The induction of the dehydration-responsive Arabidopsis gene, rd29B, is mediated mainly by abscisic acid (ABA). Promoter analysis of rd29B indicated that two ABA-responsive elements (ABREs) are required for the dehydration-responsive expression of rd29B as cisacting elements. Three cDNAs encoding basic leucine zipper (bZIP)-type ABRE-binding proteins were isolated by using the yeast onehybrid system and were designated AREB1, AREB2, and AREB3 (ABA-responsive element binding protein). Transcription of the AREB1 and AREB2 genes is up-regulated by drought, NaCl, and ABA treatment in vegetative tissues. In a transient transactivation experiment using Arabidopsis leaf protoplasts, both the AREB1 and AREB2 proteins activated transcription of a reporter gene driven by ABRE. AREB1 and AREB2 required ABA for their activation, because their transactivation activities were repressed in aba2 and abi1 mutants and enhanced in an era1 mutant. Activation of AREBs by ABA was suppressed by protein kinase inhibitors. These results suggest that both AREB1 and AREB2 function as transcriptional activators in the ABA-inducible expression of rd29B, and further that ABA-dependent posttranscriptional activation of AREB1 and AREB2, probably by phosphorylation, is necessary for their maximum activation by ABA. Using cultured Arabidopsis cells, we demonstrated that a specific ABA-activated protein kinase of 42-kDa phosphorylated conserved N-terminal regions in the AREB proteins.

bZIP-type transcription factors AREBs͞ABFs bind an abscisic acid (ABA)-responsive cis-acting element named ABRE and transactivate downstream gene expression in Arabidopsis. Because AREB1 overexpression could not induce downstream gene expression, activation of AREB1 requires ABA-dependent posttranscriptional modification. We confirmed that ABA activated 42-kDa kinase activity, which, in turn, phosphorylated Ser͞Thr residues of R-X-X-S͞T sites in the conserved regions of AREB1. Amino acid substitutions of R-X-X-S͞T sites to Ala suppressed transactivation activity, and multiple substitution of these sites resulted in almost complete suppression of transactivation activity in transient assays. In contrast, substitution of the Ser͞Thr residues to Asp resulted in high transactivation activity without exogenous ABA application. A phosphorylated, transcriptionally active form was achieved by substitution of Ser͞Thr in all conserved R-X-X-S͞T sites to Asp. Transgenic plants overexpressing the phosphorylated active form of AREB1 expressed many ABA-inducible genes, such as RD29B, without ABA treatment. These results indicate that the ABA-dependent multisite phosphorylation of AREB1 regulates its own activation in plants.transactivation activity ͉ transcription factor AREB1 ͉ transgenic Arabidopsis T he phytohormone abscisic acid (ABA) plays important roles in seed maturation and dormancy and is also involved in the adaptation of vegetative tissues to abiotic environmental stresses, such as drought and high salinity. ABA promotes stomatal closure in guard cells and regulates the expression of many genes, the products of which may function in dehydration tolerance in both vegetative tissues and seeds. Many ABA-inducible genes contain a conserved element named ABA-responsive element (ABRE) (Py-ACGTGG͞TC) in their promoter regions. The ABRE functions as a cis-acting element and is involved in ABA-responsive gene expression (for reviews, see refs. 1 and 2).The RD29B promoter region contains two ABREs, and analyses with the ABA-deficient and insensitive mutants aba1 and abi1 revealed that the drought-inducible expression of RD29B is controlled mainly by ABA (3-6). Yeast one-hybrid screening with the RD29B promoter including ABREs enabled us to clone three independent cDNAs encoding ABRE-binding proteins (AREB1, AREB2, and AREB3) in Arabidopsis (7). Each AREB protein contained a single bZIP-type DNA-binding domain, and expression of AREB1 and AREB2 was up-regulated by ABA, drought, and high-salinity stresses, shown to function as trans-acting activators by using transient expression in protoplasts (7). Choi et al. (8) also reported the cloning of four independent cDNAs for ABREbinding factors (ABF1, ABF2, ABF3, and ABF4) from Arabidopsis. ABF2 and ABF4 were identical to AREB1 and AREB2, respectively.In the Arabidopsis genome, 75 distinct bZIP transcription factors exist, and nine members are classified as a homologous subfamily of AREBs that contain three N-terminal (C1, C2, and C3) and one C-terminal (C4) conserved domains (9-...

;Protein phosphorylation has pivotal roles in ABA and osmotic stress signaling in higher plants. Two protein phosphatase genes, ABI1 and ABI2, are known to regulate these signaling pathways in Arabidopsis. The identity of ABAactivated protein kinases required for the ABA signaling, however, remains to be elucidated. Here we demonstrate that two protein kinases, p44 and p42, were activated by ABA in Arabidopsis T87 cultured cells, and at least one protein kinase, p44, was activated not only by ABA but also by low humidity in Arabidopsis plants. Analysis of T-DNA knockout mutants and biochemical analysis using a specific antibody revealed that the p44 is encoded by a SnRK2-type protein kinase gene, SRK2E. The srk2e mutation resulted in a wilty phenotype mainly due to loss of stomatal closure in response to a rapid humidity decrease. ABAinducible gene expression of rd22 and rd29B was suppressed in srk2e. These results show that SRK2E plays an important role in ABA signaling in response to water stress.

ABI1 and ABI2 encode PP2C-type protein phosphatases and are thought to negatively regulate many aspects of abscisic acid (ABA) signaling, including stomatal closure in Arabidopsis. In contrast, SRK2E/OST1/SnRK2.6 encodes an Arabidopsis SnRK2 protein kinase and acts as a positive regulator in the ABA-induced stomatal closure. SRK2E/OST1 is activated by osmotic stress as well as by ABA, but the independence of the two activation processes has not yet been determined. Additionally, interaction between SRK2E/OST1 and PP2C-type phosphatases (ABI1 and ABI2) is not understood. In the present study, we demonstrated that the abi1-1 mutation, but not the abi2-1 mutation, strongly inhibited ABA-dependent SRK2E/OST1 activation. In contrast, osmotic stress activated SRK2E/OST1 even in abi1-1 and aba2-1 plants. The C-terminal regulatory domain of SRK2E/OST1 was required for its activation by both ABA and osmotic stress in Arabidopsis. The C-terminal domain was functionally divided into Domains I and II. Domain II was required only for the ABA-dependent activation of SRK2E/OST1, whereas Domain I was responsible for the ABA-independent activation. Full-length SRK2E/OST1 completely complemented the wilty phenotype of the srk2e mutant, but SRK2E/OST1 lacking Domain II did not. Domain II interacted with the ABI1 protein in a yeast two-hybrid assay. Our results suggested that the direct interaction between SRK2E/OST1 and ABI1 through Domain II plays a critical role in the control of stomatal closure.

SummaryMitogen-activated protein kinase (MAP kinase, MAPK) cascades play pivotal roles in signal transduction of extracellular stimuli, such as environmental stresses and growth regulators, in various organisms. Arabidopsis thaliana MAP kinases constitute a gene family, but stimulatory signals for each MAP kinase have not been elucidated. Here we show that environmental stresses such as low temperature, low humidity, hyper-osmolarity, touch and wounding induce rapid and transient activation of the Arabidopsis MAP kinases ATMPK4 and ATMPK6. Activation of ATMPK4 and ATMPK6 was associated with tyrosine phosphorylation but not with the amounts of mRNA or protein. Kinetics during activation differ between these two MAP kinases. These results suggest that ATMPK4 and ATMPK6 are involved in distinct signal transduction pathways responding to these environmental stresses.