TRANSPORT OF ASCORBIC AND DEHYDROASCORBIC ACIDS ACROSS PROTOPLAST ANDVACUOLE MEMBRANES ISOLATED FROM BARLEY (HORDEUM-VULGARE L CV GERBEL) LEAVES

Protoplasts, vacuoles, and chloroplasts were isolated from leaves of 8 -d-old barley (Hordeum vulgare L. cv Gerbel) seedlings. Transport of a scorbate and dehydroascorbate into protoplasts and vacuoles was invest igated. Contents of ascorbic acid, glutathione, and cr-tocopherol and ascorbate peroxidase activity and glutathione reductase activity were analyzed in protoplasts, vacuoles, and chloroplasts. Uptake of ascorba te and dehydroascorbate by protoplasts showed saturation kinetics (Km = 90 mu M reduced ascorbic acid, 20 mu M dyhydroascorbic acid). Effect s of various membrane transport inhibitors suggested that transport wa s carrier mediated and driven by a proton electrochemical gradient. Tr anslocation of ascorbate and dehydroascorbate into vacuoles did not sh ow saturation kinetics. Neither was it influenced by effectors or by A TP but only by Mg2+, suggesting that translocation did not occur by ca rrier. Ascorbic acid was predominantly localized in the cytosol. Conte nts in the chloroplasts and vacuoles were low. The results are consist ent with the view that ascorbate is synthesized in the cytosol and rel eased to chloroplasts, apoplast, and vacuole following a concentration gradient. Translocation from the apoplast into the cytosol is against a steep gradient and appears to control the concentration of ascorbic acid in the apoplast. In its function as an antioxidant, ascorbate in the apoplast may be oxidized to dehydroascorbate, which can be effici ently transported back into the cytosol for regeneration to ascorbate.