The MYC-like sequence CATGTG plays an important role in the dehydration-inducible expression of the Arabidopsis thaliana EARLY RESPONSIVE TO DEHYDRATION STRESS 1 (ERD1) gene, which encodes a ClpA (ATP binding subunit of the caseinolytic ATP-dependent protease) homologous protein. Using the yeast one-hybrid system, we isolated three cDNA clones encoding proteins that bind to the 63-bp promoter region of erd1, which contains the CATGTG motif. These three cDNA clones encode proteins named ANAC019, ANAC055, and ANAC072, which belong to the NAC transcription factor family. The NAC proteins bound specifically to the CATGTG motif both in vitro and in vivo and activated the transcription of a β-glucuronidase (GUS) reporter gene driven by the 63-bp region containing the CATGTG motif in Arabidopsis T87 protoplasts. The expression of ANAC019, ANAC055, and ANAC072 was induced by drought, high salinity, and abscisic acid. A histochemical assay using PNAC-GUS fusion constructs showed that expression of the GUS reporter gene was localized mainly to the leaves of transgenic Arabidopsis plants. Using the yeast one-hybrid system, we determined the complete NAC recognition sequence, containing CATGT and harboring CACG as the core DNA binding site. Microarray analysis of transgenic plants overexpressing either ANAC019, ANAC055, or ANAC072 revealed that several stress-inducible genes were upregulated in the transgenic plants, and the plants showed significantly increased drought tolerance. However, erd1 was not upregulated in the transgenic plants. Other interacting factors may be necessary for the induction of erd1 in Arabidopsis under stress conditions.

A myriad of drought stress-inducible genes have been reported, and many of these are activated by abscisic acid (ABA). In the promoter regions of such ABA-regulated genes, conserved cis-elements, designated ABA-responsive elements (ABREs), control gene expression via bZIP-type AREB/ABF transcription factors. Although all three members of the AREB/ABF subfamily, AREB1, AREB2, and ABF3, are upregulated by ABA and water stress, it remains unclear whether these are functional homologs. Here, we report that all three AREB/ABF transcription factors require ABA for full activation, can form hetero- or homodimers to function in nuclei, and can interact with SRK2D/SnRK2.2, an SnRK2 protein kinase that was identified as a regulator of AREB1. Along with the tissue-specific expression patterns of these genes and the subcellular localization of their encoded proteins, these findings clearly indicate that AREB1, AREB2, and ABF3 have largely overlapping functions. To elucidate the role of these AREB/ABF transcription factors, we generated an areb1 areb2 abf3 triple mutant. Large-scale transcriptome analysis, which showed that stress-responsive gene expression is remarkably impaired in the triple mutant, revealed novel AREB/ABF downstream genes in response to water stress, including many LEA class and group-Ab PP2C genes and transcription factors. The areb1 areb2 abf3 triple mutant is more resistant to ABA than are the other single and double mutants with respect to primary root growth, and it displays reduced drought tolerance. Thus, these results indicate that AREB1, AREB2, and ABF3 are master transcription factors that cooperatively regulate ABRE-dependent gene expression for ABA signaling under conditions of water stress.

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.

Summary: Arabidopsis 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 ABA‐inducible gene expression under abiotic stress in plants.