pH and buffer capacities of apoplastic and cytoplasmic cell compartments in leaves

After opening the stomata in CO(2)-free air, darkened leaves of several plant species were titrated with CO(2) at concentrations between 1 and 16%, in air in order to reversibly decrease cellular pH values and to calculate buffer capacities from pH changes and bicarbonate accumulation using both gas-exchange and fluorescence methods for analysis. After equilibration with CO(2) for times ranging between 4.4 and 300 s, fast CO(2) release from bicarbonate indicated catalysis by highly active carbonic anhydrase. Its time constant was below 2.5 s. Additional CO(2) was released with time constants of about 5, 15 and approximately 300 s. With CO(2) as the acidifying agent, calculated buffer capacities depend on assumptions regarding initial pH in the absence of an acid load. At an initial stroma pH of 7.7, the stromal buffer capacity was about 20 mM pH-unit(-1 )in darkened spinach leaves. At an initial pH of 7.5 it would be only 12 mM pH-unit(-1), i.e. not higher than expected solely on the basis of known stromal concentrations of phosphate and phosphate esters, disregarding the contribution of other solutes. At a concentration of 16%, CO(2) reduced the stromal pH by about 1 pH unit. Buffering of the cytosol was measured by the CO(2)-dependent quenching of the fluorescence of pyranine which was fed to spinach leaves via the petiole. Brief exposures to high CO(2) minimized interference by effective cytosolic pH regulation. Cytosolic buffering appeared to be similar to or only somewhat higher than chloroplast buffering if the initial cytosolic pH was assumed to be 7.25, which is in accord with published cytosolic pH values. The difference from chloroplast pH values indicates the existence of a pH gradient across the chloroplast envelope even in darkened leaves. Apoplastic buffering was weak as measured by the CO(2)-dependent quenching of dextran-conjugated fluorescein isothiocyanate which was infiltrated together with sodium vanadate into potato leaves. In the absence of vanadate, the kinetics of apoplastic fluorescence quenching were more complex than in its presence, indicating fast apoplastic pH regulation which strongly interfered with the determination of apoplastic buffering capacities. At an apoplastic pH of 6.1 in potato leaves, apoplastic buffering as determined by CO(2) titration with and without added buffer was somewhat below 4 mM pH-unit(-1). Thus the apoplastic and cytosolic pH responses to additions of CO(2 )indicated that the observed cytoplasmic pH regulation under acid stress involves pumping of protons from the cytosol into the vacuole of leaf cells, but not into the apoplast.