Changes of citrate acid contents and expression of related genes in Majiayou pomelo fruit during room temperature storage

Abstract:【Objective】Majiayou pomelo [Citrus grandis (L.) Osbeck] is a local characteristic variety in Guangfeng district, Shangrao City, Jiangxi province. Majiayou pomelo is popular among consumers because of its large fruit size, high nutritional value, unique favor and easy storability. Organic acid is an important component to determine the intrinsic quality of citrus fruit. Excessive or low acid contents will affect the favor and storage performance of citrus fruit. At present, the research on organic acids of Majiayou pomelo fruit mainly focuses on the development process, but the changes of its organic acid and molecular regulation mechanism during postharvest storage are less studied. In this experiment, the contents of organic acid components in Majiayou pomelo pulp during a long storage period (150 d) were determined, and the expression levels of genes regulating citrate acid that was dramatically accumulated at early storage period (0-70 d) were analyzed. The purpose of this study was to clarify the changes of the content and molecular regulation mechanism of citrate acid accumulation at early storage period, so as to provide theoretical basis for identifying key genes regulating citrate acid accumula-tion and improving storage quality of Majiayou pomelo.【Methods】The experiment material was Majiayou pomelo fruit harvested from 12 years old trees. Fruits with the same maturity and size, and without diseases and pests as well as mechanical surface damage were selected for storage at room temperature for 150 days. Samples were taken every 10 days during storage period, and three biological replicates were set for each sample. Three fruits were taken from each replicate for pulp separation, and then quickly frozen in liquid nitrogen. The contents of citric acid, malic acid, quinic acid and tartaric acid in pulp were determined by the high performance liquid chromatography (HPLC). The HPLC test was performed on Shimadzu SPD-M20A with a C18 column (4.6 mm×250 mm, 5 μm) at 30 ℃, the mobile phase was 0.01 mol·L-1 H2SO4 and flow rate was 0.5 mL·min-1 . The diode array detector was used for detection at wavelength of 210 nm. Malic acid, citric acid, quinic acid and tartaric acid standards were chromatographically pure. Real-time quantitative fluorescent PCR (qRT-PCR) was used to analyze the relative expression levels of genes related to citric acid synthesis, transport and degradation in Majiayou pomelo pulp after the sample fruits were stored for 0, 20, 40 and 70 days. RNA was extracted with the kit, and the integrity of RNA was detected by the agarose gel electrophoresis. The first cDNA strand was synthesized using the reverse transcription test kit (Cat.# RR047A). RT-qPCR gene expression level was analyzed using SYBR®Preminx Ex TaqTM kit. Three biological replicates were set up for each sample, and the data were analyzed by 2-∆∆Ct method. Finally, the relationship between the change pattern of citric acid content and the expression levels of related genes was analyzed.【Results】The organic acids in the pulp of Majiayou pomelo mainly included citric acid, malic acid, tartaric acid and quinic acid, among which citric acid was the most important component, accounting for 73.4% of the total organic acids. With the increase of storage time, the content of total organic acids showed a trend of first increasing and then decreasing, that is, it gradually increased to a peak value (7.0 mg · g- 1 ) at 0- 40 d, then showed a significant downward trend at 40-70 d, reached a minimum value (3.8 mg · g- 1 ) at 70 d, and then recovered to a stable level at 80 d. The change pattern of citric acid contents was basically consistent with that of the total organic acid contents, which decreased to the lowest value of 2.7 mg·g-1 at 70 d, and did not change significantly until the end of storage (150 d) after a short increase from 70 d to 80 d. The contents of malic acid, quinic acid and tartaric acid changed steadily throughout the storage period. The expression of genes related to citric acid metabolism in the pulp of Majiayou pomelo fruits stored for 0 d, 20 d, 40 d and 70 d was analyzed. The results showed that the expressions of citric acid synthetic genes CmPEPC1 and CmCS1/2 increased at 0 d to 40 d, while CmPEPC1/2 and CmCS2 decreased at 40 d to 70 d. The relative expression of proton pump gene CmVHA-c4 first increased (0 d to 40 d) and then decreased (40 d to 70 d). However, the relative expression of CmVHP2 decreased significantly only from 40 d to 70 d. The relative expression of mitochondrial dicarboxylic acid carrier gene CmDIC decreased gradually from 0 d to 40 d after storage, and there was no significant difference between 40 d and 70 d. The relative expression levels of CmGS2 and CmGAD5 decreased gradually during 0-40 d storage, which was opposite to that of citric acid content, and the difference was not significant at 40 d to 70 d. The expression levels of other genes related to citric acid degradation, such as CmACL1, CmACL3, CmACO3, CmNAD-IDH2, CmNAD-IDH3 and CmGABA-T, were inconsistent with the trend of citric acid degradation.【Conclusion】The content of organic acid in Majiayou pomelo pulp was mainly affected by the change of citric acid content during storage, and the significant change of citrate acid content in early storage period may be co- regulated by genes related to its synthesis (CmPEPC1/2 and CmCS1/2), transport (CmDIC, CmVHP2 and CmVHA-c4) and degradation (CmGS2 and CmGAD5).