PeCS1 And PeGS2 Regulate The Citric Acid Content Of Passion Fruit(Passiflora Edulis S.)

Passion fruit（Passiflora edulis Sims.）is one of the important cash crops in southern China.The fruit has unique flavor,anti-inflammatory,antibacterial,antitussive,expectorant and antioxidant effects,and has high medicinal value.Citric acid is the main organic acid in passion fruit and an important factor affecting the fruit quality of passion fruit.There are significant differences in citric acid content among different varieties.For example,’Taiwan Giant Yellow passion fruit’is too acidic to be eaten,and’Small Yellow passion fruit’is too sweet without sour taste.At present,the mechanism of accumulation and degradation of citric acid in passion fruit is still unclear.Therefore,in this study,8 different varieties of passion fruit,including’Taiwan Giant Yellow passion fruit’and’Small Yellow passion fruit’,were used as experimental materials,and the appearance quality and titratable acid and other internal qualities of the fruits were determined.The organic acids such as citric acid was detected and analyzed by UPLC.The activities of related enzymes in citric acid metabolism pathway of passion fruit were analyzed and compared.Based on the transcriptome data of passion fruit,the genes related to citric acid metabolism were screened and analyzed in different varieties and development stages.PeCS1 and PeGS2 genes were cloned,and overexpressionvectorsofpCAMBIA1301-PeCS1and pCAMBIA1301-PeGS2 were constructed,which were stably expressed in Arabidopsis thaliana.The main results are as follows:1.The titratable acid content of different varieties of passion fruit was between 1.66%and 3.74%,and there were significant differences in acidity quality.The titratable acid content of’Taiwan Province Big Huang Jinguo’was 3.74%,which was about twice that of’Small Yellow passion fruit’（1.83%）.Through the detection of organic acid components of passion fruit,it was found that the citric acid content of different varieties of passion fruit ranged from 1500μg m L^-1to 2000μg m L^-1,which was nearly 40 times that of other organic acid components.Moreover,the change trend of citric acid content during the development of’Taiwan Giant Yellow passion fruit’and’Small Yellow passion fruit’was the same as that of titratable acid,both of which increased first and then decreased,indicating that the difference of citric acid content was the main reason for the difference of organic acids in passion fruit.2.Through the detection of enzyme activities related to citric acid metabolism,it was found that the activity of phosphoenolpyruvate carboxylase（PEPC）decreased first and then increased during the development of’Taiwan Giant Yellow passion fruit’and’Small Yellow passion fruit’,which was contrary to the change trend of citric acid content.There is a positive correlation between the activity of citrate synthase（CS）and the content of citric acid in the two varieties.The difference of CS activity in different varieties was similar to the difference of citric acid content.There was no obvious correlation between the change of cis-aconitic acid（Mit-ACO）activity in mitochondria and the content of citric acid,but there was a positive correlation between the change of cis-aconitase（Cyt-ACO）activity in cytoplasm and the content of citric acid.The cyt-ACO activity was higher than mit-ACO activity in each period,suggesting that cytoplasm may be the main site for citric acid degradation of passion fruit,and cyt-ACO plays an important role in the process of citric acid metabolism.The change of glutamine synthetase（GS）activity in’Small Yellow passion fruit’was negatively correlated with citric acid content,while the change trend of GS activity in’Taiwan Province Big Huang Jinguo’was firstly increased and then decreased,which was obviously different from that in’Small Yellow passion fruit’fruit development.To sum up,CS,Cyt-ACO and GS may play an important role in citric acid accumulation in passion fruit.3.According to the transcriptome data of passion fruit measured in our laboratory and combined with previous research,18 structural genes related to citric acid metabolism of passion fruit（corresponding to citric acid metabolism related enzymes:phosphoenolpyruvate carboxylase（PEPC）,citric acid synthase（CS）,mitochondrial cis-aconitic