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

After opening the stomata in CO2-free air, darkened leaves of several plant species were titrated with CO2 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-exc hange and fluorescence methods for analysis. After equilibration with CO2 f or times ranging between 4.4 and 300 s, fast CO2 release from bicarbonate i ndicated catalysis by highly active carbonic anhydrase. Its time constant w as below 2.5 s. Additional CO2 was released with time constants of about 5, 15 and approximately 300 s. With CO2 as the acidifying agent, calculated b uffer 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 capacit y 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 so lely on the basis of known stromal concentrations of phosphate and phosphat e esters, disregarding the contribution of other solutes. At a concentratio n of 16%, CO2 reduced the stromal pH by about 1 pH unit. Buffering of the c ytosol was measured by the CO2-dependent quenching of the fluorescence of p yranine which was fed to spinach leaves via the petiole. Brief exposures to high CO2 minimized interference by effective cytosolic pH regulation. Cyto solic buffering appeared to be similar to or only somewhat higher than chlo roplast buffering if the initial cytosolic pH was assumed to be 7.25, which is in accord with published cytosolic pH values. The difference from chlor oplast pH values indicates the existence of a pH gradient across the chloro plast envelope even in darkened leaves. Apoplastic buffering was weak as me asured by the CO2-dependent quenching of dextran-conjugated fluorescein iso thiocyanate which was infiltrated together with sodium vanadate into potato leaves. In the absence of vanadate, the kinetics of apoplastic fluorescenc e quenching were more complex than in its presence, indicating fast apoplas tic pH regulation which strongly interfered with the determination of apopl astic buffering capacities. At an apoplastic pH of 6.1 in potato leaves, ap oplastic buffering as determined by CO2 titration with and without added bu ffer was somewhat below 4 mM pH-unit(-1). Thus the apoplastic and cytosolic pH responses to additions of CO2 indicated that the observed cytoplasmic p H regulation under acid stress involves pumping of protons from the cytosol into the vacuole of leaf cells, but not into the apoplast.