Cloning And Functional Analysis Of CBF/DREB1 Homologous Genes In Mei Cultivars With Constrating Levels Of Freezing Tolerance

Mei(Prunus mume Sieb. et Zucc.), an important traditional ornamental flower, originated in China, has been domesticated in China for more than 3000 years. However, its growth and development are severely restricted by the early coming, long-standing winter and the cold-dry wind, especially in Beijing and regions of above 40 degrees north latitude. The C-repeat binding factor/Dehydration responsive element binding factors(CBF/DREB1) pathway has important roles in plant responses to cold stress. However,knowledge on the function of CBF/DREB1 genes from Mei and its regulatory roles in freezing and other abiotic stresses is limited. To elucidate the molecular mechanism underlying the response to cold stress in Mei, 11 Mei cultivars with constrasting in freezing tolerance, including ‘XueMei’, ‘XiaoGongfen’, ‘XiaolvEr’, ‘Beijing Xiaomei’, ‘XueYuehua’, ‘SongChun’, ‘FengHou’, ‘WuZangye’, ‘HuaHudie’, ‘YangGuifei’ and ‘MeirenMei’ were used in this study. The PmCBFa, PmCBFb and PmCBFc were isolated from 10 Mei cultivars. The sequence alignment was performed to analyze the difference among 11 Mei cultivars. The difference of the expression patterns of PmCBFa, PmCBFb and PmCBFc under cold stress was analyzed among 5 Meicultivars including ‘Xue Mei’, ‘XiaolvEr’, ‘Songchun’, ‘HuaHudie’ and ‘Meiren Mei’. The expression anlysis of the other five CBFs from ‘XiaolvEr’, ‘HuaHudie’ and ‘MeirenMei’ under cold stress was also examined. In addition, the expression patterns of PmCBFa, PmCBFb and PmCBFc under various abiotic stresses from ‘HuaHudie’ were detected. The functional differences of 12 PmCBFs under freezing stress in transgenic Arabidopsis were compared. Finally, the molecular mechanism of PmhCBFc overexpression increased freezing stress tolerance in transgenic Arabidopsis was further deciphered. The main results are as follows.1. Three PmCBFs from 10 Mei cultivars were isolated by using homology cloning strategy and FNPI-PCR approach, and named PmCBFa, PmCBFb and PmCBFc, respectively. Sequence analysis showed that these PmCBFs exhibited all the typical features of CBF transcription factors including a putative nuclear localization signal(NLS), AP2 domain, DSAWR and LWSY domain. Further analysis displayed that the sequences of PmCBFs were highly conserved and minor differences existed in several single amino acid sites althoughcold hardiness varied between the tested cultivars. Thisindicates that the coding area of PmCBFs can withstandstrong selective pressure during the evolutionary progress.Phylogenetic analysis of PmCBFs and DREB1/CBF peptide sequences from Prunus plants displayed that the PmCBFs were clustered into three distinct subgroups, named PmCBFa, PmCBFb, and Pm CBFc, respectively, while Arabidopsis CBFs were grouped into another clade.2. qRT-PCR was used to compare the difference of the expression patterns of PmCBFa, PmCBFb and PmCBFc under cold stress among 5 Mei cultivars including ‘Xue Mei’, ‘XiaolvEr’, ‘SongChun’, ‘HuaHudie’ and ‘MeirenMei’. The results appeared that there was no relationship between the expression level of PmCBFa、PmCBFband PmCBFcwith constrasting levels of freezing tolerance in Mei cultivars. The expression level of the other five PmCBFs from ‘XiaolvEr’, ‘HuaHudie’ and ‘MeirenMei’ under cold stress was also investigated. The results indicated the expression level of these five PmCBFs in high freezing tolerance in ‘SongChun’and ‘MeirenHei’was significantly higher than that of these five PmCBFs in middle freezing tolerance ‘XiaolvEr’during cold treatment. Besides, the expression patterns of PmhCBFsfrom ‘Huahudie’under various abiotic stresses displayed that none of PmCBFs was specific to one stress, but PmhCBFa and PmhCBFb preferred to be induced by cold stress while PmhCBFc appeared to be more induced by H2O2.3. Based on the amino acid sequence analysis, the overexpression vector of PmCBFa、PmCBFband PmCBFc from 4 Mei cultivars with constrasting levels of freezing tolerance was constructed and translated into Arabidopsis to identify their functional differences under cold stress. Overexpression of PmCBFa、PmCBFband PmCBFc from 4 Mei cultivars all conferred tolerance to freezing stress in Arabidopsis. The increased freezing tolerance in Arabidopsis was correlated with the expression level of PmCBFs, while not correlated with this PmCBF gene isolated from certain a Mei cultivar.4. The relationship between PmhCBFc and ROS under freezing stress was investigated. Characterization of overexpressing PmhCBFc plants by ROS staining, MV treatment and determination of antioxidant enzymatic activities during freezing-thawing cycles support the functioning of PmhCBFc during anti-oxidative response. Moreover, we demonstrated that overexpression of PmhCBFc in Arabidopsis enhanced tolerance to both freezing and oxidative stresses. RT-PCR analysis showed that the activation of antioxidant enzymes was consistent with up-regulation of AtCSD2 in the transgenic lines during freezing-thawing cycles. The results suggested that PmhCBFc may function in freezing tolerance, at least in part, by the capacities for ROS scavenging in the transgenic plants.