acid（Mit-ACO）,cytoplasmic cis-aconitic acid（Cyt-ACO）,NADP-dependent isocitrate dehydrogenase（NADP-IDH）and ATP-citrate lyase（ACL））were screened.The expression patterns of these genes were analyzed.The results showed that there were significant differences in the expression of PeCS1 between’Taiwan Giant Yellow passion fruit’and’Small Yellow passion fruit’,and the expression of PeCS1 in’Small Yellow passion fruit’was similar to the citric acid content.The expression levels of PeACO4 and PeACO5 were also significantly different during the development of the two varieties,which were similar to the trend of Cyt-ACO activity and citric acid degradation.The expression of PeNADP-IDH3 was negatively correlated with the citric acid content of two varieties of passion fruit,and the expression of PeNADP-IDH3 of’Small Yellow passion fruit’increased significantly after the color change period,which was consistent with the difference of citric acid content between the two varieties.There was a significant difference in the expression of PeGS2 between the two varieties.There was a positive correlation between the expression of PeGS2 and GS activity during the fruit development of’Small Yellow passion fruit’.In’Taiwan Province Big Huang Jinguo’,the expression of PeGS2 was positively correlated with GS activity before color change and negatively correlated with GS activity after color change.To sum up,PeACO4,PeACO5,PeNADP-IDH2,PeNADP-IDH3 and PeGS2 may play important roles in regulating citric acid anabolism,and PeGS2 may play different roles or participate in different regulatory mechanisms in’Taiwan Province Greater Huang Jinguo’and’Small Yellow passion fruit’.4.In this study,the genes PeCS1 and PeGS2 of passion fruit were cloned,and the overexpression vectors pCAMBIA1301-PeCS1 and pCAMBIA1301-PeGS2 were successfully constructed,and were over-expressed in Arabidopsis thaliana by soaking in flowers.After screening,the overexpressed plants were obtained.Compared with wild-type Arabidopsis thaliana,the plants with PeCS1 were taller,the stems were thicker,the leaves were more and larger,and the vegetative growth was more vigorous.In terms of reproduction,the number of flowers and pods in Arabidopsis thaliana overexpressed by PeCS1 was more,and compared with wild Arabidopsis thaliana,In terms of reproduction,the number of flowers and pods in Arabidopsis thaliana overexpressed by PeCS1 was more,and compared with wild Arabidopsis thaliana,;Arabidopsis thaliana overexpressed by PeGS2 also shows a certain degree of vigorous growth.Compared with wild Arabidopsis thaliana,the growth of plant lateral branches was more vigorous,with more stems,but fewer fruits,and a small number of pods were empty,The citric acid content of PeGS2 transgenic Arabidopsis thaliana is similar to that of wild Arabidopsis thaliana.To sum up,it is speculated that PeCS1 is involved in regulating citric acid synthesis and plays an important role in plant growth and development.PeGS2 has little effect on the regulation of citric acid in leaves,but more on the regulation of plant reproductive growth.Over-expression of Arabidopsis thaliana may be related to the influence of citric acid cycle in Arabidopsis thaliana.5.Difference analysis of citric acid between’Taiwan Giant Yellow passion fruit’and’Small Yellow passion fruit’:The enzyme activities of CS,Mit-ACO,and Cyt-ACO in the early stage of’Small Yellow passion fruit’were higher than those of’Taiwan Giant Yellow passion fruit’.It is speculated that there is a high level of citric acid synthesis and degradation activity in’Small Yellow passion fruit’before the color transition period,so the citric acid content in’Small Yellow passion fruit’is higher than that in the early stage of fruit development.After the color transition period,the CS enzyme activity of’Small Yellow passion fruit’decreased,while the CS enzyme activity of’Taiwan Giant Yellow passion fruit’continued to increase,resulting in a large amount of citric acid synthesis;After the green fruit stage,the activity of GS enzyme in’Small Yellow passion fruit’gradually increases and exceeds that of’Taiwan Giant Yellow passion fruit’in the near mature stage.The citric acid degradation activity is enhanced,resulting in a higher citric acid content in’Taiwan Giant Yellow passion fruit’in the later stage compared to’Small Yellow passion fruit’.