Secondary Cell Wall Formation Research Articles

Gene regulatory networks for lignin biosynthesis in switchgrass (Panicum virgatum).

SummaryCell wall recalcitrance is the major challenge to improving saccharification efficiency in converting lignocellulose into biofuels. However, information regarding the transcriptional regulation of secondary cell wall biogenesis remains poor in switchgrass (Panicum virgatum), which has been selected as a biofuel crop in the United States. In this study, we present a combination of computational and experimental approaches to develop gene regulatory networks for lignin formation in switchgrass. To screen transcription factors (TFs) involved in lignin biosynthesis, we developed a modified method to perform co‐expression network analysis using 14 lignin biosynthesis genes as bait (target) genes. The switchgrass lignin co‐expression network was further extended by adding 14 TFs identified in this study, and seven TFs identified in previous studies, as bait genes. Six TFs (PvMYB58/63, PvMYB42/85, PvMYB4, PvWRKY12, PvSND2 and PvSWN2) were targeted to generate overexpressing and/or down‐regulated transgenic switchgrass lines. The alteration of lignin content, cell wall composition and/or plant growth in the transgenic plants supported the role of the TFs in controlling secondary wall formation. RNA‐seq analysis of four of the transgenic switchgrass lines revealed downstream target genes of the secondary wall‐related TFs and crosstalk with other biological pathways. In vitro transactivation assays further confirmed the regulation of specific lignin pathway genes by four of the TFs. Our meta‐analysis provides a hierarchical network of TFs and their potential target genes for future manipulation of secondary cell wall formation for lignin modification in switchgrass.

The plant hormone auxin directs timing of xylem development by inhibition of secondary cell wall deposition through repression of secondary wall NAC-domain transcription factors.

Wood formation in higher plants is a complex and costly developmental process regulated by a complex network of transcription factors, short peptide signals and hormones. Correct spatiotemporal initiation of differentiation and downstream developmental stages is vital for proper wood formation. Members of the NAC (NAM, ATAF1/2 and CUC) family of transcription factors are described as top level regulators of xylem cell fate and secondary cell wall (SCW) deposition, but the signals initiating their transcription have yet to be elucidated. We found that treatment of Populus stems with auxin repressed transcription of NAC transcription factors associated with fiber and SCW formation and induced vessel‐specific NACs, whereas gibberellic acid (GA) induced the expression of both classes of NAC domain transcription factors involved in wood formation. These transcriptional changes were reflected in alterations of stem anatomy, i.e. auxin treatment reduced cell wall thickness, whereas GA had a promotive effect on SCW deposition and on the rate of wood formation. Similar changes were observed on treatment of Arabidopsis thaliana stems with GA or the synthetic auxin NAA. We also observed corresponding changes in PIN5 overexpressing lines, where interference with auxin transport leads to premature SCW deposition and formation of additional fiber bundles. Together, this suggests wood formation is regulated by an integrated readout of both auxin and GA, which, in turn, controls expression of fiber and vessel specific NACs.

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.

Transcriptional alterations during proliferation and lignification in Phyllostachys nigra cells

Highly-lignified culms of bamboo show distinctive anatomical and mechanical properties compared with the culms of other grass species. A cell culture system for Phyllostachys nigra has enabled investigating the alterations in cellular states associated with secondary cell wall formation during its proliferation and lignification in woody bamboos. To reveal transcriptional changes related to lignification in bamboo, we analyzed transcriptome in P. nigra cells treated with the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) and the synthetic cytokinin benzylaminopurine (BA) by RNA-seq analysis. We found that some genes putatively involved in cell wall biogenesis and cell division were up-regulated in response to the 2,4-D treatment, and the induction of lignification by the BA treatment was correlated with up-regulation of genes involved in the shikimate pathway. We also found that genes encoding MYB transcription factors (TFs) show correlated expression patterns with those encoding cinnamyl alcohol dehydrogenase (CAD), suggesting that MYB TFs presumably regulate secondary cell wall formation in the bamboo cells. These findings suggest that cytokinin signaling may regulate lignification in P. nigra cells through coordinated transcriptional regulation and metabolic alterations. Our results have also produced a useful resource for better understanding of secondary cell wall formation in bamboo plants.

