Three ABA-responsive SnRK2s function redundantly to phosphorylate AKSs. A, In vitro phosphorylation of AKS1 by recombinant proteins of SnRK2.2, SnRK2.3, and SnRK2.6. His-AKS1 and GST-SnRK2s were incubated with or without ATP for 30 min, and AKS1 phosphorylation was detected by protein-blot analysis. B, In vitro pull-down of AKS1 by SnRK2s. GST-SnRK2s were incubated with His-AKS1 and collected by glutathione sepharose beads. Coprecipitated His-AKS1 was detected by immunoblotting with an antibody against His-tag. C, Phosphorylation of AKS1 in snrk2 mutant plants. Guard cell protoplasts from snrk2.6, snrk2.2/2.3, or snrk2.2/2.3/2.6 mutants were incubated with or without 10 µm  ABA for 10 min. Phosphorylation of AKSs was detected by protein-blot analysis. Each experiment has been repeated at least three times with similar results.