PPi Formation by Reversal of the Tonoplast-bound H+-pyrophosphatase from `Valencia' Orange Juice Cells

Abstract

Tonoplast vesicles isolated from juice cells of mature `Valencia' oranges [ Citrus sinensis (L.) Osbeck] showed similar tonoplast-bound vacuolar ATP ase (V-ATP ase ) and inorganic pyrophosphatase (V-PP iase ) activity as measured by product formation. Both proton pumps were able to generate a similar pH gradient, although steady-state was reached faster with ATP as substrate. When a ΔpH of 3 units was imposed (vesicle lumen pH of 4.5 and incubation medium of 7.5), tonoplast-bound PP iase was not able to significantly amplify the existing ΔpH. Although not able to function as a H + pump, V-PP iase effectively synthesized PP i in the presence of inorganic phosphate (P i ). Formation of PP i by V-PP iase was enhanced by ATP but inhibited by NaF, gramicidin, and by antibodies raised against V-PP iase from mung bean [ Vigna radiata (L.) R. Wilcz. (Syn. Phaseolus aureus Roxb.)]. Immunological analysis demonstrated an increase in V-PP iase protein with fruit maturity. Data indicate that under in vivo conditions, the V-PP iase of mature orange juice cells acts as a source of inorganic pyrophosphate (PP i) but not as a H + pump. We propose that synthesis of PP i provides a mechanism for recovery of stored energy in the form of the pH gradient across the vacuole during later stages of development and postharvest storage.