ZmCPK1, a calcium-independent kinase member of the Zea mays CDPK gene family, functions as a negative regulator in cold stress signalling

Calcium-dependent protein kinases (CDPKs) have been shown to play important roles in plant environmental stress signal transduction. We report on the identification of ZmCPK1 as a member of the maize (Zea mays) CDPK gene family involved in the regulation of the maize cold stress response. Based upon in silico analysis of the Z.mays cv. B73 genome, we identified that the maize CDPK gene family consists of 39 members. Two CDPK members were selected whose gene expression was either increased (Zmcpk1) or decreased (Zmcpk25) in response to cold exposure. Biochemical analysis demonstrated that ZmCPK1 displays calcium-independent protein kinase activity. The C-terminal calcium-binding domain of ZmCPK1 was sufficient to mediate calcium independency of a previously calcium-dependent enzyme in chimeric ZmCPK25-CPK1 proteins. Furthermore, co-transfection of maize mesophyll protoplasts with active full-length ZmCPK1 suppressed the expression of a cold-induced marker gene, Zmerf3 (ZmCOI6.21). In accordance, heterologous overexpression of ZmCPK1 in Arabidopsis thaliana yielded plants with altered acclimation-induced frost tolerance. Our results identify ZmCPK1 as a negative regulator of cold stress signalling in maize. Our manuscript extends the characterization of the maize CDPK gene family by the identification of so far undescribed isoforms as well as by compiling all existing information about this gene family (TableI). Two CDPK isoforms, ZmCPK1 and ZmCPK25, were functionally described in the maize cold stress response where Zmcpk1 gene expression is up-regulated by cold exposure while Zmcpk25 is repressed. Upon ectopic expression of the biochemical active, but not of kinase-deficient, ZmCPK1 protein cold-inducible marker gene Zmerf3 is repressed. Consistent with a role as negative regulator of the plant cold stress response transgenic ZmCPK1 Arabidopsis display altered acclimation-induced freezing tolerance. Biochemical analysis reveals low but constitutive and calcium-independent protein kinase activity for ZmCPK1. Our data identify ZmCPK1 as a negative regulator of the maize cold stress response whose function is controlled on transcriptional level rather than by post-translational modification through calcium binding.