RESUMO
For the first time, the (31)P nuclear magnetic resonance technique has been used to study the properties of isolated vacuoles of plant cells, namely the vacuolar pH and the inorganic phosphate content. Catharanthus roseus cells incubated for 15 hours on a culture medium enriched with 10 millimolar inorganic phosphate accumulated large amounts of inorganic phosphate in their vacuoles. Vacuolar phosphate ions were largely retained in the vacuoles when protoplasts were prepared from the cells and vacuoles isolated from the protoplasts. Vacuolar inorganic phosphate concentrations up to 150 millimolar were routinely obtained. Suspensions prepared with 2 to 3 x 10(6) vacuoles per milliliter from the enriched C. roseus cells have an internal pH value of 5.50 +/- 0.06 and a mean trans-tonoplast DeltapH of 1.56 +/- 0.07. Reliable determinations of vacuolar and external pH could be made by using accumulation times as low as 2 minutes. These conditions are suitable to follow the kinetics of H(+) exchanges at the tonoplast. The (31)P nuclear magnetic resonance technique also offered the possibility of monitoring simultaneously the stability of the trans-tonoplast pH and phosphate gradients. Both appeared to be reasonably stable over several hours. The buffering capacity of the vacuolar sap around pH 5.5 has been estimated by several procedures to be 36 +/- 2 microequivalents per milliliter per pH unit. The increase of the buffering capacity due to the accumulation of phosphate in the vacuoles is, in large part, compensated by a decrease of the intravacuolar malate content.
RESUMO
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.