title: Mutational Evidence for the Critical Role of CBF Transcription Factors in Cold Acclimation in Arabidopsis.
abstract: The three tandemly arranged CBF genes, CBF1, CBF2, and CBF3, are involved in cold acclimation. Due to the lack of stable loss-of-function Arabidopsis (Arabidopsis thaliana) mutants deficient in all three CBF genes, it is still unclear whether the CBF genes are essential for freezing tolerance and whether they may have other functions besides cold acclimation. In this study, we used the CRISPR/Cas9 system to generate cbf single, double, and triple mutants. Compared to the wild type, the cbf triple mutants are extremely sensitive to freezing after cold acclimation, demonstrating that the three CBF genes are essential for cold acclimation. Our results show that the three CBF genes also contribute to basal freezing tolerance. Unexpectedly, we found that the cbf triple mutants are defective in seedling development and salt stress tolerance. Transcript profiling revealed that the CBF genes regulate 414 cold-responsive (COR) genes, of which 346 are CBF-activated genes and 68 are CBF-repressed genes. The analysis suggested that CBF proteins are extensively involved in the regulation of carbohydrate and lipid metabolism, cell wall modification, and gene transcription. Interestingly, like the triple mutants, cbf2 cbf3 double mutants are more sensitive to freezing after cold acclimation compared to the wild type, but cbf1 cbf3 double mutants are more resistant, suggesting that CBF2 is more important than CBF1 and CBF3 in cold acclimation-dependent freezing tolerance. Our results not only demonstrate that the three CBF genes together are required for cold acclimation and freezing tolerance, but also reveal that they are important for salt tolerance and seedling development.
authors.affiliation: Shanghai Center for Plant Stress Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China (C.Z., Z.Z., S.X., J.-K.Z.);Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, Indiana 47907 (C.Z., S.X., T.S., Y.L., J.-K.Z.);Key Laboratory of Crop Physiology and Ecology in Southern China, Ministry of Agriculture/Hi-Tech Key Laboratory of Information Agriculture of Jiangsu Province, Nanjing Agricultural University, Nanjing 210095, China (T.S.); andCollege of Life Science, Yunnan University, Kunming 650091, China (Y.L.).
authors.forename: Chunzhao
authors.identifier: 0000-0003-0284-2095
authors.initials: C
authors.lastname: Zhao
authors.affiliation: Shanghai Center for Plant Stress Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China (C.Z., Z.Z., S.X., J.-K.Z.);Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, Indiana 47907 (C.Z., S.X., T.S., Y.L., J.-K.Z.);Key Laboratory of Crop Physiology and Ecology in Southern China, Ministry of Agriculture/Hi-Tech Key Laboratory of Information Agriculture of Jiangsu Province, Nanjing Agricultural University, Nanjing 210095, China (T.S.); andCollege of Life Science, Yunnan University, Kunming 650091, China (Y.L.).
authors.forename: Zhengjing
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Shanghai Center for Plant Stress Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China (C.Z., Z.Z., S.X., J.-K.Z.);Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, Indiana 47907 (C.Z., S.X., T.S., Y.L., J.-K.Z.);Key Laboratory of Crop Physiology and Ecology in Southern China, Ministry of Agriculture/Hi-Tech Key Laboratory of Information Agriculture of Jiangsu Province, Nanjing Agricultural University, Nanjing 210095, China (T.S.); andCollege of Life Science, Yunnan University, Kunming 650091, China (Y.L.).
authors.forename: Shaojun
authors.identifier: 0000-0002-6719-9814
authors.initials: S
authors.lastname: Xie
authors.affiliation: Shanghai Center for Plant Stress Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China (C.Z., Z.Z., S.X., J.-K.Z.);Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, Indiana 47907 (C.Z., S.X., T.S., Y.L., J.-K.Z.);Key Laboratory of Crop Physiology and Ecology in Southern China, Ministry of Agriculture/Hi-Tech Key Laboratory of Information Agriculture of Jiangsu Province, Nanjing Agricultural University, Nanjing 210095, China (T.S.); andCollege of Life Science, Yunnan University, Kunming 650091, China (Y.L.).
authors.forename: Tong
authors.identifier: 0000-0003-4002-6232
authors.initials: T
authors.lastname: Si
authors.affiliation: Shanghai Center for Plant Stress Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China (C.Z., Z.Z., S.X., J.-K.Z.);Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, Indiana 47907 (C.Z., S.X., T.S., Y.L., J.-K.Z.);Key Laboratory of Crop Physiology and Ecology in Southern China, Ministry of Agriculture/Hi-Tech Key Laboratory of Information Agriculture of Jiangsu Province, Nanjing Agricultural University, Nanjing 210095, China (T.S.); andCollege of Life Science, Yunnan University, Kunming 650091, China (Y.L.).
authors.forename: Yuanya
authors.initials: Y
authors.lastname: Li
authors.affiliation: Shanghai Center for Plant Stress Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China (C.Z., Z.Z., S.X., J.-K.Z.);Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, Indiana 47907 (C.Z., S.X., T.S., Y.L., J.-K.Z.);Key Laboratory of Crop Physiology and Ecology in Southern China, Ministry of Agriculture/Hi-Tech Key Laboratory of Information Agriculture of Jiangsu Province, Nanjing Agricultural University, Nanjing 210095, China (T.S.); andCollege of Life Science, Yunnan University, Kunming 650091, China (Y.L.) jkzhu@sibs.ac.cn.
authors.forename: Jian-Kang
authors.identifier: 0000-0001-5134-731X
authors.initials: JK
authors.lastname: Zhu
mesh_terms: D000064:Acclimatization / Q000187:drug effects / Q000235:genetics*; D017360:Arabidopsis / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development / Q000502:physiology*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D003080:Cold Temperature; D005615:Freezing; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D017343:Genes, Plant; D018525:Germination / Q000187:drug effects / Q000235:genetics; D009154:Mutation / Q000235:genetics*; D009997:Osmotic Pressure / Q000187:drug effects; D010641:Phenotype; D012639:Seeds / Q000187:drug effects / Q000235:genetics / Q000254:growth & development; D012965:Sodium Chloride / Q000494:pharmacology; D013312:Stress, Physiological / Q000187:drug effects; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D029681:Arabidopsis Proteins; D014157:Transcription Factors; D012965:Sodium Chloride