NAC SECONDARY WALL THICKENING PROMOTING Research Articles

NST3 induces ectopic transdifferentiation, forming secondary walls with diverse patterns and composition in Arabidopsis thaliana.

The master transcription factor NAC SECONDARY WALL THICKENING PROMOTING FACTOR3 (NST3), also known as SND1, plays a pivotal role in regulating secondary cell wall (SCW) development in interfascicular and xylary fibers in Arabidopsis thaliana. Despite progress in understanding SCW assembly in xylem vessel-like cells, the mechanisms behind its assembly across different cell types remain unclear. Overexpressing NST3 or its homolog NST1 leads to reduced fertility, posing challenges for studying their impact on secondary wall formation. This study aimed at developing a tightly regulated dexamethasone (DEX)-inducible expression system for NST3 and NST1 to elucidate the structure and assembly of diverse SCWs. Using the DEX-inducible system, we characterized ectopically formed SCWs for their diverse patterns, mesoscale organization, cellulose microfibril orientation, and molecular composition using spinning disk confocal microscopy, field emission scanning electron microscopy (FESEM), vibrational sum-frequency generation (SFG) spectroscopy and, histochemical staining and time-of-flight secondary ion mass spectrometry (ToF-SIMS), respectively. Upon DEX treatment, NST3 and NST1 transgenic hypocotyls underwent time-dependent transdifferentiation, progressing from protoxylem-like to metaxylem-like cells. NST3-induced plants exhibited normal growth but had rough secondary wall surfaces with delaminating S2 and S3 layers. Mesoscale examination of induced SCWs in epidermal cells revealed that macrofibril thickness and orientation were comparable to xylem vessels, while wall thickness resembled that of interfascicular fibers. Additionally, induced epidermal cells formed SCWs with altered cellulose and lignin contents. These findings suggest NST3 and/or NST1 induce SCWs with shared characteristics of both xylem and fiber-like cells forming loosely arranged cell wall layers and cellulose organized at multiple angles relative to the cell growth axis and with varied cellulose and lignin abundance. This inducible system opens avenues to explore ectopic SCWs for bioenergy and bioproducts, offering valuable insights into SCW patterning across diverse cell types and developmental stages.

HbNST1 is a positive regulator of the lignin accumulation in strawflower bracts

Strawflower (Helichrysum bracteatum) capitula have papery bracts and thus have the qualities of a naturally dried flower. The involucral bract cells have a secondary cell wall (SCW) of which a crucial component is lignin. Although the constituents of SCWs have been studied extensively in plants, little is known of the mechanism regulating SCW formation, especially lignin biosynthesis in the involucral bracts of strawflower. In this study, a homolog of NAC SECONDARY WALL THICKENING PROMOTING FACTOR 1, designated HbNST1, was identified as a positive regulator of lignin biosynthesis in strawflower. The transcript level of HbNST1 was the highest in the involucral bracts. Subcellular localization analysis indicated that HbNST1 was localized to the nucleus. Overexpression of HbNST1 in Chrysanthemum indicum promoted the expression of a gene related to lignin biosynthesis, a homolog of cinnamyl alcohol dehydrogenase, designated CiCAD, suggesting that HbNST1 was associated with the accumulation of lignin in the SCW of the involucral bracts. Taken together, the results suggested that HbNST1 positively regulated lignin accumulation in the involucral bracts and mediated the expression of lignin biosynthesis-related genes in strawflower.

High-transcriptional activation ability of bamboo SECONDARY WALL NAC transcription factors is derived from C-terminal domain.

The secondary cell wall, which is mainly composed of cellulose, hemicellulose, and lignin, constitutes woody tissues and gives physical strength and hydrophobic properties for resistance against environmental stresses. We cloned and functionally analyzed the homologous transcription factor (TF) genes of SECONDARY WALL NAC (SWN) proteins from Hachiku bamboo (Phyllostachys nigra; PnSWNs). An RT-PCR analysis showed that PnSWNs are expressed in young tissues in bamboo. Their transcriptional activation activities were higher than that of the Arabidopsis NAC SECONDARY WALL THICKENING PROMOTING FACTOR 3 (NST3) TF, which was equivalent to SWN TFs in monocot. PnSWNs preferred to activate the genes related to secondary cell wall formation but not the genes related to programmed cell death. When PnSWNs were expressed in Arabidopsis, they highly induced secondary cell wall formation, like previously-shown rice SWN1. Dissection analysis revealed that this high activity largely depends on C-terminal domain. These results demonstrate that the cloned bamboo SWNs function as regulators of secondary cell wall formation with strong activation ability derived from C-terminal domain, and could be served as new genetic tools for secondary cell wall manipulation.

