Distribution of Vacuolar H+-Pyrophosphatase and a Membrane Integral Protein in a Variety of Green Plants

Vacuolar membranes isolated from several species including fernand moss exhibited pyro-phosphate-dependent H⁺ transport activity.On immunoblot analysis, H⁺ -pyrophosphatase was detected inthe vacuolar membranes. A membrane integral protein of 23,000daltons was not found in the membranes of Chara, Conocephalum,or Kalanchoë. Thus, H⁺-pyrophosphatase may be a universalenzyme among green plants, but the 23-kDa protein is not a commonprotein of central vacuoles. (Received September 10, 1993; Accepted November 29, 1993)     

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)     

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; )     

Electrophysiological Evidence for an Electrogenic Proton Pump and the Proton Symport of Glucose in the Marine Fungus Dendryphiella salina

The marine hyphomycete Dendryphiella salina (Suth.) Nicot &Pugh has a resting membrane potential of –250 mV (insidenegative). The respiratory inhibitors sodium azide and FCCPinduced a rapid but reversible depolarization of the membraneof at least 180 mV; sodium azide also caused alkalinizationof the medium. Vanadate brought about significant depolarizationbut this was not always reversible. EDTA induced depolarizationthough to a lesser extent. DIDS and SITS caused a depolarizationof around 30–70 mV which was readily reversible, N-ethylmaleimideirreversibly depolarized the membrane by 180–200 mV. Ouabainhad no effect. When external concentrations of H⁺ , K⁺ , Na⁺or Cl^– were changed singly, only changes in H⁺ affectedmembrane potential, with shifts decreasing with increasing pH.Glucose and 3-O-methyl glucose depolarized the membrane in aconcentration-dependent manner which was enhanced by starvationof the hyphae. Recovery occurred in the presence of the hexose.Glucose caused an alkalinization of the medium, with time characteristicssimilar to the membrane potential changes. It is concluded thatthere is an electrogenic proton pump and a proton—glucosesymporter in D. salina. The retention of proton-based transportsystems suggests a terrestrial origin for the fungus. Key words: Marine fungi, Dendryphiella salina, membrane potential, electrogenic proton pump, proton symport, hexose     

Effects of Cations on the Cytoplasmic pH of Chara corallina

Smith, F. A. and Gibson, J.–L. 1985. Effects of cationson the cytoplasmic pH of Chara corallina.—J.exp. Bot.36: 1331–1340 Removal of external Ca²⁺ from cells of Chara corallina lowersthe cytoplasmic pH, as determined by the intracellular distributionof the weak acid 5,5–dimethyloxazolidine2–,4–dione(DM0), when the external pH is below about 60. This effect isreversed, at least partially, by addition of the following cationsto Ca²⁺-free solutions: tetraethylammonium (TEA⁺) and Na⁺ at5 or 10 mol m^-3, Li⁺ and Cs⁺ (10 mol m^-3), or Mg²⁺, Mn²⁺ andLa³⁺ (02 or 05 mol m^-3). Under the same conditions, increasesin pH sometimes, but not always, occur in the presence of 10mol m^-3 K⁺ or Rb⁺ The results are discussed in relation to the major transportprocesses that determine pH and the electric potential differenceacross the plasma membrane, namely fluxes of H⁺ and of K⁺. Thesimplest explanation of the effects of the various cations testedin this study is that they primarily affect pHi_c via changesin influx of H⁺ but direct effects on the H⁺ pump or on K⁺ fluxesmay also be involved Key words: Chara corallina, cytoplasmic pH, cations, H⁺transport     

Is Calmodulin Involved in Electrophysiology of Chara corallina?

The role of calmodulin in Chara was investigated using the antipsychoticdrug trifluoperazine (TFP), which is known to bind to the calmodulin/Ca⁺⁺complex preventing it from carrying out its normal functions. At low concentration (4.0 µM), TFP had profound and irreversibleeffects on the electrophysiology of Chara plasmalemma. The restingp.d. depolarized and the membrane conductance decreased suggestingan inhibition of the proton pump. After several hours of exposurethe membrane became leaky, as cells deteriorated. The excitation was also affected by TFP. Spontaneous repetitivefiring was observed. The excitation increased in duration andthe action potential peak depolarized to + 20 mV. The cytoplasmic streaming was unaffected by TFP; the streamingrate at the resting potential remained unchanged when TFP wasadded to the medium, stoppage occurred at the time of excitationand the streaming slowly resumed. Key words: Calmodulin, Chara corallina, Proton pump, Cytoplasmic streaming, Current, voltage characteristics     

