Xyloglucan Side Chains Research Articles

Identification of a xyloglucan beta-xylopyranosyltransferase from Vaccinium corymbosum.

Plant cell walls contain the hemicellulose xyloglucan, whose fine structure may vary depending on cell type, tissue, and/or plant species. Most but not all of the glycosyltransferases involved in the biosynthesis of xyloglucan sidechains have been identified. Here, we report the identification of several functional glycosyltransferases from blueberry (Vaccinium corymbosum bluecrop). Among those transferases is a hitherto elusive Xyloglucan:Beta-xylosylTransferase (XBT). Heterologous expression of VcXBT in the Arabidopsis thaliana double mutant mur3 xlt2, where xyloglucan consists only of an unsubstituted xylosylated glucan core structure, results in the production of the xylopyranose-containing "U" sidechain as characterized by mass spectrometry, glycosidic linkage, and NMR analysis. The introduction of the additional xylopyranosyl residue rescues the dwarfed phenotype of the untransformed Arabidopsis mur3 xlt2 mutant to wild-type height. Structural protein analysis using Alphafold of this and other related xyloglucan glycosyltransferase family 47 proteins not only identifies potential domains that might influence the regioselectivity of these enzymes but also gives hints to specific amino acids that might determine the donor-substrate specificity of these glycosyltransferases.

Escherichia coli O157:H7 F9 Fimbriae Recognize Plant Xyloglucan and Elicit a Response in Arabidopsis thaliana.

Fresh produce is often a source of enterohaemorrhagic Escherichia coli (EHEC) outbreaks. Fimbriae are extracellular structures involved in cell-to-cell attachment and surface colonisation. F9 (Fml) fimbriae have been shown to be expressed at temperatures lower than 37 °C, implying a function beyond the mammalian host. We demonstrate that F9 fimbriae recognize plant cell wall hemicellulose, specifically galactosylated side chains of xyloglucan, using glycan arrays. E. coli expressing F9 fimbriae had a positive advantage for adherence to spinach hemicellulose extract and tissues, which have galactosylated oligosaccharides as recognized by LM24 and LM25 antibodies. As fimbriae are multimeric structures with a molecular pattern, we investigated whether F9 fimbriae could induce a transcriptional response in model plant Arabidopsis thaliana, compared with flagella and another fimbrial type, E. coli common pilus (ECP), using DNA microarrays. F9 induced the differential expression of 435 genes, including genes involved in the plant defence response. The expression of F9 at environmentally relevant temperatures and its recognition of plant xyloglucan adds to the suite of adhesins EHEC has available to exploit the plant niche.

Xyloglucan O-acetyltransferases from Arabidopsis thaliana and Populus trichocarpa catalyze acetylation of fucosylated galactose residues on xyloglucan side chains.

