Plasmalemma Redox Chain and H Extrusion: II. Respiratory and Metabolic Changes Associated with Fusicoccin-Induced and with Ferricyanide-Induced H Extrusion

Ferricyanide reduction by Elodea densa leaves is associated with a release of protons in the cytoplasm, a fraction of the increase in protons being then extruded by the ATP-driven proton pump (20). The data presented here show that ferricyanide induces a marked increase in O₂ uptake, additive to that induced by fusicoccin plus K⁺, and here interpreted as depending on the utilization of ATP by the H⁺ pump. Glucose 6-phosphate and malate levels are markedly increased by fusicoccin plus K⁺. The simultaneous presence of ferricyanide reduces by about 50% the increase of malate, while it completely suppresses that of glucose 6-phosphate. The ferricyanide-induced decrease of malate is interpreted as due to the acidification of the cytosol associated with ferricyanide reduction, while the more marked decrease of glucose 6-phosphate might depend in part on the pH change and in part on a faster oxidation of this substrate. In fact, ferricyanide reduction is accompanied by a marked decrease of the incorporation into RNA ribose of C-1 as compared with C-2 of [¹⁴C]glucose. This suggests a stimulation of the release of C-1 as CO₂ at the level of the glucose 6-phosphate oxidation pathway, as expected if NADPH was the electron donor for ferricyanide reduction. These results are interpreted as confirming that the H⁺ efflux associated with ferricyanide reduction depends on the activation of the ATP-driven plasmalemma H⁺ pump. They also suggest that NADPH is used as an electron donor to some initial component of the plasmalemma redox system.     

Proton Fluxes as a Response to External Salinity in Wild Type and NaCl-Adapted Nicotiana Cell Lines

Addition of 100 millimolar KCl, NaCl, or Na₂SO₄ strongly promoted acidification of the medium by cells of Nicotiana tabacum/gossii in suspension culture. Acidification was greater in the case of NaCl-adapted than in that of wild type cells, and strikingly so in KCl medium when fusicoccin (FC) was present. Back-titration indicated that net proton secretion in KCl medium was increased 4-fold by FC treatment in the case of adapted cells; but was not even doubled in wild type cells. Membrane potential was higher in NaCl-adapted cells. FC treatment hyperpolarized wild, but not NaCl-adapted cells, suggesting a higher degree of coupling between H⁺ efflux and K⁺ influx in adapted cells; FC enhanced net K⁺ uptake in adapted but not in wild cells. Acidification by cells suspended in 10 millimolar KCl was highly sensitive to vanadate, but that after addition of 100 millimolar KCl or NaCl was much less sensitive. Addition of 100 millimolar NaCl to wild type cells already provided with 10 millimolar KCl briefly accelerated, then slowed down the rate of acidification. If the addition was made after acidification had already ceased, alkalization was observed, particularly in the presence of FC. The results are consistent with the operation of a Na⁺-H⁺ antiporter.     

Relationship of Transplasmalemma Redox Activity to Proton and Solute Transport by Roots of Zea mays

Transplasmalemma redox activity, monitored in the presence of exogenous ferricyanide stimulates net H⁺ excretion and inhibits the uptake of K⁺ and α-aminoisobutyric acid by freshly cut or washed, apical and subapical root segments of corn (Zea mays L. cv “Seneca Chief”). H⁺ excretion is seen only following a lag of about 5 minutes after ferricyanide addition, even though the reduction of ferricyanide occurs before 5 minutes and continues linearly. Once detected, the enhanced rate of H⁺ excretion is retarded by the ATPase inhibitors N,N′-dicyclohexylcarbodiimide, diethylstilbestrol, and vanadate. A model is presented in which plasmalemma redox activity in the presence of ferricyanide involves the transport only of electrons across the plasmalemma, resulting in a depolarization of the membrane potential and activation of an H⁺-ATPase. Such a model implies that this class of redox activity does not provide an additional and independent pathway for H⁺ transport, but that the activity may be an important regulator of H⁺ excretion. The 90% inhibition of K⁺ (⁸⁶Rb⁺) uptake within 2 minutes after ferricyanide addition can be contrasted with the 5 to 15% inhibition of uptake of α-aminoisobutyric acid. The possibility exists that a portion of the K⁺ and most of the α-aminoisobutyric acid uptake inhibitions are related to the ferricyanide-induced depolarization of the membrane potential, but that the redox state of some component of the K⁺ uptake system may also regulate K⁺ fluxes.     

