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     

Dependency of H+ efflux on ATP in cells of Chara australis

The internodal cell of Chara australis was made tonoplast-freeby internal perfusion with a medium containing a Ca²⁺-chelatorEGTA and the net H⁺ efflux across the plasma membrane was estimatedeither by titration of the external medium or by measuring thechange in pH in the external medium. The amount of H⁺ effluxwas high in the presence of internal ATP and low in its absence.The ATP-dependent net H⁺ efflux was about 40 nmoles/m²/sec.This amount is smaller than that estimated for the pump currenton the basis of electrical data obtained earlier (3). This discrepancyis discussed. (Received June 18, 1980; )     

Net and Steady-state Cation Fluxes in Chlorella pyrenoidosa

The addition of K⁺ to Chlorella cells grown so as to be abnormallyrich in Na⁺ induces a net Na⁺ efflux and a concomitant uptakeof K⁺. The net Na⁺ extrusion shows first-order kinetics withtime constants of about 10 min for illuminated cells, and occursat rates in the region of 10 to 15 pmol cm1² s. The correspondingtime course for the net K⁺ influx also approximates to first-orderkinetics but is more complicated because it not only involvesa K⁺/Na⁺ component but also a K⁺/H⁺ exchange. The H⁺ extrusionusually represents less than 20 per cent of the net cation movementand may account both in magnitude and in rate for the differencebetween K⁺ and Na⁺ movements. The magnitudes of the net K⁺ andNa⁺ fluxes differed from steady-state flux rates in normal highK⁺-containing cells being as much as 20 times greater for K⁺and over 100 times greater for Na⁺. There is some indicationthat K⁺ competes for Na⁺ entry into Na⁺-rich cells, suggestingthat both the Na⁺/Na⁺ and K⁺/Na⁺ exchanges may share the sameentry site. The K⁺/Na⁺ exchange rates saturate at low externalK⁺ concentrations; the half-maximum rate was at about 0.2 mMK⁺. The Na⁺/K⁺ exchange is sensitive to temperature and between0 and 25 °C an activation energy of about 25 k cal/molewas calculated from the Arrhenius equation.     

Possible Linkage between NADH-Oxidation and Proton Secretion in Zea mays L. Roots

A possible involvement of two different systems in proton translocationand the correlation of this factor to growth rates were measuredsimultaneously by means of a pH stat and an optical system.Ferricyanide, which can accept electrons at the plasmalemma,led to an immediate increase of net H⁺ -efflux but also decreasedroot growth rate. The reduced form, ferrocyanide, inhibitednet H⁺ -effluxwithout changing the growth rate. Thus, corn rootgrowth was not determined by proton secretion exclusively. Vanadatestrongly inhibited net H⁺ -efflux by the roots but did not preventthe stimulating effect of fcrricyanide. Moreover, the extentof enhancement of net H⁺ -effluxby ferricyanide was exactlythe same in vanadate pretreated as in untreated roots. Alcoholswere used to try to increase the intracellular NADH level throughthe action of the cytoplasmic alcohol dehydrogenase presentin the roots and coleoptiles. Alcohols, known to be substratesfor alcohol dehydrogenase such as propan- 1-ol, ethanol andbutan-l -ol increased net H⁺ -effluximmediately but methanoland secondary alcohols which are not substrates had no effecton proton secretion. The K_m values of alcohol dehydrogenasefor the alcohols correspond only partly to their effects onproton secretion. However, the specificlty observed suggeststhat increased H⁺ -efflux arose from reduction of endogenousNAD by ADH and consequent increased membrane NADH-oxidasc activitytrans locating protons and electrons out of the cells. Decreased oxygen concentrations slowed proton secretion at valuesfar higher than are necessary to saturate cytochrome c oxidase.The results of these experiments suggest two distinct systemscontributing to proton efflux. Key words: ADH, proton transport, redox chain     

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     

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)     

Ion Transport and the Effects of Acetic Acid in White Clover: II. POTASSIUM ABSORPTION

