Plasmalemma redox activity and h extrusion: I. Activation of the h-pump by ferricyanide-induced potential depolarization and cytoplasm acidification

Ferricyanide reduction by Elodea densa leaves, in the dark, is associated with: (a) acidification of the medium; (b) decrease (about 0.2-0.3 units) of intracellular pH (measured in cell sap, cytoplasm, and vacuole); (c) depolarization of the transmembrane potential; (d) net efflux of K⁺ to the medium. Ferricyanide-induced acid secretion is markedly increased by the presence of fusicoccin (FC), and this effect is severely inhibited by the proton pump inhibitors erythrosine B and vanadate. In the presence of ferricyanide FC-induced H⁺ extrusion no longer requires the presence of K⁺ in the medium. The (ferricyanide reduced)/(H⁺ extruded) ratio varies from about 2, in the absence of FC, to about 1 when the toxin is present, and to more than 4, when ATP-driven H⁺ extrusion is inhibited by erythrosine B or by vanadate. Fusicoccin markedly reduces K⁺ release to the medium. The ratio (ferricyanide reduced)/(H⁺ extruded + K⁺ released) approaches unity under all of the three conditions considered. These results indicate that ferricyanide reduction depends on a plasmalemma system transporting only electrons to the extracellular acceptor, with consequent potential depolarization and cytoplasm acidification. Most of the protons released in the cytoplasm would be secondarily extruded by the ATP-driven pump, stimulated by both intracellular acidification and depolarization. K⁺ efflux would depend on potential depolarization.     

Cytoplasmic pH Regulation in Acer pseudoplatanus Cells: II. Possible Mechanisms Involved in pH Regulation during Acid-Load

Qualitative and quantitative aspects of the mechanisms involved in the regulation of cytoplasmic pH during an acid-load have been studied in Acer pseudoplatanus cells. Two main processes, with about the same relative importance, account for the removal of H⁺ from the cytoplasm, namely a `metabolic consumption' of protons and the excretion of protons or proton-equivalents out of the cells. The metabolic component corresponds to a change in the equilibrium between malate synthesis and degradation leading to a 30% decrease of the malate content of the cells during the period of cytoplasmic pH regulation. Various conditions which severely inhibit the activity of the plasmalemma proton pump ATPase reduce, at most by 50%, the excretion of H⁺. This suggests that, besides the plasmalemma proton-pump, other systems are involved in the excretion of proton-equivalents. Indirect information on qualitative and quantitative features of these systems is described, which suggests the involvement of Na⁺ and HCO₃⁻ exchanges in the regulation of cytoplasmic pH of acid-loaded cells.     

ATP-dependent and ionophore-induced proton translocation in pea stem microsomal vesicles

The presence of an electrogenic pump in pea stem microsomal vesicles has already been demonstrated, but no evidence on the nature of the electrogenic ion has been presented (Rasi-Caldogno, F., De Michelis, M.I. and Pugliarello, M.C. (1981) Biochim. Biophys. Acta 642, 37–45). In this work we tested the usefulness of the ΔpH probe Acridine orange to monitor both ATP-dependent and ionophore-induced H⁺ fluxes in pea stem microsomal vesicles. The H⁺/K⁺ exchanger nigericin causes a marked uptake of protons into the vesicles that can be followed, with similar results, both as Acridine orange absorbance changes and pH changes of the external medium. ATP induces an uptake of Acridine orange into the vesicles which is reversed by FCCP and abolished by the presence of Triton X-100 in the incubation medium, thus indicating an inward, ATP-driven, H⁺ translocation. The ATP-dependent acridine orange uptake is Mg²⁺-requiring and KCl-stimulated. Such activity is inhibited by two specific ATPase inhibitors, dicyclohexylcarbodiimide and diethylstilbestrol, while it is unaffected by oligomycin and Na₃VO₄. These results show that Acridine orange is a useful probe to measure pH gradients in our membrane system and are consistent with the hypothesis that an ATPase of plasmalemma may act as a proton pump.     