AXY4/XGOAT1, AXY4L/XGOAT2 and PtrXGOATs are O-acetyltransferases acetylating fucosylated galactose residues on xyloglucan and AXY9 does not directly catalyze O-acetylation of xyloglucan but exhibits weak acetylesterase activity. Xyloglucan is a major hemicellulose that cross-links cellulose in the primary walls of dicot plants and the galactose (Gal) residues on its side chains can be mono- and di-O-acetylated. In Arabidopsis thaliana, mutations of three AXY (altered xyloglucan) genes, AXY4, AXY4L and AXY9, have previously been shown to cause a reduction in xyloglucan acetylation, but their biochemical functions remain to be investigated. In this report, we demonstrated that recombinant proteins of AXY4/XGOAT1 (xyloglucan O-acetyltransferase1), AXY4L/XGOAT2 and their close homologs from Populus trichocarpa, PtrXGOATs, displayed O-acetyltransferase activities transferring acetyl groups from acetyl CoA onto xyloglucan oligomers. Structural analysis of XGOAT-catalyzed reaction products revealed that XGOATs mediated predominantly 6-O-monoacetylation and a much lesser degree of 3-O and 4-O-monoacetylation and 4,6-di-O-acetylation of Gal residues on xyloglucan side chains. XGOATs appeared to preferentially acetylate fucosylated Gal residues with little activity toward non-fucosylated Gal residues. Mutations of the conserved amino acid residues in the GDS and DXXH motifs in AXY4/XGOAT1 resulted in a drastic reduction in its ability to transfer acetyl groups onto xyloglucan oligomers. In addition, although recombinant AXY9 was unable to transfer acetyl groups from acetyl CoA onto xyloglucan oligomers, it was catalytically active as demonstrated by its weak acetylesterase activity that was also exhibited by AXY4/XGOAT1 and AXY4L/XGOAT2. Furthermore, we showed that the AXY8 fucosidase was able to hydrolyze fucosyl residues from both non-acetylated and acetylated xyloglucan oligomers. These findings provide biochemical evidence that AXY4/XGOAT1, AXY4L/XGOAT2 and PtrXGOATs are xyloglucan O-acetyltransferases catalyzing acetyl transfer onto fucosylated Gal residues on xyloglucan side chains and the defucosylation of these acetylated side chains by apoplastic AXY8 generates side chains with acetylated, non-fucosylated Gal residues.

Identification of an arabinopyranosyltransferase from Physcomitrella patens involved in the synthesis of the hemicellulose xyloglucan.

The hemicellulose xyloglucan consists of a backbone of a β‐1,4 glucan substituted with xylosyl moieties and many other, diverse side chains that are important for its proper function. Many, but not all glycosyltransferases involved in the biosynthesis of xyloglucan have been identified. Here, we report the identification of an hitherto elusive xyloglucan:arabinopyranosyltransferase. This glycosyltransferase was isolated from the moss Physcomitrella patens, where it acts as a xyloglucan “D”‐side chain transferase (XDT). Heterologous expression of PpXDT in the Arabidopsis thaliana double mutant mur3.1 xlt2, where xyloglucan consists of a xylosylated glucan without further glycosyl substituents, results in the production of the arabinopyranose‐containing “D” side chain as characterized by oligosaccharide mass profiling, glycosidic linkage analysis, and NMR analysis. In addition, expression of a related Physcomitrella glycosyltransferase ortholog of PpXLT2 leads to the production of the galactose‐containing “L” side chain. The presence of the “D” and “L” xyloglucan side chains in the Arabidopsis double mutant Atmur3.1 xlt2 expressing PpXDT and PpXLT2, respectively, rescues the dwarfed phenotype of untransformed Atmur3.1 xlt2 mutants to nearly wild‐type height. Expression of PpXDT and PpXLT2 in the Atmur3.1 xlt2 mutant also enhanced root growth.

Structural, mutagenic and insilico studies of xyloglucan fucosylation in Arabidopsis thaliana suggest a water-mediated mechanism.

The mechanistic underpinnings of the complex process of plant polysaccharide biosynthesis are poorly understood, largely because of the resistance of glycosyltransferase (GT) enzymes to structural characterization. In Arabidopsis thaliana, a glycosyl transferase family 37 (GT37) fucosyltransferase 1 (AtFUT1) catalyzes the regiospecific transfer of terminal 1,2-fucosyl residues to xyloglucan side chains - a key step in the biosynthesis of fucosylated sidechains of galactoxyloglucan. We unravel the mechanistic basis for fucosylation by AtFUT1 with a multipronged approach involving protein expression, X-ray crystallography, mutagenesis experiments and molecular simulations. Mammalian cell culture expressions enable the sufficient production of the enzyme for X-ray crystallography, which reveals the structural architecture of AtFUT1 in complex with bound donor and acceptor substrate analogs. The lack of an appropriately positioned active site residue as a catalytic base leads us to propose an atypical water-mediated fucosylation mechanism facilitated by an H-bonded network, which is corroborated by mutagenesis experiments as well as detailed atomistic simulations.