Dunlop, J., Knighton, M. V. and White, D. W. R. 1988. Ion transportand the effects of acetic acid in white clover. II. Potassiumabsorption.—J. exp. Bot. 39: 89–96. Acetic acid stimulated K⁺ influx into cells of white cloverin suspension culture and net K⁺ influx by roots of intact whiteclover plants. At concentrations of acetic acid up to 2·0mol m^–3 the stimulation continued unabated for at least2 h. However, at higher concentrations the rate of absorptiondeclined to near zero values after 2 h. When acetic acid wasremoved from the solution there was a net efflux of K⁺. Thestimulation was pH dependent with a maximum at pH 5·0.There was maximum stimulation at an acetic acid concentrationof 2·0 mol m^–3 Net H⁺ efflux was reduced by 1·0 mol m^–3 aceticacid. When Rb⁺ uptake and H⁺ efflux were measured at a rangeof RbCl concentrations Rb⁺ uptake increased with concentrationwhereas H⁺ efflux was maximum in the absence of Rb⁺ (and K⁺)and decreased as RbCl concentration was increased Acetic acid caused a hyperpolarization of the membrane electricalpotential difference (E) of about 25 mV. In 1·0 mol m^–3acetic acid the hyperpolarization persisted for at least 2 hwhereas at 10 mol m^–3 d E subsequently depolarized tovalues around –80 mV. With a slight lag, the time courseof the stimulation of the rate of K⁺ absorption followed thepolarization and depolarization of E. These results imply thatthe linkage between K⁺ and H⁺ movements is probably throughE. Key words: Proton efflux, membrane electrical potential difference, Trifolium repens     

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     

Characteristics of light-induced H+ transport in spinach chloroplast at lower temperatures I. Relationship between H+ transport and physical changes of the microenvironment in chloroplast membranes

Light-induced H⁺ transport of spinach chloroplasts was investigatedin the temperature range from 5° to 30°C with a glasselectrode. The rate of H⁺ transport was reduced by lowering the temperature.Addition of 1 µM phenazine methosulfate (PMS) considerablystimulated the H⁺ uptake in chloroplasts. PMS was also effectivein stimulating the H⁺ efflux when the illumination was turnedoff. The latter effect became more marked at lower temperatures.These results indicate that electron transfer reactions in thechloroplast not only drive the forward process of H⁺ gradientformation, but also participate in the backward H⁺ efflux. The Arrhenius plot applied to the first-order rate constantof the H⁺ efflux showed a discontinuity at about 20°C. Nohysteresis was detected with the temperature dependence andits discontinuity in the H⁺ transport. On the other hand, theaddition of PMS abolished the discontinuity and a linear relationshipwas observed in the Arrhenius plot. Probably, temperature-dependentphysical changes in the microenvironment of the chloroplastlamellae are responsible for determining the characteristicsof the H⁺transport. (Received September 11, 1975; )     

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     

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)     

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     

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)     

K+Nutrition and Na+Toxicity: The Basis of Cellular K+/Na+Ratios

The capacity of plants to maintain a high cytosolic K⁺/Na⁺ratiois likely to be one of the key determinants of plant salt tolerance.Important progress has been made in recent years regarding theidentification and characterization of genes and transportersthat contribute to the cytosolic K⁺/Na⁺ratio. For K⁺uptake,K⁺efflux and K⁺translocation to the shoot, genes have been isolatedthat encode K⁺uptake and K⁺release ion channels and K⁺carriersthat are coupled to either a H⁺or Na⁺gradient. Although thepicture is less clear for the movement of Na⁺, one pathway,in the form of non-selective ion channels, is likely to playa role in Na⁺uptake, whereas Na⁺efflux and compartmentationare likely to be mediated by H⁺-coupled antiport. In addition,several proteins have been characterized that play prominentroles in the regulation of K⁺and/or Na⁺fluxes. In this BotanicalBriefing we will discuss the functions and interactions of thesegenes and transporters in the broader context of K⁺nutritionand Na⁺toxicity. Copyright 1999 Annals of Botany Company Salinty, K⁺/N⁺ratio, transporter, membrane.     