Effect of Cytoplasmic Ca2+ on the Membrane Potential and Membrane Resistance of Chara Plasmalemma

Effects of cytoplasmic Ca²⁺ on the electrical properties ofthe plasma membrane were investigated in tonoplast-free cellsof Chara australis that had been internally perfused with media,containing either 1 mM ATP to fuel the electrogenic pump orhexokinase and glucose to deplete the ATP and stop the pump. In the presence of ATP, cytoplasmic Ca²⁺ up to 2.5?10^–5M did not affect the membrane potential (about -190 mV), butmembrane resistance decreased uniformly with increasing [Ca²⁺]_i.In the absence of ATP, the membrane potential, which was onlyabout -110 mV, was depolarized further by raising [Ca²⁺]_i from1.4?10^–6 to 2.5?10^–5 M. Membrane resistance, whichwas nearly the twofold that of ATP-provided cells, decreasedmarkedly with an increase in [Ca²⁺]_i from zero to 1.38?10^–6M, but showed no change for further increases. Internodal cellsof Nitellopsis obtusa were more sensitive to intracellular Ca²⁺with respect to membrane potential than were those of Charaaustralis, reconfirming the results obtained by Mimura and Tazawa(1983). The effect of cytoplasmic Ca²⁺ on the ATP-dependent H⁺ effluxwas measured. No marked difference in H⁺ effluxes was detectedbetween zero and 2.5?10^–5 M [Ca²⁺]_i; but, at 10^–4M the ATP-dependent H⁺ efflux was almost zero. Ca²⁺ efflux experimentswere done to investigate dependencies on [Ca²⁺]_i and [ATP]_i.The efflux was about 1 pmol cm^–2 s^–1 at all [Ca²⁺]_iconcentrations tested (1.38?10^–6, 2.5?10^–5, 10^–4M).This value is much higher than the influx reported by Hayamaet al. (1979), and this efflux was independent of [ATP]_i. Thepossibility of a Ca²⁺-extruding pump is discussed. ¹ Present address: Botanisches Institut der Universit?t Bonn,Venusbergweg 22, 5300 Bonn, F.R.G. (Received September 22, 1984; Accepted February 19, 1985)     

Regulation of the Cytoplasmic pH of Chara corallina in the Absence of External Ca2+: Its Significance in Relation to the Activity and Control of the H+ Pump

Effects of removal of external Ca²⁺ on the cytoplasmic pH (pH_c)of Chara corallina have been measured with the weak acid 5,5-dimethyl-oxazolidine-2,4-dione(DMO) as a function of external pH (pH₀) and of the externalconcentration of K⁺. Removal of Ca²⁺ always decreased pH_c whenpH₀ was below about 6.0; the decrease was about 0.2–0.4units at pH₀ 5.0, increasing to about 0.5 units at pH₀ 4.3.When pH₀ was 6.0 or higher the removal of Ca²⁺ had little orno effect on pH_c. This situation was not altered by changingthe concentration of K⁺, though in some experiments at pH₀ 5.0–5.2there was a slight decrease in pH₀ (about 0.2 units) when K⁺was increased from 0.2 to 2.0 mol m^–3, an effect apparentlyreversed when K⁺ was higher (5.0 or 10.0 mol m^–3). Theresults suggest that H⁺ transport continues in the absence ofexternal Ca²⁺, despite previous suggestions to the contrary,and that the H⁺ pump does not necessarily run near thermodynamicequilibrium with its chemical driving reaction. They indicate,rather, that the H⁺ pump is under kinetic control and providefurther evidence for the inadequacy of present models for theoperation of the H⁺ pump in charophyte cells, especially inrelation to its proposed role in regulating pH_c. Key words: Chara corallina, Cytoplasmic pH, Calcium     

Abscisic Acid-Induced Membrane Potential Changes in Barley Aleurone Protoplasts: a Possible Relevance for the Regulation of rab Gene Expression

