SECONDARY WALL-ASSOCIATED NAC DOMAIN Research Articles

PagMYB128 regulates secondary cell wall formation by direct activation of cell wall biosynthetic genes during wood formation in poplar.

The biosynthesis of cellulose, lignin, and hemicelluloses in plant secondary cell walls (SCWs) is regulated by a hierarchical transcriptional regulatory network. This network features orthologous transcription factors shared between poplar and Arabidopsis, highlighting a foundational similarity in their genetic regulation. However, knowledge on the discrepant behavior of the transcriptional-level molecular regulatory mechanisms between poplar and Arabidopsis remains limited. In this study, we investigated the function of PagMYB128 during wood formation and found it had broader impacts on SCW formation compared to its Arabidopsis ortholog, AtMYB103. Transgenic poplar trees overexpressing PagMYB128 exhibited significantly enhanced xylem development, with fiber cells and vessels displaying thicker walls, and an increase in the levels of cellulose, lignin, and hemicelluloses in the wood. In contrast, plants with dominant repression of PagMYB128 demonstrated the opposite phenotypes. RNA sequencing and reverse transcription - quantitative polymerase chain reaction showed that PagMYB128 could activate SCW biosynthetic gene expression, and chromatin immunoprecipitation along with yeast one-hybrid, and effector-reporter assays showed this regulation was direct. Further analysis revealed that PagSND1 (SECONDARY WALL-ASSOCIATED NAC-DOMAIN PROTEIN1) directly regulates PagMYB128 but not cell wall metabolic genes, highlighting the pivotal role of PagMYB128 in the SND1-driven regulatory network for wood development, thereby creating a feedforward loop in SCW biosynthesis.

Enhancement of Secondary Cell Wall Formation in Poplar Xylem Using a Self-Reinforced System of Secondary Cell Wall-Related Transcription Factors.

The secondary cell wall (SCW) in the xylem is one of the largest sink organs of carbon in woody plants, and is considered a promising sustainable bioresource for biofuels and biomaterials. To enhance SCW formation in poplar (Populus sp.) xylem, we developed a self-reinforced system of SCW-related transcription factors from Arabidopsis thaliana, involving VASCULAR-RELATED NAC-DOMAIN7 (VND7), SECONDARY WALL-ASSOCIATED NAC-DOMAIN PROTEIN 1/NAC SECONDARY WALL THICKENING-PROMOTING FACTOR3 (SND1/NST3), and MYB46. In this system, these transcription factors were fused with the transactivation domain VP16 and expressed under the control of the Populus trichocarpa CesA18 (PtCesA18) gene promoter, creating the chimeric genes PtCesA18pro::AtVND7:VP16, PtCesA18pro::AtSND1:VP16, and PtCesA18pro::AtMYB46:VP16. The PtCesA18 promoter is active in tissues generating SCWs, and can be regulated by AtVND7, AtSND1, and AtMYB46; thus, the expression levels of PtCesA18pro::AtVND7:VP16, PtCesA18pro::AtSND1:VP16, and PtCesA18pro::AtMYB46:VP16 are expected to be boosted in SCW-generating tissues. In the transgenic hybrid aspens (Populus tremula × tremuloides T89) expressing PtCesA18pro::AtSND1:VP16 or PtCesA18pro::AtMYB46:VP16 grown in sterile half-strength Murashige and Skoog growth medium, SCW thickening was significantly enhanced in the secondary xylem cells, while the PtCesA18pro::AtVND7:VP16 plants showed stunted xylem formation, possibly because of the enhanced programmed cell death (PCD) in the xylem regions. After acclimation, the transgenic plants were transferred from the sterile growth medium to pots of soil in the greenhouse, where only the PtCesA18pro::AtMYB46:VP16 aspens survived. A nuclear magnetic resonance footprinting cell wall analysis and enzymatic saccharification analysis demonstrated that PtCesA18pro::AtMYB46:VP16 influences cell wall properties such as the ratio of syringyl (S) and guaiacyl (G) units of lignin, the abundance of the lignin β-aryl ether and resinol bonds, and hemicellulose acetylation levels. Together, these data indicate that we have created a self-reinforced system using SCW-related transcription factors to enhance SCW accumulation.

