Independent recruitment of glycosyltransferase family 61 members for xylan substitutions in conifers

Main conclusion Several pine members of the gymnosperm-specific GT61 clades were demonstrated to be arabinosyltransferases and xylosyltransferases catalyzing the transfer of 2- O -Ara f , 3- O -Ara f and 2- O -Xyl side chains onto xylooligomer acceptors, indicating their possible involvement in Ara f and Xyl substitutions of xylan in pine. Abstract Xylan in conifer wood is substituted at O -2 with methylglucuronic acid (MeGlcA) as well as at O -3 with arabinofuranose (Ara f ), which differs from xylan in dicot wood that is typically decorated with MeGlcA but not Ara f . Currently, glycosyltransferases responsible for conifer xylan arabinosylation have not been identified. Here, we investigated the roles of pine glycosyltransferase family 61 (GT61) members in xylan substitutions. Biochemical characterization of four pine wood-associated GT61 members showed that they exhibited three distinct glycosyltransferase activities involved in xylan substitutions. Two of them catalyzed the addition of 2- O -α-Ara f or 3- O -α-Ara f side chains onto xylooligomer acceptors and thus were named Pinus taeda xylan 2- O -arabinosyltransferase 1 (PtX2AT1) and 3- O -arabinosyltransferase 1 (PtX3AT1), respectively. Two other pine GT61 members were found to be xylan 2- O -xylosyltransferases (PtXYXTs) adding 2- O -β-Xyl side chains onto xylooligomer acceptors. Furthermore, sequential reactions with PtX3AT1 and the PtGUX1 xylan glucuronyltransferase demonstrated that PtX3AT1 could efficiently arabinosylate glucuronic acid (GlcA)-substituted xylooligomers and likewise, PtGUX1 was able to add GlcA side chains onto 3- O -Ara f -substituted xylooligomers. Phylogenetic analysis revealed that PtX2AT1, PtX3AT1 and PtXYXTs resided in three gymnosperm-specific GT61 clades that are separated from the grass-expanded GT61 clade harboring xylan 3- O -arabinosyltransferases and 2- O -xylosyltransferases, suggesting that they might have been recruited independently for xylan substitutions in gymnosperms. Together, our findings have established several pine GT61 members as xylan 2- O - and 3- O -arabinosyltransferases and 2- O -xylosyltransferases and they indicate that pine xylan might also be substituted with 2- O -Ara f and 2- O -Xyl side chains.