Structure of Arabidopsis thaliana FUT1 Reveals a Variant of the GT-B Class Fold and Provides Insight into Xyloglucan Fucosylation.

The plant cell wall is a complex and dynamic network made mostly of cellulose, hemicelluloses, and pectins. Xyloglucan, the major hemicellulosic component in Arabidopsis thaliana, is biosynthesized in the Golgi apparatus by a series of glycan synthases and glycosyltransferases before export to the wall. A better understanding of the xyloglucan biosynthetic machinery will give clues toward engineering plants with improved wall properties or designing novel xyloglucan-based biomaterials. The xyloglucan-specific α2-fucosyltransferase FUT1 catalyzes the transfer of fucose from GDP-fucose to terminal galactosyl residues on xyloglucan side chains. Here, we present crystal structures of Arabidopsis FUT1 in its apoform and in a ternary complex with GDP and a xylo-oligosaccharide acceptor (named XLLG). Although FUT1 is clearly a member of the large GT-B fold family, like other fucosyltransferases of known structures, it contains a variant of the GT-B fold. In particular, it includes an extra C-terminal region that is part of the acceptor binding site. Our crystal structures support previous findings that FUT1 behaves as a functional dimer. Mutational studies and structure comparison with other fucosyltransferases suggest that FUT1 uses a SN2-like reaction mechanism similar to that of protein-O-fucosyltransferase 2. Thus, our results provide new insights into the mechanism of xyloglucan fucosylation in the Golgi.

Structural dissection of a complex Bacteroides ovatus gene locus conferring xyloglucan metabolism in the human gut.

The human gastrointestinal tract harbours myriad bacterial species, collectively termed the microbiota, that strongly influence human health. Symbiotic members of our microbiota play a pivotal role in the digestion of complex carbohydrates that are otherwise recalcitrant to assimilation. Indeed, the intrinsic human polysaccharide-degrading enzyme repertoire is limited to various starch-based substrates; more complex polysaccharides demand microbial degradation. Select Bacteroidetes are responsible for the degradation of the ubiquitous vegetable xyloglucans (XyGs), through the concerted action of cohorts of enzymes and glycan-binding proteins encoded by specific xyloglucan utilization loci (XyGULs). Extending recent (meta)genomic, transcriptomic and biochemical analyses, significant questions remain regarding the structural biology of the molecular machinery required for XyG saccharification. Here, we reveal the three-dimensional structures of an α-xylosidase, a β-glucosidase, and two α-l-arabinofuranosidases from the Bacteroides ovatus XyGUL. Aided by bespoke ligand synthesis, our analyses highlight key adaptations in these enzymes that confer individual specificity for xyloglucan side chains and dictate concerted, stepwise disassembly of xyloglucan oligosaccharides. In harness with our recent structural characterization of the vanguard endo-xyloglucanse and cell-surface glycan-binding proteins, the present analysis provides a near-complete structural view of xyloglucan recognition and catalysis by XyGUL proteins.

The galactoside 2-α-L-fucosyltransferase FUT1 from Arabidopsis thaliana: crystallization and experimental MAD phasing.

The plant cell wall is a complex network of polysaccharides made up of cellulose, hemicelluloses and pectins. Xyloglucan (XyG), which is the main hemicellulosic component of dicotyledonous plants, has attracted much attention for its role in plant development and for its many industrial applications. The XyG-specific fucosyltransferase (FUT1) adds a fucose residue from GDP-fucose to the 2-O position of the terminal galactosyl residues on XyG side chains. Recombinant FUT1 from Arabidopsis thaliana was crystallized in two different crystal forms, with the best diffracting crystals (up to 1.95 Å resolution) belonging to the monoclinic space group P21, with unit-cell parameters a = 87.6, b = 84.5, c=150.3 Å, β = 96.3°. Ab initio phases were determined using a two-wavelength anomalous dispersion experiment on a tantalum bromide-derivatized crystal with data collected at the rising and descending inflection points of the Ta white line. An interpretable electron-density map was obtained after elaborate density modification. Model completion and structural analysis are currently under way.

