A novel positive feedback mechanism of ABI5 phosphorylation by MPK3 regulates ABA signaling in Arabidopsis

Abstract Seed germination is a crucial physiological process regulated by both environmental and endogenous phytohormones. ABA negatively regulates seed germination, post-germination growth and floral transition, however, the cross talks between multiple regulatory pathways involved in seed germination are still unclear. Here, we show that ABA activates two MAP kinases, AtMPK3/AtMPK6 and selectively regulates the transcript of AtMPK3 through ABI5, a master regulator of ABA signaling in Arabidopsis thaliana. As a feedback loop, AtMPK3 interacts and phosphorylates ABI5 at the serine-314 position.ABI5 phosphorylation by MAP kinase positively regulates ABI5 nuclear localization and negatively regulates its dimerization. Further, subcellular localization of ABI5 phospho-null protein suggests the role of phosphorylation in regulation of its cytoplasmic stability and its nuclear dimerization. Overexpression of phospho-null ABI5 in abi5-8 mutant restored the ABA sensitivity during seed germination and delayed the floral transition as compared to phospho-mimeticABI5. Additionally, overexpression of constitutive phosphorylated form of ABI5 in abi5-8 mutant suggests that phosphorylation makes ABI5 partially inactive. Furthermore, phospho-null ABI5 plants showed drought sensitive phenotype whereas, mpk3, mkk4,mkk5, abi5-8 and phospho-mimetic ABI5 plants showed drought tolerant phenotype. Our findings present a new insight between MAP kinase cascade and ABA signaling which collectively regulates the ABA response through ABI5 phosphorylation.