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PLANT PHYSIOLOGY , Vol 106, Issue 1 187-193, Copyright © 1994 by American Society of Plant Biologists
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METABOLISM AND ENZYMOLOGY |
Transport of Ascorbic and Dehydroascorbic Acids across Protoplast and Vacuole Membranes Isolated from Barley (Hordeum vulgare L. cv Gerbel) Leaves
AAF. Rautenkranz, L. Li, F. Machler, E. Martinoia and J. J. Oertli
Institut fur Pflanzenwissenschaften, Eidgenossische Technische Hochschule, Versuchsstation Eschikon, CH-8315 Lindau (ZH), Switzerland (A.A.F.R., L.L., F.M., J.J.O.)
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.
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