AUXIN BINDING PROTEIN1 Links Cell Wall Remodeling, Auxin Signaling, and Cell Expansion in Arabidopsis

Cell expansion is an increase in cell size and thus plays an essential role in plant growth and development. Phytohormones and the primary plant cell wall play major roles in the complex process of cell expansion. In shoot tissues, cell expansion requires the auxin receptor AUXIN BINDING PROTEIN1 (ABP1), but the mechanism by which ABP1 affects expansion remains unknown. We analyzed the effect of functional inactivation of ABP1 on transcriptomic changes in dark-grown hypocotyls and investigated the consequences of gene expression on cell wall composition and cell expansion. Molecular and genetic evidence indicates that ABP1 affects the expression of a broad range of cell wall-related genes, especially cell wall remodeling genes, mainly via an SCF(TIR/AFB)-dependent pathway. ABP1 also functions in the modulation of hemicellulose xyloglucan structure. Furthermore, fucosidase-mediated defucosylation of xyloglucan, but not biosynthesis of nonfucosylated xyloglucan, rescued dark-grown hypocotyl lengthening of ABP1 knockdown seedlings. In muro remodeling of xyloglucan side chains via an ABP1-dependent pathway appears to be of critical importance for temporal and spatial control of cell expansion.

Molecular dynamics simulation study of xyloglucan adsorption on cellulose surfaces: effects of surface hydrophobicity and side-chain variation

The effect of surface hydrophobicity and side-chain variation on xyloglucan adsorption onto cellulose microfibrils (CMF) is investigated via molecular dynamics simulations. A molecular model of CMF with (100), (010), (1–10), (110) and (200) crystal faces was built. We considered xylogluco-oligosaccharides (XGO) with three repeating units, namely (XXXG)3, (XXLG)3, and (XXFG)3 (where each (1,4)-β-d-glucosyl residue in the backbone is given a one-letter code according to its substituents: G = β-d-Glc; X = α-d-Xyl-(1,6)-β-d-Glc; L = β-d-Gal-(1,2)-α-d-Xyl-(1,6)-β-d-Glc; F = α-l-Fuc-(1,2)-β-d-Gal-(1,2)-α-d-Xyl-(1,6)-β-d-Glc). Our work shows that (XXXG)3 binds more favorably to the CMF (100) and (200) hydrophobic surfaces than to the (110), (010) and (1–10) hydrophilic surfaces. The origin of this behavior is attributed to the topography of hydrophobic CMF surface, which stabilizes (XXXG)3 in flat conformation. In contrast, on the rough hydrophilic CMF surface (XXXG)3 adopts a less favorable random-coil conformation to facilitate more hydrogen bonds with the surface. Extending the xyloglucan side chains from (XXXG)3 to (XXLG)3 hinders their stacking on the CMF hydrophobic surface. For (XXFG)3, the interaction with the hydrophobic surface is as strong as (XXXG)3. All three XGOs have similar binding to the hydrophilic surface. Steered molecular dynamics simulation was performed on an adhesive model where (XXXG)3 was sandwiched between two CMF hydrophobic surfaces. Our analysis suggests that this sandwich structure might help provide mechanical strength for plant cell walls. Our study relates to a recently revised model of primary cell walls in which extensibility is largely determined by xyloglucan located in limited regions of tight contact between CMFs.

