Regulation of cytoplasmic pH under extreme acid conditions in suspension cultured cells of Catharanthus roseus: a possible role of inorganic phosphate

Changes in cytoplasmic pH of suspension-cultured cells of Catharanthus roseus under extreme acid conditions were measured with the pH-dependent fluorescence dye; 2',7'-bis-(2-carboxyethyl)-5 (and-6) carboxyfluorescein (-acetoxymethylester) (BCECF). When cells were treated with 1 mM HCl (pH 3 solution), the cytoplasmic pH first decreased then returned to the original level. Treatment with 10 mM HCl (pH 2 solution) acidified the cytoplasm to a greater extent, and the acidification continued at a constant level throughout the measurement. Treatment with a pH 2 solution resulted in a gradual decrease of the malate content, indicating the operation of biochemical pH regulation mechanism. The pH 2 treatment also caused a sudden decrease of the intracellular level of Pi. The cellular content of total phosphorus did not change during the acidification. The Pi was converted to the organic phosphate form. The ATP level was not increased by the pH 2 treatment, but slightly decreased. The role of Pi, which might be functioning as a regulatory factor of cytoplasmic pH, a non-competitive inhibitor of the H+-pumps of both the plasma membrane and tonoplast is discussed.