CARK1 phosphorylates subfamily III members of ABA receptors

Abscisic acid (ABA) plays a vital role in responses to abiotic stresses that allow plants to cope with environmental challenges. In this study, we analyzed ABA receptors of subfamily III as the potential targets of Cytosolic ABA Receptor Kinase 1 (CARK1). We previously found that CARK1 phosphorylated the subfamily III member RCAR11 at a distinct threonine residue (T78). Our study now shows the physical interaction of CARK1 with the receptors RCAR12/13/14 in vitro and in vivo. The catalytically inactive form CARK1-N204A did not interact with the receptors. Phosphorylation of these ABA receptors in vitro occurred at a serine/threonine amino acid residue corresponding to T78 in RCAR11, which is located in the loop of β3 within a conserved site. Further analysis revealed that the phosphorylation of RCAR11T78 could increase the sensitivity of the pyr1pyl1pyl2pyl4 quadruple mutant (1124) to ABA, including the inhibition of root elongation and increasing drought tolerance. The analysis of CARK1:1124 complementation and the expression of ABA-related genes indicated that CARK1 could rescue the insensitivity of 1124 to ABA. Our results indicate that CARK1 tends to phosphorylate subfamily III ABA receptors, and the phosphosites RCAR11T78, RCAR12T105, RCAR13T101, and RCAR14S81 are the major sites involved in the activation of the ABA response pathway.