Light-dependent reduction of dehydroascorbate and uptake of exogenous ascorbate by spinach chloroplasts

A reconstituted spinach chloroplast system containing thylakoids, stroma and 0.1 mM NADPH supported O2 evolution in the presence of oxidised glutathione (GSSG). The properties of the reaction were consistent with light-coupled GSSG-reductase activity involving H2O as eventual electron donor. The reconstituted system also supported dehydroascorbate-dependent O2 evolution in the presence of 0.6 mM reduced glutathione (GSH) and 0.1 mM NADPH with the concomitant production of ascorbate. The GSSG could replace GSH in which case the production of GSH preceded the accumulation of ascorbate. The data are consistent with the light-dependent reduction of dehydroascorbate using H2O as eventual electron donor via the sequence H2O→NADP→GSSG→dehydroascorbate. Approximately 30% of the GSH-dehydrogenase activity of spinach leaf protoplasts is localised in chloroplasts: this could not be attributed to contamination of chloroplasts by activity from the extrachloroplast compartment. Washed intact chloroplasts supported the uptake of ascorbate but the uptake mechanism had a very low affinity for ascorbate (Km approximately 20 mM). The rate of uptake of ascorbate was less than the rate of light-dependent reduction of dehydroascorbate and too slow to account for the rate of H2O2 reduction by washed intact chloroplasts.