High-Throughput Analysis of Arabidopsis Stem Vibrations to Identify Mutants With Altered Mechanical Properties.

Mechanical properties are rarely used as quantitative indices for the large-scale mutant screening of plants, even in the model plant Arabidopsis thaliana. The mechanical properties of plant stems generally influence their vibrational characteristics. Here, we developed Python-based software, named AraVib, for the high-throughput analysis of free vibrations of plant stems, focusing specifically on Arabidopsis stem vibrations, and its extended version, named AraVibS, to identify mutants with altered mechanical properties. These programs can be used without knowledge of Python and require only an inexpensive handmade setting stand and an iPhone/iPad with a high-speed shooting function for data acquisition. Using our system, we identified an nst1 nst3 double-mutant lacking secondary cell walls in fiber cells and a wrky12 mutant displaying ectopic formation of secondary cell wall compared with wild type by employing only two growth traits (stem height and fresh weight) in addition to videos of stem vibrations. Furthermore, we calculated the logarithmic decrement, the damping ratio, the natural frequency and the stiffness based on the spring-mass-damper model from the video data using AraVib. The stiffness was estimated to be drastically decreased in nst1 nst3, which agreed with previous tensile test results. However, in wrky12, the stiffness was significantly increased. These results demonstrate the effectiveness of our new system. Because our method can be applied in a high-throughput manner, it can be used to screen for mutants with altered mechanical properties.

Cover Image: Role of KNAT7 in secondary cell wall formation

KNAT7 transcription factor plays an important role in secondary cell wall biosynthesis. However, the exact function of KNAT7 is still unclear. In this issue, He et al. (514–528) demonstrate that KNAT7 positively regulates xylan biosynthesis, which provides a new clue to the regulation network of secondary cell wall formation.

Expression Analysis of Cell Wall-Related Genes in Cannabis sativa: The “Ins and Outs” of Hemp Stem Tissue Development

Textile hemp (Cannabis sativa L.) is a multipurpose crop producing biomass with uses in e.g., the textile, biocomposite, and construction sectors. It was previously shown that the hypocotyl of hemp is useful to study the kinetics of secondary tissue development, where primary and secondary growths are temporally uncoupled. We here sought to demonstrate that the stem of adult hemp plants is an additional suitable model to study the heterogeneous lignification of the tissues and the mechanisms underlying secondary cell wall formation in bast fibres. A targeted quantitative PCR analysis carried out on a set of twenty genes involved in cell wall biosynthesis clearly showed differences in expression in the core and cortical tissues along four stem regions spanning from elongation to cell wall thickening. Genes involved in phenylpropanoid biosynthesis and secondary cell wall cellulose synthases were expressed at higher levels in core tissues at the bottom, while specific genes, notably a class III peroxidase and a gene partaking in lignan biosynthesis, were highly expressed in the cortex of elongating internodes. The two systems, the hypocotyl and the adult stem of textile hemp, are equally valid and complementary to address questions related to lignification and secondary cell wall deposition.

Molecular Characterization, Evolution, and Expression Profiling of the Dirigent (DIR) Family Genes in Chinese White Pear (Pyrus bretschneideri).

