Summary Calcium is a ubiquitous second messenger that mediates plant responses to developmental and environmental cues. Calcium‐dependent protein kinases (CDPKs) are key actors of plant signaling that convey calcium signals into physiological responses by phosphorylating various substrates including ion channels, transcription factors and metabolic enzymes. This large diversity of targets confers pivotal roles of CDPKs in shoot and root development, pollen tube growth, stomatal movements, hormonal signaling, transcriptional reprogramming and stress tolerance. On the one hand, specificity in CDPK signaling is achieved by differential calcium sensitivities, expression patterns, subcellular localizations and substrates. On the other hand, CDPKs also target some common substrates to ensure key cellular processes indispensable for plant growth and survival in adverse environmental conditions. In addition, the CDPK‐related protein kinases (CRKs) might be closer to some CDPKs than previously anticipated and could contribute to calcium signaling despite their inability to bind calcium. This review highlights the regulatory properties of Arabidopsis CDPKs and CRKs that coordinate their multifaceted functions in development, immunity and abiotic stress responses.

Calcium signaling mediates most developmental processes and stress responses in plants.Among plant calcium sensors, the calcium-dependent protein kinases display a unique structure harboring both calcium sensing and kinase responding activities. AtCPK5 is an essential member of this family in Arabidopsis that regulates immunity and abiotic stress tolerance. To understand the underlying molecular mechanisms, we implemented a biochemical approach to identify in vivo substrates of AtCPK5. We generated transgenic lines expressing a constitutively active form of AtCPK5 under the control of a dexamethasoneinducible promoter. Lines expressing a kinase-dead version were used as a negative control.By comparing the phosphoproteome of the kinase-active and kinase-dead lines upon dexamethasone treatment, we identified 5 phosphopeptides whose abundance increased specifically in the kinase-active lines. Importantly, we showed that all 5 proteins were phosphorylated in vitro by AtCPK5 in a calcium-dependent manner, suggesting that they are direct targets of AtCPK5. We also detected several interaction patterns between the kinase and the candidates in the cytosol, membranes or nucleus, consistent with the ubiquitous localization of AtCPK5. Finally, we further validated the two phosphosites S245 and S280 targeted by AtCPK5 in the E3 ubiquitin ligase ATL31. Altogether, those results open new perspectives to decipher AtCPK5 biological functions.