Understanding How Noncatalytic Carbohydrate Binding Modules Can Display Specificity for Xyloglucan

Plant biomass is central to the carbon cycle and to environmentally sustainable industries exemplified by the biofuel sector. Plant cell wall degrading enzymes generally contain noncatalytic carbohydrate binding modules (CBMs) that fulfil a targeting function, which enhances catalysis. CBMs that bind β-glucan chains often display broad specificity recognizing β1,4-glucans (cellulose), β1,3-β1,4-mixed linked glucans and xyloglucan, a β1,4-glucan decorated with α1,6-xylose residues, by targeting structures common to the three polysaccharides. Thus, CBMs that recognize xyloglucan target the β1,4-glucan backbone and only accommodate the xylose decorations. Here we show that two closely related CBMs, CBM65A and CBM65B, derived from EcCel5A, a Eubacterium cellulosolvens endoglucanase, bind to a range of β-glucans but, uniquely, display significant preference for xyloglucan. The structures of the two CBMs reveal a β-sandwich fold. The ligand binding site comprises the β-sheet that forms the concave surface of the proteins. Binding to the backbone chains of β-glucans is mediated primarily by five aromatic residues that also make hydrophobic interactions with the xylose side chains of xyloglucan, conferring the distinctive specificity of the CBMs for the decorated polysaccharide. Significantly, and in contrast to other CBMs that recognize β-glucans, CBM65A utilizes different polar residues to bind cellulose and mixed linked glucans. Thus, Gln(106) is central to cellulose recognition, but is not required for binding to mixed linked glucans. This report reveals the mechanism by which β-glucan-specific CBMs can distinguish between linear and mixed linked glucans, and show how these CBMs can exploit an extensive hydrophobic platform to target the side chains of decorated β-glucans.

AtBGAL10 Is the Main Xyloglucan β-Galactosidase in Arabidopsis, and Its Absence Results in Unusual Xyloglucan Subunits and Growth Defects

In growing cells, xyloglucan is thought to connect cellulose microfibrils and regulate their separation during wall extension. In Arabidopsis (Arabidopsis thaliana), a significant proportion of xyloglucan side chains contain β-galactose linked to α-xylose at O2. In this work, we identified AtBGAL10 (At5g63810) as the gene responsible for the majority of β-galactosidase activity against xyloglucan. Xyloglucan from bgal10 insertional mutants was found to contain a large proportion of unusual subunits, such as GLG and GLLG. These subunits were not detected in a bgal10 xyl1 double mutant, deficient in both β-galactosidase and α-xylosidase. Xyloglucan from bgal10 xyl1 plants was enriched instead in XXLG/XLXG and XLLG subunits. In both cases, changes in xyloglucan composition were larger in the endoglucanase-accessible fraction. These results suggest that glycosidases acting on nonreducing ends digest large amounts of xyloglucan in wild-type plants, while plants deficient in any of these activities accumulate partly digested subunits. In both bgal10 and bgal10 xyl1, siliques and sepals were shorter, a phenotype that could be explained by an excess of nonreducing ends leading to a reinforced xyloglucan network. Additionally, AtBGAL10 expression was examined with a promoter-reporter construct. Expression was high in many cell types undergoing wall extension or remodeling, such as young stems, abscission zones, or developing vasculature, showing good correlation with α-xylosidase expression.

Subcompartment localization of the side chain xyloglucan-synthesizing enzymes within Golgi stacks of tobacco suspension-cultured cells

