PMC

Gene ontology (GO) analysis of the down-regulated genes in the srk2d/e/i triple mutant. Bar graphs separately display the numbers of genes classified into each GO term among the genes down-regulated in the srk2d/e/i, areb1 areb2 abf3 abf1-2 and areb1 areb2 abf3 mutants, and the genes down-regulated only in the srk2d/e/i mutant. GO terms are based upon the PageMan profiling tool (Usadel et al. ) and the Arabidopsis Functional Modules Supporting Data (Heyndrickx & Vandepoele ). The numbers of genes in each group are indicated in parentheses.

Four AREB/ABFs are involved in most of active gene expression downstream of SRK2D/E/I. (a) The over-represented promoter motifs found in the two gene groups; the genes commonly down-regulated in the srk2d/e/i, the areb1 areb2 abf3 and the areb1 areb2 abf3 abf1-2 mutants, and the genes down-regulated only in the srk2d/e/i mutant. The 6-mer motifs were extracted using the Element web tool (Mockler et al. ). The numbers of over-represented motifs are indicated in parentheses. (b) Scatter plot of the downstream genes of SRK2D/E/I. The genes were divided into two groups as in (a). The x- and y-axes represent mRNA fold change in wild-type (WT) treated with abscisic acid (ABA), dehydration and NaCl and mRNA intensities in WT without the treatments, respectively, determined by microarray analyses (Fujita et al. ).

Most downstream genes of SRK2D/E/I were down-regulated in the areb1 areb2 abf3 abf1-2 quadruple mutant. (a) Venn diagrams show the number of ABA-, dehydration- or NaCl-responsive genes down-regulated in the areb1 areb2 abf3, the srk2d/e/i triple mutants (Fujita et al. ; Yoshida et al. ) and the areb1 areb2 abf3 abf1-2 quadruple mutant in comparison with the wild-type (WT) expression level after 6 h of treatment with abscisic acid (ABA), dehydration or NaCl, respectively. Among significantly (greater than fourfold) down-regulated genes in the srk2d/e/i triple mutant, the genes down-regulated by at least twofold either in the areb1 areb2 abf3 triple mutant or in the areb1 areb2 abf3 abf1-2 quadruple mutant are indicated as purple. Bar graphs show the numbers of ABRE sequences in each 1 kb upstream region of genes down-regulated by at least fourfold in the srk2d/e/i triple mutant. Each bar is colour-coded according to the Venn diagrams. (b) Gene expression profiles of the down-regulated genes in the srk2d/e/i triple mutant. The genes are listed from top down in the order of their expression changes in WT treated with ABA, dehydration or NaCl.

Number of down-regulated genes in the areb1 areb2 abf3 triple mutant was increased by lack of ABF1. (a) Microarray data from the areb1 areb2 abf3 triple mutant (Fujita et al. ; Yoshida et al. ) were compared with those from the areb1 areb2 abf3 abf1-2 quadruple mutant. Genes induced by dehydration, high salinity or abscisic acid (ABA) in wild-type (WT) plants (Fujita et al. ) were used for further analyses. Venn diagrams show the number of dehydration-, NaCl- or ABA-responsive genes showing reduced expression levels in each multiple mutant in comparison with the WT expression level after 6 h of treatment with dehydration, 250 mm NaCl or 50 μm ABA, respectively. Solid and dashed lines indicate the number of genes reduced more than fourfold and two- to fourfold, respectively. (b) Experimental validation of selected AREB/ABF downstream genes by real-time quantitative reverse transcription-PCR (qRT-PCR) analysis, where the expression level in the WT plants under non-stressed conditions was defined as 1.0. Data represent means and SDs of three replicate reactions.

The areb1 areb2 abf3 abf1 quadruple mutant shows increased sensitivity to drought and reduced sensitivity to abscisic acid (ABA) in primary root growth compared with the areb1 areb2 abf3 triple mutant. (a, b) Plants before and after stress treatment. Watering was withheld from 4-week-old plants for 11–12 d, and then plants were re-watered for 1 week before the photograph was taken. Wild-type (WT) and the mutant plants (n = 5 each) were grown in soil in independent 6 cm pots (a) or a 9 cm pot (b). Survival rates were calculated from the results of four independent experiments (a; n ≥ 10 for each experiment) and three independent experiments (b; n ≥ 20 for each experiment). Similar results were obtained for two quadruple mutant lines in (b), thus representative results from one experiment are shown. (c) Seedlings at 7 and 10 d after transfer to control agar plates (GM with 1% sucrose) or plates containing 150 μm ABA. Seedlings were 4 d old at the time of transfer. (d, e) Relative root growth (d) and fresh weight (e) of seedlings, which were treated as described in (c) at the indicated number of days after transfer, were calculated from the results of three independent experiments (n ≥ 5 for each experiment). Similar results were obtained for two quadruple mutant lines, thus representative results from one experiment are shown. Bars indicate the SD; n ≥ 17. *P < 0.05 (t-test).

ABF1 was induced by stress treatments in vegetative tissues at lower levels compared with AREB1, AREB2 and ABF3. (a) Gene expression profiles of AREB/ABFs and ABI5 analysed using real-time quantitative reverse transcription-PCR (qRT-PCR). cDNAs were synthesized from 1 μg total RNA prepared from 12-day-old whole seedlings with or without dehydration stress treatments, and from the root and aerial parts of 12-day-old seedlings with or without abscisic acid (ABA) and NaCl treatments. For each gene, the expression level was quantified from standard curves derived from each pre-quantified CDS fragment as template for qRT-PCR. The expression levels of AREB1 under non-stressed conditions or AREB1 in the root under non-stressed conditions were defined as 100. Data represent means and standard deviations of three replicate reactions. (b–d) Cellular localization of GFP-ABF1 proteins in the roots and leaf epidermis. Confocal images of GFP fluorescence in root tips (b), root elongation zone (c) and leaf epidermal tissues (d) of ABF1pro:GFP-ABF1 and 35Spro:GFP plants. The GFP fluorescence images and images merged with Nomarski images are shown. Bars = 50 μm. (e) Transient expression analysis of ABF1. Schemes of the effector and reporter constructs used in the transactivation analysis are shown on the left. The effector constructs contain the cauliflower mosaic virus (CaMV) 35S promoter and tobacco mosaic virus Ω sequence fused to ABF1, AREB1, AREB2 or ABF3 cDNA fragments. The reporter construct RD29B-GUS contains five tandem repeats of a 77 bp fragment of the RD29B promoter. The promoters were fused to the −51 RD29B minimal TATA promoter-GUS construct. Protoplasts were co-transfected with the RD29B-GUS reporter and the effector construct. To normalize for transfection efficiency, the pBI35SΩ-ELUC reporter was co-transfected as a control in each experiment. Transactivation experiments were performed three times, of which a representative result is shown. Bars indicate the SD; n = 3. ‘Relative activity’ indicates the multiples of expression compared with the value obtained with the vector control. Nos-T, nopaline synthase terminator. (f) Bimolecular fluorescence complementation (BiFC) visualization of interaction of ABF1 with SRK2D, SRK2E and SRK2I following transient expression in onion epidermal cells. Yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP) fluorescence and merged images from the same field of transfected cells are shown for each transfection combination. A 35Spro:CFP (pGKX-CFP) control plasmid was always co-bombarded to identify transformed cells prior to the analysis of YFP fluorescence. In each transfected cell, YFP fluorescence was normalized against CFP fluorescence. Bars = 50 μm.