Stone cells content and size are the key factors determining the internal quality of the pear fruit. Synthesis of lignin and thickening of secondary cell wall are the keys to the development of stone cells. The polymerization of monolignols and secondary cell wall formation requires the participation of dirigent proteins (DIRs). In recent years, DIR family have been studied in higher plants, but lack of comprehensive study in the pear DIR (PbDIR) family. This study focuses on the identification and analysis of PbDIR family for the first time. We identified 35 PbDIRs from the pear genome, 89% of which are intronless genes. Phylogenetic tree and chromosome localization analysis showed that 35 PbDIRs were divided into four subfamilies (DIR-a, -b/d, -e, and -g) and irregularly distributed among 10 chromosomes. In addition, we identified 29, 26, and 14 DIRs from the other three Rosids (peach, Mei, and grape), respectively. Interspecies microsynteny analysis revealed the collinear gene pairs between pear and peach are the most. Temporal expression analysis showed that the expression changes of seven PbDIRs (DIR-a subfamily: PbDIR4 and PbDIR5; DIR-b/d subfamily: PbDIR11; DIR-g subfamily: PbDIR19; DIR-e subfamily: PbDIR23, 25 and 26) in fruits were consistent with the changes of fruit lignin and stone cells contents. In addition, the subfamily of PbDIRs in fruits showed significant responses after treatment with ABA, SA, and MeJA. According to the protein tertiary structure, key amino acid residues and expression patterns analysis found that PbDIR4 might be involved in the metabolism of lignin and related to stone cells contents in pear fruits. In this study, we systematically analyzed the structure, evolution, function and expression of PbDIR family, which not only confirmed the characteristics of PbDIR family, but also laid the foundation for revealing the role of DIR in pear stone cell development and lignin polymerization.

Ethylene-Related Gene Expression Networks in Wood Formation.

Thickening of tree stems is the result of secondary growth, accomplished by the meristematic activity of the vascular cambium. Secondary growth of the stem entails developmental cascades resulting in the formation of secondary phloem outwards and secondary xylem (i.e., wood) inwards of the stem. Signaling and transcriptional reprogramming by the phytohormone ethylene modifies cambial growth and cell differentiation, but the molecular link between ethylene and secondary growth remains unknown. We addressed this shortcoming by analyzing expression profiles and co-expression networks of ethylene pathway genes using the AspWood transcriptome database which covers all stages of secondary growth in aspen (Populus tremula) stems. ACC synthase expression suggests that the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) is synthesized during xylem expansion and xylem cell maturation. Ethylene-mediated transcriptional reprogramming occurs during all stages of secondary growth, as deduced from AspWood expression profiles of ethylene-responsive genes. A network centrality analysis of the AspWood dataset identified EIN3D and 11 ERFs as hubs. No overlap was found between the co-expressed genes of the EIN3 and ERF hubs, suggesting target diversification and hence independent roles for these transcription factor families during normal wood formation. The EIN3D hub was part of a large co-expression gene module, which contained 16 transcription factors, among them several new candidates that have not been earlier connected to wood formation and a VND-INTERACTING 2 (VNI2) homolog. We experimentally demonstrated Populus EIN3D function in ethylene signaling in Arabidopsis thaliana. The ERF hubs ERF118 and ERF119 were connected on the basis of their expression pattern and gene co-expression module composition to xylem cell expansion and secondary cell wall formation, respectively. We hereby establish data resources for ethylene-responsive genes and potential targets for EIN3D and ERF transcription factors in Populus stem tissues, which can help to understand the range of ethylene targeted biological processes during secondary growth.

Ethylene signaling induces gelatinous layers with typical features of tension wood in hybrid aspen.

