Uptake of l-Ascorbate by Intact Spinach Chloroplasts

Abstract

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...

Evidence for the Presence of the Ascorbate-Glutathione Cycle in Mitochondria and Peroxisomes of Pea Leaves

“…However, the presence of ascorbate / DHA transporters has been demonstrated in chloroplasts (Beck et al, 1983) and in protoplasts and vacuole membranes (Rautenkranz et al, 1994). In this case, if in the latency assays ascorbate is transported rapidly across the membrane, the APX would have access to its substrate regardless of its location.…”

A Specific Ascorbate Free Radical Reductase Isozyme Participates in the Regeneration of Ascorbate for Scavenging Toxic Oxygen Species in Potato Tuber Mitochondria

“…This would fulfill both the requirement of protecting mitochondria against H 2 O 2 and of being transported across the mitochondrial membrane in the oxidized form. Whether the oxidized form of AsA is the only one transported across membranes is still an open question, since evidence for the transport of AsA in the reduced form into intact chloroplasts has been provided (Beck et al, 1983). In this work we have studied AFR reductase, which catalyzes the regeneration of AsA from its first oxidation product, AFR.…”

The Ascorbate Carrier of Higher Plant Plasma Membranes Preferentially Translocates the Fully Oxidized (Dehydroascorbate) Molecule

“…Transport of ASC in plant cells was first demonstrated in purified intact chloroplasts of spinach (Anderson et al, 1983;Beck et al, 1983). Beck et al (1983) suggested that this is a carrier-mediated process that translocates ASC across the chloroplast envelope by a facilitated diffusion mechanism.…”

“…Beck et al (1983) suggested that this is a carrier-mediated process that translocates ASC across the chloroplast envelope by a facilitated diffusion mechanism. The chloroplast ASC carrier shows a rather low affinity (approximately 20 mM) and is inhibited by the addition of the sulfhydryl reagent p-chloromercuribenzene sulfonic acid and by the oxidized DHA but not by Glc (Beck et al, 1983;Foyer and Lelandais, 1996). The latter effect led to the suggestion that different carriers for Glc and ASC exist in the chloroplast membrane (Foyer and Lelandais, 1996).…”