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.

Summary: The redundancy of genes for plant transcription factors often interferes with efforts to identify the biologic functions of such factors. We show here that four different transcription factors fused to the EAR motif, a repression domain of only 12 amino acids, act as dominant repressors in transgenic Arabidopsis and suppress the expression of specific target genes, even in the presence of the redundant transcription factors, with resultant dominant loss‐of‐function phenotypes. Chimeric EIN3, CUC1, PAP1, and AtMYB23 repressors that included the EAR motif dominantly suppressed the expression of their target genes and caused insensitivity to ethylene, cup‐shaped cotyledons, reduction in the accumulation of anthocyanin, and absence of trichomes, respectively. This chimeric repressor silencing technology (CRES‐T), exploiting the EAR‐motif repression domain, is simple and effective and can overcome genetic redundancy. Thus, it should be useful not only for the rapid analysis of the functions of redundant plant transcription factors but also for the manipulation of plant traits via the suppression of gene expression that is regulated by specific transcription factors.

In plants, secondary wall thickenings play important roles in various biological processes, although the factors regulating these processes remain to be characterized. We show that expression of chimeric repressors derived from NAC SECONDARY WALL THICKENING PROMOTING FACTOR1 (NST1) and NST2 in Arabidopsis thaliana resulted in an anther dehiscence defect due to loss of secondary wall thickening in anther endothecium. Plants with double, but not single, T-DNA–tagged lines for NST1 and NST2 had the same anther-indehiscent phenotype as transgenic plants that expressed the individual chimeric repressors, indicating that NST1 and NST2 are redundant in regulating secondary wall thickening in anther walls. The activity of the NST2 promoter was particularly strong in anther tissue, while that of the NST1 promoter was detected in various tissues in which lignified secondary walls develop. Ectopic expression of NST1 or NST2 induced ectopic thickening of secondary walls in various aboveground tissues. Epidermal cells with ectopic thickening of secondary walls had structural features similar to those of tracheary elements. However, among genes involved in the differentiation of tracheary elements, only those related to secondary wall synthesis were clearly upregulated. None of the genes involved in programmed cell death were similarly affected. Our results suggest NAC transcription factors as possible regulators of secondary wall thickening in various tissues.

We reported previously that three ERF transcription factors, tobacco ERF3 (NtERF3) and Arabidopsis AtERF3 and AtERF4, which are categorized as class II ERFs, are active repressors of transcription. To clarify the roles of these repressors in transcriptional regulation in plants, we attempted to identify the functional domains of the ERF repressor that mediates the repression of transcription. Analysis of the results of a series of deletions revealed that the C-terminal 35 amino acids of NtERF3 are sufficient to confer the capacity for repression of transcription on a heterologous DNA binding domain. This repression domain suppressed the intermolecular activities of other transcriptional activators. In addition, fusion of this repression domain to the VP16 activation domain completely inhibited the transactivation function of VP16. Comparison of amino acid sequences of class II ERF repressors revealed the conservation of the sequence motif ^L/_FDLN^L/_F(x)P. This motif was essential for repression because mutations within the motif eliminated the capacity for repression. We designated this motif the ERF-associated amphiphilic repression (EAR) motif, and we identified this motif in a number of zinc-finger proteins from wheat, Arabidopsis, and petunia plants. These zinc finger proteins functioned as repressors, and their repression domains were identified as regions that contained an EAR motif.

Summary: In Arabidopsis, MYB transcription factors regulate flavonoid biosynthesis via the formation of protein complexes with a basic helix‐loop‐helix (bHLH) transcription factor and a WD40 repeat protein. Several R3‐type single‐MYB proteins (R3‐MYB), such as CPC and TRY, act as negative regulators of the development of epidermal cells. However, such regulators of flavonoid biosynthesis have not yet been reported, to our knowledge. We show here that an R3‐MYB protein, AtMYBL2, acts as a transcriptional repressor and negatively regulates the biosynthesis of anthocyanin in Arabidopsis. In an AtMYBL2 knockout line (mybl2), the expression of the DFR and TT8 genes was enhanced and resulted in the ectopic accumulation of anthocyanin, while ectopic expression of AtMYBL2 or of a chimeric repressor that is a dominant negative form of AtMYBL2 suppressed the expression of DFR and TT8, and the biosynthesis of anthocyanin. The expression of AtMYBL2 was detected in various tissues but not in those in which anthocyanin accumulated or TT8 was expressed. The minimal repression domain of AtMYBL2 was found to be the six amino acids (TLLLFR) at the carboxyl terminus, and TLLLFR appears to be a novel repression motif that is different from the ERF‐associated amphiphilic repression (EAR) motif. The defective phenotype of mybl2 mutants was complemented by 35S:AtMYBL2 but enhanced by a truncated form of AtMYBL2 from which the repression domain had been deleted. AtMYBL2 bound directly to TT8 protein and this complex suppressed the expression of DFR and TT8. The repression activity of AtMYBL2 appears to play a critical role in the regulation of anthocyanin biosynthesis.

Many eukaryotes have from one to three heat shock factors (Hsfs), but plants have more than 20 Hsfs, designated class A, B, and C. Class A Hsfs are activators of transcription, but details of the roles of individual Hsfs have not been fully characterized. We show here that Arabidopsis (Arabidopsis thaliana) HsfB1 and HsfB2b, members of class B, are transcriptional repressors and negatively regulate the expression of heat-inducible Hsfs (HsfA2, HsfA7a, HsfB1, and HsfB2b) and several heat shock protein genes. In hsfb1 hsfb2b double mutant plants, the expression of a large number of heat-inducible genes was enhanced in the non-heat condition (23°C) and the plants exhibited slightly higher heat tolerance at 42°C than the wild type, similar to Pro35S:HsfA2 plants. In addition, under extended heat stress conditions, expression of the heat-inducible Hsf genes remained consistently higher in hsfb1 hsfb2b than in the wild type. These data indicate that HsfB1 and HsfB2b suppress the general heat shock response under non-heat-stress conditions and in the attenuating period. On the other hand, HsfB1 and HsfB2b appear to be necessary for the expression of heat stress-inducible heat shock protein genes under heat stress conditions, which is necessary for acquired thermotolerance. We show that the heat stress response is finely regulated by activation and repression activities of Hsfs in Arabidopsis.