The phytohormone ethylene impacts secondary stem growth in plants by stimulating cambial activity, xylem development and fiber over vessel formation. We report the effect of ethylene on secondary cell wall formation and the molecular connection between ethylene signaling and wood formation. We applied exogenous ethylene or its precursor 1-aminocyclopropane-1-carboxylic acid (ACC) to wild-type and ethylene-insensitive hybrid aspen trees (Populus tremula×tremuloides) and studied secondary cell wall anatomy, chemistry and ultrastructure. We furthermore analyzed the transcriptome (RNA Seq) after ACC application to wild-type and ethylene-insensitive trees. We demonstrate that ACC and ethylene induce gelatinous layers (G-layers) and alter the fiber cell wall cellulose microfibril angle. G-layers are tertiary wall layers rich in cellulose, typically found in tension wood of aspen trees. A vast majority of transcripts affected by ACC are downstream of ethylene perception and include a large number of transcription factors (TFs). Motif-analyses reveal potential connections between ethylene TFs (Ethylene Response Factors (ERFs), ETHYLENE INSENSITIVE 3/ETHYLENE INSENSITIVE3-LIKE1 (EIN3/EIL1)) and wood formation. G-layer formation upon ethylene application suggests that the increase in ethylene biosynthesis observed during tension wood formation is important for its formation. Ethylene-regulated TFs of the ERF and EIN3/EIL1 type could transmit the ethylene signal.

Transcriptome analysis provides insights into xylogenesis formation in Moso bamboo (Phyllostachys edulis) shoot

Maturation-related changes in cell wall composition and the molecular mechanisms underlying cell wall changes were investigated from the apical, middle and basal segments in moso bamboo shoot (MBS). With maturation extent from apical to basal regions in MBS, lignin and cellulose content increased, whereas heteroxylan exhibited a decreasing trend. Activities of phenylalanine amonnialyase (PAL), cinnamyl alcohol dehydrogenase (CAD) and cinnamate-4-hydroxylase (C4H), which are involved in lignin biosynthesis, increased rapidly from the apex to the base sections. The comparative transcriptomic analysis was carried out to identify some key genes involved in secondary cell walls (SCW) formation underlying the cell wall compositions changes including 63, 8, 18, and 31 functional unigenes encoding biosynthesis of lignin, cellulose, xylan and NAC-MYB-based transcription factors, respectively. Genes related to secondary cell wall formation and lignin biosynthesis had higher expression levels in the middle and basal segments compared to those in the apical segments. Furthermore, the expression profile of PePAL gene showed positive relationships with cellulose-related gene PeCESA4, xylan-related genes PeIRX9 and PeIRX10. Our results indicated that lignification occurred in the more mature middle and basal segments in MBS at harvest while lignification of MBS were correlated with higher expression levels of PeCESA4, PeIRX9 and PeIRX10 genes.

Transcriptional Roadmap to Seasonal Variation in Wood Formation of Norway Spruce.

Seasonal cues influence several aspects of the secondary growth of tree stems, including cambial activity, wood chemistry, and transition to latewood formation. We investigated seasonal changes in cambial activity, secondary cell wall formation, and tracheid cell death in woody tissues of Norway spruce (Picea abies) throughout one seasonal cycle. RNA sequencing was performed simultaneously in both the xylem and cambium/phloem tissues of the stem. Principal component analysis revealed gradual shifts in the transcriptomes that followed a chronological order throughout the season. A notable remodeling of the transcriptome was observed in the winter, with many genes having maximal expression during the coldest months of the year. A highly coexpressed set of monolignol biosynthesis genes showed high expression during the period of secondary cell wall formation as well as a second peak in midwinter. This midwinter peak in expression did not trigger lignin deposition, as determined by pyrolysis-gas chromatography/mass spectrometry. Coexpression consensus network analyses suggested the involvement of transcription factors belonging to the ASYMMETRIC LEAVES2/LATERAL ORGAN BOUNDARIES and MYELOBLASTOSIS-HOMEOBOX families in the seasonal control of secondary cell wall formation of tracheids. Interestingly, the lifetime of the latewood tracheids stretched beyond the winter dormancy period, correlating with a lack of cell death-related gene expression. Our transcriptomic analyses combined with phylogenetic and microscopic analyses also identified the cellulose and lignin biosynthetic genes and putative regulators for latewood formation and tracheid cell death in Norway spruce, providing a toolbox for further physiological and functional assays of these important phase transitions.

