In Vitro [14C]Citrate Uptake by Tonoplast Vesicles of Acidless Citrus Juice Cells

Abstract

The acidless phenotype of the pummelo 2240 [Citrus maxima [Burro.] Merrill] is caused by a mutation affecting a key element of the citric acid accumulation pathway. To test the functionality of the tonoplast citrate transport mechanism, we obtained a tonoplast-enriched membrane fraction from juice tissues of acidless fruit by centrifugation through a discontinuous Ficoll gradient. The isolated tonoplast vesicles incorporated radioactively labeled citrate at a higher rate than vesicles from similarly fractionated high-acid fruit juice. Uptake of [14C]citrate occurred against a concentration gradient was stimulated by nitrate-sensitive ATP hydrolysis, but not by hydrolysis of PPi, and was not affected by the ionophore nigericin. Uptake was not inhibited by malate and only slightly by isocitrate. We did not find evidence of a defective citrate transport mechanism at the tonoplast of juice cells of acidless fruit. We propose that citric acid accumulation in the fruit of citrus is mediated by a carrier that uses energy produced during hydrolysis of ATP to transport citrate into the vacuole actively and specifically.