Modulation of the vacuolar H+-ATPase by adenylates as basis for the transient CO2-dependent acidification of the leaf vacuole upon illumination

Abstract

Using tonoplast vesicles, we have investigated the activity of the vacuolar H+-ATPase which is the dominant proton pump at the tonoplast of mesophyll cells. Bafilomycin-sensitive ATP hydrolysis or acidification of tonoplast vesicles in the presence of ATP were measured at varying ATP, ADP and Pi concentrations, and in the presence of oxidized or reduced glutathione. Increased ATP/ADP ratios as reported for the extrachloroplast cytoplasm during the induction phase of photosynthesis at high or low CO2 (P. Gardeström, Biochim. Biophys. Acta 1183 (1993) 327–332) increased the activity of the V-ATPase in simulation experiments with vesicles. Depending on reported subsequent decreases in cytoplasmic ATP/ADP ratios in the presence of high or low CO2, the ATPase activity of tonoplast vesicles changed in simulation experiments to lower values. More than 10 mM phosphate was required to decrease the ATPase activity in vesicles significantly at ATP/ADP ratios of 3 or higher, indicating that ATPase activity is controlled more by ratios of ATP to ADP than by phosphorylation potentials (ATP)/(ADP)(Pi). Oxidized glutathione was inhibitory. The results permit interpretation of the observation that on illumination of previously darkened leaves the pH of the vacuoles of mesophyll cells decreases indicating energized transport of protons across the tonoplast into acidic vacuoles, and that the extent of vacuolar acidification depends on the CO2 concentration of the surrounding air (Z.-H. Yin, S. Neimanis, U. Heber, Planta 182 (1990) 253–261). We conclude that short term control of tonoplast ATPase activity in leaves during dark/light transients can essentially be understood on the basis of reported changes in cytoplasmic ATP/ADP ratios, with a possible participation of redox modulation.