Genome-Wide Investigation of CPK-Related Kinase (CRK) Gene Family in Arabidopsis thaliana

Calcium-dependent protein kinase (CPK), representing a group of typical Ca²⁺ sensors in plants, has been well characterized in plants. CPK is capable of binding to Ca²⁺, which sequentially activates CPK. CPK-related kinase (CRK) shows protein structures similar to CPK but only contains degenerative EF-hands, which likely makes the activation of CRK Ca²⁺ independent. Compared with CPK, CRK is barely functionally analyzed. In this study, we systematically investigated <i>CRK</i> genes in the Arabidopsis genome. We found that <i>CRK</i> appeared to emerge in land plants, suggesting <i>CPK</i> and <i>CRK</i> are divided at very early stages during plant evolution. In Arabidopsis, the detailed analysis of the calmodulin-like domain of CRK indicated the substitutions of key amino acid residues in its EF-hands result in disrupted Ca²⁺ association. Next, by using a YFP tag, we found that all Arabidopsis CRK proteins were localized at the plasma membrane. After cloning the promoters of all eight <i>CRK</i> genes, we found that <i>CRK</i>s were widely expressed at all stages of Arabidopsis by using GUS staining. Furthermore, the kinase activity of CRK was examined by using phospho-antibody and Pro-Q staining. CRK was shown to possess high autophosphorylation, which was not affected by the presence of Ca²⁺. Moreover, we analyzed the cis-elements of <i>CRK</i> promoters and discovered that stress signals potentially regulate the expression of <i>CRK</i> genes. Consistently, by using quantitative real-time PCR (qPCR), we found a number of <i>CRK</i> genes were regulated by a variety of biotic and abiotic treatments such as flg22, ABA, drought, salt, and high and low temperatures. Furthermore, by utilizing proteomic approaches, we identified more than 100 proteins that interacted with CRK5 <i>in planta</i>. Notably, RLK and channels/transporters were found in CRK5-containing complexes, suggesting they function upstream and downstream of CRK, respectively.