Protocols for isolating and characterizing polysaccharides from plant cell walls: a case study using rhamnogalacturonan-II.

BACKGROUND: In plants, a large diversity of polysaccharides comprise the cell wall. Each major type of plant cell wall polysaccharide, including cellulose, hemicellulose, and pectin, has distinct structures and functions that contribute to wall mechanics and influence plant morphogenesis. In recent years, pectin valorization has attracted much attention due to its expanding roles in biomass deconstruction, food and material science, and environmental remediation. However, pectin utilization has been limited by our incomplete knowledge of its structure. Herein, we present a workflow of principles relevant for the characterization of polysaccharide primary structure using nature's most complex polysaccharide, rhamnogalacturonan-II (RG-II), as a model. RESULTS: We outline how to isolate RG-II from celery and duckweed cell walls and from red wine using chemical or enzymatic treatments coupled with size-exclusion chromatography. From there, we applied mass spectrometry (MS)-based techniques to determine the glycosyl residue and linkage compositions of the intact RG-II and derived oligosaccharides including special considerations for labile monosaccharides. In doing so, we demonstrated that in the duckweed Wolffiella repanda the arabinopyranosyl (Arap) residue of side chain B is substituted at O-2 with rhamnose. We used electrospray-MS techniques to identify non-glycosyl modifications including methyl-ethers, methyl-esters, and acetyl-esters on RG-II-derived oligosaccharides. We then showed the utility of proton nuclear magnetic resonance spectroscopy (1H-NMR) to investigate the structure of intact RG-II and to complement the RG-II dimerization studies performed using size-exclusion chromatography. CONCLUSIONS: The complexity of pectic polysaccharide structures has hampered efforts aimed at their valorization. In this work, we used RG-II as a model to demonstrate the steps necessary to isolate and characterize polysaccharides using chromatographic, MS, and NMR techniques. The principles can be applied to the characterization of other saccharide structures and will help inform researchers on how saccharide structure relates to functional properties in the future.

Cross species multi-omics reveals cell wall sequestration and elevated global transcript abundance as mechanisms of boron tolerance in plants.

Boron toxicity is a world-wide problem for crops, yet we have a limited understanding of the genetic responses and adaptive mechanisms to this stress in plants. We employed a cross-species comparison between boron stress-sensitive Arabidopsis thaliana and its boron stress-tolerant extremophyte relative Schrenkiella parvula, and a multi-omics approach integrating genomics, transcriptomics, metabolomics and ionomics to assess plant responses and adaptations to boron stress. Schrenkiella parvula maintains lower concentrations of total boron and free boric acid than Arabidopsis when grown with excess boron. Schrenkiella parvula excludes excess boron more efficiently than Arabidopsis, which we propose is partly driven by SpBOR5, a boron transporter that we functionally characterize in this study. Both species use cell walls as a partial sink for excess boron. When accumulated in the cytoplasm, excess boron appears to interrupt RNA metabolism. The extremophyte S. parvula facilitates critical cellular processes while maintaining the pool of ribose-containing compounds that can bind with boric acid. The S. parvula transcriptome is pre-adapted to boron toxicity. It exhibits substantial overlaps with the Arabidopsis boron-stress responsive transcriptome. Cell wall sequestration and increases in global transcript levels under excess boron conditions emerge as key mechanisms for sustaining plant growth under boron toxicity.

Locating Methyl-Etherified and Methyl-Esterified Uronic Acids in the Plant Cell Wall Pectic Polysaccharide Rhamnogalacturonan II.

Rhamnogalacturonan II (RG-II) is a structurally complex pectic polysaccharide that exists as a borate ester cross-linked dimer in the cell walls of all vascular plants. The glycosyl sequence of RG-II is largely conserved, but there is evidence that galacturonic acid (GalA) methyl etherification and glucuronic acid (GlcA) methyl esterification vary in the A sidechain across plant species. Methyl esterification of the galacturonan backbone has also been reported but not confirmed. Here we describe a new procedure, utilizing aq. sodium borodeuteride (NaBD4)-reduced RG-II, to identify the methyl esterification status of backbone GalAs. Our data suggest that up to two different GalAs are esterified in the RG-II backbone. We also adapted a procedure based on methanolysis and NaBD4 reduction to identify 3-, 4-, and 3,4-O-methyl GalA in RG-II. These data, together with matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF) MS analysis of sidechain A generated from selected RG-IIs and their NaBD4-reduced counterparts, suggest that methyl etherification of the ß-linked GalA and methyl esterification of the GlcA are widespread. Nevertheless, the extent of these modifications varies between plant species. Our analysis of the sidechain B glycoforms in RG-II from different dicots and nonpoalean monocots suggests that this sidechain has a minimum structure of an O-acetylated hexasaccharide (Ara-[MeFuc]-Gal-AceA-Rha-Api-). To complement these studies, we provide further evidence showing that dimer formation and stability in vitro is cation and borate dependent. Taken together, our data further refine the primary sequence and sequence variation of RG-II and provide additional insight into dimer stability and factors controlling dimer self-assembly.