A natural mutation of the NST1 gene arrests secondary cell wall biosynthesis in the seed coat of a hull-less pumpkin accession.

Hull-less pumpkins (Cucurbita pepo L.) are naturally occurring novel variants known as oilseed or naked-seeded pumpkins, and are characterized by the absence of a normal lignified seed coat. Due to a specialized seed coat structure, these variants serve as a good model for studying seed coat formation and simplify the processing of pumpkin seeds. However, causal genes for this hull-less trait still remain unknown. Here, by bulked segregant analysis and fine mapping, we found that mutation of a single gene, NAC SECONDARY WALL THICKENING PROMOTING FACTOR 1 (NST1), accounts for the hull-less trait. A 14-bp sequence insertion in the CpNST1 gene causes premature termination of CpNST1 translation, leading to lack of secondary cell wall (SCW) biosynthesis in hull-less seed coats. In situ hybridization analysis provided further evidence for the role of CpNST1 in pumpkin seed coat SCW biosynthesis. Interestingly, through secondary cell wall compositional analysis, we found that the main SCW components differed among cell layers in the seed coat. RNA-seq analysis indicated an upstream role of CpNST1 in the SCW biosynthesis network. Collectively, our findings provide mechanistic insight into seed coat SCW biosynthesis, and a target gene for breeders to introduce this hull-less trait for commercial exploitation.

Abscisic acid regulates secondary cell-wall formation and lignin deposition in Arabidopsis thaliana through phosphorylation of NST1

Plant secondary cell-wall (SCW) deposition and lignification are affected by both seasonal factors and abiotic stress, and these responses may involve the hormone abscisic acid (ABA). However, the mechanisms involved are not clear. Here we show that mutations that limit ABA synthesis or signaling reduce the extent of SCW thickness and lignification in Arabidopsis thaliana through the core ABA-signaling pathway involving SnRK2 kinases. SnRK2.2. 3 and 6 physically interact with the SCW regulator NAC SECONDARY WALL THICKENING PROMOTING FACTOR 1 (NST1), a NAC family transcription factor that orchestrates the transcriptional activation of a suite of downstream SCW biosynthesis genes, some of which are involved in the biosynthesis of cellulose and lignin. This interaction leads to phosphorylation of NST1 at Ser316, a residue that is highly conserved among NST1 proteins from dicots, but not monocots, and is required for transcriptional activation of downstream SCW-related gene promoters. Loss of function of NST1 in the snd1 mutant background results in lack of SCWs in the interfascicular fiber region of the stem, and the Ser316Ala mutant of NST1 fails to complement this phenotype and ABA-induced lignin pathway gene expression. The discovery of NST1 as a key substrate for phosphorylation by SnRK2 suggests that the ABA-mediated core-signaling cascade provided land plants with a hormone-modulated, competitive desiccation-tolerance strategy allowing them to differentiate water-conducting and supporting tissues built of cells with thicker cell walls.

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.

A WRKY transcription factor WRKY184 from Brassica napus L. is involved in flowering and secondary wall development in transgenic Arabidopsis thaliana

Flowering is one of the important developmental processes, and the timing of flowering can affect seed yield. Lignin is one of the main components of plant cell walls. Lack of lignin in crops will reduce its resistance to stress, which in turn threatens the production of crops. The WRKY transcription factor family is a large family in higher plants and is widely involved in various biological processes. In this study, we isolated the WRKY transcription factor WRKY184 from Brassica napus L. that can simultaneously regulate the development of stems and flowers. BnWRKY184 is a member of the IIc subfamily, localized in the nucleus and has no transcriptional activation capability. The tissue expression analysis showed that BnWRKY184 had the highest expression level in stems. Overexpression of BnWRKY184 in Arabidopsis thaliana led to early flowering and weaker stem phenotypes. The expression levels of the flowering-related genes FLOWERING LOCUS T, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1, LEAFY and FRUITFULL were up-regulated in transgenic plants. The gene expression levels of secondary wall synthesis-related genes, such as NAC SECONDARY WALL THICKENING PROMOTING FACTOR 2, CELLULOSE SYNTHASE 7 and CELLLOSE SYNTHASE 8, were down-regulated in transgenic plants. Yeast one-hybrid and promoter-LUC showed that BnWRKY184 can directly bind to the promoter region of AtNST2 and inhibit its expression. Thus, BnWRKY184 simultaneously affected plant flowering and secondary wall development by regulating the transcriptional levels of genes involved in both processes, and BnWRKY184 can directly regulate the expression of AtFUL and AtNST2, suggesting that BnWRKY184 play roles in certain signaling pathways during plant development.