H extrusion and potassium uptake associated with potential hyperpolarization in maize and wheat root segments treated with permeant weak acids

The rapid uptake of weak acids permeant in the uncharged form is accompanied in maize and wheat root segments by a hyperpolarization of the transmembrane electrical potential and an increase in K⁺ uptake, suggesting a stimulation of the plasmalemma H⁺ pump. The evaluation of weak acid-induced H⁺ extrusion must take into account the alkalinization of the medium due to the rapid uptake of the uncharged form of the acid, partially masking the proton pump-mediated extrusion of H⁺. The data corrected for this interference show that the lipophilic butyric acid and trimethyl acetic acid induce in maize and in wheat root segments a significant increase in `real' H⁺ extrusion, roughly matching the increase in net K⁺ uptake. The presence of K⁺ significantly increases the rate of uptake of the weak acid, possibly as a consequence of an alkalinization of the cytosol associated with K⁺ absorption. In maize root segments, the effects of fusicoccin and those of butyric acid on both K⁺ uptake and H⁺ extrusion are clearly synergistic, thus suggesting distinct modes of action. These results support the view that the activity of the plasmalemma H⁺ pump is regulated by the value of cytosolic pH.     

Acidification of Deionized Water by Roots of Intact Rice Seedlings

This investigation was designed to examine whether or not deionizedwater could be acidified by roots of intact rice seedlings.Roots of intact rice seedlings caused significant acidificationof the deionized water in which they were immersed and thisacidification could be repeated after replacement of acidifiedwater with fresh deionized water. The addition of K⁺, Na⁺, andMg²⁺ to the deionized water significantly increased the rateand extent of acidification. However, no such increase was foundwhen Ca²⁺ was present in the water. The inhibition of acidificationby vanadate and its promotion by fusicoccin indicated that theacidification of water by roots of intact rice seedlings originatedfrom an ATP-driven proton pump located in the plasmalemma. Ferricyanide was effectively reduced by the roots of intactrice seedlings. This reduction was associated with the acidificationof the bathing solution. 8-Hydroxyquinoline and p-nitrophenyl-acetateinhibited both the reduction of ferricyanide and ferricyanide-inducedacidification. Vanadate, although it slightly inhibited thereduction of ferricyanide, did not inhibit the ferricyanide-stimulateddecrease in pH. It seems that the involvement of redox activityassociated with the plasmalemma in the acidification of deionizedwater cannot be excluded. (Received August 30, 1989; Accepted April 5, 1990)     

The proton pumps of the plasmalemma and the tonoplast of higher plants

Studies on intact cells, membrane vesicles, and reconstituted proteoliposomes have demonstrated in higher plants the existence of an ATP-driven electrogenic proton pump operating at the plasmalemma. There is also evidence of a second ATP-driven H⁺ pump localized at the tonoplast. The characteristics of both these ATP-driven pumps closely correspond to those of the plasmalemma and tonoplast proton pumps ofNeurospora and yeasts.     

Potassium Transport and Regulation of Intracellular pH in Elodea densa Leaves

The effects of extracellular K⁺ concentration ([K⁺]_o) on the pH of cell sap, “bulk cytoplasm” and vacuole have been investigated in Elodea densa leaves under conditions of either low or high activity of the plasmalemma electrogenic H⁺ pump. Cell sap pH was evaluated directly in the cell sap expressed after freezing and thawing. Cytoplasmic and vacuolar pH were calculated by the weak base and weak acid distribution method, DMO and benzylamine appearing to be a suitable acid and base, respectively, for this purpose in this material. When added to the basal medium (no rapidly permeating ions present), 5 mM K⁺ induced an increase in intracellular pH, larger for the cell sap and the vacuole (about 0.2 units), and smaller but still significant for the cytoplasm (0.07 units). This alkalinizing effect of K⁺ was thus associated with a significant decrease in the pH difference across the tonoplast. The alkalinizing effect of K⁺ was markedly and synergistically enhanced by the presence of fusicoccin, a condition inducing a marked activation of H⁺ extrusion and of K⁺ uptake. The correlation between these effects of [K⁺]_o on intracellular pH and those on H⁺ extrusion indicates that changes in extracellular K⁺ concentration, and thus in K⁺ influx, can influence cytoplasmic and vacuolar pH by modulating the rate of H⁺ extrusion by the plasmalemma H⁺ pump.     