Analytical Techniques for Determining the Role of Domain of Unknown Function 579 Proteins in the Synthesis of O-Methylated Plant Polysaccharides.

Matrix polysaccharides are a diverse group of structurally complex carbohydrates and account for a large portion of the biomass consumed as food or used to produce fuels and materials. Glucuronoxylan and arabinogalactan protein are matrix glycans that have sidechains decorated with 4-O-methyl glucuronosyl residues. Methylation is a key determinant of the physical properties of these wall glycopolymers and consequently affects both their biological function and ability to interact with other wall polymers. Indeed, there is increasing interest in determining the distribution and abundance of methyl-etherified polysaccharides in different plant species, tissues, and developmental stages. There is also a need to understand the mechanisms involved in their biosynthesis. Members of the Domain of Unknown Function (DUF) 579 family have been demonstrated to have a role in the biosynthesis of methyl-etherified glycans. Here we describe methods for the analysis of the 4-O-methyl glucuronic acid moieties that are present in sidechains of arabinogalactan proteins. These methods are then applied toward the analysis of loss-of-function mutants of two DUF579 family members that lack this modification in muro. We also present a procedure to assay DUF579 family members for enzymatic activity in vitro using acceptor oligosaccharides prepared from xylan of loss-of-function mutants. Our approach facilitates the characterization of enzymes that modify glycosyl residues during cell wall synthesis and the structures that they generate.

Suppression of Arabidopsis GGLT1 affects growth by reducing the L-galactose content and borate cross-linking of rhamnogalacturonan-II.

Boron is a micronutrient that is required for the normal growth and development of vascular plants, but its precise functions remain a subject of debate. One established role for boron is in the cell wall where it forms a diester cross-link between two monomers of the low-abundance pectic polysaccharide rhamnogalacturonan-II (RG-II). The inability of RG-II to properly assemble into a dimer results in the formation of cell walls with abnormal biochemical and biomechanical properties and has a severe impact on plant productivity. Here we describe the effects on RG-II structure and cross-linking and on the growth of plants in which the expression of a GDP-sugar transporter (GONST3/GGLT1) has been reduced. In the GGLT1-silenced plants the amount of L-galactose in side-chain A of RG-II is reduced by up to 50%. This leads to a reduction in the extent of RG-II cross-linking in the cell walls as well as a reduction in the stability of the dimer in the presence of calcium chelators. The silenced plants have a dwarf phenotype, which is rescued by growth in the presence of increased amounts of boric acid. Similar to the mur1 mutant, which also disrupts RG-II cross-linking, GGLT1-silenced plants display a loss of cell wall integrity under salt stress. We conclude that GGLT1 is probably the primary Golgi GDP-L-galactose transporter, and provides GDP-L-galactose for RG-II biosynthesis. We propose that the L-galactose residue is critical for RG-II dimerization and for the stability of the borate cross-link.

Changes in the abundance of cell wall apiogalacturonan and xylogalacturonan and conservation of rhamnogalacturonan II structure during the diversification of the Lemnoideae.