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.

Modulation of NAC transcription factor NST1 activity by XYLEM NAC DOMAIN1 regulates secondary cell wall formation in Arabidopsis.

In Arabidopsis, secondary cell walls (SCW) are formed in fiber cells and vessel cells in vascular tissue for providing plants with mechanical strength and channels for the long distance transportation of water and nutrients. NAC SECONDARY WALL THICKENING PROMOTING FACTOR1 (NST1) acts as a key gene for the initiation of SCW formation through a hierarchical transcription network. In this study, we report that NST activity is modulated by the NAC domain transcription factor XYLEM NAC DOMAIN1 (XND1) during plant growth. Using yeast two-hybrid screening and in vivo protein interaction analysis, XND1 was identified as an NST-interacting protein that modulates NST1 activity. XND1 and NST1 were co-localized in the nucleus and the interaction of XND1 with NST1 resulted in inhibition of NST1 transactivation activity. In the process of inflorescence growth, XND1 was expressed with a similar pattern to NST1. Up-regulation of XND1 in fiber cells repressed SCW formation. The study demonstrates that NST1 activity is modulated by XND1 in the regulation of secondary cell walls formation.

Knockdown of Arabidopsis ROOT UVB SENSITIVE4 Disrupts Anther Dehiscence by Suppressing Secondary Thickening in the Endothecium

ROOT UV-B SENSITIVE4 (RUS4) encodes a protein with no known function that contains a conserved Domain of Unknown Function 647 (DUF647). The DUF647-containing proteins RUS1 and RUS2 have previously been associated with root UV-B-sensing pathway that plays a major role in Arabidopsis early seedling morphogenesis and development. Here, we show that RUS4 knockdown Arabidopsis plants, referred to as amiR-RUS4, were severely reduced in male fertility with indehiscent anthers. Light microscopy of anther sections revealed a significantly reduced secondary wall thickening in the endothecium of amiR-RUS4 anthers. We further show that the transcript abundance of the NAC domain genes NAC SECONDARY WALL THICKENING PROMOTING FACTOR1 (NST1) and NST2, which have been shown to regulate the secondary cell wall thickenings in the anther endothecium, were dramatically reduced in the amiR-RUS4 floral buds. Expression of the secondary cell wall-associated MYB transcription factor genes MYB103 and MYB85 were also strongly reduced in floral buds of the amiR-RUS4 plants. Overexpression of RUS4 led to increased secondary thickening in the endothecium. However, the rus4-2 mutant exhibited no obvious phenotype. Promoter-GUS analysis revealed that the RUS4 promoter was highly active in the anthers, supporting its role in anther development. Taken together, these results suggest that RUS4, probably functions redundantly with other genes, may play an important role in the secondary thickening formation in the anther endothecium by indirectly affecting the expression of secondary cell wall biosynthetic genes.

Populus NST/SND orthologs are key regulators of secondary cell wall formation in wood fibers, phloem fibers and xylem ray parenchyma cells.

