Transport of Ascorbic and Dehydroascorbic Acids across Protoplast and Vacuole 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 ascorbate and dehydroascorbate into protoplasts and vacuoles was investigated. Contents of ascorbic acid, glutathione, and [alpha]-tocopherol and ascorbate peroxidase activity and glutathione reductase activity were analyzed in protoplasts, vacuoles, and chloroplasts. Uptake of ascorbate and dehydroascorbate by protoplasts showed saturation kinetics (Km = 90 [mu]M reduced ascorbic acid, 20 [mu]M dyhydroascorbic acid). Effects of various membrane transport inhibitors suggested that transport was carrier mediated and driven by a proton electrochemical gradient. Translocation of ascorbate and dehydroascorbate into vacuoles did not show saturation kinetics. Neither was it influenced by effectors or by ATP but only by Mg2+, suggesting that translocation did not occur by carrier. Ascorbic acid was predominantly localized in the cytosol. Contents in the chloroplasts and vacuoles were low. The results are consistent with the view that ascorbate is synthesized in the cytosol and released 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 efficiently transported back into the cytosol for regeneration to ascorbate.