Regulation of Vacuolar pH of Plant Cells: II. A P NMR Study of the Modifications of Vacuolar pH in Isolated Vacuoles Induced by Proton Pumping and Cation/H Exchanges.

The vacuolar pH and the trans-tonoplast DeltapH modifications induced by the activity of the two proton pumps H(+)-ATPase and H(+)-PPase and by the proton exchanges catalyzed by the Na(+)/H(+) and Ca(2+)/H(+) antiports at the tonoplast of isolated intact vacuoles prepared from Catharanthus roseus cells enriched in inorganic phosphate (Y Mathieu et al 1988 Plant Physiol [in press]) were measured using the (31)P NMR technique. The H(+)-ATPase induced an intravacuolar acidification as large as 0.8 pH unit, building a trans-tonoplast DeltapH up to 2.2 pH units. The hydrolysis of the phosphorylated substrate and the vacuolar acidification were monitored simultaneously to estimate kinetically the apparent stoichiometry between the vectorial proton pumping and the hydrolytic activity of the H(+)-ATPase. A ratio of H(+) translocated/ATP hydrolyzed of 1.97 +/- 0.06 (mean +/- standard error) was calculated. Pyrophosphate-treated vacuoles were also acidified to a significant extent. The H(+)-PPase at 2 millimolar PPi displayed hydrolytic and vectorial activities comparable to those of the H(+)-ATPase, building a steady state DeltapH of 2.1 pH units. Vacuoles incubated in the presence of 10 millimolar Na(+) were alkalinized by 0.4 to 0.8 pH unit. It has been shown by using (23)Na NMR that sodium uptake was coupled to the H(+) efflux and occurred against rather large concentration gradients. For the first time, the activity of the Ca(2+)/H(+) antiport has been measured on isolated intact vacuoles. Ca(2+) uptake was strongly inhibited by NH(4)Cl or gramicidin. Vacuoles incubated with 1 millimolar Ca(2+) were alkalinized by about 0.6 pH unit and this H(+) efflux was associated to a Ca(2+) uptake as demonstrated by measuring the external Ca(2+) concentration with a calcium specific electrode. Steady state accumulation ratios of Ca(2+) as high as 100 were reached for steady state external concentrations about 200 micromolar. The rate of Ca(2+) uptake appeared markedly amplified in intact vacuoles when compared to tonoplast vesicles but the antiport displayed a much lower affinity for calcium. The different behavior of intact vacuoles compared to vesicles appears mainly to be due to differences in the surface to volume ratio and in the rates of dissipation of the pH gradient. Despite its low affinity, the Ca(2+)/H(+) antiport has a high potential capacity to regulate cytoplasmic concentration of calcium.