Wood fibers form thick secondary cell wall (SCW) in xylem tissues to give mechanical support to trees. NAC SECONDARY WALL THICKENING PROMOTING FACTOR3/SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN 1 (NST3/SND1) and NST1 were identified as master regulators of SCW formation in xylem fiber cells in the model plant Arabidopsis thaliana. In Populus species, four NST/SND orthologs have been conserved and coordinately control SCW formation in wood fibers and phloem fibers. However, it remains to be elucidated whether SCW formation in other xylem cells, such as ray parenchyma cells and vessel elements, is regulated by NST/SND orthologs in poplar. We knocked out all NST/SND genes in hybrid aspen using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system and investigated the detailed histological appearance of stem tissues in the knockout mutants. Observation by light microscopy and transmission electron microscopy showed that SCW was severely suppressed in wood fibers, phloem fibers and xylem ray parenchyma cells in the knockout mutants. Although almost all wood fibers lacked SCW, some fiber cells formed thick cell walls. The irregularly cell wall-forming fibers retained primary wall and SCW, and were mainly located in the vicinity of vessel elements. Field emission-scanning electron microscope observation showed that there were no apparent differences in the structural features of pits such as the shape and size between irregularly SCW-forming wood fibers in the knockout mutants and normal wood fibers in wild-type. Cell wall components such as cellulose, hemicellulose and lignin were deposited in the cell wall of irregularly SCW-forming wood fibers in quadruple mutants. Our results indicate that four NST/SND orthologs are master switches for SCW formation in wood fibers, xylem ray parenchyma cells and phloem fibers in poplar, while SCW is still formed in limited wood fibers, which are located at the region adjacent to vessel elements in the knockout mutants.

Secondary Wall Regulating NACs Differentially Bind at the Promoter at a CELLULOSE SYNTHASE A4 Cis-eQTL.

Arabidopsis thaliana CELLULOSE SYNTHASE A4/7/8 (CESA4/7/8) are three non-redundant subunits of the secondary cell wall cellulose synthase complex. Transcript abundance of these genes can vary among genotypes and expression quantitative trait loci (eQTL) were identified in a recombinant population of the accessions Bay-0 and Shahdara. Genetic mapping and analysis of the transcript levels of CESAs between two distinct near isogenic lines (NILs) confirmed a change in CESA4 expression that segregates within that interval. We sequenced the promoters and identified 16 polymorphisms differentiating CESA4Sha and CESA4Bay. In order to determine which of these SNPs could be responsible for this eQTL, we screened for transcription factor protein affinity with promoter fragments of CESA4Bay, CESA4Sha, and the reference genome CESA4Col. The wall thickening activator proteins NAC SECONDARY WALL THICKENING PROMOTING FACTOR2 (NST2) and NST3 exhibited a decrease in binding with the CESA4Sha promoter with a tracheary element-regulating cis-element (TERE) polymorphism. While NILs harboring the TERE polymorphisms exhibited significantly different CESA4 expression, cellulose crystallinity and cell wall thickness were indistinguishable. These results suggest that the TERE polymorphism resulted in differential transcription factor binding and CESA4 expression; yet A. thaliana is able to tolerate this transcriptional variability without compromising the structural elements of the plant, providing insight into the elasticity of gene regulation as it pertains to cell wall biosynthesis and regulation. We also explored available DNA affinity purification sequencing data to resolve a core binding site, C(G/T)TNNNNNNNA(A/C)G, for secondary wall NACs referred to as the VNS element.

ERECTA-family receptor kinase genes redundantly prevent premature progression of secondary growth in the Arabidopsis hypocotyl.

Secondary growth is driven by continuous cell proliferation and differentiation of the cambium that acts as vascular stem cells, producing xylem and phloem to expand vascular tissues laterally. During secondary growth of hypocotyls in Arabidopsis thaliana, the xylem undergoes a drastic phase transition from a parenchyma-producing phase to a fiber-producing phase at the appropriate time. However, it remains to be fully elucidated how progression of secondary growth is properly controlled. We focused on phenotypes of hypocotyl vasculatures caused by double mutation in ERECTA (ER) and ER-LIKE1 (ERL1) receptor-kinase genes to elucidate their roles in secondary growth. ER and ERL1 redundantly suppressed excessive radial growth of the hypocotyl vasculature during secondary growth. ER and ERL1 also prevented premature initiation of the fiber differentiation process mediated by the NAC SECONDARY WALL THICKENING PROMOTING FACTORs in the hypocotyl xylem. Upon floral transition, the hypocotyl xylem gained a competency to respond to GA in a BREVIPEDICELLUS-dependent manner, which was a prerequisite for fiber differentiation. However, even after the floral transition, ER and ERL1 prevented precocious initiation of the GA-mediated fiber formation. Collectively, our findings reveal that ER and ERL1 redundantly prevent premature progression of sequential events in secondary growth.