The transcription factor PagLBD3 contributes to the regulation of secondary growth in Populus

LATERAL ORGAN BOUNDARIES DOMAIN (LBD) genes encode plant-specific transcription factors that participate in regulating various developmental processes. In this study, we genetically characterized PagLBD3 encoding an important regulator of secondary growth in poplar (Populus alba × Populus glandulosa). Overexpression of PagLBD3 increased stem secondary growth in Populus with a significantly higher rate of cambial cell differentiation into phloem, while dominant repression of PagLBD3 significantly decreased the rate of cambial cell differentiation into phloem. Furthermore, we identified 1756 PagLBD3 genome-wide putative direct target genes (DTGs) through RNA sequencing (RNA-seq)-coupled DNA affinity purification followed by sequencing (DAP-seq) assays. Gene Ontology analysis revealed that genes regulated by PagLBD3 were enriched in biological pathways regulating meristem development, xylem development, and auxin transport. Several central regulator genes for vascular development, including PHLOEM INTERCALATED WITH XYLEM (PXY), WUSCHEL RELATED HOMEOBOX4 (WOX4), Secondary Wall-Associated NAC Domain 1s (SND1-B2), and Vascular-Related NAC-Domain 6s (VND6-B1), were identified as PagLBD3 DTGs. Together, our results indicate that PagLBD3 and its DTGs form a complex transcriptional network to modulate cambium activity and phloem/xylem differentiation.

Xylem vessel-specific SND5 and its homologs regulate secondary wall biosynthesis through activating secondary wall NAC binding elements.

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.

Histone methyltransferase ATX1 dynamically regulates fiber secondary cell wall biosynthesis in Arabidopsis inflorescence stem.

Secondary wall thickening in the sclerenchyma cells is strictly controlled by a complex network of transcription factors in vascular plants. However, little is known about the epigenetic mechanism regulating secondary wall biosynthesis. In this study, we identified that ARABIDOPSIS HOMOLOG of TRITHORAX1 (ATX1), a H3K4-histone methyltransferase, mediates the regulation of fiber cell wall development in inflorescence stems of Arabidopsis thaliana. Genome-wide analysis revealed that the up-regulation of genes involved in secondary wall formation during stem development is largely coordinated by increasing level of H3K4 tri-methylation. Among all histone methyltransferases for H3K4me3 in Arabidopsis, ATX1 is markedly increased during the inflorescence stem development and loss-of-function mutant atx1 was impaired in secondary wall thickening in interfascicular fibers. Genetic analysis showed that ATX1 positively regulates secondary wall deposition through activating the expression of secondary wall NAC master switch genes, SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN1 (SND1) and NAC SECONDARY WALL THICKENING PROMOTING FACTOR1 (NST1). We further identified that ATX1 directly binds the loci of SND1 and NST1, and activates their expression by increasing H3K4me3 levels at these loci. Taken together, our results reveal that ATX1 plays a key role in the regulation of secondary wall biosynthesis in interfascicular fibers during inflorescence stem development of Arabidopsis.

Transcriptional Regulation of Sorghum Stem Composition: Key Players Identified Through Co-expression Gene Network and Comparative Genomics Analyses.