MAIN CONCLUSION: The diversification of the Lemnoideae was accompanied by a reduction in the abundance of cell wall apiogalacturonan and an increase in xylogalacturonan whereas rhamnogalacturonan II structure and cross-linking are conserved. The subfamily Lemnoideae is comprised of five genera and 38 species of small, fast-growing aquatic monocots. Lemna minor and Spirodela polyrhiza belong to this subfamily and have primary cell walls that contain large amounts of apiogalacturonan and thus are distinct from the primary walls of most other flowering plants. However, the pectins in the cell walls of other members of the Lemnoideae have not been investigated. Here, we show that apiogalacturonan decreased substantially as the Lemnoideae diversified since Wolffiella and Wolffia walls contain between 63 and 88% less apiose than Spirodela, Landoltia, and Lemna walls. In Wolffia, the most derived genus, xylogalacturonan is far more abundant than apiogalacturonan, whereas in Wolffiella pectic polysaccharides have a high arabinose content, which may arise from arabinan sidechains of RG I. The apiose-containing pectin rhamnogalacturonan II (RG-II) exists in Lemnoideae walls as a borate cross-linked dimer and has a glycosyl sequence similar to RG-II from terrestrial plants. Nevertheless, species-dependent variations in the extent of methyl-etherification of RG-II sidechain A and arabinosylation of sidechain B are discernible. Immunocytochemical studies revealed that pectin methyl-esterification is higher in developing daughter frond walls than in mother frond walls, indicating that methyl-esterification is associated with expanding cells. Our data support the notion that a functional cell wall requires conservation of RG-II structure and cross-linking but can accommodate structural changes in other pectins. The Lemnoideae provide a model system to study the mechanisms by which wall structure and composition has changed in closely related plants with similar growth habits.

Boron-bridged RG-II and calcium are required to maintain the pectin network of the Arabidopsis seed mucilage ultrastructure.

The structure of a pectin network requires both calcium (Ca2+) and boron (B). Ca2+ is involved in crosslinking pectic polysaccharides and arbitrarily induces the formation of an "egg-box" structure among pectin molecules, while B crosslinks rhamnogalacturonan II (RG-II) side chain A apiosyl residues in primary cell walls to generate a borate-dimeric-rhamnogalacturonan II (dRG-II-B) complex through a boron-bridge bond, leading to the formation of a pectin network. Based on recent studies of dRG-II-B structures, a hypothesis has been proposed suggesting that Ca2+is a common component of the dRG-II-B complex. However, no in vivo evidence has addressed whether B affects the stability of Ca2+ crosslinks. Here, we investigated the L-fucose-deficient dwarf mutant mur1, which was previously shown to require exogenous B treatment for phenotypic reversion. Imbibed Arabidopsis thaliana seeds release hydrated polysaccharides to form a halo of seed mucilage covering the seed surface, which consists of a water-soluble outer layer and an adherent inner layer. Our study of mur1 seed mucilage has revealed that the pectin in the outer layer of mucilage was relocated to the inner layer. Nevertheless, the mur1 inner mucilage was more vulnerable to rough shaking or ethylene diamine tetraacetic acid (EDTA) extraction than that of the wild type. Immunolabeling analysis suggested that dRG-II-B was severely decreased in mur1 inner mucilage. Moreover, non-methylesterified homogalacturonan (HG) exhibited obvious reassembly in the mur1 inner layer compared with the wild type, which may imply a possible connection between dRG-II-B deficiency and pectin network transformation in the seed mucilage. As expected, the concentration of B in the mur1 inner mucilage was reduced, whereas the distribution and concentration of Ca2+in the inner mucilage increased significantly, which could be the reason why pectin relocates from the outer mucilage to the inner mucilage. Consequently, the disruption of B bridges appears to result in the extreme sensitivity of the mur1 mucilage pectin complex to EDTA extraction, despite the reinforcement of the pectin network by excessive Ca2+. Therefore, we propose a hypothesis that B, in the form of dRG-II-B, works together with Ca2+to maintain pectin network crosslinks and ultimately the mucilage ultrastructure in seed mucilage. This work may serve to complement our current understanding of mucilage configuration.

DGE-seq analysis of MUR3-related Arabidopsis mutants provides insight into how dysfunctional xyloglucan affects cell elongation.