The basis of pod dehiscence: anatomical traits of the dehiscence zone and expression of eight pod shatter-related genes in four species of Brassicaceae

Members of the Brassicaceae family disperse their seeds through a mechanism commonly referred to as fruit dehiscence or pod shatter. Pod shatter is influenced by variations in valve margin structure and by the molecular control pathways related to valve development. Anatomical patterns of the dehiscence zone from Brassica napus L., Brassica rapa L., Brassica carinata L., and Sinapis alba L., representing fruit types differing in pod shatter resistance, were compared using histological staining. The pod shatter-susceptible plant B. napus showed an increased lignin deposition at the vascular bundle of the replum as well as an increased separation of cell layers. In pod shatter-resistant plants S. alba, B. rapa, and B. carinata, we observed two layers of lignified valve margin cells. From these four species, we isolated and identified homologs of SHATTERPROOF (SHP1, SHP2), INDEHISCENT (IND), ALCATRAZ (ALC), FRUITFULL (FUL), AGAMOUS (AG), NAC SECONDARY WALL THICKENING PROMOTING FACTOR1 (NST1), and SEEDSTICK (STK) genes involved in fruit development and pod shatter in Arabidopsis. Transcriptional analysis of these eight genes was performed by real-time polymerase chain reaction and the results demonstrate that differences in the expression patterns of the eight genes may be associated with dehiscence variation within these four species.

VASCULAR‐RELATED NAC‐DOMAIN 7 directly regulates the expression of a broad range of genes for xylem vessel formation

The Arabidopsis thaliana NAC domain transcription factor, VASCULAR-RELATED NAC-DOMAIN7 (VND7), acts as a key regulator of xylem vessel differentiation. In order to identify direct target genes of VND7, we performed global transcriptome analysis using Arabidopsis transgenic lines in which VND7 activity could be induced post-translationally. This analysis identified 63 putative direct target genes of VND7, which encode a broad range of proteins, such as transcription factors, IRREGULAR XYLEM proteins and proteolytic enzymes, known to be closely associated with xylem vessel formation. Recombinant VND7 protein binds to several promoter sequences present in candidate direct target genes: specifically, in the promoter of XYLEM CYSTEINE PEPTIDASE1, two distinct regions were demonstrated to be responsible for VND7 binding. We also found that expression of VND7 restores secondary cell wall formation in the fiber cells of inflorescence stems of nst1 nst3 double mutants, as well as expression of NAC SECONDARY WALL THICKENING PROMOTING FACTOR3 (NST3, however, the vessel-type secondary wall deposition was observed only as a result of VND7 expression. These findings indicated that VND7 upregulates, directly and/or indirectly, many genes involved in a wide range of processes in xylem vessel differentiation, and that its target genes are partially different from those of NSTs.

An NAC transcription factor orchestrates multiple features of cell wall development in Medicago truncatula

To identify genes controlling secondary cell wall biosynthesis in the model legume Medicago truncatula, we screened a Tnt1 retrotransposon insertion mutant population for plants with altered patterns of lignin autofluorescence. From more than 9000 R1 plants screened, four independent lines were identified with a total lack of lignin in the interfascicular region. The mutants also showed loss of lignin in phloem fibers, reduced lignin in vascular elements, failure in anther dehiscence and absence of phenolic autofluorescence in stomatal guard cell walls. Microarray and PCR analyses confirmed that the mutations were caused by the insertion of Tnt1 in a gene annotated as encoding a NAM (no apical meristem)-like protein (here designated Medicago truncatula NAC SECONDARY WALL THICKENING PROMOTING FACTOR 1, MtNST1). MtNST1 is the only family member in Medicago, but has three homologs (AtNST1-AtNST3) in Arabidopsis thaliana, which function in different combinations to control cell wall composition in stems and anthers. Loss of MtNST1 function resulted in reduced lignin content, associated with reduced expression of most lignin biosynthetic genes, and a smaller reduction in cell wall polysaccharide content, associated with reduced expression of putative cellulose and hemicellulose biosynthetic genes. Acid pre-treatment and cellulase digestion released significantly more sugars from cell walls of nst1 mutants compared with the wild type. We discuss the implications of these findings for the development of alfalfa (Medicago sativa) as a dedicated bioenergy crop.