Effects of salt-adaptation and salt-stress on extracellular acidification and microsome phosphohydrolase activities in tomato cell suspensions

The effects of NaCl-adaptation and NaCl-stress on in vivo H⁺ extrusion and microsomal vanadate- and bafilomycin-sensitive ATPase and PPase activities were studied in tomato cell suspensions. Acidification of the external medium by 50 mM NaCl-adapted and non-adapted (control) tomato cells was similar. Extracellular acidification by both types of cells during the first hour of incubation with 2 μM fusicoccin (FC) in the presence of 100 mM NaCl was lightly increased while in the presence of 100 mM KCl it was increased by 3 (control)- and 6.5 (adapted)-fold. Extracellular alkalinization after 2 h of cell incubation in 100 mM NaCl indicated the possibility that a Na⁺/H⁺ exchange activity could be operating in both types of cells. Moreover, acidification induced by adding 100 mM NaCl + FC to non-adapted cells was relatively less affected by vanadate than that induced by 5 mM KCl + FC, which suggested that salt stress could induce some component other than H⁺ extrusion by H⁺-ATPase. In addition, no differences were observed in microsomal vanadate-sensitive ATPase activity among control, NaCl-adapted and NaCl-stressed cells, while K⁺-stimulated H⁺-PPase and bafilomycin-sensitive H⁺-ATPase activities were higher in microsomes from NaCl-adapted than in those from control cells. Likewise, the stimulation of in vivo H⁺ extrusion in NaCl adapted cells under NaCl or KCl stress in the presence of FC occurred with an inhibition of H⁺-PPase and bafilomycin-sensitive H⁺-ATPase activities and without changes in the vanadate-sensitive H⁺-ATPase activity. These results suggest that the stimulation of tonoplast proton pumps in NaCl-adapted cells, without changes in plasmalemma H⁺-ATPase, could serve to energize Na⁺ efflux across the plasmalemma and Na⁺ fluxes into vacuoles catalyzed by the Na⁺/H⁺ antiports. This revised version was published online in June 2006 with corrections to the Cover Date.     

Charge and acidity compensation during proton-sugar symport in Chlorella: The H+-ATPase does not fully compensate for the sugar-coupled proton influx

This study was undertaken in order to demonstrate the extent to which the activity of the plasmalemma H⁺-ATPase compensates for the charge and acidity flow caused by the sugar-proton symport in cells of chlorella vulgaris Beij.. Detailed analysis of H⁺ and K⁺ fluxes from and into the medium together with measurements of respiration, cytoplasmic pH, and cellular ATP-levels indicate three consecutive phases after the onset of H⁺ symport. Phase 1 occurred immediately after addition of sugar, with an uptake of H⁺ by the hexoseproton symport and charge compensation by K⁺ loss from the cells and, to a smaller degree, by loss of another ion, probably a divalent cation. This phase coincided with strong membrane depolarization. Phase 2 started approximately 5 s after addition of sugar, when the acceleration of the H⁺-ATPase caused a slow-down of the K⁺ efflux, a decrease in the cellular ATP level and an increase in respiration. The increased respiration was most probably responsible for a pronounced net acidification of the medium. This phase was inhibited in deuterium oxide. In phase 3, finally, a slow rate of net H⁺ uptake and K⁺ loss was established for several further minutes, together with a slight depolarization of the membrane. There was hardly any pH change in the cytoplasm, because the cytoplasmic buffering capacity was high enough to stabilize the pH for several minutes despite the net H⁺ fluxes. The quantitative participation of the several phases of H⁺ and K⁺ flow depended on the pH of the medium, the ambient Ca²⁺ concentration, and the metabolic fate of the transported sugar. The results indicate that the activity of the H⁺-ATPase never fully compensated for H⁺ uptake by the sugar-symport system, because at least 10% of symport-caused charge inflow was compensated for by K⁺ efflux. The restoration of pH in the cytoplasm and in the medium was probably achieved by metabolic reactions connected to increased glycolysis and respiration.Abbreviations DMO dimethyloxazolidinedione - EDTA ethylcnediaminetetraacetic acid - p.c. packed cell volume     

Ion fluxes and pH changes induced by trans-plasmalemma electron transfer and fusicoccin inLemna gibba L. (strain G1)