Our previous study of the Arabidopsis mur3-3 mutant and mutant plants in which the mur3-3 phenotypes are suppressed (xxt2mur3-3, xxt5mur3-3, xxt1xxt2mur3-3 and 35Spro:XLT2:mur3-3) showed that hypocotyl cell elongation is decreased in plants that synthesize galactose-deficient xyloglucan. To obtain genome-wide insight into the transcriptome changes and regulatory networks that may be involved in this decreased elongation, we performed digital gene expression analyses of the etiolated hypocotyls of wild type (WT), mur3-3 and the four suppressor lines. Numerous differentially expressed genes (DEGs) were detected in comparisons between WT and mur3-3 (1423), xxt2mur3-3 and mur3-3 (675), xxt5mur3-3 and mur3-3 (1272), xxt1xxt2mur3-3 and mur3-3 (1197) and 35Spro:XLT2:mur3-3 vs mur3-3 (121). 550 overlapped DEGs were detected among WT vs mur3-3, xxt2mur3-3 vs mur3-3, xxt5mur3-3 vs mur3-3, and xxt1xxt2mur3-3 vs mur3-3 comparisons. These DEGs include 46 cell wall-related genes, 24 transcription factors, 6 hormone-related genes, 9 protein kinase genes and 9 aquaporin genes. The expression of all of the 550 overlapped genes is restored to near wild-type levels in the four mur3-3 suppressor lines. qRT-PCR of fifteen of these 550 genes showed that their expression levels are consistent with the digital gene expression data. Overexpression of some of these genes (XTH4, XTH30, PME3, EXPA11, MYB88, ROT3, AT5G37790, WAG2 and TIP2;3) that are down-regulated in mur3-3 partially rescued the short hypocotyl phenotype but not the aerial phenotype of mur3-3, indicating that different mechanisms exist between hypocotyl cell elongation and leaf cell elongation.

Corrigendum: Complex pectin metabolism by gut bacteria reveals novel catalytic functions.

This corrects the article DOI: 10.1038/nature21725.

Functional Characterization of UDP-apiose Synthases from Bryophytes and Green Algae Provides Insight into the Appearance of Apiose-containing Glycans during Plant Evolution.

Apiose is a branched monosaccharide that is present in the cell wall pectic polysaccharides rhamnogalacturonan II and apiogalacturonan and in numerous plant secondary metabolites. These apiose-containing glycans are synthesized using UDP-apiose as the donor. UDP-apiose (UDP-Api) together with UDP-xylose is formed from UDP-glucuronic acid (UDP-GlcA) by UDP-Api synthase (UAS). It was hypothesized that the ability to form Api distinguishes vascular plants from the avascular plants and green algae. UAS from several dicotyledonous plants has been characterized; however, it is not known if avascular plants or green algae produce this enzyme. Here we report the identification and functional characterization of UAS homologs from avascular plants (mosses, liverwort, and hornwort), from streptophyte green algae, and from a monocot (duckweed). The recombinant UAS homologs all form UDP-Api from UDP-glucuronic acid albeit in different amounts. Apiose was detected in aqueous methanolic extracts of these plants. Apiose was detected in duckweed cell walls but not in the walls of the avascular plants and algae. Overexpressing duckweed UAS in the moss Physcomitrella patens led to an increase in the amounts of aqueous methanol-acetonitrile-soluble apiose but did not result in discernible amounts of cell wall-associated apiose. Thus, bryophytes and algae likely lack the glycosyltransferase machinery required to synthesize apiose-containing cell wall glycans. Nevertheless, these plants may have the ability to form apiosylated secondary metabolites. Our data are the first to provide evidence that the ability to form apiose existed prior to the appearance of rhamnogalacturonan II and apiogalacturonan and provide new insights into the evolution of apiose-containing glycans.

Structural diversity of xylans in the cell walls of monocots.

MAIN CONCLUSION: Xylans in the cell walls of monocots are structurally diverse. Arabinofuranose-containing glucuronoxylans are characteristic of commelinids. However, other structural features are not correlated with the major transitions in monocot evolution. Most studies of xylan structure in monocot cell walls have emphasized members of the Poaceae (grasses). Thus, there is a paucity of information regarding xylan structure in other commelinid and in non-commelinid monocot walls. Here, we describe the major structural features of the xylans produced by plants selected from ten of the twelve monocot orders. Glucuronoxylans comparable to eudicot secondary wall glucuronoxylans are abundant in non-commelinid walls. However, the α-D-glucuronic acid/4-O-methyl-α-D-glucuronic acid is often substituted at O-2 by an α-L-arabinopyranose residue in Alismatales and Asparagales glucuronoxylans. Glucuronoarabinoxylans were the only xylans detected in the cell walls of five different members of the Poaceae family (grasses). By contrast, both glucuronoxylan and glucuronoarabinoxylan are formed by the Zingiberales and Commelinales (commelinids). At least one species of each monocot order, including the Poales, forms xylan with the reducing end sequence -4)-ß-D-Xylp-(1,3)-α-L-Rhap-(1,2)-α-D-GalpA-(1,4)-D-Xyl first identified in eudicot and gymnosperm glucuronoxylans. This sequence was not discernible in the arabinopyranose-containing glucuronoxylans of the Alismatales and Asparagales or the glucuronoarabinoxylans of the Poaceae. Rather, our data provide additional evidence that in Poaceae glucuronoarabinoxylan, the reducing end xylose residue is often substituted at O-2 with 4-O-methyl glucuronic acid or at O-3 with arabinofuranose. The variations in xylan structure and their implications for the evolution and biosynthesis of monocot cell walls are discussed.

