugar donor, UDP-xylose. UDP-xylose is synthesized through conversion of UDP-glucuronic acid (UDP-GlcA) by the activities of UDP-xylose synthase (UXS). There exist six UXS genes in the Arabidopsis thaliana genome; three of them (UXS1, UXS2 and UXS4) encode Golgi-localized enzymes and the other three (UXS3, UXS5 and UXS6) encode cytosol-localized enzymes. In this report, we investigated the contributions of these UXS genes in supplying UDP-xylose for the biosynthesis of xylan and xyloglucan. Expression analyses revealed that the six UXS genes exhibited distinct and overlapping expression patterns in different cell types of stems, root-hypocotyls and young seedlings, and that the relative enzymatic activity of UXS in the cytosol was 17 times higher than that in the Golgi. Among the six UXS genes, UXS3, UXS5 and UXS6 showed the highest expression in stems and were expressed predominantly in xylem cells and interfascicular fibers. Their predominant expression in secondary wall-forming cells was consistent with the finding that the expression of UXS3, UXS5 and UXS6 was directly activated by the secondary wall NAC master switches. Although simultaneous mutations of UXS1, UXS2 and UXS4 did not cause any apparent effects on plant growth and xylan biosynthesis, simultaneous down-regulation/mutations of UXS3, UXS5 and UXS6 led to a drastic reduction in secondary wall thickening, a severe deformation of xylem vessels, a significant decrease in xylan content without an apparent reduction in its chain length and an absence of GlcA side chains in xylan, which are reminiscent of the phenotypes of some known xylan-deficient mutants. Moreover, Immunolocalization with two xyloglucan monoclonal antibodies, LM15 and LM25, revealed a significant reduction in the amount of xylogulcan in the primary walls. These results demonstrate that the cytosol-localized UXS3, UXS5 and UXS6 play a predominant role in the supply of UDP-xylose for the biosynthesis of xylan and xyloglucan.

Feruloylation and structure of arabinoxylan in wheat endosperm cell walls from RNAi lines with suppression of genes responsible for backbone synthesis and decoration.

Arabinoxylan (AX) is the major component of the cell walls of wheat grain (70% in starchy endosperm), is an important determinant of end-use qualities affecting food processing, use for animal feed and distilling and is a major source of dietary fibre in the human diet. AX is a heterogeneous polysaccharide composed of fractions which can be sequentially extracted by water (WE-AX), then xylanase action (XE-AX) leaving an unextractable (XU-AX) fraction. We determined arabinosylation and feruloylation of AX in these fractions in both wild-type wheat and RNAi lines with decreased AX content (TaGT43_2 RNAi, TaGT47_2 RNAi) or decreased arabinose 3-linked to mono-substituted xylose (TaXAT1 RNAi). We show that these fractions are characterized by the degree of feruloylation of AX, <5, 5-7 and 13-19 mg bound ferulate (g-1 AX), and their content of diferulates (diFA), <0.3, 1-1.7 and 4-5 mg (g-1 AX), for the WE, XE and XU fractions, respectively, in all RNAi lines and their control lines. The amount of AX and its degree of arabinosylation and feruloylation were less affected by RNAi transgenes in the XE-AX fraction than in the WE-AX fraction and largely unaffected in the XU-AX fraction. As the majority of diFA is associated with the XU-AX fraction, there was only a small effect (TaGT43_2 RNAi, TaGT47_2 RNAi) or no effect (TaXAT1 RNAi) on total diFA content. Our results are compatible with a model where, to maintain cell wall function, diFA is maintained at stable levels when other AX properties are altered.

Asparagus IRX9, IRX10, and IRX14A Are Components of an Active Xylan Backbone Synthase Complex that Forms in the Golgi Apparatus.

