The calcium-regulated kinase MsCIPK4 confers drought and salt stress tolerance in alfalfa by enhancing ROS scavenging

Protein kinases play important roles in regulating the response to various abiotic stress in plants, of which Calcineurin B-like protein-interacting protein kinases (CIPKs) are important components of Ca^2 + signaling pathway under abiotic stress. Here we characterized an abiotic stress-induced CIPK gene (MsCIPK4) from alfalfa (Medicago sativa), which was predominantly expressed in the roots. The deduced MsCIPK4 protein encodes 410 amino acids, and contains four conserved domains (e.g. ATP binding site, NAF motif, activation loop, and PPI motif). Subcellular localization assay revealed that MsCIPK4 was targeted to the endoplasmic reticulum. Furthermore, yeast two-hybrid assays showed that MsCIPK4 was able to interact with several CBL proteins (e.g. MsCBL2, MsCBL6, MsCBL7, and MsCBL10). Over-expression of MsCIPK4 in Arabidopsis elevated root length, seed germination and plant growth under NaCl and drought treatment as well as increased SOD, POD and CAT activity and decreased MDA content. Elevated expression of stress-related genes (e.g. ATPase, P5CS, CYP705A5, COR47, HAK5, and RD2) was also observed in the MsCIPK4-over-expressing alfalfa lines. In the transgenic alfalfa, the enzymatic activity of SOD, POD, and CAT increased, whereas MDA content decreased under 200 mM NaCl and 20 % PEG treatments. Collectively, these results suggested that MsCIPK4 was positively associated with the abiotic stress tolerance, which provides valuable reference for molecular breeding of stress tolerance alfalfa.