Recent evidence demonstrates in plants the operation of a plasmalemma redox system transporting reducing equivalents from intracellular substrates to extracellular electron acceptors such as ferricyanide and ferric ion. The finding that ferricyanide reduction is associated with the acidification of the extracellular medium suggested a possible role of this system in ion transport across the plasma membrane, and in particular in H+ extrusion (6,11,12,14,18,29). H+ extrusion in the presence of ferricyanide might occur at the level of the redox chain, similarly to that known for the mitochondria and thylakoid membranes: then the plasmalemma redox chain would represent a mechanism of H+ extrusion alternate to the better known ATP-driven proton pump (14,23). A second possibility is that the reducing agent at the external plasma membrane surface is of the XH2/X type, which would donate electrons to Fe(CN)3-and release H+ into the medium (3, 6). According to a third, alternative interpretation the electron transport to ferricyanide would activate the ATP driven pump, which would be the main mechanism responsible for H+ extrusion (17,27).In an attempt to discriminate between these alternatives we assumed that they might result in a different behavior of the intracellular pH. The direct extrusion of H + by the redox system should not influence cytoplasmic pH, inasmuch as the two hydrogen atoms (2 electrons + 2 protons) coming from the oxidation of a respiratory substrate (XH2 to X) would be transported out of the cell: the two electrons would reduce two ferricyanide molecules and the two protons would be secreted as such into the extracellular medium. In contrast, the model coupling the electron efflux by the redox system with the extrusion of protons by the ATP-driven pump would implicate at least an initial acidification of the cytoplasm by the H + released in the cytosol during the transport of electrons to ferricyanide. This acidification would then be partially compensated by a secondary increase in activity of the ATP driven H+ pump. Thus, we measured the changes in intracellular pH (in cell sap, 'bulk cytoplasm' and vacuole; 1, 2, 22) associated with ferricyanide reduction and acidification of the external medium.The relative roles of the redox chain and of the H+ pump in ferricyanide-induced H + extrusion were investigated by influencing the activity of ATP-driven H + extrusion. As an activator of the ATP-drivep pump we used fusicoccin (recently shown to stimulate ATP-dependent electrogenic H+ transport in plasmalemma preparations; 24, 25). To inhibit the pump, we used erythrosine B and vanadate, two relatively specific inhibitors of the H+-ATPase of plasmalemma (10), both very active on H+ extrusion in vivo in Elodea leaves (19).As a plant material we chose Elodea densa leaves, very active in reducing ferricyanide (14) and also endowed with an efficient mechanism of electrogenic H + extrusion, presumably depending on the operation of the ATP-driven fusicoccin-stimulated and vanadate-inhibited H+ pump (1,19).All ...
