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Xylans constitute the main non-cellulosic polysaccharide in the secondary cell walls of plants. Several genes predicted to encode glycosyltransferases are required for the synthesis of the xylan backbone even though it is a homopolymer consisting entirely of β-1,4-linked xylose residues. The putative glycosyltransferases IRX9, IRX14, and IRX10 (or the paralogs IRX9L, IRX14L, and IRX10L) are required for xylan backbone synthesis in Arabidopsis. To investigate the function of IRX9, IRX9L, and IRX14, we identified amino acid residues known to be essential for catalytic function in homologous mammalian proteins and generated modified cDNA clones encoding proteins where these residues would be mutated. The mutated gene constructs were used to transform wild-type Arabidopsis plants and the irx9 and irx14 mutants, which are deficient in xylan synthesis. The ability of the mutated proteins to complement the mutants was investigated by measuring growth, determining cell wall composition, and microscopic analysis of stem cross-sections of the transgenic plants. The six different mutated versions of IRX9 and IRX9-L were all able to complement the irx9 mutant phenotype, indicating that residues known to be essential for glycosyltransferases function in homologous proteins are not essential for the biological function of IRX9/IRX9L. Two out of three mutated IRX14 complemented the irx14 mutant, including a mutant in the predicted catalytic amino acid. A IRX14 protein mutated in the substrate-binding DxD motif did not complement the irx14 mutant. Thus, substrate binding is important for IRX14 function but catalytic activity may not be essential for the function of the protein. The data indicate that IRX9/IRX9L have an essential structural function, most likely by interacting with the IRX10/IRX10L proteins, but do not have an essential catalytic function. Most likely IRX14 also has primarily a structural role, but it cannot be excluded that the protein has an important enzymatic activity.

Plant cells are surrounded by a rigid wall made up of cellulose microfibrils, pectins, hemicelluloses, and lignin. This cell wall provides structure and protection for plant cells. In grasses and in dicot secondary cell walls, the major hemicellulose is a polymer of β-(1,4)-linked xylose units called xylan. Unlike cellulose--which is synthesized by large complexes at the plasma membrane--xylan is synthesized by enzymes in the Golgi apparatus. Xylan synthesis thus requires the coordinated action and regulation of these synthetic enzymes as well as others that synthesize and transport substrates into the Golgi. Recent research has identified several genes involved in xylan synthesis, some of which have already been used in engineering efforts to create plants that are better suited for biofuel production., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Xylan is a major component of the plant cell wall and the most abundant noncellulosic component in the secondary cell walls that constitute the largest part of plant biomass. Dicot glucuronoxylan consists of a linear backbone of β(1,4)-linked xylose residues substituted with α(1,2)-linked glucuronic acid (GlcA). Although several genes have been implicated in xylan synthesis through mutant analyses, the biochemical mechanisms responsible for synthesizing xylan are largely unknown. Here, we show evidence for biochemical activity of GUX1 (for GlcA substitution of xylan 1), a member of Glycosyltransferase Family 8 in Arabidopsis (Arabidopsis thaliana) that is responsible for adding the glucuronosyl substitutions onto the xylan backbone. GUX1 has characteristics typical of Golgi-localized glycosyltransferases and a K(m) for UDP-GlcA of 165 μm. GUX1 strongly favors xylohexaose as an acceptor over shorter xylooligosaccharides, and with xylohexaose as an acceptor, GlcA is almost exclusively added to the fifth xylose residue from the nonreducing end. We also show that several related proteins, GUX2 to GUX5 and Plant Glycogenin-like Starch Initiation Protein6, are Golgi localized and that only two of these proteins, GUX2 and GUX4, have activity as xylan α-glucuronosyltransferases.