Xyloglucan is the dominant hemicellulosic polysaccharide of the primary cell wall of dicotyledonous plants that plays a key role in plant development. It is well established that xyloglucan is assembled within Golgi stacks and transported in Golgi-derived vesicles to the cell wall. It is also known that the biosynthesis of xyloglucan requires the action of glycosyltransferases including α-1,6-xylosyltransferase, β-1,2-galactosyltransferase and α-1,2-fucosyltransferase activities responsible for the addition of xylose, galactose and fucose residues to the side chains. There is, however, a lack of knowledge on how these enzymes are distributed within subcompartments of Golgi stacks. We have undertaken a study aiming at mapping these glycosyltransferases within Golgi stacks using immunogold-electron microscopy. To this end, we generated transgenic lines of tobacco (Nicotiana tabacum) BY-2 suspension-cultured cells expressing either the α-1,6-xylosyltransferase, AtXT1, the β-1,2-galactosyltransferase, AtMUR3, or the α-1,2-fucosyltransferase AtFUT1 of Arabidopsis thaliana fused to green-fluorescent protein (GFP). Localization of the fusion proteins within the endomembrane system was assessed using confocal microscopy. Additionally, tobacco cells were high pressure-frozen/freeze-substituted and subjected to quantitative immunogold labelling using anti-GFP antibodies to determine the localization patterns of the enzymes within subtypes of Golgi cisternae. The data demonstrate that: (i) all fusion proteins, AtXT1-GFP, AtMUR3-GFP and AtFUT1-GFP are specifically targeted to the Golgi apparatus; and (ii) AtXT1-GFP is mainly located in the cis and medial cisternae, AtMUR3-GFP is predominantly associated with medial cisternae and AtFUT1-GFP mostly detected over trans cisternae suggesting that initiation of xyloglucan side chains occurs in early Golgi compartments in tobacco cells.

Characterization and Three-dimensional Structures of Two Distinct Bacterial Xyloglucanases from Families GH5 and GH12

The plant cell wall is a complex material in which the cellulose microfibrils are embedded within a mesh of other polysaccharides, some of which are loosely termed "hemicellulose." One such hemicellulose is xyloglucan, which displays a beta-1,4-linked d-glucose backbone substituted with xylose, galactose, and occasionally fucose moieties. Both xyloglucan and the enzymes responsible for its modification and degradation are finding increasing prominence, reflecting both the drive for enzymatic biomass conversion, their role in detergent applications, and the utility of modified xyloglucans for cellulose fiber modification. Here we present the enzymatic characterization and three-dimensional structures in ligand-free and xyloglucan-oligosaccharide complexed forms of two distinct xyloglucanases from glycoside hydrolase families GH5 and GH12. The enzymes, Paenibacillus pabuli XG5 and Bacillus licheniformis XG12, both display open active center grooves grafted upon their respective (beta/alpha)(8) and beta-jelly roll folds, in which the side chain decorations of xyloglucan may be accommodated. For the beta-jelly roll enzyme topology of GH12, binding of xylosyl and pendant galactosyl moieties is tolerated, but the enzyme is similarly competent in the degradation of unbranched glucans. In the case of the (beta/alpha)(8) GH5 enzyme, kinetically productive interactions are made with both xylose and galactose substituents, as reflected in both a high specific activity on xyloglucan and the kinetics of a series of aryl glycosides. The differential strategies for the accommodation of the side chains of xyloglucan presumably facilitate the action of these microbial hydrolases in milieus where diverse and differently substituted substrates may be encountered.

Xyloglucan Is Recognized by Carbohydrate-binding Modules That Interact with β-Glucan Chains

Enzyme systems that attack the plant cell wall contain noncatalytic carbohydrate-binding modules (CBMs) that mediate attachment to this composite structure and play a pivotal role in maximizing the hydrolytic process. Although xyloglucan, which includes a backbone of beta-1,4-glucan decorated primarily with xylose residues, is a key component of the plant cell wall, CBMs that bind to this polymer have not been identified. Here we showed that the C-terminal domain of the modular Clostridium thermocellum enzyme CtCel9D-Cel44A (formerly known as CelJ) comprises a novel CBM (designated CBM44) that binds with equal affinity to cellulose and xyloglucan. We also showed that accommodation of xyloglucan side chains is a general feature of CBMs that bind to single cellulose chains. The crystal structures of CBM44 and the other CBM (CBM30) in CtCel9D-Cel44A display a beta-sandwich fold. The concave face of both CBMs contains a hydrophobic platform comprising three tryptophan residues that can accommodate up to five glucose residues. The orientation of these aromatic residues is such that the bound ligand would adopt the twisted conformation displayed by cello-oligosaccharides in solution. Mutagenesis studies confirmed that the hydrophobic platform located on the concave face of both CBMs mediates ligand recognition. In contrast to other CBMs that bind to single polysaccharide chains, the polar residues in the binding cleft of CBM44 play only a minor role in ligand recognition. The mechanism by which these proteins are able to recognize linear and decorated beta-1,4-glucans is discussed based on the structures of CBM44 and the other CBMs that bind single cellulose chains.

