ABSTRACIUptake of l1-4Cjascorbate by intact ascorbate-free spinach (Spinacia okracca L. cv Vital) chloroplasts has been investigated using the technique of silicone oil filtering. Rates greater than 100 micromoles per milligrm chlorophyll per hour (external concentration, 10 millimolar) of ascorbate transport were observed. Ascorbate uptake into the sorbitolimpermeable space (stroma) followed the Michaelis-Menten-type characteristic for substrate saturation. A K. of 18 to 40 millimolar was determined. Transport of ascorbate across the chloroplast envelope resulted in an equilibrium of the ascorbate concentrations between stroma and medium. A pH optimum of 7.0 to 7.5 and the lack of alkalization of the medium upon ascorbate uptake suggest that only the monovalent ascorbate anion is able to cross the chloroplast envelope. The (6), whereas the latter may be localized predominantly in the cytoplasm (7). However, GSH-and light-dependent reduction of dehydroascorbate has also been observed with chloroplasts (27). With respect to the Km for ascorbate of the various peroxidative systems mentioned above (0.12-3 mM), the high chloroplastic concentrations of ascorbate having been found are sufficient for the reduction of H202. However, the provenance of ascorbate in the chloroplast is completely unclear. Although the biosynthesis of L-ascorbic acid in plants has not yet been completely elucidated, it is well established that it originates from D-glucose. In contrast to the biosynthetic route in animals or algae (17), ascorbate biosynthesis in higher plants proceeds via oxidation at C-1, epimerization at C-5, and a second oxidation at C-2 or C-3 (12, 16; for earlier references, see 9). The enzymes catalyzing this reaction sequence have not yet been localized in the cell. However, in that ripening strawberries (for reference, see 9) and potato tubers (21) are good systems for the study of ascorbate biosynthesis, this process should not occur in chloroplasts. If ascorbic acid originates outside the chloroplast, this organelle must be capable of reasonable rates of uptake of exogenous ascorbate.Inasmuch as ascorbic acid is present as a monovalent anion at physiological pH, uptake into the stroma solely by diffusion is unlikely. Evidence for a catalyzed ascorbate uptake by intact chloroplasts is shown in the present study.Considerable amounts of L-ascorbic acid are found in algae (10,17,33) and in fruits, storage organs, and leaves of higher plants (26). Inasmuch as 30 to 40% of the ascorbate content of spinach leaf protoplasts was found to be associated with the chloroplast fraction (8), a considerable portion of the ascorbate of green leaves appears to be localized within the chloroplasts. On this basis, a stromal concentration of 15 mM can be calculated but concentrations up to 50 mm have been reported (10). Walker (35), when citing earlier literature, ascribed a Chl to ascorbate ratio of 1:1 (w/w) to chloroplasts of field-grown summer leaves which corresponds to a concentration of even 75 mM. Chloroplastic ascorbate ma...