Three Arabidopsis DUF579 Domain-Containing GXM Proteins are Methyltransferases Catalyzing 4-O-Methylation of Glucuronic Acid on Xylan

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Abstract
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.

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