Improved mathematical model for estimating H+ influx and H+ efflux in plant vacuolar vesicles acidified by ATPase or pyrophosphatase

To adapt to environmental changes, plant cells very likely possess a biochemical system, using vacuoles, for maintaining cytoplasmic pH homeostasis. A simple approach is to estimate the active H+ influx and H+ efflux of isolated vacuolar vesicles, although there is no good mathematical model to describe H+ flux. To establish a new quantitative model, vacuolar vesicles were isolated from hypocotyls of mung bean (Vigna radiata L.), and pyrophosphate (PPi)- or ATP-dependent acidification was monitored using acridine orange. The change of pH inside the vesicles (pHin) was calculated using a pH calibration curve relating fluorescence quenching with ΔpH. After formation of a steady state ΔpH, passive H+ efflux was monitored after terminating pumping with ethylenediaminetetraacetate, and the relative H+ permeability coefficient (pH+) was calculated. The H+ efflux simulated using the pH+ corresponded to the H+ efflux determined experimentally. H+ influx was then calculated by subtracting the predicted H+ efflux from the experimental net H+ influx. H+ influx into vesicles driven by H+-PPase or H+-ATPase decreased exponentially as the intravesicular pHin decreased, suggesting modulation of pumping by ΔpH, pHin, or both. Finally, the PPi- or ATP-dependent H+ accumulation determined experimentally was closely simulated by the predicted H+ influx and H+ efflux. The ability to predict H+ flux under different conditions provides a powerful tool for studying pH homeostasis.