Preliminary Analysis Of Molecular Mechanism Of Medicago Falcata NAC3 And NAC4 Involved In The Cold And Salt Stresses Respectively

As one of the most important leguminous forages,Medicago are among the primary global crops.Medicago truncatula is widely used in molecular and genetic studies as a leguminous model plant,and the whole-genome sequencing of M.truncatula has been completed.Environmental conditions,such as temperature and soil,are the main reasons which affect the growth of different species of Medicago.Medicago falcata exhibits strong tolerance against drought,cold and soil infertility.Understanding the mechanism of M.falcata under different abiotic stresses could provide us different direction of creating new germplasm.Low temperature and high salinity can evidently change the molecular,biochemical,physiological and morphological behaviors of plants.These stresses can lead to significant crop reduction and huge economic losses.Thus,it is very important to study the molecular mechanism of plants' response and regulation with cold and salinity,and construction of new germplasm with stronger ability of stress-tolerance,which had an important significance for the development of agricultural production.M.falcata is a cultivated species with multiple ploidy levels.PI502449,which is used in this study,is diploid and exhibits strong tolerance against cold.In a previous work,we have built an online database to store the transcriptome data regarding changes in the transcription levels of genes under abiotic stresses,which support us to analyze the molecular mechanism of Medicago in abiotic stresses.Based on our transcriptiome data,we found that NAC and NIP are two key families of transcription factors which situated at the core of the regulatory network.We cloned and named tow NAC family genes,MfNAC3 and MfNAC4.MfNAC3 is significantly induced under cold,drought and salt stresses,and MfNAC4 is significantly induced under salt stress.NAC is one of the largest families of plant-specific transcription factors,many members of this family have been found to respond to the abiotic stress-related regulations,while it is still not clearly of the way which NAC works under cold and salt stresses in Medicago.We generated MfNAC3 transgenic lines and found that the survival rates of MfNAC3 transgenic plants are significantly higher than the wild type plants,which are the same after cold acclimation,and the electrolyte leakage is opposite.These results demonstrated that MfVAC3 transgenic plants have enhanced freezing tolerance under both cold-acclimated and non-acclimated conditions,and motivated us to study the cold-responsive genes in both wild-type and MfNAC3 transgenic plants.We found that the expression levels of MtCBF1-4 were significantly increased in MfNAC3 transgenic plants.The promoter sequences of these four genes were analyzed,and the results showed that the promoter region of MtCBF4 contains the NAC binding motifs.EMSA was performed and the results demonstrate that MfNAC3 proteins specifically bind to the promoter region of MtCBF4.Expression levels of MtICE3,MtCAS15 and MtCAS31 are significantly increased in MfNAC3 transgenic plants.In M.falcata,MfCBF4 was alos induced by cold.According to these results,we speculated that MfNAC3 regulates CBF4 under cold stress in both M.truncatula and M.falcata.Overexpression MtNAC3,which is the homologous gene of MfNAC3,cannot enhance the tolerance against cold stress in M.truncatula.Although there is a high similarity between MfNAC3 and MtNAC3,the differences in the TR region lead to different protein functions,thus affecting their transcription activity,which may be one reason of the different tolerance between M.falcata and M.truncatula under cold stress.We also generated MfNAC4 transgenic lines and found a significant increase in roots growth in the MfNAC4 transgenic lines compared with the wild-type plants under 120 mM NaCl conditions.The expression levels of MtZpt2-1 and MtSrlk were significantly increased in MfNAC4 transgenic plants.We found NAC binding motifs in the promoter region of MtZpt2-1,and the results of EMSA demonstrate that MfNAC4 proteins can bind to this promoter region.According to these results,we speculated that MfNAC3 regulates MtZpt2-1 under salt stress.These results indicate that MfNAC4 response to salt stress,and can enhance salt tolerance in transgenic plants.Above all,we preliminary analysis the molecular mechanism of MfNAC3 and MfNAC4 involved in the cold and salt stresses respectively in this paper,and provide new evidences of the function of NAC transcription factors in Medicago.We also provide new ideas and methods to plant molecular breeding.