Heteroxylans are abundant components of plant cell walls and provide important raw materials for the food, pharmaceutical, and biofuel industries. A number of studies in Arabidopsis (Arabidopsis thaliana) have suggested that the IRREGULAR XYLEM9 (IRX9), IRX10, and IRX14 proteins, as well as their homologs, are involved in xylan synthesis via a Golgi-localized complex termed the xylan synthase complex (XSC). However, both the biochemical and cell biological research lags the genetic and molecular evidence. In this study, we characterized garden asparagus (Asparagus officinalis) stem xylan biosynthesis genes (AoIRX9, AoIRX9L, AoIRX10, AoIRX14A, and AoIRX14B) by heterologous expression in Nicotiana benthamiana We reconstituted and partially purified an active XSC and showed that three proteins, AoIRX9, AoIRX10, and AoIRX14A, are necessary for xylan xylosyltranferase activity in planta. To better understand the XSC structure and its composition, we carried out coimmunoprecipitation and bimolecular fluorescence complementation analysis to show the molecular interactions between these three IRX proteins. Using a site-directed mutagenesis approach, we showed that the DxD motifs of AoIRX10 and AoIRX14A are crucial for the catalytic activity. These data provide, to our knowledge, the first lines of biochemical and cell biological evidence that AoIRX9, AoIRX10, and AoIRX14A are core components of a Golgi-localized XSC, each with distinct roles for effective heteroxylan biosynthesis.

A homology model of Xyloglucan Xylosyltransferase 2 reveals critical amino acids involved in substrate binding.

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 Golgi-localized 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. Site-directed 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.

Functional conservation and divergence of Miscanthus lutarioriparius GT43 gene family in xylan biosynthesis.

BACKGROUND: Xylan is the most abundant un-cellulosic polysaccharides of plant cell walls. Much progress in xylan biosynthesis has been gained in the model plant species Arabidopsis. Two homologous pairs Irregular Xylem 9 (IRX9)/9L and IRX14/14L from glycosyltransferase (GT) family 43 have been proved to play crucial roles in xylan backbone biosynthesis. However, xylan biosynthesis in grass such as Miscanthus remains poorly understood. RESULTS: We characterized seven GT43 members in M. lutarioriparius, a promising bioenergy crop. Quantitative real-time RT-PCR (qRT-PCR) analysis revealed that the expression of MlGT43 genes was ubiquitously detected in the tissues examined. In-situ hybridization demonstrated that MlGT43A-B and MlGT43F-G were specifically expressed in sclerenchyma, while MlGT43C-E were expressed in both sclerenchyma and parenchyma. All seven MlGT43 proteins were localized to Golgi apparatus. Overexpression of MlGT43A-E but not MlGT43F and MlGT43G in Arabidopsis irx9 fully or partially rescued the mutant defects, including morphological changes, collapsed xylem and increased xylan contents, whereas overexpression of MlGT43F and MlGT43G but not MlGT43A-E complemented the defects of irx14, indicating that MlGT43A-E are functional orthologues of IRX9, while MlGT43F and MlGT43G are functional orthologues of IRX14. However, overexpression of all seven MlGT43 genes could not rescue the mucilage defects of irx14 seeds. Furthermore, transient transactivation analyses of MlGT43A-E reporters demonstrated that MlGT43A and MlGT43B but not MlGT43C-E were differentially activated by MlSND1, MlMYB46 or MlVND7. CONCLUSION: The results demonstrated that all seven MlGT43s are functionally conserved in xylan biosynthesis during secondary cell wall formation but diversify in seed coat mucilage xylan biosynthesis. The results obtained provide deeper insight into xylan biosynthesis in grass, which lay the foundation for genetic modification of grass cell wall components and structure to better suit for next-generation biofuel production.

Effect of suppression of arabinoxylan synthetic genes in wheat endosperm on chain length of arabinoxylan and extract viscosity.

Arabinoxylan (AX) is the dominant component within wheat (Triticum aestivum L.) endosperm cell walls, accounting for 70% of the polysaccharide. The viscosity of aqueous extracts from wheat grain is a key trait influencing the processing for various end uses, and this is largely determined by the properties of endosperm AX. We have previously shown dramatic effects on endosperm AX in transgenic wheat by down-regulating either TaGT43_2 or TaGT47_2 genes (orthologues to IRX9 and IRX10 in Arabidopsis, respectively) implicated in AX chain extension and the TaXAT1 gene responsible for monosubstitution by 3-linked arabinose. Here, we use these transgenic lines to investigate the relationship between amounts of AX in soluble and insoluble fractions, the chain-length distribution of these measured by intrinsic viscosity and the overall effect on extract viscosity. In transgenic lines expressing either the TaGT43_2 or TaGT47_2 RNAi transgenes, the intrinsic viscosities of water-extractable (WE-AX) and of a water-insoluble alkaline-extracted fraction (AE-AX) were decreased by between 10% and 50% compared to control lines. In TaXAT1 RNAi lines, there was a 15% decrease in intrinsic viscosity of WE-AX but no consistent effect on that of AE-AX. All transgenic lines showed decreases in extract viscosity with larger effects in TaGT43_2 and TaGT47_2 RNAi lines (by up to sixfold) than in TaXAT1 RNAi lines (by twofold). These effects were explained by the decreases in amount and chain length of WE-AX, with decreases in amount having the greater influence. Extract viscosity from wheat grain can therefore be greatly decreased by suppression of single gene targets.