The reb1-1 Mutation of Arabidopsis. Effect on the Structure and Localization of Galactose-Containing Cell Wall Polysaccharides

The Arabidopsis (Arabidopsis thaliana) root epidermal bulger1-1 (reb1-1) mutant (allelic to root hair defective1 [rhd1]) is characterized by a reduced root elongation rate and by bulging of trichoblast cells. The REB1/RHD1 gene belongs to a family of UDP-D-Glucose 4-epimerases involved in the synthesis of D-Galactose (Gal). Our previous study showed that certain arabinogalactan protein epitopes were not expressed in bulging trichoblasts of the mutant. In this study, using a combination of microscopical and biochemical methods, we have investigated the occurrence and the structure of three major Gal-containing polysaccharides, namely, xyloglucan (XyG), rhamnogalacturonan (RG)-I, and RG-II in the mutant root cell walls. Our immunocytochemical data show that swollen trichoblasts were not stained with the monoclonal antibody CCRC-M1 specific for alpha-L-Fucp-(1-->2)-beta-D-Galp side chains of XyG, whereas they were stained with anti-XyG antibodies specific for XyG backbone. In addition, analysis of a hemicellulosic fraction from roots demonstrates the presence of two structurally different XyGs in reb1-1. One is structurally similar to wild-type XyG and the other is devoid of fuco-galactosylated side chains and has the characteristic of being insoluble. Similar to anti-XyG antibodies, anti-bupleuran 2IIC, a polyclonal antibody specific for galactosyl epitopes associated with pectins, stained all root epidermal cells of both wild type and reb1-1. Similarly, anti-RG-II antibodies also stained swollen trichoblasts in the mutant. In addition, structural analysis of pectic polymers revealed no change in the galactosylation of RG-I and RG-II isolated from reb1-1 root cells. These findings demonstrate that the reb1-1 mutation affects XyG structure, but not that of pectic polysaccharides, thus lending support to the hypothesis that biosynthesis of Gal as well as galactosylation of complex polysaccharides is regulated at the polymer level.

Conformational folding of xyloglucan side chains in aqueous solution from molecular dynamics simulation

Molecular dynamics simulation was carried out on xyloglucan with explicit water molecules to investigate the folding mechanism of side chains onto a main chain in aqueous solution. The model xyloglucan was composed of 12 β- d-glucopyranoses as a main chain substituted with six galactoses and three xyloses as side chains. Two conditions were set for the ribbon-like main chain; one is restricted to be ‘flat’ and the other is without restriction. The free main chain of xyloglucan has a ‘twisted’ conformation as the major one. Conformational folding of side chains onto the main chain was analyzed with dihedral angles at each glycosidic linkage. In a 5-ns calculation, the xyloglucan has a tendency to contract in both the restricted and the free systems, but the mode of contraction is different. Side chains tend to stick onto the flat surface of the main chain in the restricted system, while they do not tightly do so in the free one; instead the main chain takes a twisted and sometimes embowed conformation. This result indicates that the main chain has greater attractive forces to bind side chains when it is flat, while it loses the ability as it is twisted.

Tensile properties of Arabidopsis cell walls depend on both a xyloglucan cross-linked microfibrillar network and rhamnogalacturonan II-borate complexes.

