Dynamic and Basal Phosphorylation Landscapes of Abscisic Acid Signaling Revealed by Phosphoproteome Analysis in...

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.