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Several pine members of the gymnosperm-specific GT61 clades were demonstrated to be arabinosyltransferases and xylosyltransferases catalyzing the transfer of 2-O-Araf, 3-O-Araf and 2-O-Xyl side chains onto xylooligomer acceptors, indicating their possible involvement in Araf and Xyl substitutions of xylan in pine. Xylan in conifer wood is substituted at O-2 with methylglucuronic acid (MeGlcA) as well as at O-3 with arabinofuranose (Araf), which differs from xylan in dicot wood that is typically decorated with MeGlcA but not Araf. 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-Araf or 3-O-α-Araf 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-Araf-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-Araf and 2-O-Xyl side chains.

Multiple rice GT61 members were demonstrated to be xylan arabinosyltransferases (XATs) mediating 3-O-arabinosylation of xylan and the functions of XATs and xylan 2-O-xylosyltransferases were shown to be conserved in grass species. Xylan is the major hemicellulose in the cell walls of grass species and it is typified by having arabinofuranosyl (Araf) substitutions. In this report, we demonstrated that four previously uncharacterized, Golgi-localized glycosyltransferases residing in clade A or B of the rice GT61 family were able to mediate 3-O-arabinosylation of xylan when heterologously expressed in the Arabidopsis gux1/2/3 triple mutant. Biochemical characterization of their recombinant proteins established that they were xylan arabinosyltransferases (XATs) capable of transferring Araf residues onto xylohexaose acceptors, and thus they were named OsXAT4, OsXAT5, OsXAT6 and OsXAT7. OsXAT5 and the previously identified OsXAT2 were shown to be able to arabinosylate xylooligomers with a degree of polymerization of as low as 3. Furthermore, a number of XAT homologs from maize, sorghum, Brachypodium and switchgrass were found to exhibit activities catalyzing Araf transfer onto xylohexaose, indicating that they are XATs involved in xylan arabinosylation in these grass species. Moreover, we revealed that homologs of another GT61 member, xylan 2-O-xylosyltransferase (XYXT1), from these grass species could mediate 2-O-xylosylation of xylan when expressed in the Arabidopsis gux1/2/3 mutant. Together, our findings indicate that multiple OsXATs are involved in 3-O-arabinosylation of xylan and the functions of XATs and XYXTs are conserved in grass species.

AXY4/XGOAT1, AXY4L/XGOAT2 and PtrXGOATs are O-acetyltransferases acetylating fucosylated galactose residues on xyloglucan and AXY9 does not directly catalyze O-acetylation of xyloglucan but exhibits weak acetylesterase activity. Xyloglucan is a major hemicellulose that cross-links cellulose in the primary walls of dicot plants and the galactose (Gal) residues on its side chains can be mono- and di-O-acetylated. In Arabidopsis thaliana, mutations of three AXY (altered xyloglucan) genes, AXY4, AXY4L and AXY9, have previously been shown to cause a reduction in xyloglucan acetylation, but their biochemical functions remain to be investigated. In this report, we demonstrated that recombinant proteins of AXY4/XGOAT1 (xyloglucan O-acetyltransferase1), AXY4L/XGOAT2 and their close homologs from Populus trichocarpa, PtrXGOATs, displayed O-acetyltransferase activities transferring acetyl groups from acetyl CoA onto xyloglucan oligomers. Structural analysis of XGOAT-catalyzed reaction products revealed that XGOATs mediated predominantly 6-O-monoacetylation and a much lesser degree of 3-O and 4-O-monoacetylation and 4,6-di-O-acetylation of Gal residues on xyloglucan side chains. XGOATs appeared to preferentially acetylate fucosylated Gal residues with little activity toward non-fucosylated Gal residues. Mutations of the conserved amino acid residues in the GDS and DXXH motifs in AXY4/XGOAT1 resulted in a drastic reduction in its ability to transfer acetyl groups onto xyloglucan oligomers. In addition, although recombinant AXY9 was unable to transfer acetyl groups from acetyl CoA onto xyloglucan oligomers, it was catalytically active as demonstrated by its weak acetylesterase activity that was also exhibited by AXY4/XGOAT1 and AXY4L/XGOAT2. Furthermore, we showed that the AXY8 fucosidase was able to hydrolyze fucosyl residues from both non-acetylated and acetylated xyloglucan oligomers. These findings provide biochemical evidence that AXY4/XGOAT1, AXY4L/XGOAT2 and PtrXGOATs are xyloglucan O-acetyltransferases catalyzing acetyl transfer onto fucosylated Gal residues on xyloglucan side chains and the defucosylation of these acetylated side chains by apoplastic AXY8 generates side chains with acetylated, non-fucosylated Gal residues.

Rice xylan is predominantly monoacetylated at O-2 and O-3, and 14 rice DUF231 proteins were demonstrated to be xylan acetyltransferases. Acetylated xylans are the principal hemicellulose in the cell walls of grass species. Because xylan acetylation impedes the conversion of cellulosic biomass into biofuels, knowledge on acetyltransferases catalyzing xylan acetylation in grass species will be instrumental for a better utilization of grass biomass for biofuel production. Xylan in rice (Oryza sativa) is predominantly monoacetylated at O-2 and O-3 with a total degree of acetylation of 0.19. In this report, we have characterized 14 rice DUF231 proteins (OsXOAT1 to OsXOAT14) that are phylogenetically grouped together with Arabidopsis xylan acetyltransferases ESK1 and its close homologs. Complementation analysis demonstrated that the expression of OsXOAT1 to OsXOAT7 in the Arabidopsis esk1 mutant was able to rescue its defects in 2-O- and 3-O-monoacetylation and 2,3-di-O-acetylation. Activity assay of recombinant proteins revealed that all 14 OsXOATs exhibited acetyltransferase activities capable of transferring acetyl groups from acetyl-CoA to the xylohexaose acceptor with 10 of them having high activities. Structural analysis of the OsXOAT-catalyzed products showed that the acetylated structural units consisted mainly of 2-O- and 3-O-monoacetylated xylosyl residues with a minor amount of 2,3-di-O-acetylated xylosyl units, which is consistent with the acetyl substitution pattern of rice xylan. Further kinetic studies revealed that OsXOAT1, OsXOAT2, OsXOAT5, OsXOAT6 and OsXOAT7 had high affinity toward the xylohexaose acceptor. Our results provide biochemical evidence indicating that OsXOATs are acetyltransferases involved in xylan acetylation in rice.

The Arabidopsis thaliana (L.) Heynh. ROOT HAIR DEFECTIVE3 (RHD3) gene has previously been shown to be essential for normal cell expansion [H. Wang et al. (1997) Genes Dev 11:799-811]. In this report, we demonstrated that mutation of the RHD3 gene in the Arabidopsis fragile fiber 4 (fra4) mutant caused a dramatic reduction in the wall thickness of fibers, vessels, and pith cells in the inflorescence stems and, concomitantly, a decrease in the mechanical strength of stems. The reduced wall thickness in the fra4 mutant was accompanied by an alteration in cell wall composition. Consistent with the defective fiber and vessel wall phenotypes, the RHD3 gene exhibited a strong expression in developing fiber and xylem cells. We showed that the Arabidopsis genome contains two additional RHD3-like genes, one of which was expressed specifically in flowers. In addition, we found that mutation of the RHD3 gene caused an alteration in the organization of the actin cytoskeleton but no effects on cortical microtubules. Our findings suggest an essential role of RHD3 in cell wall biosynthesis and actin organization, both of which are known to be important for cell expansion.