Effects of Growth and Assay Temperatures on Unidirectional K+ Fluxes in Roots of Rye (Secale cereale)

The effects of growth and assay temperature on unidirectionalK⁺ fluxes in excised roots of rye (Secale cereale cv. Rheidol)were studied using ⁸⁶Rb⁺ as a tracer. Both K⁺ influx to thevacuole, estimated as K⁺ uptake between 3 and 12 h after transferof unlabelled roots to radioactive solution, and movement ofK⁺ to the xylem were determined directly. Other fluxes weredetermined on excised roots of plants, which had been labelledwith ⁸⁶Rb⁺ since germination, by conventional triple exponentialefflux analysis. When assayed at 20°C, roots of plants previously grown at20°C(WG roots) had lower rates of net K⁺ uptake than rootsof low temperature-acclimated plants, grown with a temperaturediferential between roots (87°C) and shoots (20°C) eithersince germination (DG roots) or for 3 d prior to experiments(DT roots). This resulted from a greater unidirectional K⁺ effluxacross the plasma membrane and a reduced K⁺ flux to the xylemin WG roots, compared to DG or DT roots, rather than a decreasein unidirectional K⁺ influx or a decrease in the net K⁺ fluxto the vacuole. Indeed, although WG roots had lower rates ofK⁺ influx and K⁺ efflux across the tonoplast at 20°C thanDG or DT roots, roots of plants from all growth temperaturetreatments showed an equivalent net K⁺ flux to the vacuole. Although all unidirectional K⁺ fluxes in roots from plants grownunder all temperature regimes were reduced by lowering the temperatureof the root, these fluxes were differentially affected in rootsof plants from contrasting growth temperature treatments. Rapidcooling to 8°C of WG roots resulted in a lower rate of K+influx and a transient increase in K⁺ efflux across both theplasma membrane and tonoplast, compared to DG and DT roots.Furthermore, since the K⁺ flux to the xylem was lower in WGroots, the net K⁺ uptake at 8°C into WG roots was considerablyreduced compared to DG and DT roots. These results suggest thatlow temperature-acclimation of K⁺ fluxes in rye roots may involvea reduction in the temperature sensitivity of K⁺ influx anda curtailment of K⁺ efflux across both the plasma membrane andtonoplast at low temperatures. Key words: K⁺influx, K⁺ efflux, low temperature, potassium, rye (Secale cereale cv. Rheidol)     

The Action of Divalent Zinc, Cadmium, Mercury, Copper and Lead on the Trans-Root Potential and H+, Efflux of Excised Roots

Kennedy, C. D. and Gonsalves, F. A. N. 1987. The action of divalentzinc, cadmium, mercury, copper and lead on the trans-root potentialand H⁺ efflux of excised roots.—J. exp. Bot. 38: 800–817. The action of Zn²⁺, Cd²⁺, Hg²⁺, Cu²⁺ and Pb²⁺ ions on the trans-rootpotential and H⁺ efflux of young excised maize roots has beenstudied. Micro-electrode implantations into root epidermal cellsconfirmed the root outer membranes as the major contributorin the trans-root potential changes. The effects of these ionson H⁺ efflux were studied over a period of time in a continuousflow cell apparatus, adequate controls allowing for transientinterference due to divalent cations at the pH probe. The addition of Zn²⁺, 5 to 100 µmol dm^–3, to thesolution bathing the roots reduces H⁺ efflux and depolarizesthe trans-root potential. However, in the presence of Mg²⁺,0?1 or 1?0 mmol dm^–3, not only is this depolarizationinhibited, but hyperpolarization is observed instead. Cd²⁺ affectstrans-root potential and H⁺ efflux at a much slower rate thanZn²⁺, suggesting a lower membrane permeability. Without Mg²⁺,Cd²⁺ hyperpolarizes the trans-root potential, but this is bettersustained in its presence. Hyperpolarization did not occur withHg²⁺, Cu²⁺ or Pb²⁺ whether or not Mg²⁺ was present Hg²⁺ andto a lesser extent Cu²⁺ are potent depolarizers of the trans-rootpotential and strongly inhibit H⁺ efflux. The maximum rates of depolarization observed in the absenceof Mg²⁺ increase in the order Cd PCMBS <<.lt; Zn Cu< Hg. This is similar to the relative maximum rates of H⁺inhibition, Pb Cd <<.lt; Zn < Cu < Hg, suggestingconsiderable differences in mode of action and/or membrane permeability.The lower membrane permeability of the sulphydryl reagent PCMBSapparently prevents ready access to the site(s) of action availableto Hg²⁺. The reductions in trans-root potential and H⁺ gradients inducedby this range of cations would be detrimental to the acquisitionof nutrients using these gradients as an energy source. In contrast,Zn^2+, , in the presence of adequate Mg²⁺, could be beneficialto nutrient uptake by maintaining a higher membrane potentialthan would occur in its absence. Possible modes of action for the observed effects are discussed. Key words: Trans-root potentials, H⁺ efflux, heavy metal ions     

