Effect Of Calcium Ion On Chilling Injury Of Green Pepper Under Low Temperature Stress

Green pepper is a popular vegetable,which contains rich vitamin C and other nutrients.Because the growth of green pepper is seasonal and regional,it needs long time transportation and storage.Green pepper will wilt and rot under high temperature storage,and soon lose its edible value.Therefore,low temperature transportation and storage are generally used.Under low temperature storage,green pepper will appear surface depression,discoloration,waterlogged spots and chilling injury,thus affecting the quality of green pepper fruit.In order to solve the question of chilling injury,the effects of 6%calcium chloride and 0.1mmol/L calcium antagonist chlorpromazine?CPZ?on chilling injury of green pepper stored at4 C for 20 days were studied.The chilling injury index,relative conductivity,malondialdehyde?MDA?content and proline content of green pepper were determined by basic physiological and biochemical methods,and the effects of calcium ions on the basic physiological changes of green pepper in the case of chilling injury were studied.The superoxide anion radical?O₂^-?generation rate and hydrogen peroxide?H₂O₂?content of green pepper were determined by hydroxylamine hydrochloride method,and the effects of calcium ions on the production of green pepper in chilling injury were studied.The influence of harmful oxides;the activity of superoxide dismutase?SOD?,peroxidase?POD?and catalase?CAT?of green pepper were determined by NBT photochemical reduction method,and the effect of calcium ion on the activity of antioxidant enzymes of green pepper in chilling injury was studied;the content of ascorbic acid?ASA?and reduced glutathione?GSH?of green pepper were determined by spectrophotometer.The effects of calcium ion on antioxidant content in ASA-GSH cycle of green pepper in chilling injury were studied.The activities of ascorbic acid peroxidase?APX?and glutathione reductase?GR?of green pepper were determined by ultraviolet absorption method,and the effects of calcium ion on key enzyme activities in ASA-GSH cycle of green pepper in chilling injury were studied.The phospholipids of green pepper were determined by liquid chromatography-mass spectrometry.The effects of calcium ion on membrane lipid metabolism of green pepper in chilling injury were studied by content and fatty acid content.The activities of phospholipase C?PLC?and glycerol diester kinase?DGK?of green pepper were determined by kit,and the effects of calcium ion on related phospholipase activity of green pepper under chilling injury were studied.The relative expression of PLC,DGK and transcription factor bzip-1 genes of green pepper were determined by fluorescent PCR.The effects of chilling injury on gene expression of related phospholipase and cold-related transcription factors in green pepper were studied.Finally,the interaction between transcription factor bzip-1 and PLC promoter was determined by yeast single hybridization technique,and the regulation of transcription factor bzip-1 on membrane lipid metabolism in green pepper fruit in the case of chilling injury was studied.The results are as follows:?1?Calcium chloride and CPZ regulated the activities of antioxidant enzymes SOD,POD and CAT.Calcium chloride increased their activities by 13%,35%,9%and CPZ decreased their activities by 19%,38%and 13%respectively compared with the control.Calcium chloride affected ASA-GSH cycle and regulated the activities of related enzymes APX and GR.Calcium chloride increased their activities by 36%and 31%respectively compared with the control?p<0.05?.CPZ reduced its activity by 23%and 9%compared with the control,while regulating the contents of antioxidants ASA and GSH in the ASA-GSH cycle,calcium chloride increased its activity by 18%and 16%compared with the control,and CPZ reduced its activity by 35%and 22%compared with the control,respectively.?2?Calcium chloride and CPZ affected the accumulation of oxygen and hydrogen peroxide produced by metabolic cycle.Calcium chloride decreased the rate of O₂^-formation by 33%,CPZ increased the rate of O₂^-formation by 14%,the difference is significant?p<0.05?;Calcium chloride decreased the content of H₂O₂ by 13%,CPZ increased the content of H₂O₂ by 21%,the difference is significant?p<0.05?;regulated the accumulation of MDA and proline as well as the accumulation of MDA and proline.The content of relative conductivity could alleviate the chilling injury and maintain the integrity of cell membrane,thus reduced the occurrence of chilling injury.Calcium chloride reduced its content by 44%,25%and 6%respectively,and CPZ increased its activity by 34%,10%and 23%respectively.?3?Calcium chloride and CPZ regulated membrane lipid metabolism.Calcium chloride increased the contents of phosphatidylcholine?PC?,phosphatidylethanolamine?PE?,phosphatidylglycerol?PG?,phosphatidylinositol?PI?,phosphatidylserine serine?PS?,monolactose glycerol diester?MGDG?,galactose diacylglycerol?DGDG?by 39%,12%,54%,25%,4%,41%respectively.The contents of phosphatidic acid?PA?,lysophosphatidylcholine?LPC?,lysophosphatidylethanolamine?LPE?and lysophosphatidylglycerol?LPG?decreased by25%,21%,16%and 24%respectively.CPZ reduced the contents of phosphatidylcholine?PC?,phosphatidylethanolamine?PE?,phosphatidylglycerol?PG?,phosphatidylinositol?PI?,phosphatidylserine serine?PS?and monolactose glyceride?MGDG?.Compared with the control group,the contents of DGDG decreased by 42%,2%,36%,21%,43%,27%and 45%respectively,while the contents of glycerophosphatidic acid?PA?,lysophosphatidylcholine?LPC?,lysophosphatidylcholine?LPE?,lysophosphatidylcholine?LPG?increased by 28%,7%,20%and 37%respectively,and the contents of unsaturated fatty acids were regulated by calcium chloride.The contents of linolenic acid and linoleic acid increased by 7%and 20%respectively,with significant difference?p<0.05?.The contents of saturated fatty acid stearic acid and palmitic acid decreased by 29%and 5%respectively.CPZ reduced the contents of unsaturated fatty acid linolenic acid and linoleic acid by 9%and 3%respectively,and the contents of saturated fatty acid stearic acid and palmitic acid increased by 54%and 3%respectively.?4?Calcium chloride and CPZ regulated the relative expression of phospholipase and low temperature-related transcription factor genes.Calcium chloride reduced the activity of PLC and DGK and the relative expression of their corresponding genes by 14%,6%,25%,4%respectively.CPZ increased the activity of PLC and DGK and the relative expression of their corresponding genes by 6%,6%,6%and 10%respectively.The relative expression of transcription factor bzip-1 was increased by 40%by CPZ and 50%less than that of control?p<0.05?.?5?The results of yeast single hybridization showed that the PLC/DGK pathway was regulated by bzip-1 transcription factors,which further confirmed that the occurrence of chilling injury in green pepper fruit was regulated by bzip-1 transcription factors under temperature stress.CPZ,a calcium antagonist,had the opposite effect as an experimental control,which proved that Ca²⁺effectively reduced the chilling injury of green pepper.The above experiments prove that calcium ion can alleviate the chilling injury of posthar vest green pepper fruit.Transcription factor bzip-1 interacts with PLC,regulates membrane lipid metabolism under low temperature stress,and regulates the PLC/DGK pathway,resulting in a series of adverse reactions under low temperature stress.