Analysis Of Molecular Mechanism Of The Different Cold Tolerance Between Dajiao And Cavendish Banana

Cavendish banana and Dajiao(Musa SPP.)are the most popular fruits and vital staple food aroud the globe.Low temperature is one of the key environmental stresses which greatly affects the global banana production.To understand the molecular mechanism of the different cold tolerance between Cavendish banana and Dajiao,we used comparative transcriptomics and phosphoproteomics analyses for Cavendish banana and Dajiao subjected to cold stress.And based on the results of trascriptome,MpMAPK3 gene was cloned.On the basis of efficient banana transformation system,the function of MpMAPK3 gene on the improvement of banana cold tolerance was studied.The main results were as follows:1.The rapid activation and selective induction of ICE1 and MYBS3 cold tolerance pathways in Dajiao,along with expression of other cold-specific genes,may be one of the main reasons that Dajiao has higher cold resistance than Cavendish banana.1)The results show that 10 and 68 differentially expressed genes(DEGs)are identified for 3 and 6 h of cold stress respectively in Dajiao,while 40 and 238 DEGs are identified respectively in Cavendish banana.2)GO classifications analyses show that the majority of DEGs identified in both Cavendish banana and Dajiao belong to 11 categories including regulation of transcription,response to stress signal transduction,etc.There are 17 DEGs found uniquely in cold-tolerance Dajiao,which were involved in signal transduction,abiotic stress,copper ion equilibrium,photosynthesis and photorespiration,sugar stimulation,protein modifications etc.3)Twelve early responsive genes including ICE1 and MYBS3 were selected and further assessed and confirmed by Q-PCR in the extended time course experiments(0,3,6,24 and 48 h),which revealed significant expression difference of key genes in response to cold stress,especially ICE1 and MYBS3 between cold-sensitive Cavendish banana and cold-tolerant Dajiao.We found that the cold-tolerance pathway appears selectively activated by regulation of ICE1 and MYBS3 expression in Dajiao under different stages of cold stress.We conclude that the rapid activation and selective induction of ICE1 and MYBS3 cold tolerance pathways in Dajiao,along with expression of other cold-specific genes,may be one of the main reasons that Dajiao has higher cold resistance than Cavendish banana.2.MpMAPK3 as a positive regulator of cold signaling confers the cold tolerance of Dajiao by effecting ROS pathway.1)The effect of RNAi of MpMAPK3 on cold-tolerance was evaluated in the transgenic and wild type plants exposed to cold stress.A significant phenotypic difference between transgenic and control plants under cold stress is the transgenic palnts leaves displayed severe necrosis and wilting symptoms,while WT leaves remained normal.Molecular detection showed that RNAi of MpMAPK3 resulted the significant changes of the cold responsive genes compared to WT.2)By comparing the phosphoproteome between transgenic and wild type plants under cold stress,the results showed that there were 31 phosphopeptides which associated with oxidoreductase activity showed significant changed in WT,while no changed in transgenic plants.The results indicated that the suppression expression of MAPK3 may led to the ROS disorders in transgenic palnts.MDA and PPO activity were measured against a time course of cold treatment,the content of MDA in transgenic plants was significant higher than WT,and the magnification of POD activity of transgenic plants under cold tolerance was lower than WT.These results show that the MpMAPK3 as a positive regulator of cold signaling confers the cold tolerance of Dajiao by effecting ROS pathway.3.MKK2 pathway in Dajiao,along with other cold-specific phosphoproteins,appears to be involved in the molecular mechanisms of Dajiao with high tolerance to cold stress.1)In this study,we conducted a comparative phosphoproteomic profiling of cold-sensitive Cavendish banana and relatively cold tolerant Dajiao under cold stress for 0 hour and 3 hour.Out of 679 phosphopeptides identified in Cavendish banana,180 phosphopeptides(from 147 phosphoproteins)were differentially expressed after 3 h cold stress.While in Dajiao,out of 241 phosphopeptides identified(from 63 phosphoproteins)only 83 were differentially expressed under 3 h cold stress.2)Phosphopeptide abundances of five phosphoproteins involved in mitogen-activated protein kinase kinase 2(MKK2)interaction network,including MKK2,Transcription factor HY5,calcium-sensing receptor(CaSR),Serine/threonine-protein kinase STN7 and kinesin-like protein,show a remarkable difference between Cavendish banana and Dajiao in response to cold stress.3)Western blotting of MKK2 protein and its T31 phosphorylated peptide verified the phosphoproteomic results of increased T31 phosphopeptide abundance with decreased MKK2 abundance in Daojiao for a time course of cold stress.Meanwhile increased expression of MKK2 with no detectable T31 phosphorylation was found in Cavendish banana.These results suggest that the MKK2 pathway in Dajiao,along with other cold-specific phosphoproteins,appears to be involved in the molecular mechanisms of Dajiao with high tolerance to cold stress.In summary,transcriptome,phosphoproteome and gene function identify were utilized to analyse the molecular mechanism of different cold tolerance between Dajiao and Cavendish banana.Our study will provide theoretical basis for banana cold resistance molecular breeding and improved target.