Effect of Sudden Salt Stress on Ion Fluxes in Intact Wheat Suspension Cells

Although salinity is one of the major problems limiting agriculturalproduction around the world, the underlying mechanisms of highNaCl perception and tolerance are still poorly understood. Theeffects of different bathing solutions and fusicoccin (FC),a known activator of plasma membrane ATPase, on plasma membranepotential (E_m) and net fluxes of Na⁺, K⁺and H⁺were studied inwheat suspension cells (Triticum aestivum) in response to differentNaCl treatments. E_mof cells in Murashige and Skoog (MS) mediumwas less negative than in cells exposed to a medium containing10 mM KCl + 0.1 m M CaCl₂(KSM) and to a basic salt medium (BSM),containing 1 m M KCl and 0.1 m M CaCl₂. Multiphasic Na⁺accumulationin cells was observed, peaking at 13 min after addition of 120m M NaCl to MS medium. This time scale was in good agreementwith net Na⁺flux changes measured non-invasively by moving ion-selectivemicroelectrodes (the MIFE system). When 120 m M NaCl was addedto all media studied, a quick rise of Na⁺influx was reversedwithin the first 20 min. In both 120 and 20 m M NaCl treatmentsin MS medium, net Na⁺efflux was observed, indicating that activeNa⁺transporters function in the plant cell response to saltstress. Lower external K⁺concentrations (KSM and BSM) and FCpre-treatment caused shifts in Na⁺fluxes towards net influxat 120 m M NaCl stress. Copyright 2000 Annals of Botany Company Sodium, potassium, proton, membrane potential, fusicoccin, salt stress, wheat, Triticum aestivum     

Rates of Hydrogen Ion Efflux by Nodulated Legumes Grown in Flowing Solution Culture with Continuous pH Monitoring and Adjustment

The net efflux of H⁺ from lucerne (Medicago saliva L.), redclover (Trifolium pratense L.) and white clover (Trifolium repensL.) growing in flowing solution culture and dependent upon symbioticfixation of atmospheric N, was measured over a 75 d experimentalperiod. Considerable and rapid increases in acidity of the nutrientsolution of up to 1.45 pH units were recorded when the pH wasriot held constant over a 30 h period. There was little differencein H⁺ efflux when solution pH was held constant at 4.75, 5.75or 6.75, but there was an immediate cessation when it was adjustedto 3.75. Differences in the daily net efflux of H⁺ closely followedthe pattern of daily differences in incoming radiation, andthere was also evidence of a diurnal pattern of H⁺ efflux. Althoughthere were initially distinct differences between the speciesin the calculated rate of net H⁺ efflux (µg H⁺ g^–1dry shoot d^–), by day 75 these had diminished. In allspecies, however, the maximum rate of efflux per unit of shootsoccurred during the earlier rapid phases of growth. The measuredefflux of H⁺ was well equated with the plant content of excesscations (as measured by ash alkalinity) and, on average, theratio of acidity produced to N assimilated (expressed as anequivalent) was 0-24. Medicago sativa L., Trifolium pratense L., Trifolium repens L., lucerne, red clover, white clover, acidification, cation/anion balance, flowing solution culture, H⁺ efflux, nitrogen fixation     

Salinity Effects on the Activity of Plasma Membrane H+and Ca2+Transporters in Bean Leaf Mesophyll: Masking Role of the Cell Wall

