Calcium-dependent protein kinases CPK3/4/6/11 and 27 respond to osmotic stress and activate SnRK2s in Arabidopsis

Abstract

Drought and salinity are significant environmental threats that cause hyperosmotic stress in plants, which respond with a transient elevation of cytosolic Ca²⁺ and activation of Snf1-related protein kinase 2s (SnRK2s) and downstream responses. The exact regulators decoding Ca²⁺ signals to activate downstream responses remained unclear. Here, we show that the calcium-dependent protein kinases CPK3/4/6/11 and 27 respond to moderate osmotic stress and dehydration to activate SnRK2 phosphorylation in Arabidopsis. Using quantitative phosphoproteomics in a higher-order mutant lacking 12 pyrabactin resistance 1-like (PYL) abscisic acid (ABA) receptors, we identified six CPKs that are phosphorylated under osmotic stress. CPK3/4/6/11/27 phosphorylate the SnRK2s on multiple phosphosites within the activation loop. The cpk3/4/6/11/27 mutant is defective in SnRK2 activation, seed germination, and seedling growth under mild osmotic stress. Our findings elucidate the critical roles of CPK3/4/6/11/27 in decoding Ca²⁺ signals to activate SnRK2s and demonstrate a CPK-SnRK2 kinase cascade controlling osmotic stress responses in plants.