During the reduction of extracellular [Fe(CN)₆]^3– at the plasmalemma of intact, K⁺-starvedLemna gibba L. fronds, the external medium was acidified and K⁺ released, in the absence of inhibitors with rates of 10 e^–/8.5 H⁺/1.5 K⁺ (mol·(g FW)^–1·^–1). In K⁺ plants the larger K⁺ efflux caused a lag phase in extracellular acidification and a change in rates to 10 e^–/6 H⁺/4 K⁺ and in the presence of CN^–+salicylhydroxamic acid at pH 5 to 5.2 e^–/0 H⁺/6.6 K⁺. The e^– transfer was accompanied by a membrane depolarization of up to 100 mV and a cytosolic acidification of about 0.6 pH units, but only in K⁺ plants, where the extracellular acidification was smaller. These results indicate that a stimulation of the plasmalemma H⁺-ATPase may be triggered either by a cytosolic acidification or by a strong membrane depolarization. It is concluded that the redox system catalyses only uncoupled e^– transfer without H⁺ transfer across the plasmalemma. The obligatory, but secondary charge compensation is partially achieved by the rapid K⁺ release upon membrane depolarization and partially by the activity of the plasma membrane H⁺-ATPase, but not by an e^–/anion exchange. The extracellular acidification during [Fe(CN)₆]^3– reduction is generated by the conversion of a strong trivalent into a strong tetravalent anion. This acidification is caused by changes in the concentration ratio of strong cations to strong anions. Efflux of K⁺ and not the production of organic acids or NAD(P)H oxidation is the chemical cause of the measurable cytosolic acidification. Extracellular acidification was inversely correlated with intracellular acidification. Similarly, fusicoccin-induced pH changes were correlated with changes in the strong-ion concentration difference. Extracellular ± FC-dependent acidification and intracellular alkalinization of up to 0.6 pH units were strongly dependent on K⁺ fluxes. The ferricyanide-triggered trans-plasmalemma electron-transfer system is an example of how measurable pH changes are the consequence and not the cause of charge-transfer-induced changes in strong-ion fluxes.Abbreviations DCCD dicyclohexylcarbodiimide - E_m electrical membrane potential difference - FC fusicoccin - pH_c cytosolic pH - FW fresh weight - PM plasmalemma - SHAM salicylhydroxamic acid - SID strong-ion concentration difference This work was supported by the Deutsche Forschungsgemeinschaft. We gratefully acknowledge the Alexander von Humboldt award donated to J.G. We thank Professor Ulrich Lüttge (TH Darmstadt, FRG) for his kind support and Annett Ehrhardt and Dr. Karl Fischer (TH Darmstadt, FRG) for their valuable help with Cl^– and CO₂ experiments. Special thanks are due to Professor Erasmo Marrè (Università di Milano, Italy) for continuous discussions and also to Professor Alessandro Ballio (Università di Roma, Italy) for their kind gifts of fusicoccin.     

Extra- and Intracellular pH and Membrane Potential Changes Induced by K, Cl, H(2)PO(4), and NO(3) Uptake and Fusicoccin in Root Hairs of Limnobium stoloniferum

Short-term ion uptake into roots of Limnobium stoloniferum was followed extracellularly with ion selective macroelectrodes. Cytosolic or vacuolar pH, together with the electrical membrane potential, was recorded with microelectrodes both located in the same young root hair. At the onset of chloride, phosphate, and nitrate uptake the membrane potential transiently decreased by 50 to 100 millivolts. During Cl⁻ and H₂PO₄⁻ uptake cytosolic pH decreased by 0.2 to 0.3 pH units. Nitrate induced cytosolic alkalinization by 0.19 pH units, indicating rapid reduction. The extracellular medium alkalinized when anion uptake exceeded K⁺ uptake. During fusicoccin-dependent plasmalemma hyperpolarization, extracellular and cytosolic pH remained rather constant. Upon K⁺ absorption, FC intensified extracellular acidification and intracellular alkalinization (from 0.31 to 0.4 pH units). In the presence of Cl⁻ FC induced intracellular acidification. Since H⁺ fluxes per se do not change the pH, recorded pH changes only result from fluxes of the stronger ions. The extra- and intracellular pH changes, together with membrane depolarization, exclude mechanisms as K⁺/A⁻ symport or HCO₃⁻/A⁻ antiport for anion uptake. Though not suitable to reveal the actual H⁺/A⁻ stoichiometry, the results are consistent with an H⁺/A⁻ cotransport mechanism.     

