The Molecular Mechanism Of MtCBF4 Transcription Factor In Response To Cold Stress In Medicago Truncatula

Cold stress is one of the significant negative environmental factors that constrain the growth,development, and geographical distribution of the plants. During evolution, some species of plants enhance the freezing tolerance after exposed to a period of the non-freezing low temperature, and this mechanism is called "cold acclimation". The DREB1/CBF transcription factor is one of the most important transcriptional factors regulating the expression level of genes involved in cold acclimation.After cereals, legumes are the most significant source of human food and forage worldwide. Alfalfa, as"the king of forage", possess the important economic value, thus it is very necessary that researching the molecular mechanism of legume response to low temperature. The mechanisms through which legumes resist freezing stresses have already been studied at the physiological and molecular levels. We have known that the DREB1/CBF TFs is response to cold stress in Medicago truncatula. The molecular mechanisms linking DREB1/CBF TFs to stress tolerance in legumes, including cold acclimation and freezing tolerance, nevertheless remain mostly unknown.MtCBF4, which was identified from microarray data, is transcriptionally induced by abiotic stress.Overexpressing MtCBF4 enhanced the salt stress tolerance in Medicago truncatula. In this study, we research the role of MtCBF4 in the signaling pathway of cold accliamtion in M. truncatula. The results show that MtCBF4-overexpressing plants exhibited significantly higher freezing tolerance than wild-type plants under non-acclimated and cold-acclimated conditions. And cbf4 mutants show more sensitive to the cold stress than wild-type plants. In addition, many genes involved in cold stress are identified as up-regulated genes in MtCBF4 overexpression transgenic plants using RNAseq Digital Gene Expression Profiling. Among these, we demonstrate that MtCBF4 directly activates the transcription of the COLD ACCLIMATION SPECIFIC 15 (MtCAS15) gene via qRT-PCR, EMSA and ChIP-qPCR. To gain insights into how MtCBF4 is transcriptionally regulated in response to cold, an R2R3-MYB TF, MtMYB3, was identified as binding directly to MYB cis-elements in the MtCBF4 promoter, leading to the inhibition of MtCBF4 expression previously. In this study, we determine the phenotype related to freezing tolerance of MtMYB3 overexpression transgenic plants and myb3 mutant.The results show that the survival rates of MtMYB3-overexpressing plants were decreased substantially,and the electrolyte leakage values were higher than those of wild-type plants under non-acclimated and cold-acclimated conditions. And myb3 mutants showed significantly higher freezing tolerance than wild-type plants, and increased expression level of MtCBF4 and MtCAS15. In addition, MtMYB61,another MYB TF, was identified as an interactor of MtMYB3 previously. In this study, we also determine the phenotype related to freezing tolerance of MtMYB61 overexpression transgenic plants and myb61 mutants. The results demonstrate that MtMYB61 overexpression plants also exhibited significantly higher freezing tolerance than wild-type plants under non-acclimated and cold-acclimated conditions. And myb61 mutants show more sensitive to the cold stress than wild-type plants. The further study of the molecula mechanism of MYB61 enhanced the freezing tolerance shows that MtMYB61 inhibits MtMYB3 binding to the promoter of MtCBF4 via EMSA and ChIP assay. And MtMYB3 transcript levels showed no strong change in response to cold, whereas MtMYB61 expression was rapidly and strongly induced after cold treatment, culminating at 1 h before rapidly returning to basal level. MtCBF4 also strongly responded to cold, which is reaching its highest level between 2.5 and 3.5 h,whereas MtCAS15 transcript levels showed a strong induction at 12 and 24 hIn conclusion, we identified an MYB/CBF transcriptional pathway controlling cold acclimation in M. truncatula. Under normal conditions, the expression of MtCBF4 is repressed by the binding of MtMYB3 to its promoter. Rapidly after a cold stress, MtCBF4 expression is enhanced depending on the transcriptional induction of MtMYB61, which interacts with the DNA-binding domain of MtMYB3,relieving MtMYB3-dependent transcriptional inhibition of MtCBF4. The accumulation of MtCBF4 leads to the direct transcriptional activation of cold acclimation gene expression, such as MtCAS15, and ultimately improves plant freezing tolerance. This study will provide a mechanism of MtCBF4 participate in cold acclimation and response to cold stress in M. truncatula. In this model,MtCBF4/MtCAS15 transcription is precisely controlled by cold depending on the antagonism between MtMYB3 and MtMYB61. This may provoid a theoretical basis for genetic improvements to freezing tolerance in legume.