ATG AREB1 (At1g45249) AREB2 (At3g19290) ABF3 (At4g34000) ABF1 (At1g49720) No treatmentABA, 6h Chr. 1 areb1 (SALK_002984) // Chr. 3 Chr. 4 abf1-2 (SALK_132819) areb2 (SALK_069523) abf3 (SALK_096965) (a) GTA ATG (b) abf1-1 (SALK_043079) AREB2 ABF3 ABF1 TUB1 AREB1 WT areb1 areb2 abf3 areb1 areb2 abf3 abf1-2 areb2 abf3 abf1-2 areb2 abf3 abf1-1 WT areb1 areb2 abf3 areb1 areb2 abf3 abf1-2 areb2 abf3 abf1-2areb2 abf3 abf1-1 Figure S2. Generation of an areb1 areb2 abf3 abf1 quadruple mutant. (a) Scheme of AREB1, AREB2, ABF3, and ABF1 genes. Exons are indicated as thick lines and introns as thin lines. The position of the T-DNA insertion is shown. Arrowheads indicate the positions of primers used in (b). (b) Expression levels of the AREB1, AREB2, ABF3, and ABF1 genes in each knockout mutant were determined by RT-PCR with total RNA isolated from 12-day-old wild-type and mutant seedlings with or without six-hour treatment of 50 µM ABA. TUB1, β-1 tubulin transcript as a control.

WTareb1 areb2 abf3 areb1 areb2 abf3 abf1-2 areb2 abf3 abf1-1 areb2 abf3 abf1-2 Inflorescence height （ cm ） * ** No. of rosette leaves at bolting ** WT areb1 areb2 abf3 areb1 areb2 abf3 abf1-2 areb2 abf3 abf1-1areb2 abf3 abf1-2 * * * * ** Maximum rosette radius （ mm ） (a) (c) (b) (d) (f) No. of bolting plants (e) Days after germination WTareb1 areb2 abf3areb2 abf3 abf1-1 areb2 abf3 abf1-2areb1 areb2 abf3 abf1-2 Figure S3. Growth phenotypes of areb1 areb2 abf3 abf1 quadruple mutants. (a) Photographs of knockout mutants and WT plants grown for two weeks on GM agar plates and one week on soil. (b) Maximum rosette radius of each plant in (a). Bars indicate the SD; n ≥ 8. (c) Photographs of 1-month-old knockout mutants and WT plants grown for 2 weeks on GM agar plates and then on soil. Bar = 5 cm. (d) Inflorescence heights of each plant in (c). Bars indicate the SD; n ≥ 7. *P < 0.05; **P < 0.01 (t-test). (e, f) Bolting time of knockout mutants and WT plants grown for 2 weeks on GM agar plates and then on soil under long days (16 h light, 8 h dark). The numbers of bolting plants (e) and rosette leaves (f) were counted at time of a 1-cm-high flowering stem. Bars indicate the SD; n = 15. *P < 0.05; **P < 0.01 (t-test). Each experiment was performed three times, of which a representative result is shown.

Figure S4. Analyses of plant water relations in the areb1 areb2 abf3 abf1 quadruple mutants. (a, b) Rates of water loss from 4-week-old plants at 25 ºC and 47% (a) or 25% (b) relative humidity (RH). Bars indicate SD; n ≥ 5. (c, d) Standardized water content of 4-week-old plants at 25 ºC and 47% (c) or 25% (d) RH. Bars indicate SD; n ≥ 5. (e) Stomatal aperture of knockout mutants and WT plants. Stomatal guard cells were observed based on the previously described methods (Fujita et al. 2009; Tanaka et al. 2005). Images of stomatal aperture from rosette leaves of 4-week-old plants grown on soil were obtained using Suzuki’s Universal Micro-Printing (SUMP) method. Stomatal apertures of two leaves were measured for each experiment. Bars indicate the SD; n ≥ 55. water loss rates (%) Standardized water content (%) WTareb1 areb2 abf3areb1 areb2 abf3 abf1-2 Time (h) (a) (c) (b) (d) Time (h) WT areb1 areb2 abf3 areb1 areb2 abf3 abf1-2 Stomatal aperture (µm) (e)

Figure S5. Expression intensities of the genes down-regulated either in the areb1 areb2 abf3 abf1 quadruple mutant or in the areb1 areb2 abf3 triple mutant. (a) Venn diagrams show the number of the genes down-regulated (P 2-fold) either in the areb1 areb2 abf3 abf1-2 quadruple mutant (129, 68, and 193) or in the areb1 areb2 abf3 triple mutant (13, 111, and 69), compared to WT plants treated with dehydration, NaCl, and ABA. (b) The down-regulated genes in one mutant were classified by the means of hybridization signal intensities in WT samples, which were used for each analysis of the mutants. No. of genes (%) Signal intensities areb1 areb2 abf3areb1 areb2 abf3 abf1-2 NaClABADehydration ≤ < (a)NaClABA areb1 areb2 abf3 areb1 areb2 abf3 abf (b) Dehydration ≤ < ≤ < Signal intensities overlapped

RD26 KIN No treatmentDehydrationNaClABA At3g ASFT/HHT/RWP1 Relative expression level WT areb1 areb2 abf3 areb2 abf3 abf1-1areb2 abf3 abf1-2 areb1 areb2 abf3 abf1-2 WT areb1 areb2 abf3 areb2 abf3 abf1-1areb2 abf3 abf1-2 areb1 areb2 abf3 abf1-2 WT areb1 areb2 abf3 areb2 abf3 abf1-1areb2 abf3 abf1-2 areb1 areb2 abf3 abf1-2 WT areb1 areb2 abf3 areb2 abf3 abf1-1areb2 abf3 abf1-2 areb1 areb2 abf3 abf1-2 WT areb1 areb2 abf3 areb2 abf3 abf1-1areb2 abf3 abf1-2 areb1 areb2 abf3 abf1-2 WT areb1 areb2 abf3 areb2 abf3 abf1-1areb2 abf3 abf1-2 areb1 areb2 abf3 abf1-2 WT areb1 areb2 abf3 areb2 abf3 abf1-1areb2 abf3 abf1-2 areb1 areb2 abf3 abf1-2 WT areb1 areb2 abf3 areb2 abf3 abf1-1areb2 abf3 abf1-2 areb1 areb2 abf3 abf1-2 Figure S6. Experimental validation of selected AREB/ABF downstream genes by qRT–PCR analysis. 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.

MYB41 (At4g28110) Figure S7. Expression patterns of MYB41 in response to osmotic stresses and ABA. Pictographic representation of MYB41 expression values obtained from public databases, the eFP browser (