ABSCISIC ACID-RESPONSIVE ELEMENT BINDING PROTEIN1 (AREB1) (i.e., ABF2) is a basic domain/leucine zipper transcription factor that binds to the abscisic acid (ABA)-responsive element (ABRE) motif in the promoter region of ABA-inducible genes. Here, we show that expression of the intact AREB1 gene on its own is insufficient to lead to expression of downstream genes under normal growth conditions. To overcome the masked transactivation activity of AREB1, we created an activated form of AREB1 (AREB1DQT). AREB1DQT-overexpressing plants showed ABA hypersensitivity and enhanced drought tolerance, and eight genes with two or more ABRE motifs in the promoter regions in two groups were greatly upregulated: late embryogenesis abundant class genes and ABA-and drought stress-inducible regulatory genes. By contrast, an areb1 null mutant and a dominant loss-of-function mutant of AREB1 (AREB1:RD) with a repression domain exhibited ABA insensitivity. Furthermore, AREB1:RD plants displayed reduced survival under dehydration, and three of the eight greatly upregulated genes were downregulated, including genes for linker histone H1 and AAA ATPase, which govern gene expression and multiple cellular activities through protein folding, respectively. Thus, these data suggest that AREB1 regulates novel ABRE-dependent ABA signaling that enhances drought tolerance in vegetative tissues.

SummaryThe 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 speci®c 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 speci®c transcription factors.

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.

We demonstrated that the GCC box, which is an 11-bp sequence (TAAGAGCCGCC) conserved in the 5' upstream region of ethylene-inducible pathogenesis-related protein genes in Nicotiana spp and in some other plants, is the sequence that is essential for ethylene responsiveness when incorporated into a heterologous promoter. Competitive gel retardation assays showed DNA binding activities to be specific to the GCC box sequence in tobacco nuclear extracts. Four different cDNAs encoding DNA binding proteins specific for the GCC box sequence were isolated, and their products were designated ethylene-responsive element binding proteins (EREBPs). The deduced amino acid sequences of EREBPs exhibited no homology with those of known DNA binding proteins or transcription factors; neither did the deduced proteins contain a basic leucine zipper or zinc finger motif. The DNA binding domain was identified within a region of 59 amino acid residues that was common to all four deduced EREBPs. Regions highly homologous to the DNA binding domain of EREBPs were found in proteins deduced from the cDNAs of various plants, suggesting that this domain is evolutionarily conserved in plants. RNA gel blot analysis revealed that accumulation of mRNAs for EREBPs was induced by ethylene, but individual EREBPs exhibited different patterns of expression.

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.

Summary Transcription factors belonging to the APETALA2/Ethylene Responsive Factor (AP2/ERF) family are conservatively widespread in the plant kingdom. These regulatory proteins are involved in the control of primary and secondary metabolism, growth and developmental programs, as well as responses to environmental stimuli. Due to their plasticity and to the specificity of individual members of this family, AP2/ERF transcription factors represent valuable targets for genetic engineering and breeding of crops. In this review, we integrate the evidence collected from functional and structural studies to describe their different mechanisms of action and the regulatory pathways that affect their activity.

SummaryArabidopsis thaliana RD26 cDNA, isolated from dehydrated plants, encodes a NAC protein. Expression of the RD26 gene was induced not only by drought but also by abscisic acid (ABA) and high salinity. The RD26 protein is localized in the nucleus and its C terminal has transcriptional activity. Transgenic plants overexpressing RD26 were highly sensitive to ABA, while RD26-repressed plants were insensitive. The results of microarray analysis showed that ABA-and stress-inducible genes are upregulated in the RD26-overexpressed plants and repressed in the RD26-repressed plants. Furthermore, RD26 activated a promoter of its target gene in Arabidopsis protoplasts. These results indicate that RD26 functions as a transcriptional activator in ABAinducible gene expression under abiotic stress in plants.

Ethylene-responsive element binding factors (ERFs) are members of a novel family of transcription factors that are specific to plants. A highly conserved DNA binding domain known as the ERF domain is the unique feature of this protein family. To characterize in detail this family of transcription factors, we isolated Arabidopsis cDNAs encoding five different ERF proteins (AtERF1 to AtERF5) and analyzed their structure, DNA binding preference, transactivation ability, and mRNA expression profiles. The isolated AtERFs were placed into three classes based on amino acid identity within the ERF domain, although all five displayed GCC box-specific binding activity. AtERF1, AtERF2, and AtERF5 functioned as activators of GCC box-dependent transcription in Arabidopsis leaves. By contrast, AtERF3 and AtERF4 acted as repressors that downregulated not only basal transcription levels of a reporter gene but also the transactivation activity of other transcription factors. The AtERF genes were differentially regulated by ethylene and by abiotic stress conditions, such as wounding, cold, high salinity, or drought, via ETHYLENE-INSENSITIVE2 (EIN2)-dependent or -independent pathways. Cycloheximide, a protein synthesis inhibitor, also induced marked accumulation of AtERF mRNAs. Thus, we conclude that AtERFs are factors that respond to extracellular signals to modulate GCC box-mediated gene expression positively or negatively.