The effect of ABA on the membrane potential of barley (Hordeumvulgare cv. Himalaya) aleurone protoplasts was studied by measuringthe distribution of the lipophilic cation tetraphenylphosphonium(TPP⁺). The resting membrane potential (E_m) according to ourmeasurements with TPP⁺ is about –53 mV and is in agreementwith membrane potential values as measured with intracellularmicroelectrodes (about –55 mV). The TPP⁺-measurementscould demonstrate a clear dependence of the resting E_m on theexternal pH (pH_e). Stimulation of the protoplasts with ABA induced a transienthyperpolarization of the membrane to –62 mV as measuredwith TPP⁺. The hyperpolarization was ABA-concentration dependent. Inhibition of the H⁺-ATPases with the specific proton pump inhibitorsdiethylstilbestrol (DES) or Micanozole effectively preventedhyperpolarization. This indicates that the hyperpolarizationis consistent with the activation of plasma membrane H⁺-ATPases.The K⁺-inward rectifier inhibitor BaCl₂ was able to prolongthe hyperpolarization. This result suggests that the hyperpolarizationcauses the opening of K⁺-channels. The ABA-induced proton-pump activation may be involved in ABA-inducedgene-expression, as DES was able to inhibit this gene expression.BaCl₂ did only show a slight inhibitory effect on ABA-inducedgene-expression. (Received January 4, 1994; Accepted April 12, 1994)     

Plasma membrane isolation from freshwater and salt-tolerant species of Chara: antibody cross-reactions and phosphohydrolase activities

Plasma membranes were isolated using the aqueous polymer two-phasepartition method from the algae Chara corallina and Chara longifolia,algae which differ in their ability to grow in saline environments.Enrichment of plasma membrane and depletion of tonoplast relativeto the microsomal fraction was monitored using phosphohydrolaseassays and crossreactions to antibodies raised against higherplant transporters. Antibodies to the vacuolar ATPase and pyrophosphatasecross-reacted with epitopes in the microsomal fraction, butshowed little affinity for the plasma membrane fraction. Pyrophosphataseactivity also declined in the plasma membrane fraction relativeto the microsomal fraction. The V-type H⁺ -ATPase activity,sensitive to nitrate or bafilomycin, was low in both fractions,though the cross-reaction to the antibody was reduced in theplasma membrane fraction. By contrast, the antibody recognitionof a P-type H⁺-ATPase amino acid sequence from Arabidopsis didnot occur strongly in the anticipated 90–100 kDa range.While there was enhanced recognition of a polypeptide at around140 kDa in the plasma membrane fraction, salt treatment of Charalongifolia resulted in plasma membrane fractions with reducedamounts of this epitope, but no change in vanadate-sensitiveATPase activity, suggesting that it does not represent the onlyP-type ATPase. Microsomal membranes from saltadapted C. longifoliahave higher reactivity with the antibody to the tonoplast ATPase. Key words: Chara, plasma membrane, salt tolerance, ATPase     

Potassium Channels at Chara Plasmalemma

Exposure to high K⁺ medium transforms Chara plasmalemma into[K⁺]_osensitive state (K⁺ state). The current-voltage (I/V)characteristicsunder such conditions display a negative conductance region.This feature results from the complex time and voltage dependenceof K⁺ channel opening At potentials more negative than a thresholdp.d. the channels are closed and the I/V characteristics becomelinear with a low slope conductance of 0.8 S m² and only a weakdependence on [K⁺]_o. Such behaviour is usually associated witha non-specific leak current The threshold level for K⁺ channelclosing depends on [K⁺]_o. In 2.0 mol m^–3 and 5.0 mol m^–3K⁺ medium the membrane resting p.d. follows E_K, but hyperpolarizesgradually if the [K⁺]_o is lowered. The proton pump thus appearsto be non-operative, while the cell is in the K⁺ state, andrecovers slowly as the cell is returned to a low K⁺ medium.Excitation currents decline if the cells are kept in K⁺ statefor some hours. Key words: K⁺ channels, Chara corallina, Proton pump, Current/, oltage characteristics, Conductance     

Current-Voltage Curves of Single CI- Channels which Coexist with Two Types of K+ Channel in the Tonoplast of Chara corallina