Net fluxes of H⁺and Ca²⁺were measured in the mesophyll tissueof broad bean (Vicia faba L.) leaves and in protoplasts derivedfrom these cells. NaCl at 90 m M enhanced H⁺extrusion in bothprotoplasts and tissue, but in different ways. Proton extrusionwas inhibited by vanadate, suggesting the involvement of theplasma membrane H⁺-ATPase in cell responses to salinity. Therewas virtually no effect of NaCl on the net Ca²⁺flux in protoplasts,while in the tissue a large transient Ca²⁺efflux followed thesalt treatment. Salt-induced Ca²⁺efflux was essentially independentof external Ca²⁺concentrations in the range 0.1 to 10 m M. Also,Ca²⁺flux responses were ‘saturated’ above 50 m MNaCl. It is suggested that almost all the measured Ca²⁺fluxoriginates from Na⁺/Ca²⁺and H⁺/Ca²⁺ion exchange in the cellwall. This conclusion was supported by the results of modellingcation exchange in the cell wall. Copyright 2000 Annals of BotanyCompany Salinity, membrane transporters, wall ion exchange, proton, calcium, Vicia faba     

Respiration-Dependent H+ Efflux from Intact Cells of Cyanidium caldarium

An active H⁺ efflux depending on respiration was found in anacidophilic unicellular alga, Cyanidium caldarium. Alkalizationof the medium due to passive H⁺ transport into the cells wasobserved when the respiratory activity was inhibited by addingrespiratory poisons, such as rotenone or antimycin A, or byintroducing pure nitrogen into the cell suspension. The extentof the H⁺ influx increased as the pH of the medium was loweredto 2.9, indicating that H⁺ leaks into the cells according tothe pH gradient across the plasma membrane. The medium pH whichhad increased under anaerobic condition returned to the originallevel with aeration of the cell suspension. This suggests thatan active H⁺ transport, related to respiration, pumps out theexcess H⁺ accumulated in the cells during anaerobic preincubation.The pH changes in the cell suspension were related to the intracellularATP level. From these results it was concluded that active H⁺efflux dependent upon oxidative phosphorylation functions inthe dark to maintain a constant intracellular pH against passiveH⁺ leakage through the plasma membrane. The light-induced H⁺ efflux and the respiration-dependent H⁺efflux were also compared in relation to the physiological roleof the active H⁺ efflux, especially with respect to the intracellularpH regulation in this alga. ¹The data in this paper are included in the Ph. D. dissertationsubmitted by M. Kura-Hotta to Tokyo Metropolitan University. (Received February 3, 1984; Accepted June 14, 1984)     

Intracellular pH Regulation in an Acidophilic Unicellular Alga, Cyanidium caldarium: 31P-NMR Determination of Intracellular pH

The intracellular pH of an acidophilic unicellular alga, Cyanidiumcaldarium, was determined as a function of external pH by ³¹Pnuclear magnetic resonance. The algal cells incubated underaerobic conditions or under anaerobic and illuminated conditionsmaintained the intracellular pH in the range from 6.8 to 7.0even when the external pH was changed from 1.2 to 8.4. Underanaerobic and dark conditions, however, the intracellular pHacidified at the acidic pH region of the external medium. Theacidified intracellular pH reversibly returned to neutral eitheron aeration or illumination. The results indicate that, in Cyanidiumcells growing in extremely acidic environments, an active H⁺efflux (H⁺ pump) which depends on metabolic activity (respirationor photosynthesis) is essential to maintain the intracellularpH at a constant physiological level against the passive H⁺leakage due to the steep pH gradient across the cell membrane. (Received March 19, 1986; Accepted July 17, 1986)     

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)     

Electrophysiological Measurements on the Root of Atriplex hastata

The ion contents and membrane potentials of the cells of young,hydroponically cultured seedlings of Atriplex hastata L. var.salina, Wallr. have been measured at several different NaClconcentrations. The total tissue concentrations of Na⁺ and Cl^–increase as external NaCl increases, but there is always a markedexcess of internal Na⁺ over Cl^–; this is balanced by endogenousorganic anion formation with a concomitant extrusion of H⁺ tothe bathing solution. Membrane potentials of the root cells remain essentially invariantwith changes in external NaCl at approx. –130 mV; thereis no evidence of a radial gradient of potential across theroot. The potential seems to contain a cyanide-sensitive electrogeniccomponent, also invariant with NaCl concentration, of about–70 mV, and a diffusion component. The electrogenic componentseems likely to be a H⁺ efflux, probably through a H⁺ uniportATPase.     

