Study On The Mechanism Of Calmodulin And Calmodulin Transcription Factors Synergistically Regulating Chilling Resistance Of Peach Fruit

’Baifeng’ peach(Prunus persica Batsch ’Baifeng’)is popular for its juicy taste and high nutritional value,which deeply favoured by consumers.’Baifeng’ peach fruit matures in early July in summer,with high temperature and humidity.Peach fruit suffers rapid ripening and softening after harvest,leading to a short postharvest life.Low temperature storage is considered as one of the most effective methods to prolong the shelf life of peach fruit.However,as a cold-sensitive fruit,peach fruit is highly susceptible to chilling injury(CI)during cold storage at an unsuitable temperature,causing deterioration of quality and economic loss.In this study,using ’Baifeng’ peach fruit as the experimental material,the effects of different temperature storage and exogenous calcium treatment on the quality,CI,expression of calmodulin(CaM),and calmodulin-binding transcription factors(CAMTAs)of peach fruit during storage were investigated.The molecular structure characteristics of genes such as CaM and CAMTAs were studied by bioinformatics analysis technology.The roles of CaM and CAMTAs in regulating target genes in different metabolic pathways were conducted by electrophoretic mobility shift assay(EMSA),dual luciferase reporter assay(DLR)and transient overexpression assay.The mechanisms of CaM and CAMTAs synergistically regulating the chilling resistance of peach fruit were further elucidated under different temperatures storage and exogenous calcium treatment,which provided a theoretical basis for the study of postharvest low-temperature storage and preservation technology of peach fruit.The main results were as follows:1.The effects of different temperatures and exogenous calcium treatments on CI and reactive oxygen species(ROS)metabolism of peach fruit during low temperature storage were studied.The results showed that 0℃ and calcium chloride(CaCl2)treatments could maintain higher firmness and TSS content,inhibit the increase of PPO activity,browning index,relative electrical conductivity and MDA content.Furthermore,0℃ and CaCl2 treatment increased enzyme activities and gene expression of SOD,APX,CAT and POD,reduced the ROS accumulation and cell damage,thereby improving chilling resistance and maintaining quality in peach fruit during cold storage.2.The mechanism of PpCaM7 and PpCAMTAs in synergistically regulating ROS metabolism in peach fruit was investigated.Bioinformatics analysis indicated that PpCaM7 protein possesses four EF-hands that have Ca2+-binding helix-turn-helix structures.PpCaM7 is a hydrophilic and stable protein located in the nucleus and cell membrane.PpCAMTA4,PpCAMTA5 and PpCBF5 proteins are hydrophilic unstable proteins,while PpNAC25 protein is a hydrophilic stable protein,all of which are localized in the nucleus.Further yeast two-hybrid(Y2H)assay found that PpCAMTA4 and PpCAMTA5 could interact with PpCaM7,but PpNAC25 and PpCBF5 could not interact with PpCAMTA5.PpCAMTA4 can bind to the promoters of PpSOD(Fe),PpAPX2,PpPAO and PpRBOHA genes,and activate the expression of PpPAO and PpRBOHA genes,but inhibit the expression of PpSOD(Fe)and PpAPX2.The interaction between PpCaM7 and PpCAMTA4 weakened PpCAMTA4-mediated transcriptional activiation of PpPAO and PpRBOHA genes as well as PpCAMTA4-mediated transcriptional repression of PpSOD(Fe)and PpAPX2 genes.PpCAMTA5 can bind to the promoters of PpNAC25,PpCBF5 and PpCAT1 to activate the expression of PpCBF5 and PpCAT1,and to inhibit the expression of PpNAC25.PpCaM7 enhanced PpCAMTA5-mediated transcriptional activation of PpCATl and PpCAMTA5-mediated transcriptional repression of PpNAC25 genes.Additionally,PpNAC25 can bind to the promoters of PpPAO,PpRBOHA,PpRBOHC and PpRBOHD to activate their transcription.PpCBF5 can bind to the promoters of PpPOD31,PpAPX2 and PpRBOHD to positively regulate PpPOD31 and PpAPX2 expression,whereas to negatively modulate the expression of PpRBOHD.Furthermore,0℃ and CaCl2 treatment increased gene expression of PpCaM7,PpCAMTA5 and PpCBF5,inhibited the gene expression of PpCAMTA4,PpNAC25,PpPAO,PpRBOHA,PpRBOHC and PpRBOHD.PpCaM7 and PpCAMTAs synergistically regulated the transcription of ROS metabolism-related genes,maintained the balance of ROS system and reduced cell membrane peroxidation damage,thereby improving chilling resistance in peach fruit.3.The mechanism of PpCaM7 and PpCAMTAs in synergistically regulating energy metabolism and GABA metabolism of peach fruit was investigated.PpCAMTA5 can bind to the promoter of PpCa2+-ATPase10 gene to activate its expression.PpCaM7 enhanced PpCAMTA5-mediated transcriptional activiation of PpCa2+-ATPase10 gene.PpCAMTA4 can bind to the promoter of PpGAD4 gene to repress its expression.PpCaM7 weakened PpCAMTA4 negatively regulation of PpGAD4 expression.Moreover,0℃ and CaCl2 treatment enhanced GABA metabolism related enzyme activities and gene expression and GABA content,and sufficient substrates for energy metabolism.0℃ and CaCl2 treatment increased enzyme activities and gene expression of H+-ATPase,Ca2+-ATPase,SDH,as well as CCO,ATP contents,and energy charge level,and provide sufficient energy for the normal physiological activities of peach fruit,leading to enhancing cold tolerance in peach fruit.In conclusion,0℃ and CaCl2 treatment up-regulated the expression of PpCaM7,PpCAMTA5 and PpCBF5,and inhibited the expression of PpCAMTA4 and PpNAC25.PpCaM7 and PpCAMTAs synergistically regulate ROS metabolism,energy metabolism,and GABA metabolism,thereby alleviating the CI symptoms in peach fruit.