CBF Genes are Essential for Cold Acclimation. Arabidopsis thaliana

Many studies support that the CBF genes encoding cold-induced transcription factors can confer freezing tolerance in plants. However, due to a lack of Arabidopsis mutant plants deficient in all three linked CBF genes, a requirement for the CBF genes in cold acclimation and the extent of their contribution to freezing tolerance remain to be determined. In this work, we used the CRISPR/Cas9 technology to generate cbf single, double, and triple mutants. Our results show that the three CBF genes are required for freezing tolerance in both cold acclimation-dependent and -independent manners. Comparison of the freezing tolerance among cbf single, double, and triple mutants indicates that the three CBF genes have redundant functions in cold acclimation-independent freezing tolerance, but CBF2 plays a more important role than CBF1 and CBF3 in cold acclimation-dependent freezing tolerance. In addition, we discovered that the CBF genes are also important for early seedling development and for salt tolerance. Using RNA-Seq data, we identified hundreds of CBF-regulated genes, which are mainly involved in carbohydrate and lipid metabolism, cell wall modification, gene transcription, cell signalling and hormonal responses. These findings help us understand the critical role of CBF genes in cold acclimation and also reveal new functions of these important transcription factors.