Mechanism of Photosynthate Efflux from Vicia faba L. Seed Coats

In order to develop a tentative model of the mechanism of photosynthateefflux from the vascular region of Vicia faba L. seed coats,wash-out experiments were performed after removal of the embryo. The sulphydryl group modifiers, pCMBS and NEM, reduced ¹⁴C-photosynthateefflux by 40% and 50%, respectively. Their inhibitory effectcould be prevented or reduced (in the latter case) by includingDTT in the bathing solution. Maltose competed with sucrose forefflux; a concentration of 300 mol m^–3 inhibited ₁₄C-photosynthaterelease by 35%. The cations K⁺ , Na⁺ Mg²⁺ and TPP⁺ enhancedefflux significantly, whereas the countenon Cl^– had noeffect. The presence of the protonophore CCCP (0·1 molm^–3) led to a reduction of efflux by 50% net proton extrusiondropped by 34%. To a lesser extent, an efflux inhibition wasalso achieved by decreasing the cytoplasmic pH with the weakacid DM0. In contrast, alterations in the external pH causedonly a feeble response. The ATPase inhibitor, EB, decreasedphotosynthate efflux and H⁺ extrusion. DES reduced efflux slightly,presumably by affecting ATPase activity as well as energy metabolism. Based on these findings, it is proposed that a sucrose/protonantiport mechanism could be responsible for photosynthate effluxfrom Vicia faba seed coats. Key words: Photosynthate efflux, proton extrusion, proton/sucrose antiport, seed coat, Vicia faba L.     

Effect of Divalent Cations on Na+ Permeability of Chara corallina and Freshwater Grown Chara buckellii

The effect of elevated Na⁺ concentration on Na⁺ permeability(P_Na) and Na⁺ influx in the presence of two levels of externaldivalent cations was determined in Chara corallina and freshwater-culturedChara buckellii. When Na⁺ in the medium was increased from 1.0to 70 mol m^–3, Na⁺ influx increased in both species ifCa²⁺ was low (0.1 mol m–3). If Ca²⁺ was increased to 7.0mol m^–3 when Na⁺ was increased, Na⁺ influx remained atthe low control level in C. corallina, and showed only a temporaryincrease in C. buckellii. Mg²⁺ was a better substitute for Ca²⁺in C. buckellii than in C. corallina. Na⁺ permeability data suggest that when the external Ca²⁺ concentrationis low, P_Na does not increase in the presence of elevated NaCl;the increase in Na⁺ influx appears to be due to the increasein external Na⁺ concentration alone. Ca^{2 +} supplementation appearsto decrease P_Na whereas supplemental Mg²⁺ has no effect. Na⁺ effluxes were computed from previously determined net fluxesand the influxes. It was found that for both species, fluxesin both directions were stimulated in response to all experimentaltreatments, but Na⁺ influx always exceeded efflux. This resultedin net Na⁺ accumulation in the vacuoles of both species. The results are discussed with reference to net flux and electrophysiologicaldata obtained previously under identical conditions, as wellas the comparative salinity tolerance of both species and theNa⁺/divalent cation ratio. Key words: Na⁺ influx, Na⁺ tolerance, membrane potential, permeability, Chara     

Quantitative Analysis of ATP-Dependent H+ Efflux and Pump Current Driven by an Electrogenic Pump in Nitellopsis obtusa