Light-induced Activation of Electrogenic H+ Extrusion and K+ Uptake in Elodea densa Depends on Photosynthesis and is Mediated by the Plasma membrane H+ ATPase

In Elodea densa leaves light strongly stimulates electrogenic,K ⁺-dependent, vanadate- and erythrosin B-sensitive H⁺ extrusionand hyperpolarizes the transmembrane electrical potential. Theseeffects of light are suppressed by treatment with DCMU, an inhibitorof photosynthesis, which has no effect on H⁺ extrusion in thedark. Light-induced H⁺ extrusion requires the presence of K⁺in the medium and is associated with increased K⁺ uptake andalkalinization of the cell sap. Light-induced H⁺ extrusion increaseswith increased CO₂ concentration. At constant CO₂ concentration(10⁴ parts 10^–6) the rate of H⁺ extrusion is stronglyenhanced by an increased light intensity up to 30 W m^–2.Different wavelengths, between 400 and 730 nm, induce a significantstimulation of both proton secretion and transmembrane potentialhyperpolarization. The stimulating effects of light on H⁺ extrusion, K⁺ uptakeand cell sap pH are very similar to those induced in the darkby fusicoccin, a toxin known to stimulate strongly ATP-driven,vanadate- and erythrosin B-sensitive H⁺ transport. In the light,the effects of fusicoccin are only partially additive to thoseof light, thus suggesting that the two factors influence thesame system. The identification of this system with the plasmamembrane H⁺-ATPase is indicated by the observed inhibition ofthe effects of either light or fusicoccin by the H⁺-ATPase inhibitorsvanadate and erythrosin B. These data indicate that the activation of electrogenic H⁺ extrusionand of K⁺ uptake by light is mediated by some products of photosynthesis.The mechanism and the possible physiological implications ofthis phenomenon are discussed. Key words: Photosynthesis, H⁺ pump, K⁺ uptake, Elodea densa     

Some Properties of a Functional Reconstituted Plasmalemma H-ATPase Activated by Fusicoccin

Fusicoccin was shown to stimulate the ATP-driven, intravesicular acidification of liposomes reconstituted with crude fusicoccin receptors and the H⁺-translocating ATPase, both solubilized from maize (Zea mays L.) plasma membrane. The present paper reports optimal conditions for dual reconstitution and fusicoccin activation as well as the biochemical characterization of the effect of fusicoccin on this system. Fusicoccin stimulation of proton pumping was dependent on pH and fusicoccin concentration. Its specificity was demonstrated by the positive effect of two cotylenins that have a high affinity for fusicoccin receptors and by the negative response to 7,9-epideacetylfusicoccin, an inactive fusicoccin derivative. Kinetic measurements at different ATP concentrations showed that fusicoccin increases the V_max of the enzyme. Fusicoccin stimulation of maize H⁺-ATPase was also maintained when receptors from maize were substituted by those from spinach (Spinacia oleracea L.).     