Change in lignin structure, but not in lignin content, in transgenic poplar overexpressing the rice master regulator of secondary cell wall biosynthesis

We previously succeeded in enhancing wood formation of wood in transgenic poplar plants by overexpressing secondary wall NAM/ATAF/CUC (NAC) domain protein 1 from Oryza sativa (OsSWN1), a transcription factor 'master regulator' of secondary cell wall formation in rice, under control of the fiber preferential NST3/SND1 promoter from Arabidopsis. Transgenic plants had an increased cell wall thickness and cell wall density of individual cells in the secondary xylem of stems as well as an increased wood density. OsSWN1 triggers the induction of polysaccharide and lignin biosynthetic gene expressions, however, resulting in no significant impact on the lignin content in the transgenic plants. In contrast, wet and dry chemical analyses of lignin revealed changes in S/G ratio and in the composition of lignin interunit linkages in transgenic lines. The results from gene expression analysis suggest that the structural changes in lignin were due to an unbalanced induction of lignin biosynthetic genes in transgenic lines. Our present data indicate that the overexpression of the chimeric transcription factor causes accelerated deposition of secondary cell wall components including lignin and polysaccharides through an acquired mechanism.

An auxin-induced β-type endo-1,4-β-glucanase in poplar is involved in cell expansion and lateral root formation.

PtrGH9A7, a poplar β-type endo-1,4-β-glucanase gene induced by auxin, promotes both plant growth and lateral root development by enhancing cell expansion. Endo-1,4-β-glucanase (EGase) family genes function in multiple aspects of plant growth and development. Our previous study found that PtrCel9A6, a poplar EGase gene of the β subfamily, is specifically expressed in xylem tissue and is involved in the cellulose biosynthesis required for secondary cell wall formation (Yu et al. in Mol Plant 6:1904-1917, 2013). To further explore the functions and regulatory mechanism of β-subfamily EGases, we cloned and characterized another poplar β-type EGase gene PtrGH9A7, a close homolog of PtrCel9A6. In contrast to PtrCel9A6, PtrGH9A7 is predominantly expressed in parenchyma tissues of the above-ground part; in roots, PtrGH9A7 expression is specifically restricted to lateral root primordia at all stages from initiation to emergence and is strongly induced by auxin application. Heterologous overexpression of PtrGH9A7 promotes plant growth by enhancing cell expansion, suggesting a conserved role for β-type EGases in 1,4-β-glucan chains remodeling, which is required for cell wall loosening. Moreover, the overexpression of PtrGH9A7 significantly increases lateral root number, which might result from improved lateral root primordium development due to enhanced cell expansion. Taken together, these results demonstrate that this β-type EGase induced by auxin signaling has a novel role in promoting lateral root formation as well as in enhancing plant growth.

The Effect of Poplar PsnGS1.2 Overexpression on Growth, Secondary Cell Wall, and Fiber Characteristics in Tobacco.

The glutamine synthetase (GS1) is a key enzyme that catalyzes the reaction of glutamate and ammonia to produce glutamine in the nitrogen (N) metabolism. Previous studies on GS1s in several plant species suggest that overexpression of GS1s can enhance N utilization, accelerate plant vegetative growth, and change wood formation. In this study, we isolated a GS1 gene, termed PsnGS1.2, from Populus simonii × Populus nigra. This gene was expressed at a higher level in roots, and relatively lower but detectable levels in xylem, leaves and phloem of P. simonii × P. nigra. The protein encoded by PsnGS1.2 is primarily located in the cytoplasm. Overexpression of PsnGS1.2 in tobacco led to the increased GS1 activity and IAA content, the augmented N assimilation, and the enlarged leaves with altered anatomical structures. These changes presumably promoted photosynthetic, growth, and biomass productivity. It was noteworthy that the secondary cell walls and fiber characteristics changed remarkably in PsnGS1.2 transgenic tobacco. These changes aligned well with the altered expression levels of the genes involved in fiber development, secondary cell wall component biosynthesis, IAA biosynthesis, amino acid transport, and starch breakdown. Taken together, the results from our study suggest that catalytic functions of PsnGS1.2 on N assimilation and metabolism in transgenic tobacco had significant effects on vegetative growth, leaf development, and secondary cell wall formation and properties through acceleration of photosynthesis and IAA biosynthesis, and redirection of carbon flux to synthesis of more cellulose and hemicellulose.