Most sorghum biomass accumulates in stem secondary cell walls (SCW). As sorghum stems are used as raw materials for various purposes such as feed, energy and fiber reinforced polymers, identifying the genes responsible for SCW establishment is highly important. Taking advantage of studies performed in model species, most of the structural genes contributing at the molecular level to the SCW biosynthesis in sorghum have been proposed while their regulatory factors have mostly not been determined. Validation of the role of several MYB and NAC transcription factors in SCW regulation in Arabidopsis and a few other species has been provided. In this study, we contributed to the recent efforts made in grasses to uncover the mechanisms underlying SCW establishment. We reported updated phylogenies of NAC and MYB in 9 different species and exploited findings from other species to highlight candidate regulators of SCW in sorghum. We acquired expression data during sorghum internode development and used co-expression analyses to determine groups of co-expressed genes that are likely to be involved in SCW establishment. We were able to identify two groups of co-expressed genes presenting multiple evidences of involvement in SCW building. Gene enrichment analysis of MYB and NAC genes provided evidence that while NAC SECONDARY WALL THICKENING PROMOTING FACTOR NST genes and SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN gene functions appear to be conserved in sorghum, NAC master regulators of SCW in sorghum may not be as tissue compartmentalized as in Arabidopsis. We showed that for every homolog of the key SCW MYB in Arabidopsis, a similar role is expected for sorghum. In addition, we unveiled sorghum MYB and NAC that have not been identified to date as being involved in cell wall regulation. Although specific validation of the MYB and NAC genes uncovered in this study is needed, we provide a network of sorghum genes involved in SCW both at the structural and regulatory levels.

SHORTROOT-Mediated Intercellular Signals Coordinate Phloem Development in Arabidopsis Roots.

Asymmetric cell division (ACD) and positional signals play critical roles in the tissue patterning process. In the Arabidopsis (Arabidopsis thaliana) root meristem, two major phloem cell types arise via ACDs of distinct origins: one for companion cells (CCs) and the other for proto- and metaphloem sieve elements (SEs). The molecular mechanisms underlying each of these processes have been reported; however, how these are coordinated has remained elusive. Here, we report a new phloem development process coordinated via the SHORTROOT (SHR) transcription factor in Arabidopsis. The movement of SHR into the endodermis regulates the ACD for CC formation by activating microRNA165/6, while SHR moving into the phloem regulates the ACD generating the two phloem SEs. In the phloem, SHR sequentially activates NAC-REGULATED SEED MORPHOLOGY 1 (NARS1) and SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN 2 (SND2), and these three together form a positive feedforward loop. Under this regulatory scheme, NARS1, generated in the CCs of the root differentiation zone, establishes a top-down signal that drives the ACD for phloem SEs in the meristem. SND2 appears to function downstream to amplify NARS1 via positive feedback. This new regulatory mechanism expands our understanding of the sophisticated vascular tissue patterning processes occurring during postembryonic root development.plantcell;32/5/1519/FX1F1fx1.

Hierarchical Transcription Factor and Chromatin Binding Network for Wood Formation in Black Cottonwood (Populus trichocarpa).

Wood remains the world's most abundant and renewable resource for timber and pulp and is an alternative to fossil fuels. Understanding the molecular regulation of wood formation can advance the engineering of wood for more efficient material and energy productions. We integrated a black cottonwood (Populus trichocarpa) wood-forming cell system with quantitative transcriptomics and chromatin binding assays to construct a transcriptional regulatory network (TRN) directed by a key transcription factor (TF), PtrSND1-B1 (secondary wall-associated NAC-domain protein). The network consists of four layers of TF-target gene interactions with quantitative regulatory effects, describing the specificity of how the regulation is transduced through these interactions to activate cell wall genes (effector genes) for wood formation. PtrSND1-B1 directs 57 TF-DNA interactions through 17 TFs transregulating 27 effector genes. Of the 57 interactions, 55 are novel. We tested 42 of these 57 interactions in 30 genotypes of transgenic P. trichocarpa and verified that ∼90% of the tested interactions function in vivo. The TRN reveals common transregulatory targets for distinct TFs, leading to the discovery of nine TF protein complexes (dimers and trimers) implicated in regulating the biosynthesis of specific types of lignin. Our work suggests that wood formation may involve regulatory homeostasis determined by combinations of TF-DNA and TF-TF (protein-protein) regulations.

