Zheng-Hua Ye

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.

Xylan, a major constituent of secondary cell walls, is made of a linear chain of β-1,4-linked xylosyl residues that are often substituted with glucuronic acid/methylglucuronic acid side chains and acetylated at O -2 and O -3. Previous studies have shown that ESK1, an Arabidopsis DUF231 protein, is an acetyltransferase catalyzing 2- O - and 3- O -monoacetylation of xylan. However, the esk1 mutation only causes a partial loss of xylan 2- O - and 3- O -monoacetylation, suggesting that additional xylan acetyltransferase activities are involved. In this report, we demonstrated the essential roles of two other Arabidopsis DUF231 genes, TBL3 and TBL31 , in xylan acetylation. The expression of both TBL3 and TBL31 was shown to be induced by overexpression of the secondary wall master transcriptional regulator SND1 (secondary wall-associated NAC domain protein1) and down-regulated by simultaneous mutations of SND1 and its paralog NST1 , indicating their involvement in secondary wall biosynthesis. β-Glucurondase (GUS) reporter gene analysis showed that TBL3 and TBL31 were specifically expressed in the xylem and interfascicular fibers in stems and the secondary xylem in root hypocotyls. Expression of fluorescent protein-tagged TBL3 and TBL31 in protoplasts revealed their localization in the Golgi, where xylan biosynthesis occurs. Although mutation of either TBL3 or TBL31 alone did not cause any apparent alterations in cell wall composition, their simultaneous mutations were found to result in a reduction in xylan acetylation. Further structural analysis demonstrated that the tbl3 tbl31 double mutant had a specific reduction in 3- O -acetylation of xylan. In addition, the tbl3 tbl31 esk1 triple mutant displayed a much more drastic decrease in 3- O -acetylation of xylan, indicating their functional redundancy in xylan 3- O -acetylation. These findings indicate that TBL3 and TBL31 are secondary wall-associated DUF231 genes specifically involved in xylan 3- O -acetylation.

Transcriptional regulation of secondary wall biosynthesis in Arabidopsis thaliana has been shown to be mediated by a group of secondary wall NAC master switches, including NST1, NST2, SND1 and VND1 to VND7. It has been shown that VND1 to VND7 regulate secondary wall biosynthesis in vessels, NST1 and NST2 function redundantly in anther endothecium, and SND1 and NST1 are required for secondary wall thickening in fibers of stems. However, it is unknown whether NST2 is involved in regulating secondary wall biosynthesis in fibers of stems. In this report, we demonstrated that similar to SND1, NST2 together with NST1 were highly expressed in interfascicular fibers and xylary fibers but not in vessels of stems. Although simultaneous mutations of SND1 and NST1 have been shown to result in a significant impairment of secondary wall thickening in fibers, a small amount of secondary walls was deposited in fibers during the late stage of stem development. In contrast, simultaneous mutations of SND1, NST1 and NST2 led to a complete loss of secondary wall thickening in fibers. These results demonstrate that NST2 together with SND1 and NST1 regulate secondary wall biosynthesis in fibers of stems.

Secondary walls are mainly composed of cellulose, hemicelluloses (xylan and glucomannan) and lignin, and are deposited in some specialized cells, such as tracheary elements, fibers and other sclerenchymatous cells. Secondary walls provide strength to these cells, which lend mechanical support and protection to the plant body and, in the case of tracheary elements, enable them to function as conduits for transporting water. Formation of secondary walls is a complex process that requires the co-ordinated expression of secondary wall biosynthetic genes, biosynthesis and targeted secretion of secondary wall components, and patterned deposition and assembly of secondary walls. Here, we provide a comprehensive review of genes involved in secondary wall biosynthesis and deposition. Most of the genes involved in the biosynthesis of secondary wall components, including cellulose, xylan, glucomannan and lignin, have been identified and their co-ordinated activation has been shown to be mediated by a transcriptional network encompassing the secondary wall NAC and MYB master switches and their downstream transcription factors. It has been demonstrated that cortical microtubules and microtubule-associated proteins play important roles in the targeted secretion of cellulose synthase complexes, the oriented deposition of cellulose microfibrils and the patterned deposition of secondary walls. Further investigation of many secondary wall-associated genes with unknown functions will provide new insights into the mechanisms controlling the formation of secondary walls that constitute the bulk of plant biomass.

One of the most prominent features of xylem conducting cells is the deposition of secondary walls. In Arabidopsis, secondary wall biosynthesis in the xylem conducting cells, vessels, has been shown to be regulated by two VASCULAR-RELATED NAC-DOMAIN (VND) genes, VND6 and VND7. In this report, we have investigated the roles of five additional Arabidopsis VND genes, VND1 to VND5, in regulating secondary wall biosynthesis in vessels. The VND1 to VND5 genes were shown to be specifically expressed in vessels but not in interfascicular fibers in stems. The expression of VND4 and VND5 was also seen specifically in vessels in the secondary xylem of the root-hypocotyl region. When overexpressed, VND1 to VND5 were able to activate the expression of secondary wall-associated transcription factors and genes involved in secondary wall biosynthesis and programmed cell death. As a result, many normally parenchymatous cells in leaves and stems acquired thickened secondary walls in the VND1 to VND5 overexpressors. In contrast, dominant repression of VND3 function resulted in reduced secondary wall thickening in vessels and a collapsed vessel phenotype. In addition, VND1 to VND5 were shown to be capable of rescuing the secondary wall defects in the fibers of the snd1 nst1 double mutant when expressed under the SND1 promoter. Furthermore, transactivation analysis revealed that VND1 to VND5 could activate expression of the GUS reporter gene driven by the secondary wall NAC binding element (SNBE). Together, these results demonstrate that VND1 to VND5 possess functions similar to that of the SND1 secondary wall NAC and are transcriptional regulators of secondary wall biosynthesis in vessels.