Suitability of Arabidopsis for studies on intracellular pH regulation: correlation between H+ pump activity, cytosolic pH and malate level in wild-type and in a mutant partially insensitive to fusicoccin

Data are presented on the suitability of Arabidopsis thaliana seedlings for studies on intracellular pH regulation. In this material, grown in the dark in liquid medium, the determination of weak acid distribution at equilibrium provides an adequate method for calculating cytosolic pH values, in spite of the failure of benzylamine as a vacuolar pH probe. The stimulation of the H⁺ pump by K⁺ or K⁺ and fusicoccin (FC) is associated with a marked alkalinization of both cytosol and cell sap, and with a strong increase in malate level, whereas its inhibition by erythrosin B (EB) leads to the opposite effects. A good quantitative correlation is evident between the changes in net H⁺ extrusion and those in intracellular pH and malate content, in particular, with FC+K⁺. Cell sap buffer capacity is strongly influenced by the different treatments, its changes being substantially accounted for by changes in malate level. A comparison between the values of intracellular pH and malate level in wt and in the 5-2 mutant shows that in the mutant the cytosolic pH is always more acidic, and the intracellular alkalinization induced by FC+K⁺ and also by K⁺ alone is significatively lower. These results support the view that the partial insensitivity of 5-2 to FC is due to a reduced functionality of the H⁺-extruding system on which FC acts, and that the depression of the H⁺ pump activity in the mutant does not depend on a possible regulation by constitutively higher cytosolic pH values.     

Regulation of H+Pumping by Plant Plasmalemma under Osmotic Stress: The Role of 14-3-3 Proteins

All higher plants have high-specific sites for binding fusicoccin (FCBS), a metabolite of the fungus Fusicoccum amygdaliDel. These sites are localized on the plasmalemma and produced by the association of the dimers of 14-3-3 proteins with the C-terminal autoinhibitory domain of H⁺-ATPase. Considering the fusicoccin binding to the plasmalemma as an index characterizing the formation of this complex, we studied the influence of osmotic stress on the interaction between 14-3-3 proteins and H⁺-ATPase in the suspension-cultured sugar beet cells and protoplasts obtained from them. An increase in the osmolarity of the extracellular medium up to 0.3 Osm was shown to enhance proton efflux from the cells by several times. The number of FCBS in isolated plasma membranes increased in parallel, whereas 14-3-3 proteins accumulated in this membrane to a lesser degree. The amount of H⁺-ATPase molecules did not change, and the ATP-hydrolase activity changed insignificantly. The data obtained indicate that osmotic stress affects H⁺-ATPase pumping in the plasmalemma through its influence on the coupling between H⁺-transport and ATP hydrolysis; 14-3-3 proteins are involved in this coupling. The interaction between the plasmalemma and the cell wall is suggested to be very important in this process.     

H-pumping driven by the plasma membrane ATPase in membrane vesicles from radish: stimulation by fusicoccin

The effect of fusicoccin on Mg:ATP-dependent H⁺-pumping in microsomal vesicles from 24-hour-old radish (Raphanus sativus L.) seedlings was investigated by measuring the initial rate of decrease in the absorbance of the ΔpH probe acridine orange. Fusicoccin stimulated Mg:ATP-dependent H⁺-pumping when the pH of the assay medium was in the range 7.0 to 7.6 while no effect of fusicoccin was detected between pH 6.6 and pH 6.0. Both basal and fusicoccin-stimulated H⁺-pumping were completely inhibited by vanadate and almost unaffected by nitrate. Fusicoccin did not change membrane permeability to protons and fusicoccin-induced stimulation of Mg:ATP-dependent H⁺-pumping was not affected by changes in the buffer capacity of the incubation medium. Deacetylfusicoccin stimulated H⁺-pumping as much as fusicoccin, while the physiologically inactive derivative 8-oxo-9-epideacetylfusicoccin did not. Stimulation of H⁺-pumping was saturated by 100 nanomolar fusicoccin. These data indicate that fusicoccin activates the plasma membrane H⁺-ATPase by acting at the membrane level independently of the involvement of other cell components. The percent stimulation by fusicoccin was the same at all ATP concentrations tested (0.5-5.0 millimolar), thus suggesting that with fusicoccin there is an increase in V_max of the plasma membrane H⁺-ATPase rather than a decrease in its apparent K_m for Mg:ATP.     

