Mutational analysis of protein phosphatase 2C involved in abscisic acid signal transduction in higher plants

Abstract

Protein phosphatase 2C (PP2C) is a class of ubiquitous and evolutionarily conserved serine/threonine PP involved in stress responses in yeasts, mammals, and plants. Here, I present mutational analysis of two Arabidopsis thaliana PP2Cs, encoded by ABI1 and AtPP2C , involved in the plant stress hormone abscisic acid (ABA) signaling in maize mesophyll protoplasts. Consistent with the crystal structure of the human PP2C, the mutation of two conserved motifs in ABI1, predicted to be involved in metal binding and catalysis, abolished PP2C activity. Surprisingly, although the DGH177–179KLN mutant lost the ability to be a negative regulator in ABA signaling, the MED141–143IGH mutant still inhibited ABA-inducible transcription, perhaps through a dominant interfering effect. Moreover, two G to D mutations near the DGH motif eliminated PP2C activity but displayed opposite effects on ABA signaling. The G174D mutant had no effect but the G180D mutant showed strong inhibitory effect on ABA-inducible transcription. Based on the results that a constitutive PP2C blocks but constitutive Ca 2+ -dependent protein kinases (CDPKs) activate ABA responses, the MED141–143IGH and G180D dominant mutants are unlikely to impede the wild-type PP2C and cause hyperphosphorylation of substrates. In contrast, these dominant mutants could trap cellular targets and prevent phosphorylation by PKs required for ABA signaling. The equivalent mutations in AtPP2C showed similar effects on ABA responses. This study suggests a mechanism for the action of dominant PP2C mutants that could serve as valuable tools to understand protein–protein interactions mediating ABA signal transduction in higher plants.