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Secondarywalls are synthesizedin specializedcells, suchas tracheary elements andfibers, and their remarkable strength andrigidityprovide strongmechanical support tothe cells andthe plant body. The main components of secondary walls are cellulose, xylan, glucomannan and lignin. Biochemical, molecular and genetic studies have led to the discovery of most of the genes involved in the biosynthesis of secondary wall components. Cellulose is synthesized by cellulose synthase complexes in the plasma membrane and the recent success of in vitro synthesis of cellulose microfibrils by a single recombinant cellulose synthase isoform reconstituted into proteoliposomes opens new doors to further investigate the structure and functions of cellulose synthase complexes. Most genes involved in the glycosyl backbone synthesis, glycosyl substitutions and acetylation of xylan and glucomannan have been genetically characterized and the biochemical properties of some of their encoded enzymes have been investigated. The genes and their encoded enzymes participating in monolignol biosynthesis andmodification have been extensively studied both genetically and biochemically. A full understanding of how secondary wall components are synthesized will ultimately enable us to produce plants with custom-designed secondary wall composition tailored to diverse applications.

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.

• 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.

It has previously been shown that SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN1 (SND1) is a key transcription factor regulating secondary cell wall formation, including the biosynthesis of cellulose, xylan, and lignin. In this study, we show that two closely related SND1-regulated MYB transcription factors, MYB58 and MYB63, are transcriptional regulators specifically activating lignin biosynthetic genes during secondary wall formation in Arabidopsis thaliana. MYB58 and MYB63 are phylogenetically distinct from previously characterized MYBs shown to be associated with secondary wall formation or phenylpropanoid metabolism. Expression studies showed that MYB58 and MYB63 are specifically expressed in fibers and vessels undergoing secondary wall thickening. Dominant repression of their functions led to a reduction in secondary wall thickening and lignin content. Overexpression of MYB58 and MYB63 resulted in specific activation of lignin biosynthetic genes and concomitant ectopic deposition of lignin in cells that are normally unlignified. MYB58 was able to activate directly the expression of lignin biosynthetic genes and a secondary wall-associated laccase (LAC4) gene. Furthermore, the expression of MYB58 and MYB63 was shown to be regulated by the SND1 close homologs NST1, NST2, VND6, and VND7 and their downstream target MYB46. Together, our results indicate that MYB58 and MYB63 are specific transcriptional activators of lignin biosynthesis in the SND1-mediated transcriptional network regulating secondary wall formation.

Wood is the most abundant biomass produced by land plants. Dissection of the molecular mechanisms underlying the transcriptional regulation of wood formation is a fundamental issue in plant biology and has important implications in tree biotechnology. Although a number of transcription factors in tree species have been shown to be associated with wood formation and some of them are implicated in lignin biosynthesis, none of them have been demonstrated to be key regulators of the biosynthesis of all three major components of wood. In this report, we have identified a group of NAC domain transcription factors, PtrWNDs, that are preferentially expressed in developing wood of poplar (Populus trichocarpa). Expression of PtrWNDs in the Arabidopsis (Arabidopsis thaliana) snd1 nst1 double mutant effectively complemented the secondary wall defects in fibers, indicating that PtrWNDs are capable of activating the entire secondary wall biosynthetic program. Overexpression of PtrWND2B and PtrWND6B in Arabidopsis induced the expression of secondary wall-associated transcription factors and secondary wall biosynthetic genes and, concomitantly, the ectopic deposition of cellulose, xylan, and lignin. Furthermore, PtrWND2B and PtrWND6B were able to activate the promoter activities of a number of poplar wood-associated transcription factors and wood biosynthetic genes. Together, these results demonstrate that PtrWNDs are functional orthologs of SND1 and suggest that PtrWNDs together with their downstream transcription factors form a transcriptional network involved in the regulation of wood formation in poplar.

Mannans are an abundant cell wall polysaccharide in bryophytes, seedless vascular plants and gymnosperms. A previous study has shown that mannan acetylation in Arabidopsis and konjac is mediated by mannan O-acetyltransferases belonging to the Domain of Unknown Function (DUF) 231 family. However, little is known about the acetylation patterns of mannans in bryophytes and seedless vascular plants, and the evolutionary origin of mannan O-acetyltransferases in land plants has not yet been studied. Phylogenetic analysis of the DUF231 family revealed that DUF231 members were present in the charophycean green algae and evolved to form overlapped and divergent phylogenetic groups in different taxa of land plants. Acetyltransferase activity assays of recombinant proteins demonstrated that a number of group II DUF231 members from moss, Selaginella, pine, spruce, rice and poplar were mannan 2-O- and 3-O-acetyltransferases, whereas the two group I DUF231 members from the alga Klebsormidium nitens were not. Structural analysis of mannans from moss and Selaginella showed they were composed of mannosyl and glucosyl residues and the mannosyl residues were acetylated at O-2 and O-3. These findings indicate that although the DUF231 genes originated in algae, their recruitment as mannan O-acetyltransferases probably occurred in bryophytes, and the biochemical functions of these O-acetyltransferases are evolutionarily conserved throughout land plants.