The mechanical properties of plant organs depend upon anatomical structure, cell-cell adhesion, cell turgidity, and the mechanical properties of their cell walls. By testing the mechanical responses of Arabidopsis mutants, it is possible to deduce the contribution that polymers of the cell wall make to organ strength. We developed a method to measure the tensile parameters of the expanded regions of turgid or plasmolyzed dark-grown Arabidopsis hypocotyls and applied it to the fucose biosynthesis mutant mur1, the xyloglucan glycosyltransferase mutants mur2 and mur3, and the katanin mutant bot1. Hypocotyls from plants grown in the presence of increasing concentrations of dichlorobenzonitrile, an inhibitor of cellulose synthesis, were considerably weakened, indicating the validity of our approach. In order of decreasing strength, the hypocotyls of mur2 > bot1 and mur1 > mur3 were each found to have reduced strength and a proportionate reduction in modulus compared with wild type. The tensile properties of the hypocotyls and of the inflorescence stems of mur1 were rescued by growth in the presence of high concentrations of borate, which is known to cross-link the pectic component rhamnogalacturonan II. From comparison of the mechanical responses of mur2 and mur3, we deduce that galactose-containing side chains of xyloglucan make a major contribution to overall wall strength, whereas xyloglucan fucosylation plays a comparatively minor role. We conclude that borate-complexed rhamnogalacturonan II and galactosylated xyloglucan contribute to the tensile strength of cell walls.

Xyloglucan sidechains modulate binding to cellulose during in vitro binding assays as predicted by conformational dynamics simulations.

Cross-links between cellulose microfibrils and xyloglucan (XG) molecules play a major role in defining the structural properties of plant cell walls and the regulation of growth and development of dicotyledonous plants. How these cross-links are established and how they are regulated has yet to be determined. In a previous study, preliminary data were presented which suggested that the different sidechains of XG may play a role in controlling cellulose microfibril-XG interactions. In this study, this question is addressed directly by analyzing to what extent the different sidechains of pea cell wall XG and nasturtium seed storage XG affect their binding to cellulose microfibrils. Of particular importance to this study are the chemical data indicating that pea XG possesses a trisaccharide sidechain, which is not found in nasturtium XG. To this end, conformational dynamic simulations have been used to predict whether oligosaccharides representative of pea and nasturtium XG can adopt a hypothesized cellulose-binding conformation and which of these XGs exhibits a preferential ability to bind cellulose. Extensive analysis of the conformational forms populated during 300 K and high-temperature Monte Carlo simulations established that a planar, sterically accessible, glucan backbone is essential for optimal cellulose-binding. For the trisaccharide sidechain-containing oligosaccharide as found in pea XG, sidechain orientation appeared to regulate the gradual acquisition of this hypothesized cellulose binding conformation. Thus, conformational forms were identified that included the twisted backbone (non-planar) putative solution form of XG, forms in which the trisaccharide sidechain orientation enables increased backbone planarity and steric accessibility, and finally a planar, sterically accessible, backbone. By applying these conformational requirements for cellulose binding, it has been determined that pea XG possesses a two- to threefold occurrence of the cellulose binding conformation than nasturtium XG. Based on this finding, it was predicted that pea XG would bind to cellulose at a higher rate than nasturtium XG. In vitro binding assays showed that pea XG-avicel binding does indeed occur at a twofold higher rate than nasturtium XG-avicel binding. The enhanced ability of pea cell wall XG over nasturtium seed storage XG to associate with cellulose is consistent with a structural role of the former during epicotyl growth where efficient association with cellulose is a requirement. In contrast, the relatively low ability of nasturtium XG to bind cellulose is consistent with the need to enhance the accessibility of this polymer to glycanases during germination. These findings suggest potential roles for XG sidechain substitution, enabling XG to function in a variety of different biological contexts.

Models of Xyloglucan Binding to Cellulose Microfibrils1

Abstract Molecular modeling is used to investigate the ways in which plant cell wall xyloglucans might bind to the surface of cellulose microfibrils. Binding involving the xyloglucan backbone is found to be sterically restricted. Plausible models are obtained that involve hydrogen bonding between the xylose residues and one kind of cellulose surface. In such a model, the xyloglucan sidechains mediate, as well as modulate, the binding.