Xyloglucan, galactomannan, glucuronoxylan, and rhamnogalacturonan I do not have identical structures in soybean root and root hair cell walls.

MAIN CONCLUSION: Chemical analyses and glycome profiling demonstrate differences in the structures of the xyloglucan, galactomannan, glucuronoxylan, and rhamnogalacturonan I isolated from soybean ( Glycine max ) roots and root hair cell walls. The root hair is a plant cell that extends only at its tip. All other root cells have the ability to grow in different directions (diffuse growth). Although both growth modes require controlled expansion of the cell wall, the types and structures of polysaccharides in the walls of diffuse and tip-growing cells from the same plant have not been determined. Soybean (Glycine max) is one of the few plants whose root hairs can be isolated in amounts sufficient for cell wall chemical characterization. Here, we describe the structural features of rhamnogalacturonan I, rhamnogalacturonan II, xyloglucan, glucomannan, and 4-O-methyl glucuronoxylan present in the cell walls of soybean root hairs and roots stripped of root hairs. Irrespective of cell type, rhamnogalacturonan II exists as a dimer that is cross-linked by a borate ester. Root hair rhamnogalacturonan I contains more neutral oligosaccharide side chains than its root counterpart. At least 90% of the glucuronic acid is 4-O-methylated in root glucuronoxylan. Only 50% of this glycose is 4-O-methylated in the root hair counterpart. Mono O-acetylated fucose-containing subunits account for at least 60% of the neutral xyloglucan from root and root hair walls. By contrast, a galacturonic acid-containing xyloglucan was detected only in root hair cell walls. Soybean homologs of the Arabidopsis xyloglucan-specific galacturonosyltransferase are highly expressed only in root hairs. A mannose-rich polysaccharide was also detected only in root hair cell walls. Our data demonstrate that the walls of tip-growing root hairs cells have structural features that distinguish them from the walls of other roots cells.

Galactose-depleted xyloglucan is dysfunctional and leads to dwarfism in Arabidopsis.

Xyloglucan is a polysaccharide that has important roles in the formation and function of the walls that surround growing land plant cells. Many of these plants synthesize xyloglucan that contains galactose in two different side chains (L and F), which exist in distinct molecular environments. However, little is known about the contribution of these side chains to xyloglucan function. Here, we show that Arabidopsis (Arabidopsis thaliana) mutants devoid of the F side chain galactosyltransferase MURUS3 (MUR3) form xyloglucan that lacks F side chains and contains much less galactosylated xylose than its wild-type counterpart. The galactose-depleted xyloglucan is dysfunctional, as it leads to mutants that are dwarfed with curled rosette leaves, short petioles, and short inflorescence stems. Moreover, cell wall matrix polysaccharides, including xyloglucan and pectin, are not properly secreted and instead accumulate within intracellular aggregates. Near-normal growth is restored by generating mur3 mutants that produce no detectable amounts of xyloglucan. Thus, cellular processes are affected more by the presence of the dysfunctional xyloglucan than by eliminating xyloglucan altogether. To identify structural features responsible for xyloglucan dysfunction, xyloglucan structure was modified in situ by generating mur3 mutants that lack specific xyloglucan xylosyltransferases (XXTs) or that overexpress the XYLOGLUCAN L-SIDE CHAIN GALACTOSYLTRANSFERASE2 (XLT2) gene. Normal growth was restored in the mur3-3 mutant overexpressing XLT2 and in mur3-3 xxt double mutants when the dysfunctional xyloglucan was modified by doubling the amounts of galactosylated side chains. Our study assigns a role for galactosylation in normal xyloglucan function and demonstrates that altering xyloglucan side chain structure disturbs diverse cellular and physiological processes.

Generation and structural validation of a library of diverse xyloglucan-derived oligosaccharides, including an update on xyloglucan nomenclature.