Wood biomass is mainly made of secondary cell walls; hence, elucidation of the molecular mechanisms underlying the transcriptional regulation of secondary wall biosynthesis during wood formation will be instrumental to design strategies for genetic improvement of wood biomass. Here, we provide direct evidence demonstrating that the poplar (Populus trichocarpa) wood-associated NAC domain transcription factors (PtrWNDs) are master switches activating a suite of downstream transcription factors, and together, they are involved in the coordinated regulation of secondary wall biosynthesis during wood formation. We show that transgenic poplar plants with dominant repression of PtrWNDs functions exhibit a drastic reduction in secondary wall thickening in woody cells, and those with PtrWND overexpression result in ectopic deposition of secondary walls. Analysis of PtrWND2B overexpressors revealed up-regulation of the expression of a number of wood-associated transcription factors, the promoters of which were also activated by PtrWND6B and the Eucalyptus EgWND1. Transactivation analysis and electrophoretic mobility shift assay demonstrated that PtrWNDs and EgWND1 activated gene expression through direct binding to the secondary wall NAC-binding elements, which are present in the promoters of several wood-associated transcription factors and a number of genes involved in secondary wall biosynthesis and modification. The WND-regulated transcription factors PtrNAC150, PtrNAC156, PtrNAC157, PtrMYB18, PtrMYB74, PtrMYB75, PtrMYB121, PtrMYB128, PtrZF1, and PtrGATA8 were able to activate the promoter activities of the biosynthetic genes for all three major wood components. Our study has uncovered that the WND master switches together with a battery of their downstream transcription factors form a transcriptional network controlling secondary wall biosynthesis during wood formation.

Secondary walls are the major component of wood, and studies of the mechanisms regulating secondary wall synthesis is important for understanding the process of wood formation. We have previously shown that the NAC domain transcription factor SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN1 (SND1) is a key regulator of secondary wall synthesis in fibers of Arabidopsis thaliana stems and dominant repression of SND1 leads to a reduction in secondary wall thickening in fibers. However, T-DNA knockout of the SND1 gene did not cause an alteration in secondary wall thickness, suggesting that other SND1 homologs may compensate for the loss of SND1 expression. Here, we studied the effects of simultaneous inhibition of SND1 and its homolog, NAC SECONDARY WALL THICKENING PROMOTING FACTOR1 (NST1), on secondary wall synthesis in fibers. We show that simultaneous RNA interference (RNAi) inhibition of the expression of both SND1 and NST1 genes results in loss of secondary wall formation in fibers of stems. The fiber cells in the stems of SND1/NST1—RNAi plants lack all three major secondary wall components, including cellulose, xylan, and lignin, which is accompanied by a severe reduction in the expression of genes involved in their biosynthesis. In addition, inhibition of SND1 and NST1 leads to down-regulation of several fiber-associated transcription factor genes. Double T-DNA knockout mutations of SND1 and NST1 genes cause the same effects, as does simultaneous RNAi inhibition of SND1 and NST1. Our results provide first line evidence demonstrating that SND1 and NST1 function redundantly in the regulation of secondary wall synthesis in fibers.

SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN1 (SND1) is a master transcriptional switch activating the developmental program of secondary wall biosynthesis. Here, we demonstrate that a battery of SND1-regulated transcription factors is required for normal secondary wall biosynthesis in Arabidopsis thaliana. The expression of 11 SND1-regulated transcription factors, namely, SND2, SND3, MYB103, MYB85, MYB52, MYB54, MYB69, MYB42, MYB43, MYB20, and KNAT7 (a Knotted1-like homeodomain protein), was developmentally associated with cells undergoing secondary wall thickening. Of these, dominant repression of SND2, SND3, MYB103, MYB85, MYB52, MYB54, and KNAT7 significantly reduced secondary wall thickening in fiber cells. Overexpression of SND2, SND3, and MYB103 increased secondary wall thickening in fibers, and overexpression of MYB85 led to ectopic deposition of lignin in epidermal and cortical cells in stems. Furthermore, SND2, SND3, MYB103, MYB85, MYB52, and MYB54 were able to induce secondary wall biosynthetic genes. Direct target analysis using the estrogen-inducible system revealed that MYB46, SND3, MYB103, and KNAT7 were direct targets of SND1 and also of its close homologs, NST1, NST2, and vessel-specific VND6 and VND7. Together, these results demonstrate that a transcriptional network consisting of SND1 and its downstream targets is involved in regulating secondary wall biosynthesis in fibers and that NST1, NST2, VND6, and VND7 are functional homologs of SND1 that regulate the same downstream targets in different cell types.

We demonstrate that the Arabidopsis thaliana MYB46 transcription factor is a direct target of SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN1 (SND1), which is a key transcriptional activator regulating the developmental program of secondary wall biosynthesis. The MYB46 gene is expressed predominantly in fibers and vessels in stems, and its encoded protein is targeted to the nucleus and can activate transcription. MYB46 gene expression was shown to be regulated by SND1, and transactivation analysis demonstrated that SND1 as well as its close homologs were able to activate the MYB46 promoter. Electrophoretic mobility shift assays and chromatin immunoprecipitation experiments revealed that SND1 binds to the MYB46 promoter. Dominant repression of MYB46 caused a drastic reduction in the secondary wall thickening of fibers and vessels. Overexpression of MYB46 resulted in an activation of the biosynthetic pathways of cellulose, xylan, and lignin and concomitantly led to ectopic deposition of secondary walls in cells that are normally nonsclerenchymatous. In addition, the expression of two secondary wall-associated transcription factors, MYB85 and KNAT7, was highly upregulated by MYB46 overexpression. These results demonstrate that MYB46 is a direct target of SND1 and is another key player in the transcriptional network involved in the regulation of secondary wall biosynthesis in ARABIDOPSIS: