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

Abstract

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 (pH in) 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 ( p H+ ) was calculated. The H + efflux simulated using the p H+ 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 pH in decreased, suggesting modulation of pumping by ΔpH, pH in, 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.