Xylans constitute the major non-cellulosic component of plant biomass. Xylan biosynthesis is particularly pronounced in cells with secondary walls, implying that the synthesis network consists of a set of highly expressed genes in such cells. To improve the understanding of xylan biosynthesis, we performed a comparative analysis of co-expression networks between Arabidopsis and rice as reference species with different wall types. Many co-expressed genes were represented by orthologs in both species, which implies common biological features, while some gene families were only found in one of the species, and therefore likely to be related to differences in their cell walls. To predict the subcellular location of the identified proteins, we developed a new method, PFANTOM (plant protein family information-based predictor for endomembrane), which was shown to perform better for proteins in the endomembrane system than other available prediction methods. Based on the combined approach of co-expression and predicted cellular localization, we propose a model for Arabidopsis and rice xylan synthesis in the Golgi apparatus and signaling from plasma membrane to nucleus for secondary cell wall differentiation. As an experimental validation of the model, we show that an Arabidopsis mutant in the PGSIP1 gene encoding one of the Golgi localized candidate proteins has a highly decreased content of glucuronic acid in secondary cell walls and substantially reduced xylan glucuronosyltransferase activity.

Members of a large family of cellulose synthase-like genes (CSLs) are predicted to encode glycosyl transferases (GTs) involved in the biosynthesis of plant cell walls. The CSLA and CSLF families are known to contain mannan and glucan synthases, respectively, but the products of other CSLs are unknown. Here we report the effects of disrupting ATCSLD5 expression in Arabidopsis. Both stem and root growth were significantly reduced in ATCSLD5 knock-out plants, and these plants also had increased susceptibility to the cellulose synthase inhibitor isoxaben. Antibody and carbohydrate-binding module labelling indicated a reduction in the level of xylan in stems, and in vitro GT assays using microsomes from stems revealed that ATCSLD5 knock-out plants also had reduced xylan and homogalacturonan synthase activity. Expression in Nicotiana benthamiana of ATCSLD5 and ATCSLD3, fluorescently tagged at either the C- or the N-terminal, indicated that these GTs are likely to be localized in the Golgi apparatus. However, the position of the fluorescent tag affected the subcellular localization of both proteins. The work presented provides a comprehensive analysis of the effects of disrupting ATCSLD5 in planta, and the possible role(s) of this gene and other ATCSLDs in cell wall biosynthesis are discussed.

Incorporation of [(3)H]arabinose and [(14)C]ferulic acid into soluble and polymeric fractions from suspension-cultured wheat (Triticum aestivum L.) cells and the corresponding extracellular medium was studied. The major part of these products was identified as arabinoxylan and two proteins of 40 and 100 kDa. The time course suggests an intracellular synthesis of feruloylated arabinoxylan with feruloyl-glucose as substrate. In contrast, synthesis of feruloylated proteins appears to occur with feruloyl-CoA as precursor. Intracellular formation of ferulic acid dimers is limited to 8,5'-diferulic acid, while other dimers appear to be formed extracellularly. [(3)H]Arabinose was incorporated into polymeric material in both the cellular and in the medium fraction while [(14)C]ferulic was only found in polymers from the cellular fraction, indicating synthesis of both feruloylated and non-feruloylated arabinoxylan by the cells.

Arabinoxylan arabinosyltransferase (AX-AraT) activity was investigated using microsomes and Golgi vesicles isolated from wheat (Triticum aestivum) seedlings. Incubation of microsomes with UDP-[(14)C]-beta-L-arabinopyranose resulted in incorporation of radioactivity into two different products, although most of the radioactivity was present in xylose (Xyl), indicating a high degree of UDP-arabinose (Ara) epimerization. In isolated Golgi vesicles, the epimerization was negligible, and incubation with UDP-[(14)C]Ara resulted in formation of a product that could be solubilized with proteinase K. In contrast, when Golgi vesicles were incubated with UDP-[(14)C]Ara in the presence of unlabeled UDP-Xyl, the product obtained could be solubilized with xylanase, whereas proteinase K had no effect. Thus, the AX-AraT is dependent on the synthesis of unsubstituted xylan acting as acceptor. Further analysis of the radiolabeled product formed in the presence of unlabeled UDP-Xyl revealed that it had an apparent molecular mass of approximately 500 kD. Furthermore, the total incorporation of [(14)C]Ara was dependent on the time of incubation and the amount of Golgi protein used. AX-AraT activity had a pH optimum at 6, and required the presence of divalent cations, Mn(2+) being the most efficient. In the absence of UDP-Xyl, a single arabinosylated protein with an apparent molecular mass of 40 kD was radiolabeled. The [(14)C]Ara labeling became reversible by adding unlabeled UDP-Xyl to the reaction medium. The possible role of this protein in arabinoxylan biosynthesis is discussed.