Isolation and characterization of the secondary wall-related SND1 gene in hawthorn

Secondary wall-associated NAC domain protein1 (SND1) is a key regulator directly regulating the expression levels of MYB46 and MYB83 in the regulation network for secondary wall synthesis, especially in plant fibres. In this study, a SND1 gene was isolated from hawthorn (Crataegus pinnatifida) and named as CpSND1 because it has a conservative N-terminal DNA-binding domain with AtSND1. Arabidopsis plants overexpressing CpSND1 had similar phenotypes as plants overexpressing AtSND1, including inhibited growth, upward-curling leaves, sepal dysplasia and sterility. In addition, overexpressing CpSND1 in Arabidopsis also induced the expression of downstream genes, including lignin, cellulose and xylan biosynthesis genes as well as MYB genes. Our results provided functional information of CpSND1 for future genetic engineering in hawthorn.

Co-expression network of secondary cell wall biogenesis genes in Eucalyptus tereticornis

Abstract The composition of secondary cell wall determines the indust­rially relevant wood properties in tree species. Hence, its bioge­nesis is one of the most extensively studied developmental processes during wood formation. Presently, systems genetics approach is being applied to understand the biological net­works and their interactions operational during secondary development. Genome-scale analyses of secondary cell wall formation were documented and gene regulatory networks were reported in Arabidopsis, poplar, pine, spruce, rice and sug­arcane. In the present study, the expression patterns of 2651 transcripts representing different pathways governing secon­dary development was documented across four genotypes of E. tereticornis. A co-expression network was constructed with 330 nodes and 4512 edges and the degree ranged from 11 to 53. The network documented 75 (22 %) transcription factors with high degree of interaction. Secondary wall associated NAC domain transcription factor (SND2) was identified as the top hub transcript with 53 interactions. The present study revealed that functional homologs regulating secondary cell wall formation are conserved among angiosperms and gym­nosperms.

Ectopic Expression of Hawthorn SND1 Gene in Tobacco

NAC proteins are plant-specific transcription factors (TFs) and have been shown to function in plant development processes and abiotic and/or biotic stress responses. SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN1 (SND1) is one type of NAC TFs, which is a key regulator in the regulation network for secondary wall synthesis. In this study, the SND1 gene, named CpSND1 because it has a conservative N-terminal DNA-binding domain with AtSND1, was isolated from hawthorn (Crataegus pinnatifida). The full-length CDS of this gene was 1,203 bp, encoding 400 amino acids. The CpSND1 gene was transferred into tobacco (Nicotiana tobacum) by the Agrobacterium-mediated transformation method, and 20 transgenic lines were obtained. Tobacco plants overexpressing CpSND1 had typical phenotypes, including inhibited growth, upward-curling leaves. Our results provided functional information of CpSND1 for future genetic engineering.

Reciprocal cross-regulation of VND and SND multigene TF families for wood formation in Populus trichocarpa

Secondary cell wall (SCW) biosynthesis is the biological process that generates wood, an important renewable feedstock for materials and energy. NAC domain transcription factors, particularly Vascular-Related NAC-Domain (VND) and Secondary Wall-Associated NAC Domain (SND) proteins, are known to regulate SCW differentiation. The regulation of VND and SND is important to maintain homeostasis for plants to avoid abnormal growth and development. We previously identified a splice variant, PtrSND1-A2IR , derived from PtrSND1-A2 as a dominant-negative regulator, which suppresses the transactivation of all PtrSND1 family members. PtrSND1-A2IR also suppresses the self-activation of the PtrSND1 family members except for its cognate transcription factor, PtrSND1-A2, suggesting the existence of an unknown factor needed to regulate PtrSND1-A2 Here, a splice variant, PtrVND6-C1IR , derived from PtrVND6-C1 was discovered that suppresses the protein functions of all PtrVND6 family members. PtrVND6-C1IR also suppresses the expression of all PtrSND1 members, including PtrSND1-A2, demonstrating that PtrVND6-C1IR is the previously unidentified regulator of PtrSND1-A2 We also found that PtrVND6-C1IR cannot suppress the expression of its cognate transcription factor, PtrVND6-C1PtrVND6-C1 is suppressed by PtrSND1-A2IR Both PtrVND6-C1IR and PtrSND1-A2IR cannot suppress their cognate transcription factors but can suppress all members of the other family. The results indicate that the splice variants from the PtrVND6 and PtrSND1 family may exert reciprocal cross-regulation for complete transcriptional regulation of these two families in wood formation. This reciprocal cross-regulation between families suggests a general mechanism among NAC domain proteins and likely other transcription factors, where intron-retained splice variants provide an additional level of regulation.