Internodal cells of Nitellopsis were made tonoplast-free byperfusion with a medium containing EGTA. Cytoplasmic concentrationsof solutes were controlled by a second perfusion with mediaof known composition. The electrogenic pump current (I_p), whichwas calculated from electrical data obtained from cells withand without ATP, was compared with the current carried by H⁺(I_H⁺) across the plasma membrane. A close correlation betweenI_p and I_H⁺ was found under various internal and external conditions.(1) I_p and I_H⁺ depended on the internal ATP and showed Michaelis-Mententype saturation curves. For I_p, K_m was 120 µM and themaximum current V_max was 15.1 mA m^–2, while for I_H⁺, K_mwas 160 µM and V_max was 16.6 mA m^–2. (2) I_p andI_H⁺ showed almost the same I_H²⁺ dependence. The Mg²⁺-dependentI_p was 19.5 mA m^–2, while the Mg²⁺-dependent I_H²⁺ was17.7 mA m^–2. (3) I_H²⁺ was maximal at an external pH of8 and decreased both in acidic and alkaline pH ranges. I_p wasnearly equal to I_H⁺ in the pH range between 8 and 5. (4) I_H⁺became maximal at an internal pH of 7.3, which is nearly thesame as the pH for maximal electrogenecity found by Mimura andTazawa (1984). All these facts support the idea proposed in our previous paper(Takeshige et al. 1985) that the electrogenic ion pump locatedin the plasma membrane of Nitellopsis is the H⁺ pump. ¹ Dedicated to Professor Dr. Erwin Bünning on the occasionof his 80th birthday. (Received June 21, 1985; Accepted December 20, 1985)     

Down-Regulation of the Plasmalemma H+ Pump Activity by Nicotine-Induced Intracellular Alkalinization: a Balance between Base Accumulation, Biochemical pH-Stat Response and Intracellular pH Increase

Nicotine was used to induce an intracellular alkalinizationin Elodea densa leaves in order to study the regulation of theplasmalemma H⁺ pump activity by alkaline intracellular pH values.Nicotine was found to enter the cells rapidly in the unchargedform and to induce a significant intracellular pH increase,measured either directly as cell sap pH or as vacuolar and cytoplasmicpH by calculation from the distribution at equilibrium of labelledpH probes. The nicotine-induced alkalinization was associatedwith a progressive decrease in K⁺ uptake. A strong inhibitionof net H⁺ efflux was also evident in the presence of K⁺ in theexternal medium, whereas no nicotine effect on net H⁺ effluxwas detected in the absence of K⁺ (in spite of the larger accumulationof nicotine in the tissue) in agreement with a down-regulationof the activity of the K⁺-dependent plasmalemma H⁺-ATPase byalkaline intracellular pH values. The increase in vacuolar pHresulting from nicotine accumulation was small compared to thebase load calculated from the vacuolar buffer capacity and theintracellular dissociation of nicotine. Conversely, the nicotine-inducedincrease in cytoplasmic pH was considerably larger than expectedon the basis of the cytoplasmic buffer capacity and of the theoreticalaccumulation of nicotine in the experimental conditions adopted.A balance sheet between nicotine accumulation, intracellularalkalinization and malate system response was drawn up, andthe seeming discrepancies observed were discussed. (Received August 11, 1997; Accepted November 21, 1997)     

Na+ fluxes in Chara under salt stress

The influx and efflux of Na⁺ across the plasma membrane of Characorallina and Chara longifolia were examined under mild saltstress conditions. Na⁺ influx was found to be rapid in bothspecies with the freely exchangeable cytoplasmic Na⁺ cominginto isotopic equilibrium with external ²²Na⁺ within 1 h ofexposure to isotope. Cytoplasmlc Na⁺ concentration and Na⁺ influxwere greater in C. corallina than in C. longifolla under thesame conditions. Na⁺ influx across the tonoplast was much lowerthan the flux across the plasma membrane. Na⁺ efflux was stimulatedat pH 5 relative to pH 7 by 218% in C. coralllna and 320% inC. longifolia. In both species externally applied Li⁺ inhibitedNa⁺ efflux at pH 5 but not at pH 7. Na⁺ etflux was not significantlyinhibited by amiloride. Key words: Na⁺ influx, Na⁺ efflux, Na⁺/H⁺ antiport, Chara     

