NADP(+)-isocitrate dehydrogenase gene expression and isozyme activity during citrus fruit development

The accumulation of citric acid and its decline toward fruit maturation is typical of citrus fruit. We studied NADP(+)-isocitrate dehydrogenase (NADP-IDH), an enzyme involved in citrate metabolism. A cDNA encoding the enzyme was cloned from lemon (Citrus limon) juice sac cells, and is the first-reported NADP-IDH from fruits. Sequence comparisons and phylogenetic analysis indicate that it most probably belongs to a monophyletic clade of plant cytosolic enzymes. The mRNA level in the juice sac cells was induced during lemon fruit growth, and increased by about 15-fold to a peak as the fruit neared maturation. Spectrophotometric assay of the NADP-IDH activity in the pulp during fruit development showed that in young fruit, most of the activity was associated with the mitochondrial preparation and that, as the fruit grew, the activity shifted to the soluble fraction. The two activities could also be distinguished by isozyme gel electrophoresis: while one isozyme was detected in the mitochondrial preparation of young fruit and declined later, the other was induced in the soluble fraction of older fruit and increased as the fruit grew. The increasing activity of NADP-IDH in the soluble fraction throughout fruit development correlated well with the increase in gene expression, which suggests that the soluble activity is regulated by the expression of the cytosolic NADP-IDH gene. The possible role of this form of the enzyme in citric acid catabolism in the pulp is discussed.