Takahashi Y , Zhang J , Hsu PK , Ceciliato PHO , Zhang L , Dubeaux G , Munemasa S , Ge C , Zhao Y , Hauser F , Schroeder JI .

R01 GM060396 NIGMS NIH HHS , P42 ES010337 NIEHS NIH HHS , S10 OD021724 NIH HHS , R01 GM114660 NIGMS NIH HHS

	Fig. 1. Identification of MAPKK-kinases that reactivate OST1/SnRK2 kinases by phosphorylation.a Seeds of amiR-HsMYO wild-type (control line) or amiR-ax1117 mutant were sowed on 1/2 MS medium containing 2 μM ABA, or 0.02% EtOH as control, for germination assays. Representative images showing seed germination after 6 days. b The percentage of seedlings showing green cotyledons was analyzed. Data represent mean ± s.d. n = 4 experiments. Each experiment included 64 seeds for each genotype. Letters at the top of columns are grouped based on two-way ANOVA and Tukey’s test, P < 0.05. c Identification of the amiRNA sequence in amiR-ax1117 plants. Black box labels the sequence of amiR-ax1117. The amiR-ax1117 is predicted to include Raf-like protein kinase genes M3Kδ5, M3Kδ7, M3Kδ1, M3Kδ6, and M3Kδ-CTR1 kinase (see Supplementary Fig. 1). d Wild-type (WT) and m3k amiRNA seedlings were incubated with 10 µM ABA for 15 min. In-gel kinase assays were performed using histone type III-S as a substrate. e SnRK2 band intensities as shown in d were measured using ImageJ, n = 3 experiments, error bars show ±s.e.m. f, Recombinant GST-OST1/SnRK2.6 protein was dephosphorylated by alkaline phosphatase in vitro and used for in vitro phosphorylation assays after incubation with CPK6, CPK23 or MPK12 protein kinases. Note visible autophosphorylation activity of CPK6 and CPK23. g Dephosphorylated recombinant His-OST1/SnRK2.6 protein was incubated with kinase domains of seven M3Ks and used for in-gel kinase assays (phylogenetic tree: see Supplementary Fig. 1). Note lanes on the left are from the same gel as lanes in the middle section.
	Fig. 2. MAPKK-kinase-induced OST1/SnRK2.6 Ser171 phosphorylation is essential for ABA activation of OST1/SnRK2.6 activation.a The inactive M3Kδ6 kinase domain mutant (K775W) did not reactivate His-OST1/SnRK2.6 in vitro. b Inactive GST-OST1/SnRK2.6-D140A kinase protein was incubated with M3Kδ6 kinase domain, and in vitro phosphorylation assays were performed with 32P-ATP. c Recombinant inactive OST1(D140A) and M3Kδ1 kinase domains were incubated with ATP. A mass spectrum of phosphorylated OST1 peptide (SSVLHpSQPK) is shown. pS indicates phosphorylated Ser171 of OST1(D140A). d Phosphorylation at Ser171 was not detectable after in vitro autophosphorylation of OST1/SnRK2.6, but was consistently phosphorylated in the presence of M3Kδ1. e OST1(S171A)-GFP was transiently expressed in Arabidopsis mesophyll cell protoplasts. Protoplasts were incubated with 10 µM ABA or control buffer for 15 min, and OST1/SnRK2.6 activities were analyzed by in-gel kinase assays.
	Fig. 3. OST1/SnRK2.6 Ser171 is essential for ABA-induced stomatal closure and S-type anion channel activation in planta.a Stomatal conductances were analyzed in intact detached leaves of stable transgenic Arabidopsis [pUBQ10:OST1-HF/ost1-3 (OST1-comp2) and pUBQ10:OST1-S171A-HF/ost1-3 (S171A-comp2)]. 2 μM ABA was applied to petioles at 0 min. Data presented are means ± s.e.m. (n = 4 leaves from four plants for each genotype). b Leaf temperatures of Col, ost1-3, OST1-comp2 and S171A-comp2 were measured by thermal imaging. Plants were sprayed with 20 µM ABA, and thermal images were taken after 3 h. The bright field image shows where leaves from neighboring plants overlapped. c, Leaf temperatures were measured by using Fiji software (n = 5 experiments, means ± s.e.m.). Letters at the top of columns are grouped based on one-way ANOVA and Tukey’s test, P < 0.05. d ABA-activated S-type anion channel currents were investigated by patch-clamp analyses using guard cell protoplasts from the transgenic Arabidopsis lines pUBQ10:OST1-HF/ost1-3 (OST1-WT) and pUBQ10:OST1-S171A-HF/ost1-3 (OST1-S171A). e Average current-voltage relationship of S-type anion channel as shown in d. Data presented are means ± s.e.m. f Kinase activities of OST1(S171A) in mesophyll cells from stably-transformed homozygous transgenic plants were investigated by in-gel kinase assays. Protoplasts were incubated with 10 µM ABA for 15 min.