A Cl^– channel and two types of K⁺ channel have been observed,by the use of the patch-clamp technique, in the membrane surroundingcytoplasmic droplets from Chara corallina. Measurements on cell-attachedpatches showed that the channel selective for Cl^– hada chord conductance of 21 pS at the resting membrane p.d. (mean= 11 mV, n = 19) and when open, passed an outward current of1.4 pA (n = 24 patches) at the resting p.d., with reversal ofthe direction of current at –54 mV (130 mol m^–3Cl^– in the external solution). The Cl^– concentrationin the cytoplasmic droplet calculated from the reversal p.d.was 15 mol m^–3. The channel strongly rectified outwardcurrent flow, but this rectification disappeared with symmetricalCl^– concentrations across detached patches of membrane.It is concluded that rectification observed in cell-attachedpatches is primarily due to asymmetric ^Cl– concentrationsrather than an asymmetry in energy barriers to Cl^– permeationin the channel or any voltage-dependent kinetics of the channel.The channel was rarely observed in detached patches despitebeing commonly observed in cell-attached patches. However, theabsence of Ca²⁺ at the cytoplasmic face of the membrane allowedobservation of the channel in detached patches for brief periods,during which ion substitution experiments revealed a permeabilitysequence of aspartate (76:33:1). A 100 pS K⁺ channel previously described by Luhring (1986) wasfrequently observed, in some instances simultaneously, witha channel having a conductance of 60 pS and displaying outwardrectification. This rectification was due to the channel remainingopen almost continuously for positive membrane potential differences(p.d.) and remaining shut almost continuously for negative p.d.'s.The 60 pS channel, like the 100 pS K⁺ channel, reversed currentflow at the resting p.d., suggesting that it was also permeableto K⁺. Key words: Plant ion-channels, chloride channel, potassium channel, patch-clamp     

Transient light-induced changes in ion channel and proton pump activities in the plasma membrane of tobacco mesophyll protoplasts

Rapid, transient changes of the membrane potential upon lighttransitions are generally observed in microelectrode studies.In a patch-clamp study similar responses to light transitionswere found in current clamp. Corresponding with the changesof membrane potential, light-induced current changes in voltageclamp were observed. This paper evaluates the involvement ofoutward rectifying conductances and plasma membrane bound H⁺-ATpases(proton pump) to these light responses in mesophyll protoplastsof Nicotiana tabacum L. The contribution of K⁺-channels to theseresponses, could be minimized by variation of the holding potentialor addition of the K⁺-channel blocker verapamil. It was concludedthat light transitions modulate both proton pump and K⁺-channelactivity. Effects of light on membrane current were not observedin root cells and chlorophyll-deficient cells, suggesting thatthe response requires photosynthetic activity. However, blockersof photosystems I and II did not affect current changes. Key words: Light, patch-clamp, plasma membrane, tobacco, whole cell     

In Vivo Treatment of Barley Roots with Vanadate Increases Vacuolar H+-Translocating ATPase Activity of the Tonoplast-Enriched Membrane Vesicles and the Level of Endogenous ABA

Evidence for an increase in the activity of the vacuolar H⁺-translocatingATPase of barley roots, accompanied by qualitative change, inresponse to exogenously applied abscisic acid (ABA) has beenreported previously [Kasai et al. (1993b) Plant Cell Physiol.34: 1107]. In the present study, such an increase in the H⁺-translocatingATPase activity was shown to occur upon treatment of barleyroots with vanadate. Furthermore, the level of endogenous ABAin the roots was shown to increase significantly as a resultof this treatment. The present observations provide furthersupport for the proposed physiological significance of cellularABA in the regulation of the vacuolar H⁺-translocating ATPaseactivity. (Received September 3, 1993; Accepted December 16, 1993)     

Electrical Characteristics of the Tonoplast of Cham corallincr. A Study Using Permeabilised Cells

Use of permeabilised cells of Chara corallina provides a uniqueopportunity to study the electrical characteristics of the tonoplastwhilst being able to control ionic conditions on the outsideof the membrane. Current-voltage (I/V) analysis over wide voltagespans, and admittance measurements at 5 Hz showed that manypermeabilised cells had a similar conductance and capacitanceto the tonoplast of intact cells. Cells developed two regionsof negative-slope conductance upon addition of external Cl^–,which suggests the existence of potential-dependent Cl^–channels in the Chara tonoplast. With Cl^– concentrationssimilar to those expected in vivo, the resting potential wasmore sensitive to changes in external K⁺ than Cl^–; however,a decrease in external K⁺ did not significantly alter the shapeof the I/V relation. ¹Present address: Biopysics Laboratory, School of BiologicalSciences, A12, University of Sydney, Sydney, N.S.W., 2006, Australia ²Permanent address: Department of botany, Faculty of Science,University of Tokyo, Hongo, Tokyo 113, Japan (Received May 6, 1987; Accepted September 21, 1987)     