PpNAC1, a main regulator of phenylalanine biosynthesis and utilization in maritime pine

SummaryThe transcriptional regulation of phenylalanine metabolism is particularly important in conifers, long‐lived species that use large amounts of carbon in wood. Here, we show that the Pinus pinaster transcription factor, PpNAC1, is a main regulator of phenylalanine biosynthesis and utilization. A phylogenetic analysis classified PpNAC1 in the NST proteins group and was selected for functional characterization. PpNAC1 is predominantly expressed in the secondary xylem and compression wood of adult trees. Silencing of PpNAC1 in P. pinaster results in the alteration of stem vascular radial patterning and the down‐regulation of several genes associated with cell wall biogenesis and secondary metabolism. Furthermore, transactivation and EMSA analyses showed that PpNAC1 is able to activate its own expression and PpMyb4 promoter, while PpMyb4 is able to activate PpMyb8, a transcriptional regulator of phenylalanine and lignin biosynthesis in maritime pine. Together, these results suggest that PpNAC1 is a functional ortholog of the Arabidopsis SND1 and NST1 genes and support the idea that key regulators governing secondary cell wall formation could be conserved between gymnosperms and angiosperms. Understanding the molecular switches controlling wood formation is of paramount importance for fundamental tree biology and paves the way for applications in conifer biotechnology.

CORTICAL MICROTUBULE DISORDERING1 Is Required for Secondary Cell Wall Patterning in Xylem Vessels.

Proper patterning of the cell wall is essential for plant cell development. Cortical microtubule arrays direct the deposition patterns of cell walls at the plasma membrane. However, the precise mechanism underlying cortical microtubule organization is not well understood. Here, we show that a microtubule-associated protein, CORD1 (CORTICAL MICROTUBULE DISORDERING1), is required for the pitted secondary cell wall pattern of metaxylem vessels in Arabidopsis thaliana Loss of CORD1 and its paralog, CORD2, led to the formation of irregular secondary cell walls with small pits in metaxylem vessels, while overexpressing CORD1 led to the formation of abnormally enlarged secondary cell wall pits. Ectopic expression of CORD1 disturbed the parallel cortical microtubule array by promoting the detachment of microtubules from the plasma membrane. A reconstructive approach revealed that CORD1-induced disorganization of cortical microtubules impairs the boundaries of plasma membrane domains of active ROP11 GTPase, which govern pit formation. Our data suggest that CORD1 promotes cortical microtubule disorganization to regulate secondary cell wall pit formation. The Arabidopsis genome has six CORD1 paralogs that are expressed in various tissues during plant development, suggesting they are important for regulating cortical microtubules during plant development.

Transcription Factors VND1-VND3 Contribute to Cotyledon Xylem Vessel Formation.

