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

It has been proposed that the transcriptional regulation of secondary wall biosynthesis in Arabidopsis is controlled by a transcriptional network mediated by SND1 and its close homologs. Uncovering all the transcription factors and deciphering their interrelationships in the network are essential for our understanding of the molecular mechanisms underlying the transcriptional regulation of biosynthesis of secondary walls, the major constituent of wood and fibers. Here, we present functional evidence that the MYB83 transcription factor is another molecular switch in the SND1-mediated transcriptional network regulating secondary wall biosynthesis. MYB83 is specifically expressed in fibers and vessels where secondary wall thickening occurs. Its expression is directly activated by SND1 and its close homologs, including NST1, NST2, VND6 and VND7, indicating that MYB83 is their direct target. MYB83 overexpression is able to activate a number of the biosynthetic genes of cellulose, xylan and lignin and concomitantly induce ectopic secondary wall deposition. In addition, its overexpression upregulates the expression of several transcription factors involved in regulation of secondary wall biosynthesis. Dominant repression of MYB83 functions or simultaneous RNAi inhibition of MYB83 and MYB46 results in a reduction in secondary wall thickening in fibers and vessels and a deformation of vessels. Furthermore, double T-DNA knockout mutations of MYB83 and MYB46 cause a lack of secondary walls in vessels and an arrest in plant growth. Together, these results demonstrate that MYB83 and MYB46, both of which are SND1 direct targets, function redundantly in the transcriptional regulatory cascade leading to secondary wall formation in fibers and vessels.

Wood biomass is mainly made of secondary cell walls; hence, elucidation of the molecular mechanisms underlying the transcriptional regulation of secondary wall biosynthesis during wood formation will be instrumental to design strategies for genetic improvement of wood biomass. Here, we provide direct evidence demonstrating that the poplar (Populus trichocarpa) wood-associated NAC domain transcription factors (PtrWNDs) are master switches activating a suite of downstream transcription factors, and together, they are involved in the coordinated regulation of secondary wall biosynthesis during wood formation. We show that transgenic poplar plants with dominant repression of PtrWNDs functions exhibit a drastic reduction in secondary wall thickening in woody cells, and those with PtrWND overexpression result in ectopic deposition of secondary walls. Analysis of PtrWND2B overexpressors revealed up-regulation of the expression of a number of wood-associated transcription factors, the promoters of which were also activated by PtrWND6B and the Eucalyptus EgWND1. Transactivation analysis and electrophoretic mobility shift assay demonstrated that PtrWNDs and EgWND1 activated gene expression through direct binding to the secondary wall NAC-binding elements, which are present in the promoters of several wood-associated transcription factors and a number of genes involved in secondary wall biosynthesis and modification. The WND-regulated transcription factors PtrNAC150, PtrNAC156, PtrNAC157, PtrMYB18, PtrMYB74, PtrMYB75, PtrMYB121, PtrMYB128, PtrZF1, and PtrGATA8 were able to activate the promoter activities of the biosynthetic genes for all three major wood components. Our study has uncovered that the WND master switches together with a battery of their downstream transcription factors form a transcriptional network controlling secondary wall biosynthesis during wood formation.Wood, composed of cellulose, hemicelluloses, and lignin, is the most abundant biomass produced by plants. It is an important raw forest product that has traditionally been used for myriad applications, including construction, pulping, paper making, direct burning for energy, and so on. Recently, wood from tree species, such as poplar (Populus spp.), has been proposed to be a renewable source for biofuel production (Carroll and Somerville, 2009). Therefore, there is a surge of interest in elucidating the molecular mechanisms underlying the process of wood formation in the hope of developing strategies for increasing wood biomass production and/or modifying wood composition tailored for biofuel production. Since wood biomass is mainly made of secondary walls, elucidation of the mechanisms underlying the coordinated activation of secondary wall biosynthetic genes will undoubtedly contribute to our understanding of the molecular control of wood formation.Molecular and genomic studies in tree species have uncovered a number of wood-associated transcription factors that might be involved in the coordinated regulation of wood formation (Patzlaff et al

The bulk of grass biomass potentially useful for cellulose-based biofuel production is the remains of secondary wall-containing sclerenchymatous fibers. Hence, it is important to uncover the molecular mechanisms underlying the regulation of secondary wall thickening in grass species. So far, little is known about the transcriptional regulatory switches responsible for the activation of the secondary wall biosynthetic program in grass species. Here, we report the roles of a group of rice and maize NAC and MYB transcription factors in the regulation of secondary wall biosynthesis. The rice and maize secondary wall-associated NACs (namely OsSWNs and ZmSWNs) were able to complement the Arabidopsis snd1 nst1 double mutant defective in secondary wall thickening. When overexpressed in Arabidopsis, OsSWNs and ZmSWNs were sufficient to activate a number of secondary wall-associated transcription factors and secondary wall biosynthetic genes, and concomitantly result in the ectopic deposition of cellulose, xylan and lignin. It was also found that the rice and maize MYB transcription factors, OsMYB46 and ZmMYB46, are functional orthologs of Arabidopsis MYB46/MYB83 and, when overexpressed in Arabidopsis, they were able to activate the entire secondary wall biosynthetic program. Furthermore, the promoters of OsMYB46 and ZmMYB46 contain secondary wall NAC-binding elements (SNBEs), which can be bound and activated by OsSWNs and ZmSWNs. Together, our results indicate that the rice and maize SWNs and MYB46 are master transcriptional activators of the secondary wall biosynthetic program and that OsSWNs and ZmSWNs activate their direct target genes through binding to the SNBE sites.

SUMMARY Heterochromatin is integral to cell identity maintenance by impeding the activation of genes for alternate cell fates. Heterochromatic regions are associated with histone 3 lysine 9 trimethylation (H3K9me3) or H3K27me3, but these modifications are also found in euchromatic regions that permit transcription. We discovered that resistance to sonication is a reliable indicator of the heterochromatin state, and we developed a biophysical method (Gradient-seq) to discriminate subtypes of H3K9me3 and H3K27me3 domains in sonication-resistant heterochromatin (srHC) versus euchromatin. These classifications are more accurate than the histone marks alone in predicting transcriptional silence and resistance of alternate-fate genes to activation during direct cell conversion. Our proteomics of H3K9me3-marked srHC and functional screens revealed diverse proteins, including RBMX and RBMXL1, that impede gene induction during cellular reprogramming. Isolation of srHC with Gradient-seq provides a genome-wide map of chromatin structure, elucidating subtypes of repressed domains that are uniquely predictive of diverse other chromatin properties.