Dissociation of H + extrusion from K + uptake by means of lipophilic cations

128

(1 1, 12). Seedlings (4-d-old) having straight primary roots, 12 to 16 cm long, were used. In some experiments, 7 cm long root segments were employed. These segments were obtained by cutting the primary root at 1 cm and 8 cm from the tip and were washed for 4 h in aerated 0.2 mm CaSO4 before experimental use.Experimental Chamber and Conditions. The root was placed horizontally along the center of a Plexiglas chamber that was 20 cm long, 3 cm deep, and 4 mm wide. The seed and other roots, when present, were housed in an adjoining chamber. Support for the root was provided by fine stainless steel pins, positioned 5 mm above the floor of the chamber, with the surface of the bathing solution being 1.5 cm above the root. Because the corn root is slightly less dense than water, the tip was held in position by a soft plastic wedge located 5 mm from the root tip. When root segments were used, the proximal end was held down by a silk thread, looped around the root and under a stainless steel pin; attachment to the seed provided adequate anchorage for intact roots. The root was centered within the chamber by means of vertical Plexiglas guides glued to the lateral walls of the chamber.Solutions

contrasting

confidence: 53%

Fluxes of H⁺ and K⁺ in Corn Roots

Newman¹,

Kochian²,

Grusak³

et al. 1987

128

“…The observation that, under these conditions, neither the driving force for K+ influx nor K+ influx are affected by 2H20 is in agreement with a passive electrophoretic movement of the cation (1,23).…”

Section: Discussionsupporting

confidence: 56%

Effects of Deuterium Oxide on Growth, Proton Extrusion, Potassium Influx, and in Vitro Plasma Membrane Activities in Maize Root Segments

Sacchi

Cocucci²

1992

Elongation of subapical segments of maize (Zea mays) roots was greatly inhibited by 2H20 in the incubation medium. Short-term exposure (30 min) to 2H20 slightly reduced 02 uptake and significantly increased ATP levels. 2H20 inhibited H' extrusion in the presence of both low (0.05 mM) and high (5 mM) external concentrations of K' (about 30 and 53%, respectively at 50% [v/v] 2H20).Experiments on plasma membrane vesicles showed that H+-pumping and ATPase activities were greatly inhibited by 2H20 (about 35% at 50% [v/v] 2H20); NADH-ferricyanide reductase and 1,3-B-glucan synthase activities were inhibited to a lesser extent (less than 15%). ATPase activities present in both the tonoplast-enriched and submitochondrial particle preparations were not affected by 2H20. Therefore, the effect of short incubation time and low concentration of 2H20 is not due to a general action on overall cell metabolism but involves a specific inhibition of the plasma membrane H -ATPase. K+ uptake was inhibited by 2H20 only when K+ was present at a low (0.05 mM) external concentration where absorption is against its electrochemical potential. The transmembrane electric potential difference (Em) was slightly hyperpolarized by 2H20 at low K+, but was not affected at the higher K+ concentrations. These results suggest a relationship between H+ extrusion and K' uptake at low K+ external concentration.plast, probably through a direct or chaotropic effect of 2H20 (7). The effect of 2H20 on membrane functions has not yet been specifically investigated in plant materials. An effect of 2H20 on plant membrane functions might be hypothesized on the basis of the consideration that H+ translocating activities, which play a central role in the function of plant membranes (27), could also utilize 2H' even if to a lesser extent.The experiments described in this paper were aimed at studying the effects of 2H20 on H+ extrusion and related transport activities in subapical maize (Zea mays) root segments. Plasma membrane vesicles of high purity from maize roots were used to compare the in vivo effect of 2H20 on H+ transport with the in vitro effect of 2H20 on H+ pumping and ATPase activities and to clarify whether 2H20 inhibits specifically the H+-translocating activities or membrane activities as a whole. Finally, the possibility of using 2H20 as a tool to investigate the involvement of H+ in membrane transport has been analyzed. MATERIALS AND METHODS Plant MaterialIncubation in 2H20 of animal and plant tissues and organs has been used extensively for proton density-labeling experiments to study synthesis, degradation, and turnover of proteins (8), to investigate the role of water in the structure and function of biosystems (29), and to suppress solvent signal from the water in proton NMR (11). However, a toxic effect of 2H20 has been observed on growth of Escherichia coli (4), growth and development of plants (2,6,28), and seed germination (12). It has been suggested that these effects, obtained at high 2H20 concentrations and after long exposur...

“…The finding that the marked depolarization associated with Fe(CN)3-reduction completely suppresses the strict requirement for K+ by ATP-driven H+ extrusion (with or without FC) suggests that K + action on H + extrusion mainly depends on its depolarizing activity, thus confirming that Em contributes to the regulation of the activity of the H+ pump (5,8,21). On the other hand, the very marked effect of ferricyanide in inducing K+ release to the medium suggests that even in higher plants, as already demonstrated in algae, the decrease in Em below a given threshold might 'open' some type of channel to passive K + transport (4).…”

Section: Discussionmentioning

confidence: 55%

“…As shown in the model of Figure 3 the linkage between these two systems would depend on the activity of the redox system in depolarizing the potential and in acidifying the cytoplasm. In fact, an activation of the H+ pump has been demonstrated in vivo in va,rious materials as an effect of treatments either depolarizing the potential (5,8,21) or decreasing cytoplasmic pH (7,13,22,26).…”

Section: Discussionmentioning

confidence: 99%

Plasmalemma Redox Activity and H⁺ Extrusion

Marré

Moroni²,

Albergoni³

et al. 1988

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 ...