Effect of Intracellular ATP Levels on the Light-Induced H+ Efflux from Intact Cells of Cyanidium caldarium

The light-induced H⁺ efflux observed at acidic pH in Cyanidiumcells was shown to be an active H⁺ transport depending on theintracellular ATP produced by cyclic photo-phosphorylation.Triton X-100 was found to act as an effective uncoupler in intactCyanidium cells without collapsing the pH gradient across theplasma membrane. Triton X-100 at 0.015% significantly reducedthe intracellular ATP levels, stimulated the p-BQ, Hill reactionand completely inhibited the light-induced H⁺ efflux. Inhibitionof the H⁺ efflux by Triton X-100 correlated well with the depressionof the apparent rale of light-induced ATP synthesis as wellas the decrease in the intracellular ATP level in light. The light-induced H⁺ efflux was completely inhibited by diethylstilbestrol,a specific inhibitor of plasma membrane ATPase, without anychanges in the intracellular ATP level, thereby suggesting theparticipation of the plasma membrane ATPase in the light-inducedH⁺ efflux. ¹The data in this paper are included in the Ph. D. dissertationsubmitted by M. Kura-Hotta to Tokyo Metropolitan University. (Received February 3, 1984; Accepted June 14, 1984)     

H+ Efflux and Trans-Root Potential Measured while Increasing the Temperature of Solutions Bathing Excised Roots of Zea mays

Kennedy, C. D. and Gonsalves, F. A. N. 1988. H⁺ efflux and trans-rootpotential measured while increasing the temperature of solutionsbathing excised roots of Zea mays.—J. exp. Bot. 39: 37–49. Novel temperature-ramp procedures have been used to measureH⁺ efflux and trans-root potential of excised roots of Zea mays(var. Fronica). Two types of experiment were performed: (1),increasing temperature from 17°C, and (2), pre-cooling theroots to 1°C before starting the temperature ramp. The ratesof increase of temperature for H⁺ efflux and trans-root potentialexperiments were 0·5 and 2·1°C min^–1respectively The H⁺ scans revealed strong sharp maxima at 30°C and 32°C,for non-pre-cooled and pre-cooled roots respectively, the latterbeing significantly smaller. The trans-root potential scansfor the pre-cooled roots showed a corresponding maximum at 30°C,which was inhibited by KCN (1-0 mmol dm^–3) with or withoutSHAM (10 mmol dm^–3), or Hg²⁺ (1, 10, 100 µmol dm^–3)in the bathing solutions. Some of the evidence suggests thatthese maxima are associated with electrogenic H⁺ pumping, mediatedby a plasma membrane-bound ATPase. However, no correspondingmaximum was observed in the trans-root potential scans for non-pre-cooledroots, the potential remaining at about — 75 m V from20°C to 35°C. As there is a 7-fold increase in H⁺ effluxbetween 20°C and 30°C, the relationship between netH+ efflux and electrogenic proton pumping in these roots isby no means clear. Some possibilities are considered here. Pre-cooled and non-pre-cooled roots show clear maxima in thetrans-root potential scans at about 46°C, at which temperaturethere is a slight net H⁺ influx. This, and other less prominentfeatures observed, are briefly discussed. Key words: H⁺ efflux, trans-root potential, temperature-ramp procedure, Zea mays, roots     

H+ tolerance of Chara Internodal Cells and Apparent Net Influx of H+ in Weakly Acidic Solutions: Implication of the Net Flux of H+ as a Minor Component among the Total Net Flux of Ions across the Intact Cell Membrane of Chara