TBL3 and TBL31, Two Arabidopsis DUF231 Domain Proteins, are Required for 3-O-Monoacetylation of Xylan.

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.

Induced transcriptional profiling of phenylpropanoid pathway genes increased flavonoid and lignin content in Arabidopsisleaves in response to microbial products

BackgroundThe production and use of biologically derived soil additives is one of the fastest growing sectors of the fertilizer industry. These products have been shown to improve crop yields while at the same time reducing fertilizer inputs to and nutrient loss from cropland. The mechanisms driving the changes in primary productivity and soil processes are poorly understood and little is known about changes in secondary productivity associated with the use of microbial products. Here we investigate secondary metabolic responses to a biologically derived soil additive by monitoring changes in the phenlypropanoid (PP) pathway in Arabidopsis thaliana.ResultsThis study was designed to test the influence of one of these products (Soil Builder™-AF, SB) on secondary metabolism after being applied at different times. One time (TI) application of SB to Arabidopsis increased the accumulation of flavonoids compared to multiple (TII) applications of the same products. Fourteen phenolic compounds including flavonols and anothocyanins were identified by mass spectrometry. Kaempferol-3,7-O-bis-α-L-rhamnoside and quercetin 3,7-dirhamnoside, the major compounds, increased 3-fold and 4-fold, respectively compared to control in the TI treatment. The most abundant anthocyanin was cyanidin 3-rhamnoglucoside, which increased 3-fold and 2-fold in TI compared to the control and TII, respectively. Simultaneously, the expression of genes coding for key enzymes in the PP pathway (phenylalanine ammonia lyase, cinnamate 4-hydroxylase, chalcone synthase, flavonoid-3′-O-hydroxylase, flavonol synthase1 and dihydroflavonol-4-reductase) and regulatory genes (production of anthocyanin pigment2, MYB12, MYB113, MYB114, EGL3, and TT8) were up-regulated in both treatments (TI and TII). Furthermore, application of TI and TII induced expression of the lignin pathway genes (hydroxyl cinamyl transferase, caffeyl-CoA O-methyl transferase, cinnamyl alcohol dehydrogenase, cinnamyl-CoA reductase, secondary wall-associated NAC domain protein1, MYB58 and MYB63 resulting in higher accumulation of lignin content compared to the control.ConclusionsThese results indicate that the additions of microbially based soil additives have a perceptible influence on phenylpropanoid pathway gene regulation and its production of secondary metabolites. These findings open an avenue of research to investigate the mode of action of microbially-based soil additives which may assist in the sustainable production of food, feed, fuel and fiber.

SND1 Transcription Factor-Directed Quantitative Functional Hierarchical Genetic Regulatory Network in Wood Formation in Populus trichocarpa

Wood is an essential renewable raw material for industrial products and energy. However, knowledge of the genetic regulation of wood formation is limited. We developed a genome-wide high-throughput system for the discovery and validation of specific transcription factor (TF)-directed hierarchical gene regulatory networks (hGRNs) in wood formation. This system depends on a new robust procedure for isolation and transfection of Populus trichocarpa stem differentiating xylem protoplasts. We overexpressed Secondary Wall-Associated NAC Domain 1s (Ptr-SND1-B1), a TF gene affecting wood formation, in these protoplasts and identified differentially expressed genes by RNA sequencing. Direct Ptr-SND1-B1-DNA interactions were then inferred by integration of time-course RNA sequencing data and top-down Graphical Gaussian Modeling-based algorithms. These Ptr-SND1-B1-DNA interactions were verified to function in differentiating xylem by anti-PtrSND1-B1 antibody-based chromatin immunoprecipitation (97% accuracy) and in stable transgenic P. trichocarpa (90% accuracy). In this way, we established a Ptr-SND1-B1-directed quantitative hGRN involving 76 direct targets, including eight TF and 61 enzyme-coding genes previously unidentified as targets. The network can be extended to the third layer from the second-layer TFs by computation or by overexpression of a second-layer TF to identify a new group of direct targets (third layer). This approach would allow the sequential establishment, one two-layered hGRN at a time, of all layers involved in a more comprehensive hGRN. Our approach may be particularly useful to study hGRNs in complex processes in plant species resistant to stable genetic transformation and where mutants are unavailable.

