Phosphorylation dynamics of RAF12 and PP2C control SnRK2 activity under hyperosmotic stress in Arabidopsis

Hyperosmotic stress rapidly induces the activation of SNF1-related protein kinases 2 (SnRK2s) that orchestrate plant adaptive responses. However, prolonged activation can unbalance cellular homeostasis. Molecular mechanisms that manage the activation and subsequent deactivation of SnRK2s during osmotic stress signaling are poorly understood. Our findings suggest that type 2C protein phosphatases-ABI1, ABI2, HAI1, and HAI2-cooperatively suppress SnRK2 activities in Arabidopsis. Notably, abi1abi2hai1hai2 quadruple mutant displays reduced hyperosmotic stress sensitivity and partially constitutive stress responses even under normal conditions. We also discovered that B2 Raf-like MAPKKK (RAF12) inhibits HAI2 phosphatase activities through the direct phosphorylation of HAI2, releasing the SnRK2 inhibition. Interestingly, upon hyperosmotic stress, RAF12 rapidly forms reversible condensates. RAF12 condensation, driven by its intrinsically disordered region, potentially facilitates RAF12 kinase activation. Our research elucidates that the RAF-PP2C-SnRK2 phosphorylation switch is involved in perceiving hyperosmotic stress, initiating and amplifying osmotic stress signaling, and subsequently shaping plant adaptive responses.