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-...

SummaryMany abiotic stress-inducible genes contain two cis-acting elements, namely a dehydration-responsive element (DRE; TACCGACAT) and an ABA-responsive element (ABRE; ACGTGG/TC), in their promoter regions. We precisely analyzed the 120 bp promoter region (À174 to À55) of the Arabidopsis rd29A gene whose expression is induced by dehydration, high-salinity, low-temperature, and abscisic acid (ABA) treatments and whose 120 bp promoter region contains the DRE, DRE/CRT-core motif (A/GCCGAC), and ABRE sequences. Deletion and base substitution analyses of this region showed that the DRE-core motif functions as DRE and that the DRE/DRE-core motif could be a coupling element of ABRE. Gel mobility shift assays revealed that DRE-binding proteins (DREB1s/CBFs and DREB2s) bind to both DRE and the DRE-core motif and that ABRE-binding proteins (AREBs/ABFs) bind to ABRE in the 120 bp promoter region. In addition, transactivation experiments using Arabidopsis leaf protoplasts showed that DREBs and AREBs cumulatively transactivate the expression of a GUS reporter gene fused to the 120 bp promoter region of rd29A. These results indicate that DRE and ABRE are interdependent in the ABA-responsive expression of the rd29A gene in response to ABA in Arabidopsis.

We cloned a cDNA encoding a novel synGAP, syn-GAP-d (GenBank TM accession number AB016962), from a rat brain cDNA library. The clone consisted of 4801 nucleotides with a coding sequence of 3501 nucleotides, encoded a protein consisting of 1166 amino acids with >99% homology with 1092 amino acid overlaps to syn-GAP, and contained a 13-nucleotide insertion to the previously reported synGAP mRNAs, which suggested that the clone was a splice variant of synGAP. We also found that there are at least seven variants in the 3 portion of the synGAP mRNA and that they encoded five different protein isoforms. The coding sequence of these C-terminal variants were classified into ␣1, ␣2, ␤1, ␤2, ␤3, ␤4, and ␥, and synGAP-d was classified as the ␤1 form. The previously reported synGAPs (synGAP-a, -b, and -c and p135synGAP) can be classified as the ␣1 isoform. All isoforms were expressed specifically in the brain. Unexpectedly, the ␤ isoform, which lacks a C-terminal PSD-95-binding motif ((S/T)XV), was more restricted to the postsynaptic density fraction than the motif-containing ␣1 isoform. The ␤ isoform did not interact with PSD-95 but specifically interacted with a nonphosphorylated ␣ subunit of Ca 2؉

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