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

Abstract

bZIP-type transcription factors AREBs/ABFs 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 Ser/Thr residues of R-X-X-S/T sites in the conserved regions of AREB1. Amino acid substitutions of R-X-X-S/T 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 Ser/Thr residues to Asp resulted in high transactivation activity without exogenous ABA application. A phosphorylated, transcriptionally active form was achieved by substitution of Ser/Thr in all conserved R-X-X-S/T 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.

Fig. 1.

Putative target sites of protein kinases within AREB conserved regions and ABA-dependent phosphorylation of recombinant AREB proteins by in-gel kinase activity assay. (A) The conserved regions, C1, C2, C3, and C4 contain protein kinase target sequences. In the conserved regions of AREBs, the most common sequences were R-X-X-S/T (C1-1, C2-1/2, C3-1, and C4-1, putative targets for CDPK etc.) and S/T-X-X-E/D (C1-2, C2-3, C3-1, and C3-2, for CK II). Color lines with letters a (red), b (blue), and c (green) indicate the polypeptides used for the in-gel kinase activity assay, which provides data in B–D. (B) ABA-dependent phosphorylation of recombinant AREB proteins by the in-gel kinase activity assay. T87 cell extracts treated with or without 50 μM ABA treatment were resolved on 10% polyacrylamide gel containing recombinant AREB1a and AREB2a polypeptides shown diagrammatically in A. The protein kinase activities were analyzed as described in Materials and Methods. (C) The wild-type and mutated recombinant AREB1b polypeptide shown diagrammatically in A were used as substrates. (D) The wild-type and mutated recombinant AREB1c (shown diagrammatically in A) polypeptides were used as substrates.

Fig. 2.

Properties of AREB1 protein fragment phosphorylation. (A) Effects of protein kinase inhibitor staurosporine on phosphorylation of the recombinant AREB1a polypeptide. Staurosporine (20 and 100 nM) was added to reaction mixture. (B) Effects of BAPTA on phosphorylation of the recombinant AREB1b polypeptide. Na-BAPTA (5 mM final concentration) was added to reaction buffer and equilibrated for 30 min before addition of radiolabeled ATP. In the far right lane, additional kinase activity appeared near 60 kDa (see Results for details). (C) Stress-dependent phosphorylation of the recombinant AREB1b polypeptide. Protein extracts prepared from T87 cells treated for 30 min with 50 μM ABA (Ab), 0.5 M NaCl (Na), and 0.8 M mannitol (Os, high osmolality) and at low temperature (Lt, 4°C) or untreated (Ct) were used for in-gel kinase activity assay. The recombinant AREB1b polypeptide was used as a substrate. (D) ABA-activated SnRK2-type protein kinases phosphorylate the recombinant AREB1b polypeptide. Protein extracts prepared from transgenic T87 cells overexpressing each SnRK2-GFP fusion protein under control of the CaMV 35S promoter were used for in-gel kinase activity assay. Phosphorylated bands derived from SnRK2-GFP fusion proteins were indicated by circles. Arrowheads indicate the position of 42 kDa in A–D.

Fig. 3.

Transient transactivation analysis of amino acid-substituted AREB1. Transient transactivation of the RD29B promoter-GUS fusion gene by AREB1 and its amino acid-substituted proteins by using Arabidopsis protoplasts prepared from T87 suspension cells. The reporter GUS gene driven by the 77-bp DNA fragment of the RD29B promoter containing two ABREs was transfected into T87 protoplasts with each effector plasmid or the vector plasmid (pBI35SΩ) as a control. To normalize for transfection efficiency, the CaMV 35S promoter/luciferase plasmid was cotransfected in each experiment. Each effector plasmid contains the CaMV 35S promoter and TMV Ω sequence fused to wild-type or amino acid-substituted AREB1 ORF. Bars indicate the SD of triplicates. Transformed protoplasts were incubated with (open) or without (solid) 50 μM ABA in culture medium, at 22°C for 16–20 h in the dark in all transient transactivation experiments shown. (A) Effects of each single substitution of the R-X-X-S/T sites. (B) Effects of multiple amino acid substitutions (Ser/Thr to Ala). M1; S94A, M2; S(86, 94)A, M3; S(26, 86, 94)A, M4; S(26, 86, 94)A and T135A, M5; S(26, 86, 94, 413)A and T135A. (C) Effects of single or multiple amino acid substitutions (Ser/Thr to Asp). M6; S94D, M7; S(26, 86, 94, 413)D, M8; S(26, 86, 94, 413)D and T135D.

Fig. 4.

Effects of overexpression of the phosphorylated active form of AREB1 on plant growth and expression of the RD29B gene. (A) Three and one independent lines of transgenic plants harboring 35S:AREB1pa and the pBI121 vector (pBI121) were grown on germination media agar plates, respectively. The relative amount of the AREB1 and RD29B mRNAs in the transgenic plants was analyzed by quantitative RT-PCR (amount of the most abundant point was indicated as 100). Total RNA was prepared from 3-week-old seedlings of transgenic plants. Pictures of 3-day-old (B) and 9 day-old (C) seedlings. (D) Total RNA was prepared from 3-week-old seedlings of transgenic plants treated with or without 50 μM ABA. The relative amount of the RD29B mRNA in the transgenic plants was analyzed by quantitative RT-PCR and was indicated after considering the nontreated vector control as “1.”

Fig. 5.

Effects of overexpression of the phosphorylated active form of AREB1 in the ABA-deficient aba2–1 mutant. (A) Total RNA was prepared from 3-week-old seedlings of transgenic aba2–1 mutants harboring 35S:AREB1pa, 35S:AREB1wt, and pBI121. Quantitative RT-PCR was performed as described in Fig. 4D. (B) Effects of dehydration stress on the plants. Three-week-old transgenic plants were grown on germination media agar plates, transferred to filter papers and left for 30 min. (C) Electrolyte leakage was measured before and after dehydration treatment of 3-week-old plants removed from agar plates, on filter paper for 30 min, as described in ref. . Bars indicate SDs. (D) Photographs of the guard cells were taken through a color laser 3D profile microscope (Keyence, Osaka) as described in ref. . (Scale bars, 50 μm.)

Fig. 6.

Quantitative RT-PCR analysis of up-regulated genes in transgenic plants overexpressing the constitutive active form of AREB1. mRNA accumulation of up-regulated genes identified in microarray analysis were confirmed by using quantitative RT-PCR. (A) Expression of genes for LEA-class proteins [At5g52300 (RD29B), white; At3g17520, gray; At5g66400 (RAB18), black]. (B) Expression of seed-specific genes. [At2g05580, white; At5g50600 (steroleosin), gray; At3g27660 (oleosin), black]. The x axis indicates the relative amount of mRNA after considering the most abundant point as “100.”