The cold-responsive C-repeat binding factors in Betula platyphylla Suk. positively regulate cold tolerance

Cold stress is one of the most destructive abiotic stresses limiting plant growth and development. CBF (C-repeat binding factor) transcription factors and their roles in cold response have been identified in Arabidopsis as well as several other plant species. However, the biological functions and related molecular mechanisms of CBFs in birch (Betula platyphylla Suk.) remain undetermined. In this study, five cold-responsive BpCBF genes, BpCBF1, BpCBF2, BpCBF7, BpCBF10 and BpCBF12 were cloned. Via protoplast transformation, BpCBF7 was found to be localized in nucleus. The result of yeast one hybrid assay validated the binding of BpCBF7 to the CRT/DRE (C-repeat/dehydration responsive element) elements in the promoter of BpERF1.1 gene. By overexpressing and repressing BpCBFs in birch plants, it was proven that BpCBFs play positive roles in the cold tolerance. At the metabolic level, BpCBFs OE lines had lower ROS accumulation, as well as higher activities of antioxidant enzymes (SOD, POD and CAT) and higher accumulation of protective substances (soluble sugar, soluble protein and proline). Via yeast one hybrid and co-transformation of effector and reporter vectors assay, it was proven that BpCBF7 can regulate the expression of BpERF5 and BpZAT10 genes by directly binding to their promoters. An interacting protein of BpCBF7, BpWRKY17, was identified by yeast two hybrid library sequencing and the interaction was validated with in vivo methods. These results indicates that BpCBFs can increase the cold tolerance of birch plants, partly by gene regulation and protein interaction. This study provides a reference for the research on CBF transcription factors and genetic improvement of forest trees upon abiotic stresses.