Abstract
Abscisic acid (ABA) is a major phytohormone regulating plant growth and stress responses. Subclass III SnRK2 kinases and clade A type 2C protein phosphatases (PP2Cs) are core components of ABA signaling. Despite advances from phosphoproteomics, major gaps remain, particularly in mapping PP2C dephosphorylation targets and SnRK2-dependent phosphorylation dynamics under non-stress conditions. Here, we performed large-scale LC–MS/MS phosphoproteomic analyses using the subclass III SnRK2 triple mutant srk2dei and the constitutively active PP2C mutant abi1–1C, with and without ABA treatment in Arabidopsis thaliana. We identified 2757 and 2886 differentially regulated phosphopeptides in srk2dei and abi1–1C, respectively. Beyond known ABA signaling components, these datasets revealed numerous previously uncharacterized candidate proteins involved in metabolism, membrane transport, transcription, and cytoskeletal regulation. Integrative analysis uncovered a core set of candidate proteins oppositely regulated by SnRK2-mediated phosphorylation and ABI1-mediated dephosphorylation, defining a coordinated hierarchical network. These results indicate that the SnRK2–PP2C module functions not only in stress-induced ABA responses but also as a central regulator of phosphorylation homeostasis under basal conditions. This study provides a systematic framework for the global SnRK2–PP2C phosphorylation network and reframes ABA signaling as a dynamic homeostatic system.