Glucose- and K+-induced acidification in different yeast species

The process of acidification of the external medium after addition of glucose and subsequently of KCl to a suspension of yeast cells varies substantially from species to species. After glucose it is most pronounced inSaccharomyces cerevisiae andSchizosaccharomyces pombe but is very much lower inLodderomyces elongisporus, Dipodascus magnusii andRhodotorula gracilis. Both the buffering capacity and the varied effects of vanadate, suloctidil and erythrosin B indicate that the acidification is by about one-half due to the activity of plasma membrane H⁺-ATPase and by about one-half to the extrusion of acidic metabolites from cells. This is supported by the finding that a respiratory quotient greater than one (in various strains ofS. cerevisiae and inS. pombe) is indicative of a greater buffering capacity and overall acidification of the medium. Taking into account the virtually negligible buffering capacity of the medium in the pH range where the effect of K⁺ is observed, the effect of K⁺ is generally of a similar magnitude as that of adding glucose. It is clearly dependent on (anaerobic) production of metabolic energy, quite distinct from the dependence of the H⁺-ATPase-caused acidification.     

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 possible role of redox-associated protons in growth of plant cells

The protons excreted by plant cells may arise by two different mechanisms: (1) by the action of the plasma membrane H⁺-ATPase and (2) by plasma membrane redox reactions. The exact proportion from each source is not known, but the plasma membrane H⁺-ATPase is, by far, the major contributor to proton efflux. There is still some question of whether the redox-associated protons produced by NADH oxidation on the inner side of the plasma membrane traverse the membrane in a 1 : 1 relationship with electrons generated in the redox reactions. Membrane depolarization observed in the presence of ferricyanide reduction by plasma membranes of whole cells or tissues or the lag period between ferricyanide reduction and medium acidification argue that only scalar protons may be involved. The other major argument against tight coupling between protons and electrons involves the concept of strong charge compensation. When ferricyanide is reduced to ferrocyanide on the outside of cells or tissues, an extra negative charge arises, which is compensated for by the release of H⁺ or K⁺, so that the total ratio of increased H⁺ plus K⁺ equals the electrons transferred by transmembrane electron transport. These are strong arguments against a tight coupling between electrons and protons excreted by the plasma membrane. On the other hand, there is no question that inhibitor studies provide evidence for two mechanisms of proton generation by plasma membranes. When the H⁺-ATPase activity is totally inhibited, the addition of ferricyanide induces a burst of extra proton excretion, orvice versa, when plasma membrane redox reactions are inhibited, the H⁺-ATPase can function normally. Since plasma membrane redox reactions and associated H⁺ excretion are related to growth, it is possible that in plants the ATPase-generated protons have a different function from redox-associated protons. The H⁺-ATPase-generated protons have been considered for many years to be necessary for cell wall expansion, allowing elongation to take place. A special function of the redox-generated protons may be in initiating proliferative cell growth, based on the presence of a hormone-stimulated NADH oxidase in membranes of soybean hypocotyls and stimulation of root growth by low concentrations of oxidants. Here we propose that this NADH oxidase and the redox protons released by its action control growth. The mechanism for this may be the evolution of protons into a special membrane domain, from which a signal to initiate cell proliferation may originate, independent of the action of the H⁺-ATPase-generated protons. It is also possible that both expansion and proliferative growth are controlled by redox-generated protons.     

Plasmalemma redox activity and h extrusion in roots of fe-deficient cucumber plants

Cucumber plants (Cucumis sativus L.) with incipient Fe deficiency showed increased root capacity to reduce chelated Fe³⁺ compared to Fe-sufficient plants. When Fe-ethylenediaminete-traacetate was added to the root medium of the Fe-deficient plants, the reductase activity was associated with acidification of the medium and an increase in the net apparent K⁺ efflux. In the presence of the H⁺-ATPase inhibitor N,N′-dicyclohexylcarbodiimide the net apparent H⁺ efflux was completely suppressed, though some reductase activity was preserved, and the net apparent K⁺ efflux was significantly increased. The inhibition of the reductase activity by N,N′-dicyclohexylcarbodiimide was similar whether the pH of the medium was buffered or not. Anoxia and the protonophore carbonyl cyanide m-chlorophenyl hydrazone also caused a similar inhibition of the reductase activity. It is proposed that this redox system transports electrons only and that its activity is inhibited by plasmamembrane depolarization and anoxia. The H⁺ and K⁺ efflux associated with the reductase activity may be a result of the plasmamembrane depolarization it causes.     