Relation of ATPases in rat renal brush-border membranes to ATP-driven H+ secretion

Summary In the presence of inhibitors for mitochondrial H⁺-ATPase, (Na⁺+K⁺)- and Ca²⁺-ATPases, and alkaline phosphatase, sealed brush-border membrane vesicles hydrolyse externally added ATP demonstrating the existence of ATPases at the outside of the membrane (ecto-ATPases). These ATPases accept several nucleotides, are stimulated by Ca²⁺ and Mg²⁺, and are inhibited by N,N-dicyclohexylcarbodiimide (DCCD), but not by N-ethylmaleimide (NEM). They occur in both brushborder and basolateral membranes. Opening of brush-border membrane vesicles with Triton X-100 exposes ATPases located at the inside (cytosolic side) of the membrane. These detergent-exposed ATPases prefer ATP, are activated by Mg²⁺ and Mn²⁺, but not by Ca²⁺, and are inhibited by DCCD as well as by NEM. They are present in brush-border, but not in basolateral membranes. As measured by an intravesicularly trapped pH indicator, ATP-loaded brush-border membrane vesicles extrude protons by a DCCD- and NEM-sensitive pump. ATP-driven H⁺ secretion is electrogenic and requires either exit of a permeant anion (Cl^–) or entry of a cation, e.g., Na⁺ via electrogenic Na⁺/d-glucose and Na⁺/l-phenylalanine uptake. In the presence of Na⁺, ATP-driven H⁺ efflux is stimulated by blocking the Na⁺/H⁺ exchanger with amiloride. These data prove the coexistence of Na⁺-coupled substrate transporters, Na⁺/H⁺ exchanger, and an ATP-driven H⁺ pump in brush-border membrane vesicles. Similar location and inhibitor sensitivity reveal the identity of ATP-driven H⁺ pumps with (a part of) the DCCD- and NEM-sensitive ATPases at the cytosolic side of the brush-border membrane.     

Ethanol-Induced Activation of ATP-Dependent Proton Extrusion in Elodea densa Leaves

In Elodea densa leaves, ethanol up to 0.17 m stimulates H⁺ extrusion activity. This effect is strictly dependent on the presence of K⁺ in the medium and is suppressed by the presence of the plasmalemma H⁺-ATPase inhibitor vanadate. Stimulation of H⁺ extrusion is associated with (a) a decrease in cellular ATP level, (b) a marked hyperpolarization of transmembrane electrical potential, and (c) an increase in net K⁺ influx. These results suggest that ethanol-induced H⁺ extrusion is mediated by an activation of the plasma membrane ATP-dependent, electrogenic proton pump. This stimulating effect is associated with an increase of cell sap pH and of the capacity to take up the weak acid 5,5-dimethyloxazolidine-2,4-dione, which is interpretable as due to an increase of cytosolic pH. This indicates that the stimulation of H⁺ extrusion by ethanol does not depend on a cytosolic acidification by products of ethanol metabolism. The similarity of the effects of ethanol and those of photosynthesis on proton pump activity in E. densa leaves suggests that a common metabolic situation is responsible for the activation of the ATP-dependent H⁺-extruding mechanism.     

Interrelationships between trans-Plasma Membrane Electron/Proton Transfer Stoichiometry, Organic Acid Metabolism, and Nitrate Reduction in Dwarf Bean (Phaseolus vulgaris)

Iron deficiency in dwarf bean (Phaseolus vulgaris L.) induces an increased activity of a system in the rhizodermal cells, which reduces extracellular ferric salts, and an active proton efflux from the roots, which is coupled to accumulation of citrate and malate in the roots and subsequent export of these compounds in the xylem. During reduction of extracellular ferricyanide by Fe-deficient plants, the stoichiometry of electron transport to proton efflux is 2e⁻/1H⁺, and citrate and malate levels in the roots are strongly decreased. Reduction of ferricyanide by Fe-sufficient plants has no influence on root and shoot levels of citrate and malate, but in such plants the process is characterized by a e⁻/H⁺ efflux stoichiometry close to unity. Apparently, organic acid metabolism and transport are closely associated with the e⁻/H⁺ efflux ratio. To assess the significance of organic acid metabolism as one of the direct intracellular components of the induced unbalanced e⁻/H⁺ efflux by roots, we studied NO₃⁻ reduction in shoots and roots of Fe-deficient and Fe-sufficient plants. Nitrate reductase activity in the roots was positively correlated with the level of citrate and malate, whereas the enzyme activity in the leaves responded positively to the import of these organic acid anions.     

