Overexpression of an Alfalfa Gene, MsCKX5, Confers Cold Tolerance in Transgenic Arabidopsis thaliana

Background: Cytokinin oxidase/dehydrogenase (CKX) can irreversibly degrade cytokinin, regulating the growth and development of plants and helping them cope with environmental stress. Methods: To understand the expression characteristics and the biological functions of CKX in alfalfa (Medicago sativa), a novel gene, designated as MsCKX5 (GenBank: PV934228), was cloned, characterized and overexpressed in Arabidopsis thaliana. Results: qRT-PCR results showed that MsCKX5 gene expression was clearly tissue-specific and had the highest expression level in the stems. In addition, the expression level of MsCKX5 was significantly induced by cold treatments. Heterologous expression of the MsCKX5 gene in A. thaliana could enhance cold tolerance by regulating the activities of antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), and adjust the relative conductivity and malondialdehyde (MDA) content. The combined analysis of transcriptome and metabolome results indicated that flavone and flavonol biosynthesis as well as the plant hormone signal transduction pathways were the main enriched pathways between wild-type and MsCKX5 overexpressed lines. Conclusions: These results provide an important molecular basis for further elucidating the molecular mechanism of plant cold resistance and the breeding of cold-resistant crop varieties.