Chimeric repressor of PtSND2 severely affects wood formation in transgenic Populus

NAC domain transcription factors are important regulators that activate the secondary wall biosynthesis in wood formation. In this work, we investigated the possible functions of an NAC family member SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN2 (PtSND2) using chimeric repressor silencing technology. Reverse transcription-polymerase chain reaction, subcellular localization and transcriptional activation analyses indicated that PtSND2 is a wood-associated transcriptional factor with the predicted transcriptional activation activity, which could be inhibited by the repression domain SUPERMAN REPRESSION DOMAIN X (SRDX) in yeast. Wood formation was severely repressed in transgenic poplar plants overexpressing PtSND2-SRDX. Meanwhile, the secondary cell wall thickness of xylem fibers was restrained, and the contents of cellulose and lignin were obviously decreased in the stems of transgenic plants. Further studies indicated that expressions of a number of wood-associated genes were down-regulated in the stems of transgenic plants. Our results suggest that PtSND2 may play important roles during the secondary growth of stems in poplar.

The Arabidopsis DUF231 Domain-Containing Protein ESK1 Mediates 2-O- and 3-O-Acetylation of Xylosyl Residues in Xylan

Xylan, a major polysaccharide in plant lignocellulosic biomass, is acetylated at O-2 and/or O-3 and its acetylation impedes the use of biomass for biofuel production. Currently, it is not known what genes encode acetyltransferases that are responsible for xylan O-acetylation. In this report, we demonstrate an essential role for the Arabidopsis gene ESKIMO1 (ESK1) in xylan O-acetylation during secondary wall biosynthesis. ESK1 expression was found to be regulated by the secondary wall master regulator SND1 (secondary wall-associated NAC domain protein1) and specifically associated with secondary wall biosynthesis. Its encoded protein was localized in the Golgi, the site of xylan biosynthesis. The esk1 mutation caused reductions in secondary wall thickening and stem mechanical strength. Chemical analyses of cell walls revealed that although the esk1 mutation did not cause apparent alterations in the xylan chain length and the abundance of the reducing end sequence, it resulted in a significant reduction in the degree of xylan acetylation. The reduced acetylation of esk1 xylan rendered it more accessible and digestible by endoxylanase, leading to generation of shorter xylooligomers compared with the wild type. Further structural analysis of xylan showed that the esk1 mutation caused a specific reduction in 2-O- and 3-O-monoacetylation of xylosyl residues but not in 2,3-di-O-acetylation or 3-O-acetylation of xylosyl residues substituted at O-2 with glucuronic acid. Consistent with ESK1's involvement in xylan O-acetylation, an activity assay revealed that the esk1 mutation led to a significant decrease in xylan acetyltransferase activity. Together, these results demonstrate that ESK1 is a putative xylan acetyltransferase required for 2-O- and 3-O-monoacetylation of xylosyl residues and indicate the complexity of the biochemical mechanism underlying xylan O-acetylation.

