The involvement of stromal ATP in maintaining the pH gradient across the chloroplast envelope in the light

Abstract

The possible involvement of ATP in maintaining the pH gradient across the chloroplast envelope membrane was investigated by simultaneously measuring the stromal ATP concentration and the pH of the stroma and intrathylakoid spaces in intact isolated chloroplasts. Addition of exogenous ATP in the dark increased stromal pH by 0.3–0.4 pH units and increased the pH gradient across the thylakoid membrane by a similar amount. In the dark, dihydroxyacetone phosphate plus oxaloacetate increased stromal ATP to levels equal to those obtained in illuminated chloroplasts, but stromal pH was only increased by 0.1–0.3 pH units compared to an increase of 0.8–1.0 units in the light. The energy-transfer inhibitor, phlorizin, decreased stromal ATP in illuminated chloroplasts almost to dark levels, but did not decrease stromal pH. Inorganic pyrophosphate and an analog of ATP were used to exchange endogenous adenine nucleotides out of chloroplasts, and this also decreased the stromal ATP to dark levels without decreasing stromal pH in the light. Addition of 15–20 μM 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU) reduced both the stromal pH and ATP content of illuminated chloroplasts to dark levels but lower concentrations of DCMU preferentially decreased stromal pH. It is concluded that the pH gradient across the chloroplast envelope is unlikely to be maintained by an electrogenic proton pump driven by ATP hydrolysis. Photosynthetic electron transport is required to maintain the pH gradients across both the chloroplast thylakoid and chloroplast envelope membranes.