Xyloglucans are structurally complex plant cell wall polysaccharides that are involved in cell growth and expansion, energy metabolism, and signaling. Determining the structure-function relationships of xyloglucans would benefit from the availability of a comprehensive and structurally diverse collection of rigorously characterized xyloglucan oligosaccharides. Here, we present a workflow for the semi-preparative scale generation and purification of neutral and acidic xyloglucan oligosaccharides using a combination of enzymatic and chemical treatments and size-exclusion chromatography. Twenty-six of these oligosaccharides were purified to near homogeneity and their structures validated using a combination of matrix-assisted laser desorption/ionization mass spectrometry, high-performance anion exchange chromatography, and 1H nuclear magnetic resonance spectroscopy. Mass spectrometry and analytical chromatography were compared as methods for xyloglucan oligosaccharide quantification. 1H chemical shifts were assigned using two-dimensional correlation spectroscopy. A comprehensive update of the nomenclature describing xyloglucan side-chain structures is provided for reference.

Transport of boron by the tassel-less1 aquaporin is critical for vegetative and reproductive development in maize.

The element boron (B) is an essential plant micronutrient, and B deficiency results in significant crop losses worldwide. The maize (Zea mays) tassel-less1 (tls1) mutant has defects in vegetative and inflorescence development, comparable to the effects of B deficiency. Positional cloning revealed that tls1 encodes a protein in the aquaporin family co-orthologous to known B channel proteins in other species. Transport assays show that the TLS1 protein facilitates the movement of B and water into Xenopus laevis oocytes. B content is reduced in tls1 mutants, and application of B rescues the mutant phenotype, indicating that the TLS1 protein facilitates the movement of B in planta. B is required to cross-link the pectic polysaccharide rhamnogalacturonan II (RG-II) in the cell wall, and the percentage of RG-II dimers is reduced in tls1 inflorescences, indicating that the defects may result from altered cell wall properties. Plants heterozygous for both tls1 and rotten ear (rte), the proposed B efflux transporter, exhibit a dosage-dependent defect in inflorescence development under B-limited conditions, indicating that both TLS1 and RTE function in the same biological processes. Together, our data provide evidence that TLS1 is a B transport facilitator in maize, highlighting the importance of B homeostasis in meristem function.

A galacturonic acid-containing xyloglucan is involved in Arabidopsis root hair tip growth.

Root hairs provide a model system to study plant cell growth, yet little is known about the polysaccharide compositions of their walls or the role of these polysaccharides in wall expansion. We report that Arabidopsis thaliana root hair walls contain a previously unidentified xyloglucan that is composed of both neutral and galacturonic acid-containing subunits, the latter containing the ß-D-galactosyluronic acid-(1→2)-α-D-xylosyl-(1→ and/or α-L-fucosyl-(1→2)-ß-D-galactosyluronic acid-(1→2)-α-D-xylosyl-(1→) side chains. Arabidopsis mutants lacking root hairs have no acidic xyloglucan. A loss-of-function mutation in At1g63450, a root hair-specific gene encoding a family GT47 glycosyltransferase, results in the synthesis of xyloglucan that lacks galacturonic acid. The root hairs of this mutant are shorter than those of the wild type. This mutant phenotype and the absence of galacturonic acid in the root xyloglucan are complemented by At1g63450. The leaf and stem cell walls of wild-type Arabidopsis contain no acidic xyloglucan. However, overexpression of At1g63450 led to the synthesis of galacturonic acid-containing xyloglucan in these tissues. We propose that At1g63450 encodes XYLOGLUCAN-SPECIFIC GALACTURONOSYLTRANSFERASE1, which catalyzes the formation of the galactosyluronic acid-(1→2)-α-D-xylopyranosyl linkage and that the acidic xyloglucan is present only in root hair cell walls. The role of the acidic xyloglucan in root hair tip growth is discussed.

4-O-methylation of glucuronic acid in Arabidopsis glucuronoxylan is catalyzed by a domain of unknown function family 579 protein.

