Fig. 4.

(A) In vitro reconstitution analysis of the ABA signaling cascade. Immunoprecipitated SRK2E-GFP (ABA-activated) was incubated with GST-ABI1, GST-abi1–1, ABI1, or PYR1 proteins in the presence or absence of 10 μM ABA. SnRK2 activity was monitored with an in-gel phosphorylation assay (IGP) using histone as a substrate. Immunoprecipitates (IP) were checked by Western blot analysis (WB) against an anti-GFP antibody. Arrows indicate SRK2E-GFP. This result was confirmed through several replications. (B) Proposed model of the ABA signaling pathway in Arabidopsis. Left, in the absence of ABA, PP2C dephosphorylates and inactivates SnRK2. Middle, in the presence of ABA, RCAR/PYR interacts with PP2C, and SnRK2 is released from negative regulation and converted to an active form. The activated SnRK2 phosphorylates ABA-responsive transcription factors or unknown substrates to induce ABA responses. Right, even in the presence of ABA, the abi1–1 mutant of PP2C constitutively dephosphorylates SnRK2, consequently conferring dominant ABA insensitivity. In this model, PP2C constitutively binds to domain II at the C terminus of SnRK2. Red and blue colors indicate an active or inactive form, respectively.