The Role Of Phosphoenolpyruvate Carboxykinase In Citrate Degradation Of Citrus

Organic acids are a crucial determinant of the flavour of fruits,and in citrus fruits,the dominant one is citric acid（or citrate）.In this research,the pathways through which phosphoenolpyruvate carboxykinase（PEPCK）is involved in citrate degradation was studied with the material of citrus fruits and juice sacs,and the role PEPCK plays in citrate metabolic network was discussed.The main results are as follow:1.Two genes,Cit PEPCK1 and Cit PEPCK2,encoding PEPCK were obtained by searching Citrus Genome Database.Phylogenetic analysis showed that the differentiation of these two genes had happened before the divergence of the species from Rutaceae.The phosphorylation site S2 of Cit PEPCK2 has lost the ability to be phosphorylated in some cultivars of citrus.And a frameshift mutation was found in Cit PEPCK2 of Sun Chu Sha Kat mandarin resulted from the deletion of two bases.2.Relevance was found between the expression of Cit PEPCK1 and the consumption of citrate.As citrate accumulated during the early stages of development of Ougan and decreased from 90 to210 days after blossom,the expression of Cit PEPCK1 declined in the early development stages and steadily increased after 120 days after blossom.3.PEPCK participated in citrate degradation in pummelo juice sacs.Metabolic flux analysis and gene expression data showed that,in nitrogen-deficient juice sacs,the metabolic flux and gene expression of ATP-citrate lyase（ACL）,glutamate dehydrogenase（GDH）,glutamate decarboxylase（GAD）and glutamine synthetase（GS）were all down regulated,while both the flux of PEPCK and the expression of Cit PEPCK1 were up regulated.At the same time,citrate degradation was accelerated.It suggested that PEPCK had become the main path for the intermediates from citrate degradation to export from TCA cycle.4.PEPCK/malic enzyme（ME）is the key to converting citrate to sugars and completely oxidating citric acid.Elementary flux mode analysis of a central metabolic network with the core of TCA cycle gave rise to 40 potential pathways through which citrate could be degraded.They can be categorized into 6 classes according to their patterns of carbon flow.PEPCK and ME played similar roles in the network,and their reactions are alternative to each other.It was proposed that either of them is strictly necessary in a metabolic mode transforming citrate to 3-carbon products（pyruvate and phosphoenolpyruvate）or oxidizing citrate absolutely into CO₂.In conclusion,Cit PEPCK1 was found to be related to the ripening of fruits and played a role in the accelerated citrate degradation induced by nitrogen deficiency in pummelo juice sacs.Pathway analysis showed that PEPCK and ME have similar positions in metabolism,either of which is essential to degrade citrate into 3-carbon compounds or thoroughly into CO₂.