Potassium Transport Across the Membranes of Chara: II.42K FLUXES AND THE ELECTRICAL CURRENT AS A FUNCTION OF MEMBRANE VOLTAGE

Smith, J. R. 1987. Potassium transport across the membranesof Chara. II. ⁴²K fluxes and the electrical current as a functionof membrane voltage.—J. exp. Bot. 38: 752–777. The current required to clamp the trans-membrane voltage ofinternodal cells of Chara australis at different levels wasmeasured simultaneously with either the ⁴²K influx or efflux.Examination of the voltage-dependence of the ratio of the electricalcurrent to the unidirectional tracer fluxes yielded no evidenceof any amplification of the electrical driving force on theK⁺ ions. There was thus no evidence for the interaction of K⁺ions with themselves or any other species during their passageacross the membrane. These measurements allow the determinationof , the fraction of the electrical current carried by K⁺ ions.When the external [K⁺] = 10 mol m^–3, the average valueof was 0?85 for V_m > –125 mV and 07?5 for V_m <–150 mV. When the external [K⁺] = 0?1 mol m^–3, was 0?6 for V_m < –80 mV and 0?1 for V_m > –250mV. It was also found that the conductance associated with K⁺transport was inhibited by hyperpolarization. Key words: Potassium, conductance, flux-ratio     

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     

Action of Mono- and Difluorodinitrobenzene on Induced Electrical Modifications by External pH Changes in Nitella

The action of mono- (FNB) or difluorodinitrobenzene (DFNB) onion permeability is mainly attributed to its interactionwithamino groups of the membrane by dinitrophenylation. Nitella cells were dimtrophenylated at pH 7.3 and the membranepotential and electrical resistance were then measured in acidicor basic solutions. No matter what the pH value was, FNB andDFNB induced a depolarization of the membrane potential andcaused a diminution of resistance. However these effects ofFNB and DFNB were more drastic at alkaline pH and in the presenceof a weak concentration of potassium. Neither the addition of0.1 mM calcium nor the substitution of chlorides by nitratesmodified the DFNB effect. These results are compatible withthe assumption that the DFNB binding to the membrane leads toan augmentation of the negative charges of the membrane bringingabout an increased cation conductance and a modification ofthe affinity of a K⁺/H⁺exchange pump. The transient responseof the membrane potential at the time of dinitrophenylationwas used to roughly estimate the total density of amino groupsof the membrane of Nitella.     

Ionic Currents across the Plasmalemma of Chara inflata Cells: I. OSMOTIC EFFECTS OF SORBITOL ON K+, CI- AND LEAK CURRENTS

This paper describes experiments designed to investigate theeffects of increases in external osmotic pressure on the electrophysiologicalbehaviour of the plasmalemma in cells of the brackish-wateralga, Chara inflata. The electrical conductance of the plasmalemmaof these cells of, due to the diffusion of ions, consists mainlyof K⁺, Cl^– and leak components. The addition of sorbitolat concentrations in the range 40–280 mol m^–3 tothe external solution bathing the cells, progressively and reversiblydepolarized the cell membrane and increased the total membraneconductance, chiefly due to increases in each of the separateionic conductances. At concentrations higher than about 280mol m^–3 when the cells became plasmolysed, the effectsof sorbitol on the electrical properties of the cell ceasedto be fully reversible. When the membrane electrical potentialdifference is stepped in a negative direction with a voltage-clamp,the resulting inward current has voltage-dependent componentsconsisting of an inactivating K⁺ current, an activating Cl^–current and a constant leak current. The addition of sorbitolto the external solution modified these currents by increasingtheir magnitude, by increasing the half-time of the inactivationof the K⁺ current, and by decreasing the half-time of activationof the Cl^– current. Key words: Chara inflata, osmotic effects, K⁺ and Cl^– currents     

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.