Citrus fruits are characterized by the accumulation of high levels of citric acid in the juice sac cells and a decline in acid level toward maturation. It has been suggested that changes in mitochondrial aconitase (EC 4.2.1.3) activity affect fruit acidity. Recently, a cytosolic aconitase (cyt‐Aco) homologous to mammalian iron‐regulated proteins was identified in plants, leading us to re‐evaluate the role of aconitase in acid accumulation. Aconitase activity was studied in 2 contrasting citrus varieties, sweet lime (Citrus limettioides Tan., low acid) and sour lemon (Citrus limon var. Eureka, high acid). Two aconitase isozymes were detected. One declined early in sour lemon fruit development, but was constant throughout sweet lime fruit development. Its reduction in sour lemon was associated with a decrease in aconitase activity in the mitochondrial fraction. Another isozyme was detected in sour lemon toward maturation, and was associated with an increase in aconitase activity in the soluble fraction, suggesting a cytosolic localization. The cyt‐Aco was cloned from lemon juice sac cells, but in contrast to the changes in isozyme activity, its expression was constant during fruit development. We present a model, which suggests that reduction of the mitochondrial aconitase activity plays a role in acid accumulation, while an increase in the cyt‐Aco activity reduces acid level toward fruit maturation.

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Most citrus (Citrus L. spp.) fruits accumulate a considerable amount of citric acid in the vacuoles of the juice sac cells. As part of research aimed to understand the mechanism of acid accumulation, we compared the gene structures and transcript levels of citrate synthase in sour lemon (high acid, C. limon (L.) Burm.), 'Shamouti' orange (moderate acid, C. sinensis (L.) Osbeck) and sweet lime (acidless, C. limmetioides Tan.). Southern analyses suggested that a single gene for citrate synthase was present in the genomes of all three Citrus varieties. The gene structures seemed to be very similar, with minor differences in Shamouti orange. Overall, the transcript levels of citrate synthase were similar in sweet lime and sour lemon, and about 2-fold lower in Shamouti orange. The enzymatic activity of citrate synthase was compared between sour lemon and sweet lime. In sour lemon, the specific activity of the enzyme was induced early in fruit development and, in parallel with the increase in acid content, reached a maximal level, and did not diminish significantly towards fruit maturation; the pattern and level of activity detected during sweet lime fruit development were similar. These results suggest that the difference in acid accumulation between acidless and acid-containing fruits may not be attributed to changes in the activity of citrate synthase.

Arsenic compounds generate diverse effects in all living organisms. In citrus (Citrus L. sp.), they reduce acidity and improve fruit quality by unknown mechanisms. The major organic acid in citrus fruit is citric acid, which begins accumulating early in fruit development, reaches a peak in middle-sized fruit and then, in most species, declines as the fruit matures. In an attempt to understand the basis of the effect of arsenite, it was applied to `Minneola' tangelo (Citrus paradisi Macf. × C. reticulata Blanco) ≈6 weeks postanthesis, and a detailed analysis of total titratable acidity and citric acid concentration was performed throughout fruit growth. Within 35 days after arsenite application, total acid content and citrate concentration were slightly lower compared with the controls, and this difference persisted throughout fruit development. The concentrations of other organic acids were not reduced by the treatment. Sodium arsenite reduced the citrate concentration in `Eurieka' lemon callus [Citrus limon (L.) Burm.] also, without affecting tissue growth. Extractable activity of citrate synthase in treated fruit was inhibited within 1 day following arsenite spray, but recovered to a normal level a few days later. In contrast, gene expression was remarkably induced 1 day following treatment, which might explain the recovery in enzyme activity. Data suggest that reduction in acid accumulation may not be related to the initial inhibition of citrate synthase activity.

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