MYB103 is required for FERULATE‐5‐HYDROXYLASE expression and syringyl lignin biosynthesis in Arabidopsis stems

The transcription factor MYB103 was previously identified as a member of the transcriptional network regulating secondary wall biosynthesis in xylem tissues of Arabidopsis, and was proposed to act on cellulose biosynthesis. It is a direct transcriptional target of the transcription factor SECONDARY WALL ASSOCIATED NAC DOMAIN PROTEIN1 (SND1), and 35S-driven dominant repression or over-expression of MYB103 modifies secondary wall thickness. We identified two myb103 T-DNA insertion mutants and chemically characterized their lignocellulose by pyrolysis/GC/MS, 2D NMR, FT-IR microspectroscopy and wet chemistry. The mutants developed normally but exhibited a 70-75% decrease in syringyl (S) lignin. The level of guaiacyl (G) lignin was co-ordinately increased, so that total Klason lignin was not affected. The transcript abundance of FERULATE-5-HYDROXYLASE (F5H), the key gene in biosynthesis of Slignin, was strongly decreased in the myb103 mutants, and the metabolomes of the myb103 mutant and an F5H null mutant were very similar. Other than modification of the lignin S to G ratio, there were only very minor changes in the composition of secondary cell-wall polymers in the inflorescence stem. In conclusion, we demonstrate that F5H expression and hence biosynthesis of Slignin are dependent on MYB103.

Splice variant of the SND1 transcription factor is a dominant negative of SND1 members and their regulation in Populus trichocarpa

Secondary Wall-Associated NAC Domain 1s (SND1s) are transcription factors (TFs) known to activate a cascade of TF and pathway genes affecting secondary cell wall biosynthesis (xylogenesis) in Arabidopsis and poplars. Elevated SND1 transcriptional activation leads to ectopic xylogenesis and stunted growth. Nothing is known about the upstream regulators of SND1. Here we report the discovery of a stem-differentiating xylem (SDX)-specific alternative SND1 splice variant, PtrSND1-A2(IR), that acts as a dominant negative of SND1 transcriptional network genes in Populus trichocarpa. PtrSND1-A2(IR) derives from PtrSND1-A2, one of the four fully spliced PtrSND1 gene family members (PtrSND1-A1, -A2, -B1, and -B2). Each full-size PtrSND1 activates its own gene, and all four full-size members activate a common MYB gene (PtrMYB021). PtrSND1-A2(IR) represses the expression of its PtrSND1 member genes and PtrMYB021. Repression of the autoregulation of a TF family by its only splice variant has not been previously reported in plants. PtrSND1-A2(IR) lacks DNA binding and transactivation abilities but retains dimerization capability. PtrSND1-A2(IR) is localized exclusively in cytoplasmic foci. In the presence of any full-size PtrSND1 member, PtrSND1-A2(IR) is translocated into the nucleus exclusively as a heterodimeric partner with full-size PtrSND1s. Our findings are consistent with a model in which the translocated PtrSND1-A2(IR) lacking DNA-binding and transactivating abilities can disrupt the function of full-size PtrSND1s, making them nonproductive through heterodimerization, and thereby modulating the SND1 transcriptional network. PtrSND1-A2(IR) may contribute to transcriptional homeostasis to avoid deleterious effects on xylogenesis and plant growth.

Arabidopsis VASCULAR-RELATED NAC-DOMAIN6 Directly Regulates the Genes That Govern Programmed Cell Death and Secondary Wall Formation during Xylem Differentiation

Xylem consists of three types of cells: tracheary elements (TEs), parenchyma cells, and fiber cells. TE differentiation includes two essential processes, programmed cell death (PCD) and secondary cell wall formation. These two processes are tightly coupled. However, little is known about the molecular mechanisms underlying these processes. Here, we show that VASCULAR-RELATED NAC-DOMAIN6 (VND6), a master regulator of TEs, regulates some of the downstream genes involved in these processes in a coordinated manner. We first identified genes that are expressed downstream of VND6 but not downstream of SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN1 (SND1), a master regulator of xylem fiber cells, using transformed suspension culture cells in microarray experiments. We found that VND6 and SND1 governed distinct aspects of xylem formation, whereas they regulated a number of genes in common, specifically those related to secondary cell wall formation. Genes involved in TE-specific PCD were upregulated only by VND6. Moreover, we revealed that VND6 directly regulated genes that harbor a TE-specific cis-element, TERE, in their promoters. Thus, we found that VND6 is a direct regulator of genes related to PCD as well as to secondary wall formation.