Modulation of ABA Signaling by Altering VxG Phi L Motif of PP2Cs in Oryza sativa

Abstract

The abscisic acid (ABA) signaling pathway is regulated by clade A type 2C protein phosphatases (PP2CAs) in plants. In the presence of ABA, PP2Cs release stress/ABA-activated protein kinases by binding to ABA-bound receptors (PYL/RCARs) for activation. Although the wedging tryptophan in PP2Cs is critical in the interaction with PYL/RCARs in Arabidopsis and rice, it remains elusive as to how other interface regions are involved in the interaction. Here, we report the identification of a conserved region on PP2Cs, termed the VxG Phi L motif, which modulates the interaction with PYL/RCARs through its second and fourth residues. The effects of the second and fourth residues on the interaction of OsPP2C50 with several OsPYL/RCAR proteins were investigated by systematic mutagenesis. One OsPP2C50 mutant, VFGML ("FM'') mutant, lowered the affinity to OsPYL/RCAR3 by similar to 15-fold in comparison with the wild-type. Comparison of the crystal structures of wild-type OsPP2C50: ABA: OsPYL/RCAR3 with those composed of FM mutant revealed local conformational changes near the VxG Phi L motif, further supported by hydrogen-deuterium exchange mass spectrometry. In rice protoplasts, ABA signaling was altered by mutations in the VxG Phi L motif. Transgenic Arabidopsis plants overexpressing OsPP2C50 and OsPP2C50FM showed altered ABA sensitivity. Taken together, the VxG Phi L motif of PP2Cs appears to modulate the affinity of PP2Cs with PYL/RCARs and thus likely to alter the ABA signaling, leading to the differential sensitivity to ABA in planta.