The hemicellulose 4-O-methyl glucuronoxylan is one of the principle components present in the secondary cell walls of eudicotyledonous plants. However, the biochemical mechanisms leading to the formation of this polysaccharide and the effects of modulating its structure on the physical properties of the cell wall are poorly understood. We have identified and functionally characterized an Arabidopsis glucuronoxylan methyltransferase (GXMT) that catalyzes 4-O-methylation of the glucuronic acid substituents of this polysaccharide. AtGXMT1, which was previously classified as a domain of unknown function (DUF) 579 protein, specifically transfers the methyl group from S-adenosyl-L-methionine to O-4 of α-D-glucopyranosyluronic acid residues that are linked to O-2 of the xylan backbone. Biochemical characterization of the recombinant enzyme indicates that GXMT1 is localized in the Golgi apparatus and requires Co(2+) for optimal activity in vitro. Plants lacking GXMT1 synthesize glucuronoxylan in which the degree of 4-O-methylation is reduced by 75%. This result is correlated to a change in lignin monomer composition and an increase in glucuronoxylan release during hydrothermal treatment of secondary cell walls. We propose that the DUF579 proteins constitute a previously undescribed family of cation-dependent, polysaccharide-specific O-methyl-transferases. This knowledge provides new opportunities to selectively manipulate polysaccharide O-methylation and extends the portfolio of structural targets that can be modified either alone or in combination to modulate biopolymer interactions in the plant cell wall.

Structural characterization of the heteroxylans from poplar and switchgrass.

Heteroxylans are polysaccharides with a backbone composed of 1,4-linked ß-D-xylosyl residues. In hardwoods some of these xylosyl residues are substituted at O-2 with 4-O-methyl α-D-glucuronic and occasionally with α-D-glucuronic acid. In grasses, the xylan backbone is predominantly substituted with α-L-arabinofuranosyl residues (most often at O-3, but sometimes at O-2). Grass heteroxylan backbone residues may also have small amounts of α-D-glucuronic acid and/or 4-O-methyl α-D-glucuronic acid at O-2. Heteroxylans have a role in maintaining the structural integrity of the cell walls that comprise the bulk of lignocellulosic biomass. Moreover, differences in the molecular features of these hemicellulosic polysaccharides, including their degree of polymerization, degree of branching and spatial arrangement of side chains along the xylan backbone, have been correlated to altered cell wall properties (Izydorczyk MS, Biliaderis CG, Carbohydr Polym 28:33-48, 1995) and the ease with which biomass can be enzymatically converted to fermentable sugars. Thus, understanding the relationship between heteroxylan structure and biomass properties is required to engineer bioenergy crops with improved processing characteristics. In this chapter we describe some of the analytical methods we routinely use to perform in-depth structural analysis of heteroxylans from poplar and switchgrass biomass.

The Synthesis and Origin of the Pectic Polysaccharide Rhamnogalacturonan II - Insights from Nucleotide Sugar Formation and Diversity.

There is compelling evidence showing that the structurally complex pectic polysaccharide rhamnogalacturonan II (RG-II) exists in the primary cell wall as a borate cross-linked dimer and that this dimer is required for the assembly of a functional wall and for normal plant growth and development. The results of several studies have also established that RG-II structure and cross-linking is conserved in vascular plants and that RG-II likely appeared early in the evolution of land plants. Two features that distinguish RG-II from other plant polysaccharides are that RG-II is composed of 13 different glycoses linked to each other by up to 22 different glycosidic linkages and that RG-II is the only polysaccharide known to contain both apiose and aceric acid. Thus, one key event in land plant evolution was the emergence of genes encoding nucleotide sugar biosynthetic enzymes that generate the activated forms of apiose and aceric acid required for RG-II synthesis. Many of the genes involved in the generation of the nucleotide sugars used for RG-II synthesis have been functionally characterized. By contrast, only one glycosyltransferase involved in the assembly of RG-II has been identified. Here we provide an overview of the formation of the activated sugars required for RG-II synthesis and point to the possible cellular and metabolic processes that could be involved in assembling and controlling the formation of a borate cross-linked RG-II molecule. We discuss how nucleotide sugar synthesis is compartmentalized and how this may control the flux of precursors to facilitate and regulate the formation of RG-II.

Methods for structural characterization of the products of cellulose- and xyloglucan-hydrolyzing enzymes.

Structural characterization of oligosaccharide products generated by enzymatic hydrolysis of plant cell wall polysaccharides provides valuable information about the enzyme's activity and substrate specificity. In this chapter, we describe some of the chemical, chromatographic, and spectroscopic methods that we routinely use to isolate and characterize oligosaccharides formed by enzymatic fragmentation of cellulose and xyloglucan. These include techniques to determine glycosyl residue and glycosyl linkage compositions by gas chromatography and mass spectrometry. We also illustrate the use of electrospray ionization with multistage mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and nuclear magnetic resonance spectroscopy to perform detailed structural analysis of these oligosaccharides.