| `Nangguo' pear is a local produce in Liaoning province,China.The pear is generally harvested in the first half of September.When stored at ambient temperature,the fruit is prone to softening and deterioration,and losing the commodity value within approximately half a month after harvesting.In order to solve the contradiction between the concentrated harvest periods of 'Nangguo' pear and the long-term market supply demand,the refrigeration method is commonly used in practical production to delay the ripening and aging process of the fruit.However,in the actual production,we found that the peel of refrigerated 'Nanguo' pears quickly turned brown when the produce was transferred to shelf-life at room temperature.The peel of 'Nangguo' pears which experienced long time refrigeration quickly turned brown even before the fruit became soft,which has seriously affected the commodity value and price of this fruit.But the non-refrigerated 'Nangguo' pears show browning symptom only after overripe and soft,and browning parts are different from the refrigerated ones,which turn brown in core first and then the flesh,without browning in the peel.The peel browning(PB)has become the bottleneck of 'Nangguo' pear industry development.So to study on the PB mechanism of 'Nangguo' pears under refrigeration and explore appropriate controlling techniques of PB has vital significance of theory and practice for promoting the development of the 'Nangguo' pear industry.In this study,iTRAQ analysis was carried out to investigate the differences in proteins expression between the 'Nangguo' pear refrigerating for 180 d showing PB and the ones that did not undergo refrigeration,where were no significant difference on hardness between two samples.Furthermore,bioinformatics analysis,i.e.GO analysis and KEGG analysis,was conducted.On this basis,we examined the peel browning index(BI),MDA content,polyphenol oxidase(PPO)activity,phenols content,ATP content and energy charge(EC).In addition,the expression levels of the transcripts for genes which involved in oxidative phosphorylation,glutathione metabolism,pyruvate metabolism and glycerophospholipid metabolism were evaluated.The PB mechanism of refrigerated 'Nangguo' pears was discussed based on the energy and lipid metabolisms.In addition,intermittent warming(IW)and low temperature conditioning(LTC)treatments were applied to the refrigeration process of 'Nangguo' pears to explore the alleviation effect of the two poikilothermic treatments on PB and related mechanism.The main research results are as follows:The iTRAQ results showed that 2,830 differentially expressed proteins were identified from pear fruit.In all,181 proteins with a ratio>±1.2 and FDR<0.05 were identified as differentially accumulated.Among these proteins,95 were down-regulated and 86 were up-regulated.GO enrichment analysis results showed that most of the differentially expressed proteins were associated with the biological processes 'cellular process','response to stimulus',and 'cellular component organization or biogenesis';cellular component'membrane';molecular function 'binding' and 'catalytic activity'.The results of KEGG analysis indicated that the 'carbon metabolism','biosynthesis of amino acids',and 'ribosome'pathways were the most enriched of the differentially expressed proteins.After a long-term period of refrigeration,significant higher MDA content and PPO activity,and lower phenols content was detected in pears' peel during shelf-life at room temperature,accompanied with up-regulation of phospholipase D(PLD)gene involved in glycerophospholipid metabolism pathway,which demonstrated aggravation of the membrane lipid peroxidation in fruit peel and the phenolics enzymatic browning reaction.ATP content and EC value in pears' peel were significantly decreased.Combined with the down-regulation of NADH dehydrogenase(NDH),vacuolar proton-inorganic pyrophosphatase(VPP)and ATP synthase(ATPase)genes involved in oxidative phosphorylation pathway,it can be proved that there was a disorder in energy metabolism pathways and ATP synthesis in pears' peel,which caused deficiency in cellular energy.Down-regulation of acetyl-CoA carboxylase(ACC)and malate dehydrogenase(MDH)genes involved in pyruvate metabolism pathway caused disorder in fatty acid synthesis metabolic process,which affected the synthesis and the stability of cell membranes.The up-regulation of glutathione peroxidase(GPX)gene involved in glutathione metabolism pathway may be caused by the enhancing of cell resistance under cold stress.These results illustrate the energy and lipid metabolisms are closely related to PB of 'Nangguo' pears.Exogenous ATP treatment significantly decrease the BI,MDA content and increase the ATP content and EC value of pears.In addition,ATP treatment significantly increased the genes levels of NDH,VPP and ATPase,and suppressed the up-regulation of PLD gene levels.ATP treatment also significantly increased the ACC and MDH genes levels,and alleviated up-regulation of GPX gene levels.The results validated that exogenous energy supply can to some extent,promote the normalization of related genes expression involved in energy and lipid metabolisms,and delay the occurrence of the PB and decrease the degree of PB of pear fruit,which further proved the relations of energy and lipid metabolisms level and PB of 'Nanguo'pears.Both IW and LTC treatments can delay the occurrence of PB in 'Nanguo' pears during shelf-life after refrigeration,and significantly decreased the BI and MDA content,accompanied with the significant higher ATP content and EC value.The genes expression levels of NDH,VPP and ATPase were all up-regulated,and the ATP content and EC value were also increase in two treated pears'peel,which illustrated two treatments make cells maintain a relatively higher energy level to sustain the normal metabolism.Meanwhile,all of the two treatments significantly depressed the expression of PLD gene,and inhibited the increase of MDA content in pears' peel,which proved slowing in membrane lipid peroxidation level,and better maintenance in compartmentalization of cells.Moreover,the two treatments promoted the up-regulation of genes expression of ACC and MDH,which reflected the normalizing in the fatty acid synthesis pathway and maintenance in synthesis and stability of the cell membranes.The two treatments relieved the up-regulation levels of GPX,which to some extent proved the promoting effect of the treatments on the normal cellular physiological metabolism,and the activity of defense system of peel cells. |
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