Changes of citric acid content and the related gene expression at different developmental stages of fruits in 'Jing' an ponkan'

Abstract:【Objective】‘Jing'an ponkan'is an important citrus fruit in Jiangxi province. The fruits are rich in acids which are related to fruit quality. The study aimed to explore the changes of citric acid content and its relationship with the related gene expression in‘Jing'an ponkan'during fruit development.【Methods】The fruits of‘Jing'an ponkan'were picked up at 60, 80, 100, 120, 140, 180 and 200 days after full blossom (DAFB) respectively from 10 trees in an orchid of Jing'an county, Jiangxi province.A sample of 30 fruits were divided into three biological replicates, the flesh portions of the fruit were frozen with liquid nitrogen then stored at -80 ℃. The titratable acid (TA) was determined by acid-base neutralization titration, and the result was converted by the amount of citric acid. The organic acid content was determined by high performance liquid chromatography (HPLC) . The extraction of RNA was carried out by CTAB method. And it was detected by agarose gel electrophoresis. Expressiones of genes involved in citric acid biosynthesis and degradation (Cit CSs, Cit PEPCs, Cit PEPCKs, Cit Acos, Cit IDHs, Cit GADs, Cit GS2, Cit ACLs and Cit FBPases) were analyzed by real-time fluorescence quantitative PCR (Q-PCR) .【Results】Citric acid was the most important organic acid in‘Jing'an ponkan'fruits, followed by quinic acid and tartaric acid. The citric acid accounted for more than 71.15% of the total organic acid content in ponkan fruits. The quinic acid and the tartaric acid content accounted for 14.26%and 26.22% of the total organic acid content, while malic acid content was less than 0.2% of the total organic acid content. The citric acid increased from 60 DAFB to 120 DAFB, then decreased. This was consistent with the variation of TA and total organic acids content. At 120 DAFB, the citric acid content reached the maximum value of 32.91 mg· g^-1. At 200 DAFB, the citric acid, the total organic acids and the titratable acid content were 17.20 mg· g^-1, 22.49 mg· g^-1 and 1.36%. The content of the quinic acid and the tartaric acid gradually decreased during fruit development and maturation. At 200 DAFB, the quinic acid and the tartaric acid content were 4.68 mg· g^-1 and 0.56 mg· g^-1. The malic acid content was very low in the whole developmental stage, and the total content decreased slightly. The results of the gene expressiones related to the citric acid metabolism and correlation analysis revealed that the relative expression of Cit CS1 decreased slightly, and the relative expression of Cit CS2 and Cit PEPC1 increased first and then decreased. The relative expression of the Cit PEPC2 and the Cit PEPC4 increased at the late stage in‘Jing'an ponkan'fruit, both of them had no significant correlation with the citric acid content. The relative expression of Cit PEPCKs, Cit Aco1 and Cit FBPases showed a slow decline, which also had no significant correlation with the citric acid content. The relative expression of Cit Aco2/3 and Cit ACLα 1 rose slowly at early developmental stage then increases rapidly during fruit maturation, which showed a significant negative correlation with the citric acid content. The relative expression of Cit IDH1 and Cit ACLα 1/β decreased firstly then increased, but the relative expression of Cit IDH2/3, Cit GADs and Cit GS2 was reverse, which increased firstlyand then decreased. The relative expression of Cit IDH1/2 and the citric acid content showed a significant negative correlation.【Conclusion】The citric acid was the most important acid in the organic acid of‘Jing'an ponkan'fruits during fruit development. With fruit ripening, the citric acid and the organic acid content increased firstly and then decreased. The change of the citric acid content in the fruit was not directly related to the gene expression related to the citric acid synthesis it is mainly influenced by the genes related to degradation. Especially the increase of the relative expression of the Cit Aco2/3, the Cit IDH1/2 and the Cit ACLα 1 in the late mid period of fruit development might be an important factor in regulation of of organic acid in‘Jing'an ponkan'fruit developmental period in the late fall.