Proton Transport in Plasma Membrane and Tonoplast Vesicles from Red Beet (Beta vulgaris L.) Storage Tissue

Abstract

The proton transport properties of plasma membrane and tonoplast vesicles isolated from red beet (Beta vulgaris L.) storage tissue were examined and compared. Membrane vesicles isolated with 250 millimolar KCl in the homogenization media and recovered at low density following sucrose density gradient centrifugation displayed characteristics of proton transport (nitrate inhibition, no inhibition by orthovanadate, pH optimum of 7.75, pyrophosphate-driven proton transport) which were consistent with a tonoplast origin. When the KCl in the homogenization medium was replaced by 250 millimolar KI, sealed membrane vesicles were recovered at higher densities in sucrose gradients and displayed properties (orthovanadate sensitivity, no inhibition by nitrate, pH optimum of 6.5) consistent with a plasma membrane origin. A comparison of anion effects (potassium salts) upon DeltapH and DeltaPsi revealed a direct correspondence between the relative ability of anions to stimulate proton transport and reduce DeltaPsi. For tonoplast vesicles, the relative order for this effect was KI > KBr >/= KCl > KClO(3) > K(2)SO(4) while for plasma membrane vesicles, a different order KI > KNO(3) >/= KBr >/= KClO(3) > KCl > K(2)SO(4) was observed. Proton transport in plasma membrane and tonoplast vesicles was inhibited by fluoride; however, plasma membrane vesicles appeared to be more sensitive to this anion. In order to correlate anion effects in the two vesicle fractions with anion transport, the kinetics of anion stimulation of steady-state pH gradients established in the absence of monovalent ions was examined. Anions were added as potassium salts and the total potassium concentration (100 millimolar) was maintained through the addition of K(+)/Mes. For plasma membrane vesicles, chlorate and nitrate displayed saturation kinetics while chloride displayed stimulation of proton transport which followed a linear profile. For tonoplast vesicles, the kinetics of chloride stimulation of proton transport displayed a saturable component. The results of this study indicate differences in proton transport properties of these two vesicle types and provide information on conditions where proton transport in the two fractions can be optimized.