Precise measurements of the net flux of protons in Chara internodalcells were made with a recently designed high-resolution pH-meter.Survival of intact Chara internodal cells in artificial pondwater (APW) that contained HC1 at various concentrations wasalso examined. The apparent net flux of H⁺ was inward and muchsmaller than that reported so far. In APW at pH 4.005, a valuehigher than the extracellular pH expected from the values ofH⁺ efflux reported to date, all of the intact Chara internodalcells died within a day. With reference to the data on the circadianflow of ions in the pulvinus of Phaseolus [Kiyosawa (1979) PlantCell Physiol. 20: 1621–1634, Hosokawa and Kiyosawa (1983)Plant Cell Physiol. 24: 1065–1072] and ionic regulationin Chara L-cells [Kiyosawa and Okihara (1988) Plant Cell Physiol.29: 9–19], a discussion is presented of the prossiblyminor contribution of the net flux of H⁺ in the generation ofthe electrical membrane potential. Regulation of the net fluxof H⁺ in weakly acidic APW is also discussed. (Received September 4, 1989; Accepted January 25, 1990)     

Utilization of the Vibrating Probe and Ion-Selective Microelectrode Techniques to Investigate Electrophysiological Responses to Wounding in Pea Roots

This paper examines the ionic composition of wound-induced electricalcurrents in higher plant tissue, using two non-injurious electrophysiologicaltechniques. By simultaneous recording of K⁺, H⁺ , and Ca²⁺ ionfluxes with extracellular ion-selective microelectrodes, wehave determined that a Ca²⁺ influx (2.4 µA cm^–2),a small H⁺ influx (0.17 µA cm^–2) and a large K⁺efflux (16 µA cm^–2) occur immediately after woundingin roots of Pisum sativum L. var. Greenfeast. Using an extracellularvibrating probe at the wound site, net ion currents of 26 µAcm^–2 were measured 5 min after wounding. In a more concentratedbathing medium (1/4 rather than 1/16 strength Hoagland's solution),net ion currents of 59 µA cm^–2 were measured, andthese would appear to be the largest extracellular currentsthat have been measured in plants. We made a quantitative comparisonof the summed ion fluxes with the net ion currents and thisrevealed that ion fluxes, in addition to those measured here,occur after wounding. Key words: Wounding, ion flux, electric current, calcium, potassium     

Light-induced H+ Efflux from Intact Cells of Cyanidium caldarium

Light-induced pH changes in suspensions of an acidophilic unicellularalga, Cyanidium caldarium Geitler, were studied as a functionof the pH of the medium. In the neutral pH region, alkalizationof the medium due to photosynthetic CO₂ uptake was observed.In the acidic pH region, illumination caused a significant decreasein the pH of the medium, indicating the efflux of H⁺ from thecells. Both the rate and extent of the pH decrease increasedas the pH of the medium was lowered to 3.0. The H⁺ efflux wasnot affected by 3-(3',4'-dichlorophenyl)-l,l-dimethylurea, butwas inhibited by phenylmercuric acetate. The fastest H⁺ effluxoccurred at 45°C, whereas its extent was almost constantfrom 25 to 50°C. The activity decreased at temperaturesabove 50°C and was inactivated completely at 60°C. Itsaction spectrum corresponded the spectrum for chlorophyll aabsorption. Results indicate that the light-induced H⁺ effluxis driven by photosystem I and is important in the maintenanceof the intracellular pH at the functional neutral region againsta steep pH gradient across the cell membrane. (Received May 6, 1981; Accepted August 8, 1981)     

Inhibition of Malate Synthesis by Vanadate: Implications for the Cytosolic Alkalinization Induced by Vanadate Concentrations Partially Inhibiting the Plasmalemma H+ Pump

