The CDPK-related protein kinase HvCRK2 and HvCRK4 interact with HvCML32 to negatively regulate drought tolerance in transgenic Arabidopsis thaliana

Calcium-dependent protein kinase (CDPK) as one of calcium sensors were play important roles in stress responses. CDPK-related protein kinase (CRK) was identified as subgroup III of CDPK has been characterized in many plants, but the members and functions of CRK genes in hulless barley (Hordeum vulgare L.) has not been clarified. Here, we identified four HvCRK genes and named HvCRK1-4 according to chromosomes localization. Moreover, the physiological function of highly induced genes of HvCRK2 and HvCRK4 were investigated in drought stress tolerance by examining their overexpression transgenic lines functions generated in Arabidopsis thaliana. Under drought stress, both overexpression HvCRK2 and HvCRK4 displayed reduced drought resistance, and accompanied by higher accumulation levels of ROS. Notably, overexpression of HvCRK2 and HvCRK4 reduced sensitivity to exogenous ABA, meanwhile the expression of ABA-responsive genes in transgenic plants were down-regulated compared to the wild type in response to drought stress. Furthermore, the physically interaction of HvCRK2 and HvCRK4 with calmodulin (CaM) and calmodulin-like (CML) proteins were determined in vivo, the further results showed that HvCML32 binds to HvCRK2/4 S_TKC structural domains and negatively regulates drought tolerance. In summary, this study identified HvCRK members and indicated that HvCRK2 and HvCRK4 genes play negative roles in drought tolerance, and provide insight into potential molecular mechanism of HvCRK2 and HvCRK4 in response to drought stress.