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Summary: Secondary cell wall biosynthesis has been shown to be regulated by a suite of transcription factors. Here, we identified a new xylem vessel‐specific NAC domain transcription factor, secondary wall‐associated NAC domain protein5 (SND5), in Arabidopsis thaliana and studied its role in regulating secondary wall biosynthesis.We showed that the expression of SND5 and its close homolog, SND4/ANAC075, was specifically associated with secondary wall‐containing cells and dominant repression of their functions severely reduced secondary wall thickening in these cells. Overexpression of SND4/5 as well as their homologs SND2/3 fused with the activation domain of the viral protein VP16 led to ectopic secondary wall deposition in cells that are normally parenchymatous. SND2/3/4/5 regulated the expression of the same downstream target genes as do the secondary wall NAC master switches (SWNs) by binding to and activating the secondary wall NAC binding elements (SNBEs).Furthermore, we demonstrated that the poplar (Populus trichocarpa) orthologs of SND2/3/4/5 also activated SNBEs and regulated secondary wall biosynthesis during wood formation.Together, these findings indicate that SND2/3/4/5 and their poplar orthologs regulate the expression of secondary wall‐associated genes through activating SNBEs and they are positioned at an upper level in the SWN‐mediated transcriptional network. [ABSTRACT FROM AUTHOR]

Xylan and xyloglucan are the twomajor cell wall hemicelluloses in plants, and their biosynthesis requires a steady supply of the sugar donor, UDP-xylose. UDP-xylose is synthesized through conversion of UDP-glucuronic acid (UDP-GlcA) by the activities of UDP-xylose synthase (UXS). There exist six UXS genes in the Arabidopsis thaliana genome; three of them (UXS1, UXS2 and UXS4) encode Golgi-localized enzymes and the other three (UXS3, UXS5 and UXS6) encode cytosol-localized enzymes. In this report, we investigated the contributions of these UXS genes in supplying UDP-xylose for the biosynthesis of xylan and xyloglucan. Expression analyses revealed that the six UXS genes exhibited distinct and overlapping expression patterns in different cell types of stems, root-hypocotyls and young seedlings, and that the relative enzymatic activity of UXS in the cytosol was 17 times higher than that in the Golgi. Among the six UXS genes, UXS3, UXS5 and UXS6 showed the highest expression in stems and were expressed predominantly in xylem cells and interfascicular fibers. Their predominant expression in secondary wall-forming cells was consistent with the finding that the expression of UXS3, UXS5 and UXS6 was directly activated by the secondary wall NAC master switches. Although simultaneous mutations of UXS1, UXS2 and UXS4 did not cause any apparent effects on plant growth and xylan biosynthesis, simultaneous down-regulation/mutations of UXS3, UXS5 and UXS6 led to a drastic reduction in secondary wall thickening, a severe deformation of xylem vessels, a significant decrease in xylan content without an apparent reduction in its chain length and an absence of GlcA side chains in xylan, which are reminiscent of the phenotypes of some known xylan-deficient mutants. Moreover, Immunolocalization with two xyloglucan monoclonal antibodies, LM15 and LM25, revealed a significant reduction in the amount of xylogulcan in the primary walls. These results demonstrate that the cytosol-localized UXS3, UXS5 and UXS6 play a predominant role in the supply of UDP-xylose for the biosynthesis of xylan and xyloglucan. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Xylan is a major acetylated polymer in plant lignocellulosic biomass and it can be mono- and di-acetylated at O-2 and O-3 as well as mono-acetylated at O-3 of xylosyl residues that is substituted with glucuronic acid (GlcA) at O-2. Based on the finding that ESK1, an Arabidopsis thaliana DUF231 protein, specifically mediates xylan 2-O- and 3-O-monoacetylation, we previously proposed that different acetyltransferase activities are required for regiospecific acetyl substitutions of xylan. Here, we demonstrate the functional roles of TBL32 and TBL33, two ESK1 close homologs, in acetyl substitutions of xylan. Simultaneous mutations of TBL32 and TBL33 resulted in a significant reduction in xylan acetyl content and endoxylanase digestion of the mutant xylan released GlcA-substituted xylooligomers without acetyl groups. Structural analysis of xylan revealed that the tbl32 tbl33 mutant had a nearly complete loss of 3-O-acetylated, 2-O-GlcA-substituted xylosyl residues. A reduction in 3-O-monoacetylated and 2,3-di-O-acetylated xylosyl residues was also observed. Simultaneous mutations of TBL32, TBL33 and ESK1 resulted in a severe reduction in xylan acetyl level down to 15% of that of the wild type, and concomitantly, severely collapsed vessels and stunted plant growth. In particular, the S2 layer of secondary walls in xylem vessels of tbl33 esk1 and tbl32 tbl33 esk1 exhibited an altered structure, indicating abnormal assembly of secondary wall polymers. These results demonstrate that TBL32 and TBL33 play an important role in xylan acetylation and normal deposition of secondary walls. [ABSTRACT FROM AUTHOR]

Wood is mainly composed of secondary walls, which constitute the most abundant stored carbon produced by vascular plants. Understanding the molecular mechanisms controlling secondary wall deposition during wood formation is not only an important issue in plant biology but also critical for providing molecular tools to custom-design wood composition suited for diverse end uses. Past molecular and genetic studies have revealed a transcriptional network encompassing a group of wood-associated NAC and MYB transcription factors that are involved in the regulation of the secondary wall biosynthetic program during wood formation in poplar trees. Here, we report the functional characterization of poplar orthologs of MYB46 and MYB83 that are known to be master switches of secondary wall biosynthesis in Arabidopsis. In addition to the two previously-described PtrMYB3 and PtrMYB20, two other MYBs, PtrMYB2 and PtrMYB21, were shown to be MYB46/MYB83 orthologs by complementation and overexpression studies in Arabidopsis. The functional roles of these PtrMYBs in regulating secondary wall biosynthesis were further demonstrated in transgenic poplar plants showing an ectopic deposition of secondary walls in PtrMYB overexpressors and a reduction of secondary wall thickening in their dominant repressors. Furthermore, PtrMYB2/3/20/21 together with two other tree MYBs, the Eucalyptus EgMYB2 and the pine PtMYB4, were shown to differentially bind to and activate the eight variants of the 7-bp SMRE consensus sequence, composed of ACC(A/T)A(A/C)(T/C). Together, our results indicate that the tree MYBs, PtrMYB2/3/20/21, EgMYB2 and PtMYB4, are master transcriptional switches that activate the SMRE sites in the promoters of target genes and thereby regulate secondary wall biosynthesis during wood formation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]