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Xylan and xyloglucan are the twomajor 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. [ABSTRACT FROM AUTHOR]

Xylan is a major cross-linking hemicellulose in secondary walls of vascular tissues, and the recruitment of xylan as a secondary wall component was suggested to be a pivotal event for the evolution of vascular tissues. To decipher the evolution of xylan structure and xylan biosynthetic genes, we analyzed xylan substitution patterns and characterized genes mediating methylation of glucuronic acid (GlcA) side chains in xylan of the model seedless vascular plant, Selaginella moellendorffii, and investigated GT43 genes from S. moellendorffii and the model non-vascular plant, Physcomitrella patens, for their roles in xylan biosynthesis. Using nuclear magentic resonance spectroscopy, we have demonstrated that S. moellendorffii xylan consists of β-1,4- linked xylosyl residues subsituted solely with methylated GlcA residues and that xylans from both S. moellendorffii and P. patens are acetylated at O-2 and O-3. To investigate genes responsible for GlcA methylation of xylan, we identified two DUF579 genes in the S. moellendorffii genome and showed that one of them, SmGXM, encodes a glucuronoxylan methyltransferase capable of adding the methyl group onto the GlcA side chain of xylooligomers. Furthermore, we revealed that the two GT43 genes in S. moellendorffii, SmGT43A and SmGT43B, are functional orthologs of the Arabidopsis xylan backbone biosynthetic genes IRX9 and IRX14, respectively, indicating the evolutionary conservation of the involvement of two functionally non-redundant groups of GT43 genes in xylan backbone biosynthesis between seedless and seed vascular plants. Among the five GT43 genes in P. patens, PpGT43A was found to be a functional ortholog of Arabidopsis IRX9, suggesting that the recruitment of GT43 genes in xylan backbone biosynthesis occurred when non-vascular plants appeared on land. [ABSTRACT FROM AUTHOR]

Xylan is made of a linear chain of β-1,4-linked xylosyl residues, some of which are substituted with side chains, such as glucuronic acid (GlcA), methylglucuronic acid (MeGlcA) and arabinose, depending on the source of xylan. Although past studies have revealed a number of genes involved in the elongation of the xylan backbone and the addition of GlcA and arabinosyl side chains, no genes have been shown to be implicated in glucuronoxylan methylation. In this report, we investigated the roles of three Arabidopsis genes, namely GLUCURONOXYLAN METHYLTRANSFERASE1 (GXM1), GXM2 and GXM3, in xylan biosynthesis. The GXM1/2/3 genes were found to be expressed in secondary wall-forming cells and their expression was regulated by SND1, a secondary wall master transcriptional switch. Their encoded proteins were shown to be located in the Golgi, where xylan biosynthesis occurs. Chemical analysis of cell wall sugars from single and double mutants of these genes revealed that although no alterations in the amount of xylose were observed, a significant reduction in the level of MeGlcA was evident in the gxm3 single mutant and the gxm double mutants. Structural analysis of xylan demonstrated that the gxm mutations caused a specific defect in GlcA methylation on xylan without affecting the frequency of xylan substitution. Only about 10% of the GlcA residues on xylan were methylated in the gxm2/3 double mutant, whereas in the wild type 60% of the GlcA residues were methylated. Furthermore, an activity assay demonstrated that recombinant GXM proteins exhibited a methyltransferase activity capable of transferring the methyl group from S-adenosylmethionine onto GlcA-substituted xylooligomers and simultaneous mutations of GXM2/3 genes caused a loss of such a methyltransferase activity. Taken together, our results provide the first line of genetic and biochemical evidence that the three DUF579 domain-containing proteins, GXM1, GXM2 and GXM3, are methyltransferases catalyzing 4-O-methylation of GlcA side chains on xylan. [ABSTRACT FROM AUTHOR]