The effects of Na-orthovanadate, at concentrations only partiallyinhibiting net H⁺ extrusion, were determined on vacuolar andcytosolic pH by the weak base and weak acid distribution atequilibrium. Treatment with vanadate induces in Elodea densaleaves and in Arabidopsis thaliana seedlings a moderate acidificationof both cell sap and vacuole. Conversely, it induces an alkalinizationof cytosol, this effect being in apparent contrast with a conditionof reduced activity of the H⁺-transporting plasmalemma ATPase,which should be associated with a cytosolic acidification. InArabidopsis seedlings treated with vanadate, the increase inpH of both cytosol and external medium is associated with adecrease in cell sap buffer capacity, more evident for highervanadate concentrations, and particularly marked in the pH rangebetween 3·5 and 5·5. In these conditions, themalate content is strongly reduced, its decrease almost completelyaccounting for the decrease in cell sap buffer capacity. Anin vitro analysis of the vanadate effect on phosphoenolpyruvatecarboxylase indicates that the decrease in malate content seemssubstantially due to an inhibiting effect of vanadate on thisenzyme. These results stress that the in vivo use of vanadateas an inhibitor of the plasmalemma H⁺-ATPase must be taken withcaution; in particular, for studying the correlations betweenchanges in net H⁺ extrusion and changes in cytosolic pH andrelated processes. Key words: Vanadate, malate, cytosolic pH, Elodea densa, Arabidopsis thaliana     

Electrogenic Pump Current and ATP-Dependent H+ Efflux Across the Plasma Membrane of Nitellopsis obtusa

The correlation between the pump current and the ATP-dependentH⁺ efflux was examined in internodal cells of Nitellopsis obtusa.To control the cytoplasmic pH and ATP concentration, the tonoplastwas removed by intracellular perfusion with an EGTA-containingmedium. Two groups of perfused cells were prepared, one with1 mM ATP (+ATP cells) and the other without ATP but with hexokinaseand glucose (–ATP cells). The ATP-dependent H⁺ effluxwas calculated as the difference in H⁺ efflux between the +ATPand –ATP cells. Based on an electrically equivalent circuitmodel of the plasma membrane, the pump current was calculatedfrom the membrane potentials and the membrane resistances ofboth +ATP and –ATP cells. When the membrane potentialwas not too high (–220 mV), the ATP-dependent H⁺ current(19 mA m^–2) was almost equal to the pump current (20 mAm^–2) calculated from the electrical data. This indicatesthat the electrogenic pump current across the plasma membraneof Nitellopsis obtuse was mostly carried by H⁺. But when themembrane potential was high (–280 mV), the H⁺ currentwas lower than the pump current. The possible cause of thisdiscrepancy is discussed. (Received November 5, 1984; Accepted February 28, 1985)     

K+-Na+ Exchange and Selectivity in Barley Root Cells: Effect of Na+ on the Na+ Fluxes

Using the compartmental analysis the unidirectional Na⁺ fluxesin cortical cells of barley roots, the cytoplasmic and vacuolarNa⁺ contents Q_c and Q_v, and the trans-root Na⁺ transport R'have been studied as a function of the external Na⁺ concentration.Using the re-elution technique the effect of low K⁺ concentrationson the plasmalemma efflux _co of Na⁺ (K+-Na+ exchange) and onR' was investigated at different Na+ concentrations and correspondinglydifferent values of the cytoplasmic sodium content Qc. The relationof the K+-dependent Na+ efflux coK+-dep to Qc or to the cytoplasmicNa+ concentration obeyed Michaelis-Menten kinetics. This isconsistent with a linkage of co, K+-dep to K+ influx by a K⁺-Na⁺exchange system. The apparent Km corresponded to a cytoplasmicNa⁺ concentration of 28 mM at 0·2 mM K⁺ and about 0·2mM Na⁺ in the external solution. 0·2 mM K⁺ stimulatedthe plasma-lemma efflux of Na⁺ and inhibited Na⁺ transport selectivelyeven in the presence of 10 mM Na⁺ in the external medium showingthe high efficiency of the K⁺-Na⁺ exchange system. However,_co, K⁺-dep was inhibited at 10 mM Na¹ compared to lower Na¹concentrations suggesting some competition of Na¹ with K¹ atthe external site of the exchange system. The effect of theNa+ concentration on Na¹ influx _oc is discussed with respectto kinetic models of uuptake.