Auxin and Fusicoccin Enhancement of beta-Glucan Synthase in Peas : An Intracellular Enzyme Activity Apparently Modulated by Proton Extrusion

Fusicoccin (FC), like indoleacetic acid (IAA), causes Golgi-localized β-1,4-glucan synthase (GS) activity to increase when applied to pea third internode segments whose GS activity has declined after isolation from the plant. This suggests that GS activity is modulated by H⁺ extrusion; in agreement, vanadate and nigericin inhibit the GS response. The GS response is not due to acidification of the cell wall. Treatment of tissue with heavy water, which in effect raises intracellular pH, mimics the IAA/FC GS response. However, various treatments that tend to raise cytoplasmic pH directly, other than IAA- or FC-induced H⁺ extrusion, failed to increase GS activity, suggesting that cytoplasmic pH is not the link between H⁺ extrusion and increased GS activity. Although FC stimulates H⁺ extrusion more strongly than IAA does, FC enhances GS activity at most only as much as, and often somewhat less than, IAA does. This and other observations indicate that GS enhancement is probably not due to membrane hyperpolarization, stimulated sugar uptake, or changes in ATP level, but leave open the possibility that GS is controlled by H⁺ transport-driven changes in intracellular concentrations of ions other than H⁺.     

Proton efflux from corn roots induced by tripropyltin

Tripropyltin restores medium acidification by washed corn root tissue in which electrogenic H⁺ efflux has been blocked by ATPase inhibitors or injury. However, the restored H⁺ efflux is not electrogenic and will not drive K⁺ influx, and, by itself, tripropyltin is inhibitory to K⁺ influx. Tripropyltin elicits a 5-fold increase in endogenous chloride efflux, and Cl⁻/OH⁻ exchange can, thus, account for the observed acidification of the medium. This explanation cannot be applied equally to the acidification produced by the K⁺/H⁺ exchanging ionophore nigericin.     

Depolarization of Cell Membrane Potential during Trans-Plasma Membrane Electron Transfer to Extracellular Electron Acceptors in Iron-Deficient Roots of Phaseolus vulgaris L

Transfer of electrons from the cytosol of bean (Phaseolus vulgaris L.) root cells to extracellular acceptors such as ferricyanide and Fe^IIIEDTA causes a rapid depolarization of the membrane potential. This effect is most pronounced (30-40 millivolts) with root cells of Fe-deficient plants, which have an increased capacity to reduce extracellular ferric salts. Ferrocyanide has no effect. In the state of ferricyanide reduction, H⁺ (1H⁺/2 electrons) and K⁺ ions are excreted. The reduction of extracellular ferric salts by roots of Fe-deficient bean plants is driven by cellular NADPH (Sijmons, van den Briel, Bienfait 1984 Plant Physiol 75: 219-221). From this and from the membrane potential depolarization, we conclude that trans-plasma membrane electron transfer from NADPH is the primary process in the reduction of extracellular ferric salts.     

H and k electrogenic exchanges in corn roots

The membrane potential difference, the net H⁺ exchange rate, the K⁺ net flux, and the K⁺ (⁸⁶Rb⁺) influx were measured in excised corn roots as functions of the K⁺ concentration in the medium at various pH values, in the presence of poorly permeant anions. The roots behaved as a K⁺/H⁺ exchange system. By comparing the results in normal or hypoxic conditions, or in the presence of vanadate, it was possible to distinguish the active components of membrane potential and transports from the passive ones. The magnitude of the electrogenic potential was not related to the active H⁺ extrusion rate. At pH 6, the variations of the electrogenic potential resulted from variations of the stoichiometry of the active H⁺/K⁺ exchange. The same relationship between this stoichiometry and the K⁺ concentration was observed in conditions ensuring different membrane polarizations (pH 6, pH 4, or pH 6 with fusicoccin). Both metabolic and Mg-ATPase specific inhibitors stopped the active H⁺ transport and the net K⁺ influx. Nevertheless, the tracer influx in the presence of vanadate remained higher than the passive influx calculated from the permeability coefficient determined in hypoxia. It is proposed that vanadate uncouples the K⁺ moiety of the H⁺/K⁺ antiport and allows it to mediate isotopic exchanges.     