Function of reduced pyridine nucleotide-ferredoxin oxidoreductases in saccharolytic Clostridia

The physiological function of the clostridial NADH- and NADPH-ferredoxin oxidoreductases was investigated with Clostridium pasteurianum and Clostridium butyricum.The NADH-ferredoxin oxidoreductases are concluded to be catabolic enzymes required for the reduction of ferredoxin by NADH. The conclusion is based on the finding that during the entire growth phase the fermentation of glucose can be formally represented by the weighted sum of Eqns 1 and 2, Glucose + 2 H₂O → 1 butyrate^? + 2 HCO₃^? + 3 H⁺ + 2 H₂ (1) Glucose + 4 H₂O → 2 acetate^? + 2 HCO₃^? + 4 H⁺ + 4 H₂ (2) and that in these redox processes NADH rather than NADPH is specifically formed during glyceraldehyde phosphate dehydrogenation. This NADH can be consumed by substrate reduction in Process 1 only, while it must be reoxidized in Process 2 by the ferredoxin-dependent proton reduction to hydrogen which involves the NADH-ferredoxin oxidoreductases.The kinetic and regulatory properties of these enzymes are in line with their catabolic role: they are found with high specific activities typical for other catabolic enzymes; essentially they catalyze electron flow from NADH to ferredoxin only because the back reaction is very effectively inhibited by low concentrations of NADH. These enzymes have a key role in the coupling of the two partial processes and in regulating the overall thermodynamic efficiency of the fermentations.The NADPH-ferredoxin oxidoreductases are concluded to participate in anabolism; they are required for the regeneration of NADPH. The conclusion is based on the finding that in the two clostridia all catabolic oxidations-reductions are specific for NAD(H) and that the usual NADPH-producing processes such as the glucose 6-phosphate dehydrogenase or malate enzyme reactions are absent. The kinetic properties of the enzymes are in agreement with their anabolic function: the NADPH-ferredoxin oxidoreductases are found with sufficient specific activities; they preferentially catalyze electron transfer from reduced ferredoxin to NADP⁺.     

Stimulation of Weak Acid Uptake and Increase in Cell Sap pH as Evidence for Fusicoccin- and K-Induced Cytosol Alkalinization

In maize root segments fusicoccin induced a consistent increase in cell sap pH (taken as representative of vacuolar pH). This effect was markedly enhanced by the presence of K⁺ in the medium, whereas in the absence of fusicoccin K⁺ did not significantly influence cell sap pH. Treatment with a weak acid at 2 mm concentration inhibited the uptake of a different (¹⁴C-labeled) weak acid fed at a lower concentration, thus suggesting that acidification of the cytoplasm inhibits weak acid uptake. Fusicoccin and K⁺ increased the rate of uptake of 5,5-dimethyloxazolidine-2,4-dione, butyric acid, or isobutyric acid slightly when fed separately, strongly when fed in combination. The synergism between fusicoccin and K⁺ in stimulating weak acid uptake was parallel to that observed for the stimulation of H⁺ extrusion. Application of the weak acid distribution method to a condition of `quasi-equilibrium' indicated that fusicoccin induces a cytosolic pH increase of about 0.14 unit. These results are interpreted as providing circumstantial evidence that fusicoccin- and K⁺- induced stimulation of H⁺ extrusion led to an alkalinization of the cytosol, and that other early metabolic responses, such as an increase in malate level, are a consequence of the increase in cytosolic pH.     

