Facilitated diffusion drives transport of oxidised ascorbate molecules into purified plasma membrane vesicles of Phaseolus vulgaris

Recently, the presence of a carrier-mediated transport system for ascorbate was demonstrated in the plant plasma membrane. To investigate the possible physiological importance of this system in apoplastic ascorbate metabolism we further characterized this carrier. Transport of Asc was measured by incubating freshly-purified plasma membrane vesicles from hypocotylar hooks of Phaseolus vulgaris together with [C-14]-labelled Asc. In this paper we show that ascorbate transport is detectable over a relatively broad pH range (6 to 7.5) and is not affected by protonophore addition. [C-14]-Ascorbate is not taken up into vesicle fractions consisting of sealed inside-out oriented vesicles, suggesting that it is transported only from the apoplast to the cytoplasm. Asc uptake into vesicles previously loaded with ascorbate was also tested. Surprisingly, uptake of radioactive molecules was up to 3-fold higher in the ascorbate-loaded Vesicles compared to non-loaded control Vesicles (P < 0.001). The uptake of [C-14]-ascorbate in both the ascorbate-loaded as the non-loaded membrane vesicles was inhibited by addition of DTT and not by glutathione or ferricyanide. Based on various observations such as cis-inhibition, trans-stimulation and insensitivity towards proton gradients; a facilitated uptake mechanism is suggested. Our results strongly indicate that dehydroascorbate is the preferred transported species from the apoplastic to the cytoplasmic side of the membrane. This transport system is possibly involved in the regeneration of apoplastic ascorbate.