The irregular xylem9 mutant is deficient in xylan xylosyltransferase activity

Xylan is the second most abundant polysaccharide in dicot wood, and thus elucidation of the xylan biosynthetic pathway is required to understand the mechanisms controlling wood formation. Genetic and chemical studies in Arabidopsis have implicated three genes, FRAGILE FIBER8 (FRA8), IRREGULAR XYLEM8 (IRX8) and IRREGULAR XYLEM9 (IRX9), in the biosynthesis of glucuronoxylan (GX), but the biochemical functions of the encoded proteins are not known. In this study, we determined the effect of the fra8, irx8 and irx9 mutations on the activities of xylan xylosyltransferase (XylT) and glucuronyltransferase (GlcAT). We show that microsomes isolated from the stems of wild-type Arabidopsis exhibit XylT and GlcAT activities in the presence of exogenous 1,4-linked beta-d-xylooligomers. Xylooligomers ranging in size from two to six can be used as acceptors by XylT to form xylooligosaccharides with up to 12 xylosyl residues. We provide evidence that the irx9 mutation results in a substantial reduction in XylT activity but has no discernible effect on GlcAT activity. In contrast, neither XylT nor GlcAT activity is affected by fra8 and irx8 mutations. Our results provide biochemical evidence that the irx9 mutation results in a deficiency in xylan XylT activity, thus leading to a defect in the elongation of the xylan backbone.