An investigation into the role of photosynthesis in regulating ATP levels and rates of h efflux in isolated meosphyll cells

Aerated and stirred 10-ml suspensions of mechanically isolated Asparagus sprengeri Regel mesophyll cells were used for simultaneous measurements of net H⁺ efflux and steady-state ATP levels.

Initial rates of medium acidification indicated values for H⁺ efflux in the light and dark of 0.66 and 0.77 nanomoles H⁺/10⁶ cells per minute, respectively. When the medium pH was maintained at 6.5, with a pH-stat apparatus, rates of H⁺ efflux remained constant. Darkness or DCMU, however, stimulated H⁺ efflux by 100% or more. Darkness increased ATP levels by 33% and a switch from dark to light reduced ATP levels by 31%. In the absence of aeration, illumination prevented the accumulation of respiratory CO₂ and the buffering capacity of the medium was about 50% less than that found in the nonilluminated nonaerated medium. As a result, rates of pH decline were similar even though the dark rate of H⁺ efflux was approximately 50% greater.

Proposals that photosynthesis stimulates H⁺ efflux are based on changes in the rate of pH decline. The present data indicate that photosynthesis inhibits H⁺ efflux and that changes in rates of pH decline should not be equated with changes in the rate of H⁺ efflux.

     

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)     

Na(+)/H(+) exchanger NHE1 as plasma membrane scaffold in the assembly of signaling complexes

The plasma membrane Na⁺/H⁺ exchanger NHE1 has an established function in intracellular pH and cell volume homeostasis by catalyzing electroneutral influx of extracellular Na⁺ and efflux of intracellular H⁺. A second function of NHE1 as a structural anchor for actin filaments through its direct binding of the ezrin, radixin, and moesin (ERM) family of actin-binding proteins was recently identified. ERM protein binding and actin anchoring by NHE1 are necessary to retain the localization of NHE1 in specialized plasma membrane domains and to promote cytoskeleton-dependent processes, including actin filament bundling and cell-substrate adhesions. This review explores a third function of NHE1, as a plasma membrane scaffold in the assembly of signaling complexes. Through its coordinate functions in H⁺ efflux, actin anchoring, and scaffolding, we propose that NHE1 promotes protein interactions and activities, assembles signaling complexes in specialized plasma membrane domains, and coordinates divergent signaling pathways. hydrogen ion efflux; intracellular pH; molecular scaffold     

Coupling of Proton Fluxes in the Polar Leaves of Potamogeton lucens L

An attempt has been made to quantify the light-induced H⁺ effluxand influx observed in polar leaves of Potamogeton lucens.Theseproton fluxes are spatially separated. The H⁺ efflux, mediatedby a plasmalemma bound H⁺ –ATPase, occurs across theplasmamembrane at the morphological lower epidermis and is accompaniedby an H⁺ influx (or OH^– efflux) at the upper side oftheleaf. As a result, these leaves exhibit a remarkable pH–polarityin the light. The pH near the lower epidermis may drop to avalueas low as 3.5, while a pH of about 10.5 can be observed at theupper epidermis. Obviously this phenomenon requires theco–ordinationof transport processes in the different cell layers of the leaftissue. These observations led to quantitative studies oftherelation between the H⁺ fluxes at either plasmalemma. Thesefluxes were calculated from the pH values recorded at twodistancesfrom the leaf surface. Although the H⁺ influx always exceededthe efflux, a coupling between the transport processesacrosseither plasma membrane became evident from the time–coursesof the two fluxes. Key words: Potamogeton lucens, proton flux, flux coupling, pH–;polarity