Abscisic acid-dependent multisite phosphorylation regulates the activity of a transcription activator AREB1

Abstract

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-...

ABA receptor PYL9 promotes drought resistance and leaf senescence

“…Phosphorylation of ABFs and RAV1 by SnRK2s is important for their functions in ABA-induced leaf senescence (Fig. 4) and increased survival (15). When phosphorylated by SnRK2s, RAV1 and ABFs increase the expression of NAC transcription factors through ABRE motifs and/or RAV1-binding motifs ( Fig.…”

“…ABA-activated SnRK2s phosphorylate ABF transcription factors, which activate these factors and enable them to regulate expression of ABA-responsive genes (15). To identify the transcription factors involved in ABA-induced leaf senescence, we cloned several ABFs, including ABF2, ABI5, enhanced EM level (EEL), and AREB3, and coexpressed them with SAG12-LUC in Col-0 leaf protoplasts (Fig.…”

“…4D). The phosphorylation of ABF2 at amino acid residues S26, S86, S94, and T135 is important for stress-responsive gene expression in Arabidopsis, and these sites are putatively phosphorylated by SnRK2s (15,25). Expression of SnRK2.6 significantly enhanced the ability of ABF2 to increase SAG12-LUC expression in the presence of ABA.…”

“…The ABA-bound PYLs prevent clade A protein phosphatase type 2Cs (PP2Cs) from inhibiting the sucrose nonfermenting 1-related protein kinase 2s (SnRK2s). ABA-activated SnRK2s phosphorylate transcription factors, such as ABA-responsive element-binding factors (ABFs), and these phosphorylated ABFs regulate the expression of ABA-responsive genes (15). In Arabidopsis, 14 PYLs function diversely and redundantly in ABA and drought-stress signaling (16)(17)(18)(19).…”

Regulation of Leaf Starch Degradation by Abscisic Acid Is Important for Osmotic Stress Tolerance in Plants

“…Statistical significances determined by unpaired two-tailed Student's t tests: *P < 0.05 for the indicated comparison; #P < 0.05 mutant versus the wild type at the indicated time points; n.s., not significant. SNF1-related kinase 2s (SnRK2s) Furihata et al, 2006), enabling them to bind the ABRE motifs in the promoters of the target genes, thereby activating their expression (Yoshida et al, 2014;Fujita et al, 2013).…”