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: Plants respond and adapt to drought stress in order to survive under stress conditions. Several genes that respond to drought at the transcriptional level have been described, but there are few reports on genes involved in the recovery from dehydration. Analysis of rehydration‐inducible genes should help not only to understand the molecular mechanisms of stress responses in higher plants, but also to improve the stress tolerance of crops by gene manipulation. We used a full‐length cDNA microarray containing ca. 7000 Arabidopsis full‐length cDNAs and identified 152 rehydration‐inducible genes. Venn diagram analysis showed relationship of the rehydration‐inducible genes to proline‐inducible and water‐treatment‐inducible genes. Among the 152 rehydration‐inducible genes, 58 genes contained the ACTCAT sequence involved in proline‐ and hypoosmolarity‐inducible gene expression in their promoter regions, suggesting that ACTCAT sequence is a major cis‐acting element involved in rehydration‐inducible gene expression, and that some novel cis‐acting elements are involved in rehydration‐inducible gene expression. Functional analysis of rehydration‐inducible and rehydration‐repressed genes revealed their functions not only in the release from a stressed status but also in the recovery of growth in plants.

Proline (Pro) is one of the most widely distributed osmolytes in water-stressed plants. We previously isolated from Arabidopsis a gene encoding Pro dehydrogenase (ProDH), a mitochondrial enzyme involved in the first step of the conversion of Pro to glutamic acid. The ProDH gene in Arabidopsis is up-regulated by rehydration after dehydration but is down-regulated by dehydration.ProDH is also induced by l-Pro and hypoosmolarity. The induction of ProDH expression under rehydration seems to be caused by both accumulated Pro and hypoosmolarity. We analyzed a DNA region that is located 5′ to the transcription start site (a promoter region) of ProDH to identify cis-acting elements involved in l-Pro-induced and hypoosmolarity-induced expression in transgenic tobacco (Nicotiana tabacum) and Arabidopsis plants. We found that a 9-bp sequence, ACTCATCCT, in the ProDH promoter is necessary for the efficient expression of ProDH in response to l-Pro and hypoosmolarity. Moreover, ACTCAT is a core cis-acting element, which we have called Pro- or hypoosmolarity-responsive element (PRE), that is necessary forl-Pro-responsive and hypoosmolarity-responsive expression of ProDH. Microarray and RNA gel-blot analyses showed that 21 l-Pro-inducible genes have the PRE sequences in their promoter regions. These results indicate that the PRE sequence play an important role in the l-Pro-responsive gene expression.

A 6-bp sequence, ACTCAT, acts as a cis-acting element involved in hypoosmolarity- and proline-responsive expression of an Arabidopsis proline dehydrogenase (ProDH) gene. Search of the database for plant cis-acting elements revealed that the ACTCAT sequence is similar to the GCN4 motif [ATGA(C/G)TCAT] that is recognized by bZIP transcription factors. To identify transcription factor(s) for regulation of ProDH, we examined whether Arabidopsis bZIPs function as transcription factors for the ACTCAT sequence. Transient expression analysis revealed that the four proteins in Group S bZIPs, AtbZIP11/ATB2, AtbZIP44, AtbZIP2/GBF5 and AtbZIP53, formed an ATB2 subgroup that activated expression of the GUS reporter gene driven by the ACTCAT sequence while other bZIPs and different families of plant transcription factors did not. The transactivation activity of the ATB2 subgroup was enhanced in a hypoosmotic condition. In a gel mobility shift assay, the recombinant proteins of the ATB2 subgroup specifically bound to the ACTCAT sequence. RNA gel blot analysis indicated that the expression of AtbZIP2/GBF5 and AtbZIP53, as well as that of ProDH, is induced by hypoosmolarity. Moreover, we showed that the sGFP::AtbZIP11/ATB2 fusion protein is localized in the nucleus. These results suggest that the ATB2 subgroup functions as a transcriptional activator for hypoosmolarity-inducible ProDH in Arabidopsis.

The cDNA clone ERD5 (earlyresponsive to dehydration), isolated from 1-h-dehydrated Arabidopsis, encodes a precursor of proline (Pro) dehydrogenase (ProDH), which is a mitochondrial enzyme involved in the first step of the conversion of Pro to glutamic acid. The transcript of the erd5 (ProDH) gene was undetectable when plants were dehydrated, but large amounts of transcript accumulated when plants were subsequently rehydrated. Accumulation of the transcript was also observed in plants that had been incubated under hypoosmotic conditions in media that contained l- ord-Pro. We isolated a 1.4-kb DNA fragment of the putative promoter region of the ProDH gene. Theβ-glucuronidase (GUS) reporter gene driven by the 1.4-kb ProDH promoter was induced not only by rehydration but also by hypoosmolarity and l- andd-Pro at significant levels in transgenic Arabidopsis plants. The promoter of the ProDH gene directs strong GUS activity in reproductive organs such as pollen and pistils and in the seeds of the transgenic plants. GUS activity was detected in vegetative tissues such as veins of leaves and root tips when the transgenic plants were exposed to hypoosmolarity and Pro solutions. GUS activity increased during germination of the transgenic plants under hypoosmolarity. The relationship between Pro metabolism and the physiological aspects of stress response and development are discussed.