Proteomic Analysis Of Aroma Fading Mechanisms Of 'Nanguo' Pears After Refrigeration And Regulation Of Aroma Fading

�Nanguo' pears(Pyrus ussuriensis Maxim),as a unique variety in Liaoning province,characterized with a reddish bright color,an attractive unique aroma and sweet taste in mature fruit is popular with people of all ages,so it has extremely high economic value and is one of the mainly developed and promoted fruits in the fruit industry of Liaoning province.Freshly harvested �Nanguo' pears with a relatively poor taste and a low aroma content must pass through a post-ripening stage before eating.After a general half-month post-ripening stage,the quality of the fruit will change significantly,such as peel browning,increased acidity and decreased aroma,etc.,which all directly affect the edible value of the fruit.Currently,low temperature storage is the most effective method to delay the post-ripening,but �Nanguo' pears removed to the room temperature shelf after a long time low temperature storage will be accompanied with product quality degradation due to a decreasing content of aroma or a missing of some aroma components,which has a serious impact on the commercial value of �Nanguo' pears.Therefore,improving the aroma fading after low temperature storage and exploring its control technology is of great theoretical and practical significance to promote the development of �Nanguo' pears products.The study applied iTRAQ technology to compare the fruits on the day of storage and the day of delivery,as well as in the optimal taste period without cold storage and in the optimal edible period after cold storage.By use of differential proteomics analysis technique,the proteins at four storage sites were deeply excavated,and the compared results of differential proteins performed the bioinformatics analysis and functional verification like GO,KEGG and PPI so as to explore the metabolic pathways and differential proteins involved in the aroma fading of �Nanguo' pears after cold storage.Based on the above analysis and research,exogenous ATP processing was applied to control the aroma fading of �Nanguo' pears after refrigeration,and the combined method of PCR and Western was also applied to analyzed physicochemical indexes like fruit firmness,respiration intensity,contents of fatty acid and ester aroma,ATP content,oxaloacetic acid content and fumaric acid content of �Nanguo' pears on different number of days during shelf time after treated by the cold storage and other different treatments.The key gene expression and according enzyme activity change in the LOX Pathway(Lipoxygenase Pathway),Fatty acid synthesis Pathway(Fatty acid metabolism),TCA cycle(Citrate cycle),and Oxidative phosphorylation Pathway(Oxidative phosphorylation)were mainly studied.The mechanism of aroma fading of�Nanguo' pears after cold storage was discussed in terms of energy metabolism and fatty acid metabolism.In order to further prove the correlation between the expression and activity changes of related genes in the above four pathways and the aroma fadingof �Nanguo' pears after cold storage and to realize the application value of the above theory,the molecular mechanism of the enhancement of �Nanguo' pears aroma after cold storage by the intermittent warming treatment(IW)technology was further analyzed based on the above theory.Bioinformatics analysis of iTRAQ differentially expressed protein-group showed that 670 proteins related to aroma synthesis were induced by low temperature,and 290 differentially expressed proteins occurred during refrigeration with the up-regulated number at 201 and down-regulated number 89 of these differentially expressed proteins.380 kinds of differentially expressed proteins occurred in the process of post-ripening after cold storage,with the up-regulated number at 125 and down-regulated number 255.GO analysis showed that differentially expressed proteins related to aroma synthesis occurred both in low temperature storage and post-ripening at room temperature low temperature storage were involved in biological processes metabolism such as cell physiological process,development process,molecular orientation,stress reaction,biological regulation and cellular component synthesis.Differentially expressed proteins involved in cellular component mainly constitute organelles,components of cells,membranes and organelle component and cell membrane component.The differentially expressed proteins distributed in molecular function mainly bear the functions of connection structure between enzyme and molecular,molecular activity,transport activity,resistance activity and electron transport carrier.Bioinformatics KEGG analysis showed that differentially expressed proteins related to the aroma synthetic caused by low temperature were located to 61 metabolic pathways,mainly distributed in the metabolism of carbohydrate,energy and amino acids,fatty acid metabolism etc.The content of aromatic esters and total esters of �Nanguo' pears after refrigeration decreased significantly compared with those of �Nanguo' pear without treatment.The expression and activity of the key enzyme genes LOX,AAT,ADH and HPL in the LOX pathway of aroma synthesis were significantly down-regulated compared with those in the control group.The activity and expression of acetyl-coenzyme A carboxylase,the gene expression of fatty acid synthase and the content of fatty acid all decreased simultaneously.In the process of energy metabolism,the gene activity and expression of the key genes malate dehydrogenase and succinate dehydrogenase involved in the ATP synthesis in the TCA cycle way also got a negative control.The gene expression of NADH dehydrogenase in oxidative phosphorylation decreased,respiration intensity decreased significantly,the quantity of ATP synthesis decreased,and the content of fumaric acid and oxaloacetic acid as substrates of key enzyme catalysis in energy metabolism also declined synchronously,all of which above suggested that low temperature affected the energy metabolism and fatty acid metabolism.In the experimental group treated with ATP,Aroma ester type and total estercontent in �Nanguo' pears after cold storage significantly increased.The expression and activity of the key enzyme genes LOX,AAT,ADH and HPL in the LOX pathway of aroma synthesis were significantly improved compared with those in the control group.The activity and expression of acetyl-coenzyme A carboxylase,the gene expression of fatty acid synthase and the content of fatty acid all increased simultaneously.In the process of energy metabolism,the gene activity and expression of the key genes malate dehydrogenase and succinate dehydrogenase involved in the ATP synthesis in the TCA cycle way also got elevated by the exogenous ATP.The gene expression of NADH dehydrogenase in oxidative phosphorylation got an obvious rise,respiration intensity increased apparently,the quantity of ATP synthesis went up,and the content of fumaric acid and oxaloacetic acid as substrates of key enzyme catalysis in energy metabolism also got improved synchronously,all of which above suggested that the energy metabolism and fatty acid metabolism are closely related to the aroma of �Nanguo' pears.Compared with the continuous low temperature treatment,intermittent warming treatment significantly increased the species of aroma ester and total ester content in the optimum edible period.IW treatment improved the expression and activity of the key enzyme genes LOX,AAT,ADH and HPL in the LOX pathway.The activity and expression of acetyl-coenzyme A carboxylase and the gene expression of fatty acid synthase all increased significantly,and the respiration intensity increased apparently.In the process of energy metabolism,the gene activity and expression of the key genes malate dehydrogenase and succinate dehydrogenase in the TCA cycle way also got elevated.The content of the key material,oxaloacetic acid and fumaric acid increased.The gene expression of NADH dehydrogenase in oxidative phosphorylation got an obvious rise,the content of ATP went up apparently.These results further proved that energy shortage and synthesis of fatty acids are the main reasons for the aroma dilution of �Nanguo' pear during post-ripening after low temperature storage.