Xylan is the major hemicellulose present in both primary and secondary cell walls of rice vegetative tissues. Since xylan is one of the factors contributing to biomass recalcitrance, understanding how xylan is synthesized in rice will potentially provide tools to modify grass biomass composition better suited for biofuel production. Studies of xylan biosynthesis in Arabidopsis have revealed that family GT43 glycosyltransferases, which form 2 functionally nonredundant groups, IRX9/IRX9 homolog and IRX14/IRX14 homolog, are required for xylan backbone elongation. The rice genome harbors 10 genes encoding family GT43 members and it is currently unknown whether they are all involved in xylan biosynthesis. In this report, we performed biochemical analysis of xylan xylosyltransferase activity in rice stem microsomes and investigated the roles of 4 representative rice GT43 members, OsGT43A (LOC_Os05 g03174), OsGT43E (LOC_Os05 g48600), OsGT43H (LOC_Os04 g01280), and OsGT43J (LOC_Os06 g47340), in xylan biosynthesis. OsGT43 proteins were shown to be localized in the Golgi, where xylan biosynthesis occurs. Complementation analysis by expression of OsGT43s in Arabidopsis irx9 and irx14 mutants demonstrated that OsGT43A and OsGT43E but not OsGT43H and OsGT43J were able to rescue the mutant phenotypes conferred by the irx9 mutation, including defective stem mechanical strength, vessel morphology, xylan content, GlcA side chains, xylan chain length, and xylosyltransferase activity. On the other hand, OsGT43J but not OsGT43A, OsGT43E, and OsGT43H restored the defective xylan phenotype in the irx14 mutant. These results indicate that the rice GT43 family evolved to retain the involvement of 2 functionally nonredundant groups, OsGT43A and OsGT43E (IRX9 homologs) vs. OsGT43J (an IRX14 homolog), in xylan backbone biosynthesis.

Wood is one of the promising bioenergy feedstocks for lignocellulosic biofuel production. Understanding how wood components are synthesized will help us design strategies for better utilization of wood for biofuel production. One of the major wood components is xylan, in which about 10% of xylosyl residues are substituted with glucuronic acid (GlcA) side chains. All the GlcA side chains of xylan in wood of Populus trichocarpa are methylated, which is different from Arabidopsis xylan in which about 60% of GlcA side chains are methylated. Genes responsible for methylation of GlcA side chains in Populus xylan have not been identified. Here, we report genetic and biochemical analyses of four DUF579 domain-containing proteins, PtrGXM1, PtrGXM2, PtrGXM3 and PtrGXM4, from Populus trichocarpa and their roles in GlcA methylation in xylan. The PtrGXM genes were found to be highly expressed in wood-forming cells and their encoded proteins were shown to be localized in the Golgi. When overexpressed in the Arabidopsis gxm1/2/3 triple mutant, PtrGXMs were able to partially complement the mutant phenotypes including defects in glucuronoxylan methyltransferase activity and GlcA methylation in xylan, indicating that PtrGXMs most likely function as glucuronoxylan methyltransferases. Direct evidence was provided by enzymatic analysis of recombinant PtrGXM proteins showing that they possessed a methyltransferase activity capable of transferring the methyl group onto GlcA-substituted xylooligomers. Kinetic analysis showed that PtrGXMs exhibited differential affinities toward the GlcA-substituted xylooligomer acceptor with PtrGXM3 and PtrGXM4 having 10 times higher K_m values than PtrGXM1 and PtrGXM2. Together, these findings indicate that PtrGXMs are methyltransferases mediating GlcA methylation in Populus xylan during wood formation.

Xylan is the second most abundant polysaccharide in secondary walls of dicot plants and one of its structural features is the high degree of acetylation of xylosyl residues. In Arabidopsis, about 60% of xylosyl residues in xylan are acetylated and the biochemical mechanisms controlling xylan acetylation are largely unknown. A recent report by Yuan et al. (2013) revealed the essential role of a DUF231 domain-containing protein, ESKIMO1 (ESK1), in xylan acetylation in Arabidopsis as the esk1 mutation caused specific reductions in the degree of xylan 2-O or 3-O-monoacetylation and in the activity of xylan acetyltransferase. Interestingly, the esk1 mutation also resulted in an elevation of glucuronic acid (GlcA) substitutions in xylan. Since GlcA substitutions in xylan occur at the O-2 position of xylosyl residues, it is plausible that the increase in GlcA substitutions in the esk1 mutant is attributed to the reduction in acetylation at O-2 of xylosyl residues, which renders more O-2 positions available for GlcA substitutions. Here, we investigated the effect of removal of GlcA substitutions on the degree of xylan acetylation. We found that a complete loss of GlcA substitutions in the xylan of the gux1/2/3 triple mutant led to a significant increase in the degree of xylan acetylation, indicating that xylan acetyltransferases and glucuronyltransferases compete with each other for xylosyl residues for their acetylation or GlcA substitutions in planta. In addition, detailed structure analysis of xylan from the rwa1/2/3/4 quadruple mutant revealed that it had a uniform reduction of acetyl substitutions at different positions of the xylosyl residues, which is consistent with the proposed role of RWAs as acetyl coenzyme A transporters. The significance of these findings is discussed.