Regulation By Nitric Oxide And Chilling Signal On Sphingolipid Metabolism Of Peach Fruit

The peach is chilling sensitive fruit,suffers easily chilling injury during cold storage.Cell membrane is an important site to feel the cold stress.Sphingolipid is an important component of the cell membrane,and is also an important signal transduction molecule participating the processes of cell apoptosis,cell proliferation and responding to environmental stress.Sphingolipid plays an important role in maintaining normal physiological functions of cells.Phospholipase Dα（PLDα）,an important sphingolipid,is able to catalyze the hydrolysis of phosphodiester bonds and the exchange of bases.The hydrolysate of PLDα,phosphatidic acid（PA）,as a second messenger,participates the signal transduction processes of plant.PLDαplays an important role in responses to environmental stress.Nitric oxide（NO）is a kind of small molecule with bioactivity and fat-solubility;plays an important role in alleviating chilling injury of postharvest peaches during cold storage.NO is an important factor in response to cold stress,can effectively respond to cold stress and maintain storage quality postharvest fruit.However,there are few studies on the effects of NO on sphingolipid components and sphingolipid metabolism under cold stress.Therefore,ascertaining the regulation mechanism of NO and cold signal on sphingolipid metabolism,is of great importance to relieve chilling injury and maintain storage quality of fruit.In this experiment,the postharvest Feicheng peaches,as experimental material,was treated with 15μmol L^-11 NO solution and 5μmol L^-1 c-PTIO solution,and then stored at 0°C and 25°C,respectively.The full-length and amino acid sequence of PLDαof Feicheng peach was obtained by the methods of molecular biology.The activities of key enzymes in sphingolipid metabolism and the content of sphingolipids were detected by chemical methods.There were 2 HKD motifs in PLDαprotein,which were separated by 320 amino acids.The hydrophilic balance coefficient was-0.417,which indicated that PLDαwas a hydrophobic protein.Phylogentic tree revealed that PLDαprotein of peach was closest to that of strawberry in evolution.The three-dimensional structure of peach PLDαprotein showed a"cave"structure,which could well explain the hydrophobicity of the protein.Predictive analysis of protein modifications showed that glutathionylation was more easily performed than nitration for PLDαprotein.The results of qPCR showed that low temperature increased the expression level of PLDαgene,while NO decreased the expression level of PLDαgene.The results of sphingolipid metabolic enzyme activities and sphingolipid contents showed that NO increased the contents of sphingolipids and the activity of key enzymes including 3KSR,SPHK,CERS,CERK and SMS in sphingolipid metabolism in peaches during storage.The opposite results were obtained at low temperatures.The above results indicate that there was HKD motif catalyzing the hydrolyzation in the active site of PLDα.PA was regulated at the active site of PLDαand then involved in the response to environmental stress of plant.NO and cold signal were antagonistic in regulating the gene expression of PLDα.NO could reduce the production of PA by down-regulating the gene expression of PLDα,and then decrease the content of reactive oxygen species,protect cells from damage,and then decrease chilling injury of peaches during cold storage.There were antagonistic effects between NO and cold signals on cell proliferation and apoptosis.NO could maintain the dynamic balance of cell proliferation and apoptosis by regulating the contents of signal molecule during storage,and then,delayed the changes of fruit quality.NO and cold signaling were antagonistic to maintain sphingolipid metabolism.NO could maintain the synthesis of sphingolipids by maintaining the activities of key enzymes in sphingolipid metabolism,thereby sustain proper contents of sphingolipids,enhance the stability of cell membrane and delay the decrease of fruit quality.