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     

Acidification and Alkalinization of Culture Medium by Catharanthus roseus cellsIs Anoxic Production of Lactate a Cause of Cytoplasmic Acidification?

Catharanthus roseus(L.) G. Don cells acidified Mura-shige-Skoogmedium rapidly. Upon transfer to fresh medium, the medium pH(initially5.3) dropped below 4 within 2 d. This acidificationwas reversed under hypoxic conditions. The cells induced a similaracidification in a simple medium consisting of CaCl₂, KCl, andglucose: medium pH dropped below 4 within 6 h. The acidificationwas accompanied by an influx of K⁺ at a H⁺(efflux)/K⁺ ratioof ca 0.6 as well as by an expansion of endogenous organic acidpool, in which malic and citric acids were the major components.Anoxia reversed all these processes: the direction of both K⁺and H⁺ fluxes reversed with a H⁺/K⁺ ratio of 1.70. Anoxia induceda cytoplasmic acidification from pH 7.6 (aerobic) to 7.4 asmeasured by 31P-NMR, accompanied by a rapid, long-lasting lactateaccumulation at expense of malic and citric acids. Evidencesuggested that accumulation of lactic acid was not a cause ofcytoplasmic acidification under anoxia, but a result of pH regulationby the biochemical pH-stat [Davies (1973) Symp. Soc. Exp. Biol.27: 513]. The anoxic acidification of the cytoplasm was ascribedto the influx of H⁺ from the medium. (Received April 18, 1997; Accepted July 8, 1997)     

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.     

Effects of hyper-osmotic stress on K+ fluxes, H+ extrusion, transmembrane electric potential difference and comparison with the effects of fusicoccin

The stimulation of H⁺ extrusion by hyper-osmotic stress (0.2–0.3 M mannitol) in cultured cells of Arabidopsis thaliana (L.) Heynh. was shown to be associated with an inhibition of Cl^? efflux, whereas hypo-osmotic stress, inhibiting H⁺ extrusion, early and strongly stimulated Cl^? efflux. In this paper, we investigate the contribution of other factors [K⁺ transport and transmembrane electric potential difference (E_m)] to the hyper-osmotic-induced activation of the plasma membrane (PM) H⁺-ATPase. The effects of mannitol (MA) on K⁺ transport and on E_m were compared with those of fusicoccin (FC) since the modes of action of osmotica and of the toxin in stimulating H⁺-ATPase activity seem to differ at least in some steps. The changes in H⁺ extrusion induced by hyper- or hypo-osmotic stress were opposite and could be reversed by the application of the respective opposite stress. The effect of MA on H⁺ extrusion was dependent on the presence of K⁺ (or Rb⁺) similarly to that of FC, while Na⁺ and Li⁺, which also stimulated the FC effect, were ineffective on that of MA. The MA effect was independent of the anions (Cl^?, SO₄^2?, NO₃^?) accompanying K⁺. K⁺ net uptake and K⁺ influx were stimulated by both MA and FC. Tetraethylammonium (TEA⁺) and Cs⁺ inhibited both MA- and FC-induced H⁺ extrusion, suggesting the involvement of K⁺ channels. MA (0.2 M) induced a strong hyperpolarization of E_m both in the absence and in the presence of K⁺. The hyperpolarizing effect of MA was also found when the cells were already hyperpolarized by FC, and was rapidly reversed by removing the osmoticum from the medium. In the presence of the lipophilic cation tributylbenzylammonium (TBBA⁺), MA was no longer able to stimulate H⁺ extrusion, while FC still stimulated it. In cells pretreated with TBBA⁺, which strongly depolarized E_m, the subsequent addition of FC repolarized it, while the hyperpolarizing effect of MA was lacking. On the contrary, in cells pretreated with Erythrosine B (EB), E_m was strongly depolarized and the following addition of FC did not hyperpolarize it, while the hyperpolarizing effect of MA was still observed. These results suggest that the mechanism of MA in activating H⁺ extrusion and K⁺ uptake is different from that of FC. The rise in net K⁺ uptake seems to be driven by the activation of some hyperpolarizing system that does not seem to depend on a direct activation of PM H⁺-ATPase, but rather on the inhibition of Cl^? efflux induced by hyper-osmotic stress.     

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.