Masaru Ohme-Takagi

Arabidopsis Ethylene-Responsive Element Binding Factors Act as Transcriptional Activators or Repressors of GCC Box–Mediated Gene Expression

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.

Repression Domains of Class II ERF Transcriptional Repressors Share an Essential Motif for Active Repression

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/FDLNL/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.

AREB1 Is a Transcription Activator of Novel ABRE-Dependent ABA Signaling That Enhances Drought Stress Tolerance in Arabidopsis

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 (AREB1ΔQT). AREB1ΔQT-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.

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.

The NAC Transcription Factors NST1 and NST2 of Arabidopsis Regulate Secondary Wall Thickenings and Are Required for Anther Dehiscence

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.

A NOVEL MODE OF DNA RECOGNITION BY A BETA -SHEET REVEALED BY THE SOLUTION STRUCTURE OF THE GCC-BOX BINDING DOMAIN IN COMPLEX WITH DNA

The 3D solution structure of the GCC‐box binding domain of a protein from Arabidopsis thaliana in complex with its target DNA fragment has been determined by heteronuclear multidimensional NMR in combination with simulated annealing and restrained molecular dynamic calculation. The domain consists of a three‐stranded anti‐parallel β‐sheet and an α‐helix packed approximately parallel to the β‐sheet. Arginine and tryptophan residues in the β‐sheet are identified to contact eight of the nine consecutive base pairs in the major groove, and at the same time bind to the sugar phosphate backbones. The target DNA bends slightly at the central CG step, thereby allowing the DNA to follow the curvature of the β‐sheet.

Unique Mode of GCC Box Recognition by the DNA-binding Domain of Ethylene-responsive Element-binding Factor (ERF Domain) in Plant

Ethylene-responsive element-binding proteins (EREBPs)have novel DNA-binding domains (ERF domains), which are widely conserved in plants, and interact specifically with sequences containing AGCCGCC motifs (GCC box). Deletion experiments show that some flanking region at the N terminus of the conserved 59-amino acid ERF domain is required for stable binding to the GCC box. Three ERF domain-containing fragments of EREBP2, EREBP4, and AtERF1 from tobacco and Arabidopsis, bind to the sequence containing the GCC box with a high binding affinity in the pm range. The high affinity binding is conferred by a monomeric ERF domain fragment, and DNA truncation experiments show that only 11-base pair DNA containing the GCC box is sufficient for stable ERF domain interaction. Systematic DNA mutation analyses demonstrate that the specific amino acid contacts are confined within the 6-base pair GCCGCC region of the GCC box, and the first G, the fourth G, and the sixth C exhibit highest binding specificity common in all three ERF domain-containing fragments studied. Other bases within the GCC box exhibit modulated binding specificity varying from protein to protein, implying that these positions are important for differential binding by different EREBPs. The conserved N-terminal half is likely responsible for formation of a stable complex with the GCC box and the divergent C-terminal half for modulating the specificity.

AtMYBL2, a protein with a single MYB domain, acts as a negative regulator of anthocyanin biosynthesis in Arabidopsis.

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.

TCP Transcription Factors Control the Morphology of Shoot Lateral Organs via Negative Regulation of the Expression of Boundary-Specific Genes in Arabidopsis

Plants form shoot meristems in the so-called boundary region, and these meristems are necessary for normal morphogenesis of aerial parts of plants. However, the molecular mechanisms that regulate the formation of shoot meristems are not fully understood. We report here that expression of a chimeric repressor from TCP3 (TCP3SRDX), a member of TEOSINTE BRANCHED1, CYCLOIDEA, and PCF (TCP) transcription factors in Arabidopsis thaliana, resulted in the formation of ectopic shoots on cotyledons and various defects in organ development. Expression of TCP3SRDX induced ectopic expression of boundary-specific genes, namely the CUP-SHAPED COTYLEDON (CUC) genes, and suppressed the expression of miR164, whose product cleaves the transcripts of CUC genes. This abnormal phenotype was substantially reversed on the cuc1 mutant background. By contrast, gain of function of TCP3 suppressed the expression of CUC genes and resulted in the fusion of cotyledons and defects in formation of shoots. The pattern of expression of TCP3 did not overlap with that of the CUC genes. In addition, we found that eight TCPs had functions similar to that of TCP3. Our results demonstrate that the TCP transcription factors play a pivotal role in the control of morphogenesis of shoot organs by negatively regulating the expression of boundary-specific genes.

TCP Transcription Factors Regulate the Activities of ASYMMETRIC LEAVES1 and miR164, as Well as the Auxin Response, during Differentiation of Leaves in Arabidopsis

This work examines the regulatory signaling for leaf differentiation by identifying the target genes of TEOSINTE BRANCHED1, CYCLOIDEA, and PCF (TCP) transcription factors in Arabidopsis. It identifies genetically redundant and differentially regulated pathways for balancing the differentiated and undifferentiated fates in leaf development. Coordination of the maintenance of the undifferentiated fate of cells in the shoot meristem and the promotion of cellular differentiation in plant organs is essential for the development of plant shoots. CINCINNATA-like (CIN-like) TEOSINTE BRANCHED1, CYCLOIDEA, and PCF (TCP) transcription factors are involved in this coordination via the negative regulation of CUP-SHAPED COTYLEDON (CUC) genes, which regulate the formation of shoot meristems and the specification of organ boundaries. However, the molecular mechanism of the action of CIN-like TCPs is poorly understood. We show here that TCP3, a model of CIN-like TCPs of Arabidopsis thaliana, directly activates the expression of genes for miR164, ASYMMETRIC LEAVES1 (AS1), INDOLE-3-ACETIC ACID3/SHORT HYPOCOTYL2 (IAA3/SHY2), and SMALL AUXIN UP RNA (SAUR) proteins. Gain of function of these genes suppressed the formation of shoot meristems and resulted in the fusion of cotyledons, whereas their loss of function induced ectopic expression of CUC genes in leaves. Our results indicate that miR164, AS1, IAA3/SHY2, and SAUR partially but cooperatively suppress the expression of CUC genes. Since CIN-like TCP genes were revealed to act dose dependently in the differentiation of leaves, we propose that evolutionarily diverse CIN-like TCPs have important roles in the signaling pathways that generate different leaf forms, without having any lethal effects on shoots.