Reconstitution of the release of AKS1 from KAT1 gene in response to ABA in vitro. A, In vitro DNA-protein pull-down using His-AKS1 and biotin-conjugated KAT1 gene fragments in the presence of recombinant proteins of His-PYR1, His-HAB1, and GST-SnRK2.6. The KAT1 fragments were collected with the streptavidin-coupled beads after 30 min incubation with or without 100 µm  ABA. Phosphorylation of AKS1 and the amount of AKS1 bound to the KAT1 gene fragment were determined by protein-blot and immunoblot analyses, respectively. B, The relative amounts of AKS1 bound to the KAT1 gene fragment. Relative intensity of immunoblot bands of His-AKS1 in DNA-protein pull-down assay was quantified by ImageJ 1.42q. Values are means ± sd (n = 3, *P < 0.01, unpaired Student’s t test). C, A model for transcription inhibition by release of AKS1 from a target gene in response to ABA. AKS1 is bound to both SnRK2 and its target gene in the nucleus. AKS1 promotes the transcription of target genes including KAT1 in the absence of ABA. SnRK2 is activated and phosphorylates AKS1 in response to ABA, and the phosphorylation causes monomerization of AKS1. The monomerized AKS1 is released from DNA, resulting in the repression of transcription. AKS1 may also dissociate from SnRK2.6 after phosphorylation.