Changes in arabinoxylan content and composition during development of wheat seedlings were investigated. The cell walls isolated from the seedlings showed an increasing content of arabinoxylan during development, which could be correlated to increased activity of xylan synthase and arabinoxylan arabinosyltransferase. Arabinoxylan changed from initially having a high degree of arabinose substitution to a much lower degree of substitution. beta-Glucan was present in the walls at the early stages of development, but was actively degraded after day 4. Increased deposition of arabinoxylan did not take place until beta-glucan had been fully degraded. Ferulic and p-coumaric acid esters were present at all points but increased significantly from day 3 to 6, where lignification began. Ferulic acid dimers did not appear in the cell wall until day three and the different ferulic acid dimers varied in the course of accumulation. The ratio of ferulic acid dimers to free ferulic acid was maximal at the time when the wall had been depleted for beta-glucan, which had not yet been fully replaced by arabinoxylan. This pattern suggests a role for ferulic acid dimers in stabilizing the wall during the transition from a flexible to a more rigid structure. To investigate if the same changes could be observed within a single seedling, 7 day old seedlings were divided into four sections and the walls were analyzed. Some of the changes observed during the seedling development could also be observed within a single seedling, when analyzing the segments from the elongation zone at the base to the top of the leaf. However, the expanding region of older seedlings was much richer in hydroxycinnamates than the expanding region of younger seedlings. Diferulic acids are stabilizing the wall in the transition phase from an expanding to a mature wall. This transition can take place in different manners depending on the cell and tissue type.

Nucleotide sugar transport across Golgi membranes is essential for the luminal biosynthesis of glycan structures. Here we identify GDP-fucose transporter 1 (GFT1), an Arabidopsis nucleotide sugar transporter that translocates GDP-L-fucose into the Golgi lumen. Using proteo-liposome-based transport assays, we show that GFT preferentially transports GDP-L-fucose over other nucleotide sugars in vitro, while GFT1-silenced plants are almost devoid of L-fucose in cell wall-derived xyloglucan and rhamnogalacturonan II. Furthermore, these lines display reduced L-fucose content in N-glycan structures accompanied by severe developmental growth defects. We conclude that GFT1 is the major nucleotide sugar transporter for import of GDP-L-fucose into the Golgi and is required for proper plant growth and development.

Summary: Insights into the evolution of plant cell walls have important implications for comprehending these diverse and abundant biological structures. In order to understand the evolving structure–function relationships of the plant cell wall, it is imperative to trace the origin of its different components. The present study is focused on plant 1,4‐β‐xylan, tracing its evolutionary origin by genome and transcriptome mining followed by phylogenetic analysis, utilizing a large selection of plants and algae. It substantiates the findings by heterologous expression and biochemical characterization of a charophyte alga xylan synthase. Of the 12 known gene classes involved in 1,4‐β‐xylan formation, XYS1/IRX10 in plants, IRX7, IRX8, IRX9, IRX14 and GUX occurred for the first time in charophyte algae. An XYS1/IRX10 ortholog from Klebsormidium flaccidum, designated K. flaccidumXYLAN SYNTHASE‐1 (KfXYS1), possesses 1,4‐β‐xylan synthase activity, and 1,4‐β‐xylan occurs in the K. flaccidum cell wall. These data suggest that plant 1,4‐β‐xylan originated in charophytes and shed light on the origin of one of the key cell wall innovations to occur in charophyte algae, facilitating terrestrialization and emergence of polysaccharide‐based plant cell walls. [ABSTRACT FROM AUTHOR]