Attachment of Salmonella strains to a plant cell wall model is modulated by surface characteristics and not by specific carbohydrate interactions.

BACKGROUND: Processing of fresh produce exposes cut surfaces of plant cell walls that then become vulnerable to human foodborne pathogen attachment and contamination, particularly by Salmonella enterica. Plant cell walls are mainly composed of the polysaccharides cellulose, pectin and hemicelluloses (predominantly xyloglucan). Our previous work used bacterial cellulose-based plant cell wall models to study the interaction between Salmonella and the various plant cell wall components. We demonstrated that Salmonella attachment was favoured in the presence of pectin while xyloglucan had no effect on its attachment. Xyloglucan significantly increased the attachment of Salmonella cells to the plant cell wall model only when it was in association with pectin. In this study, we investigate whether the plant cell wall polysaccharides mediate Salmonella attachment to the bacterial cellulose-based plant cell wall models through specific carbohydrate interactions or through the effects of carbohydrates on the physical characteristics of the attachment surface. RESULTS: We found that none of the monosaccharides that make up the plant cell wall polysaccharides specifically inhibit Salmonella attachment to the bacterial cellulose-based plant cell wall models. Confocal laser scanning microscopy showed that Salmonella cells can penetrate and attach within the tightly arranged bacterial cellulose network. Analysis of images obtained from atomic force microscopy revealed that the bacterial cellulose-pectin-xyloglucan composite with 0.3 % (w/v) xyloglucan, previously shown to have the highest number of Salmonella cells attached to it, had significantly thicker cellulose fibrils compared to other composites. Scanning electron microscopy images also showed that the bacterial cellulose and bacterial cellulose-xyloglucan composites were more porous when compared to the other composites containing pectin. CONCLUSIONS: Our study found that the attachment of Salmonella cells to cut plant cell walls was not mediated by specific carbohydrate interactions. This suggests that the attachment of Salmonella strains to the plant cell wall models were more dependent on the structural characteristics of the attachment surface. Pectin reduces the porosity and space between cellulose fibrils, which then forms a matrix that is able to retain Salmonella cells within the bacterial cellulose network. When present with pectin, xyloglucan provides a greater surface for Salmonella cells to attach through the thickening of cellulose fibrils.

The presence of fucogalactoxyloglucan and its synthesis in rice indicates conserved functional importance in plants.

The predominant structure of the hemicellulose xyloglucan (XyG) found in the cell walls of dicots is a fucogalactoXyG with an XXXG core motif, whereas in the Poaceae (grasses and cereals), the structure of XyG is less xylosylated (XXGGn core motif) and lacks fucosyl residues. However, specialized tissues of rice (Oryza sativa) also contain fucogalactoXyG. Orthologous genes of the fucogalactoXyG biosynthetic machinery of Arabidopsis (Arabidopsis thaliana) are present in the rice genome. Expression of these rice genes, including fucosyl-, galactosyl-, and acetyltransferases, in the corresponding Arabidopsis mutants confirmed their activity and substrate specificity, indicating that plants in the Poaceae family have the ability to synthesize fucogalactoXyG in vivo. The data presented here provide support for a functional conservation of XyG structure in higher plants.

Multiple regulatory mechanisms control the expression of the Geobacillus stearothermophilus gene for extracellular xylanase.

