Studies On Citric Acid Metabolism Of Guanxi Pumelo Fruit During Postharvest Storage

The phenomenon of "sour reguigitation" during the postharvest storage of Guanxi pumelo[Citrus maxima(Burm.)Merr.'Guanximiyou']fruit has seriously affected its flavor and commodity value.In the previous study,it was found that the acidity of the fruit was caused by the abnormal accumulation of citric acid,but the mechanism has not been clear.In this study,we started with the metabolic pathway of citric acid.The main organic acids including fruit based and fruit top were determined by LC-MSn technique,the activities of enzymes related to the citric acid metabolism and the expression of enzymes related genes were studied.In addition,the transcriptome analysis of sour and normal fruit juice sac was carried out to extract other factors about the abnormal accumulation of citric acid in fruit,in order to provide a reference for the further study of Guanixi pumelo "sour reguigitation".The main results were as follows:1.The LC-MSn quantitative method for the determination of organic acids in Guanxi pumelo fruit was established.The optimum extraction method was 55?extraction temperature and 0%ethanol concentration.The concentrations of nine organic acids(pyruvate,citric acid,cis-aconitic acid,malic acid,fumaric acid,succinic acid,a-ketoglutarate,tartaric acid,quinic acid)in the juice sac were accurately determined.2.The concentration of nine organic acids during the sour reguigitation process in Guanxi pumelo fruits were determined,which in the fruit based and fruit top was compared.The results showed that the concentration of citric acid was higher than other organic acids,and it was rising in the storage process,verified the abnormal accumulation of citric acid caused the sour reguigitation of Guanxi pumelo fruit.In the citric acid synthesis pathway,the concentration of pyruvate which is the upstream of citric acid increased with the storage time and it was positively correlated with the changing of citric acid concentration,which indicated that the citric acid synthesis rate was accelerated.In citric acid degradation pathway,The concentration of aconitate,succinic acid and a-ketoglutarate which is the downstream of citric acid decreased with the increasing of storage time and it was negatively correlated with the change of citric acid concentration,which indicated that the degradation rate of citric acid was slowed down.Therefore,it is speculated that the abnormal accumulation of citric acid in Guanxi pumelo fruit is the result of accelerated synthesis and slowed degradation of citric acid.In addition,the concentration of organic acids in the fruit based juice sac was higher than the fruit top,except succinic acid,and the trend of sour reguigitation was different,it is speculated that the metabolism of other organic acids may be affected by citric acid metabolism,resulting in differences in the concentration of organic acids at the fruit based.3.The changes of enzymes-related citric acid metabolism in fruit juice sac were determined,and juice sac in fruit based and fruit top were compared.The results showed that citric acid synthase(CS)activity increased during the sour reguigitation and was positively correlated with citric acid in citric acid synthesis pathway,indicated that the synthesis rate of citric acid was accelerated.In the citrate degradation pathway,the activity of aconitase(ACO)and succinate dehydrogenase(SDH)decreased,and was negatively correlated with citric acid,indicated that the degradation rate of citric acid slowed down.Therefore,it was verified that the abnormal accumulation of citric acid in Guanxi pumelo was due to the simultaneous effect of citric acid synthesis and degradation.In addition,the activity of organic acid metabolic enzyme in the fruit top juice sac were mostly higher than fruit based and was negatively correlation with organic acid concentration,presumably because of the activity of citric acid metabolic enzyme in fruit top was high which accelerated organic acid metabolism,resulting in lower organic acid concentration.4.The expression genes about citric acid synthesis and degradation sac was analyzed,and the genes expression of juice sac at fruit based and fruit top was compared.The results showed that in citric acid synthesis pathway,the expression of CmCS2 was positively correlated with the activity of CS and the concentration of citric acid.It was presumed that with the increasing of the expression of CmCS2 gene the activity of CS was enhanced,which resulted in abnormal accumulation of citric acid.In the citric acid degradation pathway,the expression of CmAco3 was positively correlated with ACO activity and cis-aconitic acid concentration and it was negatively correlated with the concentration of citric acid.It was presumed that with the decreasing of expression of CmAco3 the activity of ACO was decreased,leading to degradation of citric acid and the rate of synthesis ACO slow down.In addition,gene expression of juice sac at fruit top was higher than fruit based,which was consistent with the results of citric acid metabolism related enzymes.The results showed that the organic acid metabolism in the fruit top was higher than fruit based,which caused the lower concentration of the citric acid at the fruit top.5.The correlation analysis between organic acids and citric acid metabolism related enzymes showed that there were different trends of organic acid metabolism in juice sac at fruit top and fruit based.In addition,TA content increased with the increasing rate of water loss,suggested that decreased of fruit water content leading to increased of organic acid concentration.6.Two trancriptome that normal and acidification juice sac of Guanxi pumelo fruit were determined by using RNA-seq technology.Enrichment analysis of differential genes showed that the iron ion binding,membrane component metabolism,oxidation reduction metabolism,metabolic pathway and biosynthesis secondary metabolic metabolism were associated with acidification.In addition,a large number of genes related to sucrose and glucose metabolism in the process of acidification of Guanxi pumelo fruit were specific expression which may be the main cause of abnormal accumulation of citric acid.Fruit aging-related genes of SOD and PRDX5 enzyme may be closely related to the acidification of Guanxi pumelo fruit.