Molecular Mechanism Of The Different Cold Tolerance Between Dajiao And Cavendish Banana Regulated By Cell Membrane And MAPK Cascade Pathway

Banana originated from the tropics and is an important fruit and food crop.Among them,the main cultivars(‘Cavendish')is a thermophilic specie,and low temperature is one of the fundamental environmental factors limiting its quality and yield.It is a complex physiological and biochemical process that the plant response to low temperature stress,involving many aspects of regulation,in which cell membrane and MAPK cascade pathway play a very important role.In order to uncover and utilize banana's own cold resistance gene,membrane proteome and lipid proteome were used to analyze the mechanism of different cold tolerance between cold resistant Dajiao and cold sensitive Cavendish,and yeast two-hybrid technology was used to preliminarily analyze the regulation mode of MAPK cascade pathway to downstream,and the MaMKK2 a overexpression strain,laying a foundation for subsequent functional research.The main results are as follows:1?Under cold stress,the peroxidases(MaPOD P7 and MaPOD 52)and aquaporins(MaPIP1;1?MaPIP2;4?MaPIP1;2 and MaP IP2;6b)which early accumulation to regulate the concentration of ROS and cell water potential in vivo may be one of the main reasons why Dajiao is superior to Cavendish in cold resistance at membrane protein level.After 6 h of low temperature treatment at 10 °C,cold injury phenotypes such as dehydration wilting,accumulation of active oxygen,accumulation of malondialdehyde and increase of relative conductivity appeared in the top leaves of Cavendish seedlings,but no obvious change was found in Dajiao seedlings.To investigate the early cold stress response of banana,here we applied comparative membrane proteomics analysis for both cold sensitive Cavendish and cold tolerant Dajiao subjected to cold stress at 10 °C for 0 h,3 h,and 6 h.A total of 82 and 137 differentially abundant membrane proteins(DAMPs)were identified in Cavendish and Dajiao,respectively.Among them,the number of membrane proteins upregulated by Dajiao(80)in 3 h is 7 times more than that of Cavendish(11).Go analysis showed that the molecular functions of DAMPS in Cavendish and Dajiao are similar,mainly involving hydrolase activity,binding activity,transport activity and antioxidant activity.However,the number of DAMPs of Dajiao in each molecular function is about twice as many as that of Cavendish,and is mainly up-expressed membrane proteins after 3 h of low temperature treatment.Combined with physiological phenotype,the peroxidases(MaPOD P7 and MaPOD 52)and aquaporins(MAPIP1;1,MaPIP2;4,MaPIP1;2 and MAPIP2;6b)selected as the key candidate membrane proteins for cold resistance.The results of subcellular localization showed that MaPOD P7 was localized on plasma membrane and chloroplast.qRT-PCR and MRM demonstrated that the above candidate membrane proteins(genes)were up-regulated by low temperature induction earlier in Dajiao than in Cavendish.This means that the membrane proteome is reliable.Pretreatment with sodium azide,a peroxidase inhibitor,can reduce the cold resistance of Dajiao,showing wilting of leaves and accumulation of hydrogen peroxide.Moreover,the enzyme activity analysis showed that the soluble POD activity of Cavendish was higher than that of Dajiao and was rapidly increased by low temperature induction,while the POD activity related to membrane in Dajiao was almost higher than that in Cavendish and was increased at 3 h and maintained at a higher level by low temperature induction,implying that membrane-related POD was very important to the cold resistance of Dajiao.Therefore,Dajiao can accumulate peroxidases and aquaporins at an early stage under cold stress,which was used to control the concentration of active oxygen and cell water potential in vivo,while Cavendish showed slower response to low temprature and cold injury phenotypes such as dehydration wilting,accumulation of active oxygen and membrane damage.2.Under cold stress,the integrity of plasma membrane,the relative stability of chloroplast photosynthesis membrane and the initiation of cyanide-resistant respiratory pathway on mitochondrial inner membrane may be the important reasons why Dajiao is superior to Cavendish in cold resistance at membrane lipid level.Both Cavendish and Dajiao belong to 18:3 plants.Combined with membrane proteomics and lipidomics analysis,the response pattern of Dajiao membrane lipids to low temperature was found as follows:(1)The increase of lipid metabolism-related enzymes will promote the conversion of PA,DAG and TAG to PC,reduce the conversion of PC to PE,PI and PS,and promote phospholipid accumulation with relatively high unsaturation.The changes of these phospholipids are beneficial to improve the membrane's spontaneous curvature,membrane fluidity and membrane nano-domain stability under cold stress.(2)The degradation of PI by MaPI-PLC is beneficial to the transmission of cold stress signals.(3)The increase of lipid transporters(MaABCA 7 and 3 MaTGD)facilitate the transport of phospholipids synthesized by endoplasmic reticulum to chloroplasts,promotes the increase of PG,MGDG and DGDGDG,and stabilizes the photosynthetic membrane.(4)The decrease of Cl in mitochondrial leads to the obstruction of cytochrome respiratory pathway,while the increase of MaAOX1 activates the cyanide-resistant respiratory pathway and reduces the accumulation of reactive oxygen species.Because the above membrane proteins and membrane lipids in Cavendish are very different from Dajiao in cold response,these results in cold damage symptoms such as decreased chlorophyll,decreased photosynthetic rate,increased relative conductivity and accumulation of reactive oxygen species.3.MaMKK2a-MaMAPK3a-MaICE1-MaPOD P7 pathway plays a positive role in regulating the cold resistance of Dajiao.The MaMKK2 a,MaMAP3a,MaICE1 and MaPOD P7 genes in Dajiao were significantly up-regulated by cold stree,and they were proved to interact with each other by yeast double hybridization.MaMKK2 a and MaMAPK3 a have been shown to be full of cells by subcellular localization,so there is a possibility of interaction.MaMAPK3 a and MaICE1 were proven to interact with each other in the nucleus by the BiFC.These mean that there may be MaMKK2a-MaMAPK3a-MaICE1-MaPOD P7 pathway in the Dajiao,which has a positive effect on its cold resistance.The results of yeast double hybridization also showed that 10 of the 20 Dajiao MAPK were able to interact with MaICE1.These include MaMAPK2b/2c and MaMAPK3a/3b,which have the highest homology to the MAPK protein which has positive and negative regulation effect on Arabidopsis thaliana cold stress,respectively.It is indicated that MAPK has a difference in the regulation of cold resistance between Dajiao and Arabidopsis thaliana.In addition,MaMKK2a and MaMKK7a can interact with multiple MAPKs,and MaMAPK3a/3b also interacts with multiple MKKs,meaning that there may be other MAPK cascade pathway members involved in the cold resistance of Dajiao.Finally,over-expression of MaMKK2a gene of Dajiao in Cavendish can prevent its embryo from germinating,and replacing T31 with A31in MaMKK2a can obtain over-expression plants of Cavendish.It lays a foundation for the functional research of MaMKK2a.