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Citric acid, the major organic acid in citrus fruit, is produced by the tricarboxylic acid (TCA) cycle and degraded through gamma-aminobutirate (GABA) shunt and glutamine synthesis (GS) pathway. In this study, Ponkan (Citrus reticulata) and navel orange (Citrus sinensis) fruits were used to investigate the effects of varieties and cultivars, ecological environment, and postharvest heat treatment on citric acid metabolism and related gene expression, and the molecular mechanisms of citric acid metabolism and its regulation in citrus fruit were discussed. The main results are as follows:1. During the development of Ponkan fruit, citric acid increased in the early stage which was followed by a later decrease until the maturation stage. Earlier-ripening mutant Ponkan (ZSPG) fruit accumulated lower concentration of citric acid than the normal Ponkan (PTPG), which was consistent with their total organic acids and TA. Results of gene expression showed that there was no significant difference in the expression of citric acid biosynthesis related genes such as CitCSs, CitPEPCs and CitPEPCKs between PTPG and ZSPG fruit. However, significant higher transcript levels of CitAco3, CitIDHl/3, CitGAD4/5and CitGS2, which took part in the degradation of citric acid, were found in ZSPG than PTPG fruits. Such results indicated that lower citric acid level in ZSPG was caused by the upregulation of cascade genes encoded enzymes involved in citric acid degradation, rather than its biosynthesis.2. Effects of ecological environment on organic acid metabolism during fruit development were investigated by using two cultivars of navel orange, i.e.’Newhall’ and ’SkaggsBonanza’, as fruit materials. Organic acids in matured navel orange cultivated in Ganzhou (GZ) city of Jiangxi province (25°26’N) was significantly lower than those in Songyang (SY) county of Zhejiang province (28°27’N), and the difference was mainly caused by citric acid contents. Similar to the result of Ponkan, the different citric acid levels in navel orange in different habitats were closely related with the degradation of citric acid, rather than its biosynthesis, since the expression levels of CitAco3, CitIDH1and CitGS2in navel orange fruits grown in GZ were significantly higher than those in SY. Meanwhile, the expression of CitGADs genes, which involved in GABA shunt, had no significantly differences between GZ and SY.3. Hot air (40℃,2d) treatment (HAT) showed significant effects on the degradation of organic acids, especially on citric acid, but it had no obvious effect on CCI or TSS. Gene expression results showed that HAT significantly up-regulated the expression of CitAco3, CitIDH2/3and CitGAD4, while it had no effect on CitGS2. Our results indicated that HAT might accelerate citric acid degradation via the GABA shunt pathway, particularly by modulating the CitAco3-CitIDH2/3-CitGAD4cascade.In conclusion, the difference of organic acids content in citrus of different cultivars, habitats and postharvest heat treatment were all determined by their citric acid levels, and degradation rate, rather than the biosynthesis rate, of citric acid showed significant effect on the total organic acids concentration in citrus fruit, though the mechanisms varied for different cases. The different citric acid levels between two Ponkan cultivars might be caused by both GS and GABA degradation pathway, while ecological environment and HAT treatment up-regulated GS and GABA degradation pathway, respectively. The results indicated that the regulatory sites of citric acid degradation varied under different circumstances, and GABA pathway is also related to environment stress.
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