Arabidopsis (Arabidopsis thaliana) VASCULAR-RELATED NAC-DOMAIN1 (VND1) to VND7 encode a group of NAC domain transcription factors that function as master regulators of xylem vessel element differentiation. These transcription factors activate the transcription of genes required for secondary cell wall formation and programmed cell death, key events in xylem vessel element differentiation. Because constitutive overexpression of VND6 and VND7 induces ectopic xylem vessel element differentiation, functional studies of VND proteins have largely focused on these two proteins. Here, we report the roles of VND1, VND2, and VND3 in xylem vessel formation in cotyledons. Using our newly established in vitro system in which excised Arabidopsis cotyledons are stimulated to undergo xylem cell differentiation by cytokinin, auxin, and brassinosteroid treatment, we found that ectopic xylem vessel element differentiation required VND1, VND2, and VND3 but not VND6 or VND7. The importance of VND1, VND2, and VND3 also was indicated in vivo; in the vnd1 vnd2 vnd3 seedlings, xylem vessel element differentiation of secondary veins in cotyledons was inhibited under dark conditions. Furthermore, the light responsiveness of VND gene expression was disturbed in the vnd1 vnd2 vnd3 mutant, and vnd1 vnd2 vnd3 failed to recover lateral root development in response to the change of light conditions. These findings suggest that VND1 to VND3 have specific molecular functions, possibly linking light conditions to xylem vessel formation, during seedling development.

The cotton (Gossypium hirsutum) NAC transcription factor (FSN1) as a positive regulator participates in controlling secondary cell wall biosynthesis and modification of fibers.

Cotton (Gossypium hirsutum) fibers are the highly elongated and thickened single-cell trichomes on the seed epidermis. However, little is known about the molecular base of fiber cell wall thickening in detail. In this study, a cotton NAC transcription factor (GhFSN1) that is specifically expressed in secondary cell wall (SCW) thickening fibers was functionally characterized. The GhFSN1 transgenic cotton plants were generated to study how FSN1 regulates fiber SCW formation. Up-regulation of GhFSN1 expression in cotton resulted in an increase in SCW thickness of fibers but a decrease in fiber length. Transcriptomic analysis revealed that GhFSN1 activates or represses numerous downstream genes. GhFSN1 has the ability to form homodimers, binds to its promoter to activate itself, and might be degraded by the ubiquitin-mediated proteasome pathway. The direct targets of GhFSN1 include the fiber SCW-related GhDUF231L1, GhKNL1, GhMYBL1, GhGUT1 and GhIRX12 genes. GhFSN1 binds directly to a consensus sequence (GhNBS), (C/T)(C/G/T)TN(A/T)(G/T)(A/C/G)(A/G)(A/T/G)(A/T/G)AAG, which exists in the promoters of these SCW-related genes. Our data demonstrate that GhFSN1 acts as a positive regulator in controlling SCW formation of cotton fibers by activating its downstream SCW-related genes. Thus, these findings give us novel insights into comprehensive understanding of GhFSN1 function in fiber development.

Regulation of secondary cell wall biosynthesis by a NAC transcription factor from Miscanthus.

Cell wall recalcitrance is a major limitation for the sustainable exploitation of lignocellulosic biomass as a renewable resource. Species and hybrids of the genus Miscanthus have emerged as candidate crops for the production of lignocellulosic feedstock in temperate climates, and dedicated efforts are underway to improve biomass yield. However, nothing is known about the molecular players involved in Miscanthus cell wall biosynthesis to facilitate breeding efforts towards tailored biomass. Here, we identify a Miscanthus sinensis transcription factor related to SECONDARY WALL‐ASSOCIATED NAC DOMAIN1 (SND1), which acts as a master switch for the regulation of secondary cell wall formation and lignin biosynthesis. Ms SND1 is expressed in growth stages associated with secondary cell wall formation, together with its potential targets. Consistent with this observation, Ms SND1 was able to complement the secondary cell wall defects of the Arabidopsis snd1 nst1 double mutant, and ectopic expression of Ms SND1 in tobacco leaves was sufficient to trigger patterned deposition of cellulose, hemicellulose, and lignin reminiscent of xylem elements. Transgenic studies in Arabidopsis thaliana plants revealed that MsSND1 regulates, directly and indirectly, the expression of a broad range of genes involved in secondary cell wall formation, including MYB transcription factors which regulate only a subset of the SCW differentiation program. Together, our findings suggest that MsSND1 is a transcriptional master regulator orchestrating secondary cell wall biosynthesis in Miscanthus.