	Fig. 4. MAPKK-kinases are essential for ABA signalling module.a, b In vitro reconstitution of ABA-induced OST1/SnRK2 activation without M3Kδ6 (a) or with M3Kδ6 (b). The recombinant proteins His-PYR1/RCAR11, His-OST1/SnRK2.6 without (a) or with (b) GST-M3Kδ6 kinase domain were mixed. After addition of His-HAB1, protein solutions were incubated for 10 min. Then, 50 µM ABA was added to the protein solution. Reactions were stopped at the indicated times. OST1/SnRK2.6 kinase activities were detected by in-gel kinase assays. c Recombinant His-PYR1/RCAR11, His-HAB1, His-OST1/SnRK2.6, His-AKS1, and GST-M3Kδ6 kinase domain were mixed as indicated above the gel. 50 µM ABA was added before (lane 5) or after (lanes 6 and 8) 10 min incubation at room temperature. Then, 100 µM ATP was added (lanes 2–8) to trigger phosphorylation reactions for 10 min. Note that M3Kδ6 is required for ABA-induced AKS1 phosphorylation when ABA is added 10 min after exposure to HAB1-PP2C-including mix (compare lanes 6 and 8). Reactions were stopped by addition of SDS-PAGE loading buffer. Phosphorylation of AKS1 is detected by binding of 14-3-3Phi (At1g35160) to the phosphorylated AKS1 protein15. AKS1 phosphorylation is shown by protein-blot (top), and protein amount is monitored by immuno-blot (bottom). d–f Reconstitution of ABA-activation of SLAC1 channels in Xenopus oocytes, in the presence or absence of M3Ks. d Representative whole-cell chloride current recordings of oocytes co-expressing the indicated proteins, without (control) or with injection of 50 μM ABA (+ABA). Currents were recorded in response to voltage pulses ranging from +40 mV to −120 mV in −20 mV steps with a holding potential at 0 mV and a final tail potential of −120 mV. e Mean current-voltage curves of oocytes co-expressing the indicated proteins, with or without injection of ABA. The symbols of H2O control, OST1 + SLAC1, PYL9/RCAR1 + ABI1 + OST1 + SLAC1, PYL9/RCAR1 + ABI1 + OST1 + SLAC1 + ABA, and PYL9/RCAR1 + ABI1 + OST1 + SLAC1 + M3Ks overlapped. Single symbols are shown for some data points for better viewing. f Average SLAC1-mediated currents at −100 mV, co-expressing the indicated proteins, in the presence or absence of 50 μM ABA. Data from three independent batches of oocytes showed similar results. One representative batch of oocytes is shown, with the number of oocytes in that batch indicated in parentheses. H2O, OST1 + SLAC1, PYL9/RCAR1 + ABI1 + OST1 + SLAC1, and PYL9/RCAR1 + ABI1 + OST1 + SLAC1 + ABA controls are the same data in both panels in e as the data are from the same oocyte batch. Error bars denote mean ± s.e.m. Means with letters (a, b, c, and d) are grouped based on one-way ANOVA and Tukey’s multiple comparisons test, P < 0.05.
	Fig. 5. MAPKK-kinases are required for plant ABA response.a Genome structures and T-DNA insertion sites of M3K genes are shown. b Genomic regions of CRISPR/Cas9-mediated M3Kδ1 and M3Kδ7 gene deletions are shown. These deletions were introduced in the m3kδ6-2 T-DNA knockout mutant as a background. c RT-PCR assays show transcripts of kinase domains of M3Ks in the m3kδ1crispr m3kδ6-2 m3kδ7crispr triple mutant. d m3kδ1crispr m3kδ6-2 m3kδ7crispr triple mutant seedlings were grown on 1/2 MS plates supplemented with 2 µM ABA or ethanol (control) for 16 days. e Seedlings showing green cotyledons as in d were counted. n = 3 (EtOH) and n = 4 (ABA) experiments, means ± s.d., 45 seeds per genotype were used in each experiment. f RT-PCR shows M3Kδ1, δ6, and δ7 expression in the indicated m3k T-DNA insertion mutants. δ6(KD) refers to primers that amplify the M3Kδ6 kinase domain in the m3kδ6-1 T-DNA line. g m3kδ1 m3kδ6-1 m3kδ7 T-DNA triple mutant plants were grown on 1/2MS plates supplemented with 0.8 µM ABA for 9 days. h Seedlings showing green cotyledons as in g were counted. n = 3, means ± s.d., 60–88 seeds were used per genotype in each assay. i Three amiRNA lines targeting M3Kδ1, δ6, and δ7 were grown on 1/2MS plates supplemented with EtOH (control) or 2 µM ABA for 9 days. As a control line, the amiRNA-HsMYO line21 was used. j Seedlings showing green cotyledons as in i were counted. n = 3 (EtOH) and 4 (ABA) experiments, means ± s.d., 81 seeds per genotype were analyzed in each experiment. (e, h, and i) Letters at the top of columns are grouped based on two-way ANOVA and Tukey’s test, P < 0.05.
	Fig. 6. MAPKK-kinases mediate ABA- and osmotic stress-induced SnRK2 activation in planta.a m3kδ1crispr m3kδ6-2 m3kδ7crispr triple mutant seedlings were incubated in 10 µM ABA or 0.3 M mannitol (Osmo) for 15 min. SnRK2 activities were tested by in-gel kinase assays. Arrowhead shows SnRK2 activity23. b Normalized band intensities as shown in a were measured by using ImageJ. n = 4, means ± s.e.m. c m3kδ1 m3kδ6-1 m3kδ7 T-DNA triple mutant seedlings were incubated in 10 µM ABA or 0.3 M mannitol (Osmo) for 15 min. SnRK2 activities were analyzed by in-gel kinase assays. d Normalized band intensities as shown in c were measured by using ImageJ. n = 4 experiments, means ± s.e.m. e, Recombinant GST-tagged Arabidopsis SnRK2 protein kinases were incubated with M3Kδ1 kinase domain. SnRK2 kinase activities were analyzed by in-gel kinase assays. f M3Kδ6-FLAG was transiently expressed in Arabidopsis mesophyll cell protoplasts. Protoplasts were incubated in 0.8 M mannitol (Osmo) for 15 min. M3Kδ6 proteins were detected by immuno-blot using anti-FLAG antibody. In the Osmo lane, the M3Kδ6 band showed a slight mobility shift as indicated by an asterisk.

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