CPK2 Enhances ABA Sensitivity in Seed Germination and Root Growth by Promoting ABA-Induced <i>ABI5</i> Expression and ABI5 Protein Stability

Abscisic acid (ABA) is a crucial phytohormone that functions as a master regulator of plant growth and development, as well as responses to diverse abiotic stresses, by integrating environmental cues with developmental programs. The transcription factor ABA INSENSITIVE 5 (ABI5) functions at the central hub of the ABA signaling pathway and mediates the expression of its target genes. Emerging evidence reveals extensive crosstalk between calcium-dependent protein kinases (CPKs)-mediated calcium signaling and the ABA-ABI5 cascade, enabling plants to balance growth and stress responses. However, the molecular mechanisms underlying the interactions between CPKs and ABA-ABI5 signaling are still elusive. In this study, we revealed that CPK2 enhances sensitivity to ABA during both seed germination and seedling root growth by promoting ABA-induced <i>ABI5</i> expression and increasing ABA-mediated ABI5 stability. Compared to the wildtype (Col-0), the <i>CPK2</i>-OE line exhibited the highest sensitivity to ABA in both seed germination and root growth, while the <i>cpk2abi5-7</i> double mutant showed the least sensitivity. The single mutants <i>cpk2</i> and <i>abi5-7</i>, as well as the <i>abi5-7CPK2-OE2</i> line, displayed intermediate phenotypes, suggesting that CPK2 acts upstream of ABI5. Biochemical and molecular biological studies revealed that CPK2 physically interacts with ABI5 and directly phosphorylates it at Ser42, Ser145, and Thr201. Moreover, both ABA-induced <i>ABI5</i> expression and protein accumulation were significantly reduced in <i>cpk2</i> mutants upon ABA treatment. Taken together, these findings provide compelling evidence that CPK2 exacerbates the ABA inhibition of seed germination and root growth by enhancing both the expression and stability of ABI5, thereby reinforcing stress adaptation during early plant development.