Organic Acid Metabolism In Pumelo Fruit During Postharvest Storage Phase

Organic acid is one of the important factors which have a great contribution to the overall organoleptic quality of the non-climacteric citrus fruit during post-harvest storage. In stored citrus fruit, a high concentration of organic acids or a low pH value is an indication of delayed fruit senescence. Pumelo (Citrus grandis), a kind of citrus fruit originated and widely cultivated in China, is very popular among consumers for its storability and flavor. The present work focuses on pumelo fruit, aiming to reveal the concentration variation of organic acids and soluble sugars in different tissues among different cultivars. Systemic works were performed at physiological, biochemical and molecular levels in order to explore the organic acid metabolism and the relationships between organic acid and senescence in Hirado Butun pumelo (HBP)(Citrus grandi) during post-harvest storage. The main results are listed below:1. Technological system of organic acid detection in citrus fruit was established based on high performance capillary electrophoresis (HPCE). The method could accurately determine four main organic acids (citric, malic, aconitic and fumaric acid) in pumelo fruit throught optimizing buffer, electroosmotic flow modifier, separation voltage and detection wavelength. Optimized parameters are as follows:buffer solution:100mM phosphate (0.5mM CTAB, pH7.0); the UV detection wavelength:200nm; the injection pressure:0.5psi for3sec; the separation voltage:12kV; column temperature:20℃.2. The changes of organic acids concentration were assayed among the stored fruits of Shatian pumelo (Citrus grandis), Guanxi pumelo (Citrus grandis), HBP (Citrus grandis), acidless pumelo (Citrus grandis) and Nankang pumelo (Citrus grandis). Regardless of cultivars, organic acids concentrations in the pulp fluctuated significantly instead of simply decreasing or increasing. The general trend of organic acids concentration in HBP showed an increase at the prior storage phase and latter decreased, with a peak value occurred at78DOS (days of storage). In addition, organic acids concentrations of Shatian pumelo, Guanxi pumelo and HBP stored at low temperature (8-10℃) were also determined. The results showed that no difference in the changes of organic acids concentration in Shatian and HBP at both ambient temperature (16-20℃) and low temperature (8-10℃), while Guanxi pumelo revealed an inverse trend at the two storage temperatures.3. The changes in concentration of organic acids and soluble sugars were identified in the albedo and flavedo among Shatian pumelo, Guanxi pumelo and HBP. There are similar change tendencies of organic acids concentration in the pulp between Shatian pumelo and HBP, i.e. organic acid concentration shows a temporary peak value during the decreasing process and four organic acids concentrations display basic consistent change tendencies, while the concentration of citric acid in the pulp of Guanxi pumelo increased, the other organic acids (malic, aconitic and fumaric acid) decreased to different extent. Quinic acid takes over the dominate position in the peel of pumelo. Each organic acid decreased with storage duration. Regardless of albedo or flavedo, soluble sugars concentrations declined among the three pumelo varieties.4. Research on organic acid metabolism in HBP during post-harvest storageIt had been reported that HBP showed’returned acidity’phenomenon in the production practice. Previous researches revealed that organic acid concentration increased at the prior storage phase and latter decreased. However, there is an obvious fluctuation during storage, at which the organic acid concentration was higher than that at the earlier storage phase. Therefore, we selected HBP as material to study the organic acid metabolism in post-harvest storage phase.(1) The concentrations of organic acids in HBP pulp during post-harvest storage were determined precisely by HPCE. Reduplicative experiments of three years confirmed that organic acids concentrations changed with fluctuation during post-harvest storage, and a peak value was observed, with a significantly higher abundance than that at the earlier stage of storage. Further research revealed that storage temperature and respiration might not be the critical factors to cause the fluctuation of organic acids concentrations.(2) Differentially expressed genes which were detected by subcellular organelle expression microarray were then verified by qRT-PCR. No correlation was found between citric acid concentration and the expression of genes which participated directly in citrate biosynthesis and degradation in TCA cycle, including citrate synthase and aconitate hydratase. We screened four genes which were involved in different malate metabolic pathways from microarray, but no correlation was found between malic acid concentration and the expressions of genes except malate dehydrogenase (peroxisome), which was obviously negatively correlated with malic acid concentration and was down-regulated with storage prolonged. The result indicated that the glyoxylate cycle might play an important role in organic acid metabolisn of HBP fruit during post-harvest storage.(3) GC/MS analysis showed that2-oxoglutaric acid content decreased with the storage time, while that of succinic acid increased. However, the gene expressions of succinate dehydrogenase and2-oxoglutarate dehydrogenase E1were not favorable for the accumulation of succinic acid. It was also noticed that the contents of both GABA and glutamine increased during post-harvest storage, and were positively correlated with the content of succinic acid and negatively with2-oxoglutaric acid. Besides TCA cycle, we inferred that other metabolic pathways might be also involved in the conversion of2-oxoglutaric acid into succinic acid.(4) For the further research on internal senescence of HBP fruit during post-harvest storage, we determined the contents of ATP and H2O2and the activities of antioxidases (POD and SOD). ATP content in HBP increased with prolonging of storage time, and its changes was positively correlated with that of organic acids concentrations. H2O2content slightly fluctuated while increasing. It was found that the activities of both antioxidases were positively correlated with H2O2content and organic acids concentrations. The opening of mitochondrial permeability transition pore (MPTP) in HBP pulp rose persistently during post-harvest storage, which indicated that the mitochondrial membrane was damaged.Taken together, different pumelo cultivars showed diverse changes in organic acids concentration during post-harvest storage due to their specific genetic backgrounds, while different tissues of pumelo possessed dissimilar types and concentrations of organic acids due to their distinct function. Our research showed that not only the concentrations of organic acids, but also the gene expressions in some subcellular organelles, as well as metabolite contents fluctuated during post-harvest storage in HBP fruit. These fluctuations are intrinsic features of the life activities of harvested citrus fruit. As flavor and commercial quality declined, these fluctuations became irregular and disordered and gradually faded away. As2-oxoglutaric acid content in the harvested fruit declined, that of GABA and glutamine rose. So, we speculated that GABA shunt is an important pathway in regulating the utilization of organic acids in post-harvest citrus fruit. Moreover, the correlation of organic acids concentrations with the enzymatic systems of scavenging ROS indicated that organic acids play an important role in delaying fruit senescence.