Geobacillus stearothermophilus T-6 produces a single extracellular xylanase (Xyn10A) capable of producing short, decorated xylo-oligosaccharides from the naturally branched polysaccharide, xylan. Gel retardation assays indicated that the master negative regulator, XylR, binds specifically to xylR operators in the promoters of xylose and xylan-utilization genes. This binding is efficiently prevented in vitro by xylose, the most likely molecular inducer. Expression of the extracellular xylanase is repressed in medium containing either glucose or casamino acids, suggesting that carbon catabolite repression plays a role in regulating xynA. The global transcriptional regulator CodY was shown to bind specifically to the xynA promoter region in vitro, suggesting that CodY is a repressor of xynA. The xynA gene is located next to an uncharacterized gene, xynX, that has similarity to the NIF3 (Ngg1p interacting factor 3)-like protein family. XynX binds specifically to a 72-bp fragment in the promoter region of xynA, and the expression of xynA in a xynX null mutant appeared to be higher, indicating that XynX regulates xynA. The specific activity of the extracellular xylanase increases over 50-fold during early exponential growth, suggesting cell density regulation (quorum sensing). Addition of conditioned medium to fresh and low cell density cultures resulted in high expression of xynA, indicating that a diffusible extracellular xynA density factor is present in the medium. The xynA density factor is heat-stable, sensitive to proteases, and was partially purified using reverse phase liquid chromatography. Taken together, these results suggest that xynA is regulated by quorum-sensing at low cell densities.

Two Arabidopsis proteins synthesize acetylated xylan in vitro.

Xylan is the third most abundant glycopolymer on earth after cellulose and chitin. As a major component of wood, grain and forage, this natural biopolymer has far-reaching impacts on human life. This highly acetylated cell wall polysaccharide is a vital component of the plant cell wall, which functions as a molecular scaffold, providing plants with mechanical strength and flexibility. Mutations that impair synthesis of the xylan backbone give rise to plants that fail to grow normally because of collapsed xylem cells in the vascular system. Phenotypic analysis of these mutants has implicated many proteins in xylan biosynthesis; however, the enzymes directly responsible for elongation and acetylation of the xylan backbone have not been unambiguously identified. Here we provide direct biochemical evidence that two Arabidopsis thaliana proteins, IRREGULAR XYLEM 10-L (IRX10-L) and ESKIMO1/TRICOME BIREFRINGENCE 29 (ESK1/TBL29), catalyze these respective processes in vitro. By identifying the elusive xylan synthase and establishing ESK1/TBL29 as the archetypal plant polysaccharide O-acetyltransferase, we have resolved two long-standing questions in plant cell wall biochemistry. These findings shed light on integral steps in the molecular pathways used by plants to synthesize a major component of the world's biomass and expand our toolkit for producing glycopolymers with valuable properties.

Family 34 glycosyltransferase (GT34) genes and proteins in Pinus radiata (radiata pine) and Pinus taeda (loblolly pine).

Using a functional genomics approach, four candidate genes (PtGT34A, PtGT34B, PtGT34C and PtGT34D) were identified in Pinus taeda. These genes encode CAZy family GT34 glycosyltransferases that are involved in the synthesis of cell-wall xyloglucans and heteromannans. The full-length coding sequences of three orthologs (PrGT34A, B and C) were isolated from a xylem-specific cDNA library from the closely related Pinus radiata. PrGT34B is the ortholog of XXT1 and XXT2, the two main xyloglucan (1→6)-α-xylosyltransferases in Arabidopsis thaliana. PrGT34C is the ortholog of XXT5 in A. thaliana, which is also involved in the xylosylation of xyloglucans. PrGT34A is an ortholog of a galactosyltransferase from fenugreek (Trigonella foenum-graecum) that is involved in galactomannan synthesis. Truncated coding sequences of the genes were cloned into plasmid vectors and expressed in a Sf9 insect cell-culture system. The heterologous proteins were purified, and in vitro assays showed that, when incubated with UDP-xylose and cellotetraose, cellopentaose or cellohexaose, PrGT34B showed xylosyltransferase activity, and, when incubated with UDP-galactose and the same cello-oligosaccharides, PrGT34B showed some galactosyltransferase activity. The ratio of xylosyltransferase to galactosyltransferase activity was 434:1. Hydrolysis of the galactosyltransferase reaction products using galactosidases showed the linkages formed were α-linkages. Analysis of the products of PrGT34B by MALDI-TOF MS showed that up to three xylosyl residues were transferred from UDP-xylose to cellohexaose. The heterologous proteins PrGT34A and PrGT34C showed no detectable enzymatic activity.