The Regulation of Aconitase, A Central Enzyme of Citric Acid Metabolism in Citrus Fruit

摘要

Accumulation of citric acid in the juicesac cell is a major determinant of maturity and fruit quality in citrus. A higher than desirable citric acid content often reduces fruit quality during harvest. On the other hand,with some mandarins,the problem is often low acidic content at maturity,resulting in a tasteless fruit,or one that is "too sweet" if sugars are markedly high. Reduced acid content sometimes correlates with increased levels of ethanol and secondary metabolites which further reduce fruit taste quality. A critical step in acid metabolism is catalyzed by aconitase, an enzyme that is present in the juice-sac cells in two forms,mitochondrial and cytosolic. Reduced activity of the mitochondrial aconitase contributes to acid accumulation early in fruit development, while increased activity of the cytosolic aconitase causes citrate decline toward fruit maturation. We recently demonstrated that these two forms respond differently to iron,a key element in aconitase regulation. In the current work,we investigated the regulation of both aconitase forms by citramalate and oxalocitrate in fruits and in calli originated from juice-sac cells. The use of these compounds significantly increased citric acid content and reduced the activity of the enzyme. Aconitase appeared to be regulated by these inhibitors at the post-translational level. Citramalate, a natural compound in the fruit pulp, was found at high levels early in fruit development,but then decreased about threefold when the increase in citric acid was detected,putting the hypothesis of citramalate's role in citrate accumulation throughout fruit development into question. The biosynthetic pathway of citramalate was investigated by the heterologous expression of its putative biosynthetic gene, isopropyl-malate synthase (IPMS), in yeast. Although IPMS is considered a key enzyme in leucine biosynthesis, the recombinant enzyme showed equal specific activities with 2oxoisovalerate, which leads to amino acid biosynthesis, and pyruvate, which leads to citramalate biosynthesis. This suggests that IPMS may act as the citramalate biosynthetic enzyme in citrus fruit.