Arabidopsis PLANT U-BOX44 down-regulates osmotic stress signaling by mediating Ca2+-DEPENDENT PROTEIN KINASE4 degradation.

Calcium (Ca2+)-dependent protein kinases (CPKs) are essential regulators of plant responses to diverse environmental stressors, including osmotic stress. CPKs are activated by an increase in intracellular Ca2+ levels triggered by osmotic stress. However, how the levels of active CPK protein are dynamically and precisely regulated has yet to be determined. Here, we demonstrate that NaCl/mannitol-induced osmotic stress promoted the accumulation of CPK4 protein by disrupting its 26S proteasome-mediated CPK4 degradation in Arabidopsis (Arabidopsis thaliana). We isolated PLANT U-BOX44 (PUB44), a U-box type E3 ubiquitin ligase that ubiquitinates CPK4 and triggers its degradation. A calcium-free or kinase-inactive CPK4 variant was preferentially degraded compared to the Ca2+-bound active form of CPK4. Furthermore, PUB44 exhibited a CPK4-dependent negative role in the response of plants to osmotic stress. Osmotic stress induced the accumulation of CPK4 protein by inhibiting PUB44-mediated CPK4 degradation. The present findings reveal a mechanism for regulating CPK protein levels and establish the relevance of PUB44-dependent CPK4 regulation in modulating plant osmotic stress responses, providing insights into osmotic stress signal transduction mechanisms.