Xylan is a major component of the plant cell wall and the most abundant noncellulosic component in the secondary cell wails that constitute the largest part of plant biomass. Dicot glucuronoxylan consists of a linear backbone of/3(1,4)-linked xylose residues substituted with a(1,2)-linked glucuronic acid (GlcA). Although several genes have been implicated in xylan synthesis through mutant analyses, the biochemical mechanisms responsible for synthesizing xylan are largely unknown. Here, we show evidence for biochemical activity of GUX1 (for GlcA substitution of xylan 1), a member of Glycosyltransferase Family 8 in Arabidopsis (Arabidopsis thaliana) that is responsible for adding the glucuronosyl substitutions onto the xylan backbone. GUX1 has characteristics typical of Golgi-localized glycosyltransferases and a Km for UDP-GlcA of 165 /ZM. GUX1 strongly favors xylohexaose as an acceptor over shorter xylooligosaccharides, and with xylohexaose as an acceptor, GlcA is almost exclusively added to the fifth xylose residue from the nonreducing end. We also show that several related proteins, GUX2 to GUX5 and Plant Glycogenin-like Starch Initiation Protein6, are Golgi localized and that only two of these proteins, GUX2 and GUX4, have activity as xylan a-glucuronosyltransferases. [ABSTRACT FROM AUTHOR]

Hemicelluloses are polysaccharides in plant cell walls that have β-(1→4)-linked backbones with an equatorial configuration. Hemicelluloses include xyloglucans, xylans, mannans and glucomannans, and β-(1→3,1→4)-glucans. These types of hemicelluloses are present in the cell walls of all terrestrial plants, except for β-(1→3,1→4)-glucans, which are restricted to Poales and a few other groups. The detailed structure of the hemicelluloses and their abundance vary widely between different species and cell types. The most important biological role of hemicelluloses is their contribution to strengthening the cell wall by interaction with cellulose and, in some walls, with lignin. These features are discussed in relation to widely accepted models of the primary wall. Hemicelluloses are synthesized by glycosyltransferases located in the Golgi membranes. Many glycosyltransferases needed for biosynthesis of xyloglucans and mannans are known. In contrast, the biosynthesis of xylans and β-(1→3,1→4)-glucans remains very elusive, and recent studies have led to more questions than answers. [ABSTRACT FROM AUTHOR]

The biosynthesis of arabinoxylan was investigated using microsomal membranes isolated from wheat seedlings. Incubation of the microsomes with UDP‐[ C]‐d‐xylose resulted in incorporation of radioactivity into polymeric product. Incorporation reached a maximum at day 3 during development of the seedlings. Treatment of the radiolabeled product with Aspergillus niger xylanase A released about 70% of the radioactivity to a 65% ethanol‐soluble fraction. The released radioactivity was shown to be in the form of xylobiose, xylotriose and xylotetraose, which are the expected hydrolysis products of endo‐xylanases. The synthesized xylan had an apparent molecular mass larger than 500 kDa. [ABSTRACT FROM AUTHOR]

A mixture of arabinoxylan oligosaccharides from wheat seedling was permethylated and analyzed by electrospray ion trap MS and GC–MS. The presence of isomeric structures differing in degree of branching and position of the branched residue along the xylose backbone was demonstrated for oligosaccharides with four and five monosaccharide residues. No isomeric structures were found for oligosaccharides with three monosaccharide residues. Linkage analysis by GC–MS reveals that xylose residues were substituted with single arabinoxyl residues at C-3. [Copyright &y& Elsevier]

Xylan is a major component of the plant cell wall and the most abundant noncellulosic component in the secondary cell wails that constitute the largest part of plant biomass. Dicot glucuronoxylan consists of a linear backbone of/3(1,4)-linked xylose residues substituted with a(1,2)-linked glucuronic acid (GlcA). Although several genes have been implicated in xylan synthesis through mutant analyses, the biochemical mechanisms responsible for synthesizing xylan are largely unknown. Here, we show evidence for biochemical activity of GUX1 (for GlcA substitution of xylan 1), a member of Glycosyltransferase Family 8 in Arabidopsis (Arabidopsis thaliana) that is responsible for adding the glucuronosyl substitutions onto the xylan backbone. GUX1 has characteristics typical of Golgi-localized glycosyltransferases and a Km for UDP-GlcA of 165 /ZM. GUX1 strongly favors xylohexaose as an acceptor over shorter xylooligosaccharides, and with xylohexaose as an acceptor, GlcA is almost exclusively added to the fifth xylose residue from the nonreducing end. We also show that several related proteins, GUX2 to GUX5 and Plant Glycogenin-like Starch Initiation Protein6, are Golgi localized and that only two of these proteins, GUX2 and GUX4, have activity as xylan a-glucuronosyltransferases. [ABSTRACT FROM AUTHOR]