Active oxygen species production in tobacco cells elicited by cryptogein*

We analyse the relationship between active oxygen species (AOS) production and pH changes induced in tobacco cells by cryptogein, a fungal proteinaceous elicitor of defence mechanisms in plants. When tobacco cells were treated with cryptogein, an intracellular acidification, an alkalinization of the extracellular medium and a transient burst of AOS (H₂O₂) were observed. Treatment of elicited cells with either diphenyleneiodonium (DPI), an inhibitor of the neutrophil NADPH oxidase, or Tiron, which scavenges O₂^˙? abolished AOS production. These data suggest the involvement of a NADPH oxidase-like enzyme leading to H₂O₂ production through O₂^˙? dismutation. Although H₂O₂ production could be, per se, the origin of the pH changes observed, we showed that it was not the main cause, since DPI and Tiron did not inhibit extracellular alkalinization. On the other hand, cryptogein-induced changes in pH could be abolished using fusicoccin (FC), which is known to stimulate the plasmalemma H⁺ ATPase. Consequently, the observed changes in pH induced by cryptogein could be mainly due to the inhibition of the plasmalemma H⁺-ATPase activity. Furthermore, changes in extracellular pH were shown to modulate the intensity of AOS production by elicited cells. The possible regulation of the NAD(P)H oxidase activity of plant cells by changes in pH is further discussed.     

Electron transport across the plasmalemma of Lemna gibba G1

Lemna gibba L., grown in the presence or absence of Fe, reduced extracellular ferricyanide with a V _max of 3.09 mol · g^-1 fresh weight · h^-1 and a K _m of 115 M. However, Fe³⁺-ethylenediaminetetraacetic acid (EDTA) was reduced only after Fe-starvation. External electron acceptors such as ferricyanide, Fe³⁺-EDTA, 2,6-dichlorophenol indophenol or methylene blue induced a membrane depolarization of up to 100 mV, but electron donors such as ferrocyanide or NADH had no effect. Light or glucose enhanced ferricyanide reduction while the concomitant membrane depolarization was much smaller. Under anaerobic conditions, ferricyanide had no effect on electrical membrane potential difference (E_m). Ferricyanide reduction induced H⁺ and K⁺ release in a ratio of 1.16 H⁺+1 K⁺/2 e^- (in +Fe plants) and 1.28 H⁺+0.8 K⁺/2 e^- (in -Fe plants). Anion uptake was inhibited by ferricyanide reduction. It is concluded that the steady-state transfer of electrons and protons proceeds by separate mechanisms, by a redox system and by a H⁺-ATPase.Abbreviations E _m electrical membrane potential difference - EDTA ethylenediaminetetraacetic acid - DCPIP dichlorophenol indophenol - +Fe control plant - -Fe iron-deficient plant - FW fresh weight - H⁺ electrochemical proton gradient     

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

Abstract Dissociation of active H⁺ extrusion (?ΔH⁺) from K⁺ uptake in pea and maize root segments was attempted by substituting K⁺ in the incubation medium with lipophilic cations assumed to enter the cell by passive, non-specific, permeation through the lipid component of the plasmalemma. Among the compounds tested, tributylbenzylammonium significantly stimulated ?ΔH⁺ in the absence of other monovalent cations in the medium. This effect was much more evident when the experiment was carried out in the presence of fusicoccin, which strongly stimulates proton extrusion and monovalent cation uptake, and hyperpolarizes the trans-membrane electric potential in these materials. Also the lipophilic cations tetraphenylphosphonium, dimethyldibenzylammonium and hexylguanidine markedly stimulated FC-promoted ?ΔH⁺. Octylguanidine at a low concentration induced an early stimulation followed by a strong inhibition of ?ΔH⁺. A complete lack of additivity was observed between the effects of lipophilic cations and that of K⁺ on H⁺ extrusion. Lipophilic cations severely inhibited K⁺ uptake. These data are interpreted as supporting the view of an electric, rather than a chemical, (namely, involving the same carrier system) nature of the coupling of active H⁺ extrusion with K⁺ influx.