SOMBRERO, BEARSKIN1, and BEARSKIN2 Regulate Root Cap Maturation inArabidopsis

The root cap has a central role in root growth, determining the growth trajectory and facilitating penetration into the soil. Root cap cells have specialized functions and morphologies, and border cells are released into the rhizosphere by specific cell wall modifications. Here, we demonstrate that the cellular maturation of root cap is redundantly regulated by three genes, SOMBRERO (SMB), BEARSKIN1 (BRN1), and BRN2, which are members of the Class IIB NAC transcription factor family, together with the VASCULAR NAC DOMAIN (VND) and NAC SECONDARY WALL THICKENING PROMOTING FACTOR (NST) genes that regulate secondary cell wall synthesis in specialized cell types. Lateral cap cells in smb-3 mutants continue to divide and fail to detach from the root, phenotypes that are independent of FEZ upregulation in smb-3. In brn1-1 brn2-1 double mutants, columella cells fail to detach, while in triple mutants, cells fail to mature in all parts of the cap. This complex genetic redundancy involves differences in expression, protein activity, and target specificity. All three genes have very similar overexpression phenotypes to the VND/NST genes, indicating that members of this family are largely functionally equivalent. Our results suggest that Class IIB NAC proteins regulate cell maturation in cells that undergo terminal differentiation with strong cell wall modifications.

NAC transcription factors NST1 and NST3 regulate pod shattering in a partially redundant manner by promoting secondary wall formation after the establishment of tissue identity

Three distinct pattern elements of the silique are thought to contribute to its dehiscence: a separation layer, cells with a secondary wall adjacent to the separation layer, and a valve endocarp layer with secondary wall. However, the role of the secondary wall has not been proven, and the factors that regulate its formation in siliques remain to be characterized. We show here that secondary wall formation in siliques is necessary for dehiscence, and that two plant-specific transcription factors, NAC SECONDARY WALL THICKENING PROMOTING FACTOR 1 and 3 (NST1 and NST3), regulate its formation in siliques of Arabidopsis. The promoters of the NST1 and NST3 genes were active in the valve endocarp layer and in cells surrounding vascular vessels in the replum, and NST1 promoter activity only was faintly detectable at valve margins. In nst1 mutants, specific loss of secondary walls was evident at valve margins, while nst1 nst3 double mutants lacked secondary walls in all parts of the siliques, with the exception of vascular vessels. These siliques were similarly indehiscent. The promoters of two tissue-identity genes, INDEHISCENT (IND) and SHATTERPROOF2 (SHP2), were as active in the nst1 nst3 mutant as in the wild-type. Moreover, the ectopic secondary wall formation that occurs in the fruitfull (ful) mutant was absent in the ful nst1 double mutant. We propose that secondary walls in valve margins are required for dehiscence, and that NST1 and NST3 regulate their formation in siliques in a partially redundant manner after the establishment of tissue identity.

Two NAC domain transcription factors, SND1 and NST1, function redundantly in regulation of secondary wall synthesis in fibers of Arabidopsis

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.

NAC transcription factors, NST1 and NST3, are key regulators of the formation of secondary walls in woody tissues of Arabidopsis.

Wood is formed by the successive addition of secondary xylem, which consists of cells with a conspicuously thickened secondary wall composed mainly of lignin and cellulose. Several genes involved in lignin and cellulose biosynthesis have been characterized, but the factors that regulate the formation of secondary walls in woody tissues remain to be identified. In this study, we show that plant-specific transcription factors, designated NAC SECONDARY WALL THICKENING PROMOTING FACTOR1 (NST1) and NST3, are key regulators of the formation of secondary walls in woody tissues of Arabidopsis thaliana. In nst1-1 nst3-1 double knockout plants, the secondary wall thickenings in interfascicular fibers and secondary xylem, except for vascular vessels, were completely suppressed without affecting formation of cells destined to be woody tissues. Conversely, as shown previously for NST1, overexpression of NST3 induced ectopic secondary wall thickenings in various aboveground tissues. Furthermore, the expression of chimeric repressors derived from NST1 and NST3 suppressed secondary wall thickenings in the presumptive interfascicular fibers. Because putative orthologs of NST1 and NST3 are present in the genome of poplar, our results suggest that they are also key regulators of the formation of secondary walls in woody plants and could be used as a tool for the genetic engineering of wood and its derivatives.