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)     

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)     

Effects of Two Plasmalemma ATPase Inhibitors on H+ Extrusion and Intracellular pH in Elodea densa Leaves

Beffagna, N. and Romani, G. 1988. Effects of two plasmalemmaATPase inhibitors on H⁺ extrusion and intracellular pH in Elodeadensa leaves.—J. exp. Bot. 39: 1033–1043. Elodea leaves in the dark show very little exchange of H⁺ withthe medium in the external pH range between 5.0 and 6.0. Thepresence of fusicoccin and potassium in the medium markedlystimulates H⁺ extrusion. Fusicoccin- and K⁺ -induced H⁺ extrusionis inhibited by either erythrosin B (EB) or Na-orthovanadate,two inhibitors of H⁺ transporting plasma membrane ATPase. EBcompletely inhibits it from the first 30 min of treatment, whensupplied at pH 5.5 at a concentration of 30 mmol m^–3.Vanadate also inhibits H⁺ extrusion, this effect becoming evidentonly after 45 min of treatment. After this time inhibition iscomplete with 250 mmol m^–3 vanadate but only partial forlower concentrations. In the presence of either inhibitor the intracellular pH, measuredas cell sap pH, is significantly lowered. When the intracellularpH changes are determined on vacuole and, separately, on cytoplasmby the weak acid and base distribution method, acidificationof both compartments is found to accompany the blocking of H⁺extrusion by either of the inhibitors. Key words: Intracellular pH, vanadate, erythrosin B, H⁺pumping     

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     

Intracellular pH and Proton-Transport in Barley Root Cells under Salt Stress: in Vivo 31P-NMR Study

Salt stress-induced changes of intracellular pH and in levelsof phosphorous compounds were monitored in intact root tipsof barley seedlings (Hordeum vulgare cv. Akashin-riki) by invivo ³¹P-nuclear magnetic resonance (NMR) spectroscopy. Vacuolaralkalization was observed after treatment with both 300 and500 mM NaCl. Much of the observed apparent alkalization of thecytoplasm was eliminated when the effect of Na⁺ ions on thetitration curve was considered. Within 1 h after the initiationof salt stress, levels of glucose-6-phosphate and UDP-glucosedecreased markedly, and such decreases might lead directly orindirectly to cell death. Simultaneous measurements of the externaland intracellular pH revealed the promotion of external acidificationand internal alkalization during salt stress. Possible mechanismsof Na⁺/H⁺ antiport at the tonoplast and the role of proton-pumpin the plasma membrane are discussed. ³Present address: Shijonawate Gakuen Women's Junior College,Daito, Osaka, 574 Japan.     

Effect of External pH on the Cytoplasmic and Vacuolar pHs in Mung Bean Root-Tip Cells: A 31P Nuclear Magnetic Resonance Study

The effect of the external pH on the intracellular pH in mungbean (Vigna mungo (L.) Hepper) root-tip cells was investigatedwith the ³¹P nuclear magnetic resonance (NMR) method. The ³¹PNMR spectra showed three peaks caused by cytoplasmic G-6-P,cytoplasmic P_i and vacuolar P_i. The cytoplasmic and vacuolarpHs could be determined by comparing the P_i chemical shiftswith the titration curve. When the external pH was changed overa range from pH 3 to 10, the cytoplasmic pH showed smaller changesthan the vacuolar pH, suggesting that the former is regulatedmore strictly than the latter. The H⁺-ATPase inhibitor, DCCD,caused the breakdown of the mechanism that regulates the intracellularpH. H⁺-ATPase appears to have an important part in the regulationof the intracellular pH. (Received January 4, 1984; Accepted August 27, 1984)     

Role for the Vacuolar H+-ATPase in Regulating the Cytoplasmic pH: An in Vivo Study Carried out in chl1, an Arabidopsis thaliana Mutant Impaired in NO-3 Transport

The effects of the growth in a medium containing NH₄NO₃ as nitrogensource were studied on cell sap pH, cytoplasmic pH and malatecontent in chl1, an Arabidopsis thaliana mutant impaired inchlorate and nitrate transport. In all the conditions testedthe pH of the cytoplasm in chl1 was more alkaline, and thatof the vacuole was more acidic as compared with those measuredin wt. Treatment with bafilomycin A1, a specific inhibitor ofthe vacuolar H⁺-ATPase, induced a small alkalinization of thevacuole, and a significant acidification of the cytoplasm, theseeffects being greater in chl1 than in wt. The greater responseof the mutant to bafilomycin Al suggests that, in the absenceof the inhibitor, the activity of the tonoplast H⁺-ATPase inchl1 is higher than in wt, this diversity being a possible reasonfor the differences in intracellular pH detected between thetwo strains. A possible role for the vacuolar H⁺-ATPase in regulatingthe cytoplasmic pH is discussed. (Received August 2, 1995; Accepted February 1, 1996)     

Uptake of Imidazole and its Effects on the Intracellular pH and Ionic Relations of Chara corallina

Ammonia (pK_a 9.25) and methylamine (pK_a, 10.65) increase cytoplasmicpH and stimulate Cl^– influx in Chara corallina, theseeffects being associated with influx of the amine cations ona specific porter. The weak base imidazole (pK_a 6.96) has similareffects but diffuses passively into the cell both as an unionizedbase and as a cation. When the external pH is greater than 6.0influx of the unionized species predominates. Imidazole accumulates to high concentrations in the vacuole,where it is protonated. Cytoplasmic pH and vacuolar pH riseby only 0.2–0.3 units, suggesting a large balancing protoninflux across the plasma membrane. Balance of electric chargeis partially maintained by net efflux of K⁺ and net influx ofCl^–. Calculation of vacuolar concentrations of imidazole(from (¹⁴C] imidazole uptake, assuming that there is no metabolism)plus K⁺ and Na⁺ indicates an excess of cations over inorganicanions (Cl^–). However, although the osmotic potentialof the cells increases, also indicating increased solute concentrations,the increase is less than that predicted by the calculated ionicconcentrations. This discrepancy remains to be resolved. Becausethe osmotic potential also increases when imidazole is absorbedfrom Cl^–-free solutions it is likely that maintenanceof charge-balance can also involve synthesis and vacuolar storageof organic or amino acids. Key words: Imidazole, potassium, intracellular pH, membrane transport, Chara     

Proton Transport and pH Control in Sinapis alba Root Hairs: A Study carried out with Double-Barrelled pH Micro-electrodes

Continuous measurements of cytoplasmic pH (pH_c) in Sinapis roothairs have been carried out with double-barrelled pH-micro-electrodesin order to gain information on translocation of protons acrossthe plasmalemma and cytoplasmic pH control. (i) The cytoplasmicpH of Sinapis (7–33 ? 0–12, standard conditions)changes no more than 0.1 pH_c, per pH_o-unit, regardless of whethercyanide is present or not. (ii) Weak acids rapidly acidify pH_cand hyperpolarize, while weak bases alkalize pH_c and depolarizethe cells, (iii) 1.0 mol M,³ NaCN acidifies the cytoplasm by0.4 to 0.7 pH-units, but alkalizes the vacuole. (iv) 20 mmolm^–3 CCCP has no significant effect on pH_c, if added atpH 9.6 or 7.2, but acidifies pH_c by 1.3 units at pH 4.3. Inthe presence of CCCP, cyanide acidifies the cytoplasm, (v) Chloridetransiently acidifies pH_c, while K⁺, Na⁺, and have no significant effects, (vi) Cytoplasmic buffer capacityforms a bell-shaped curve versus pH_c with an optimum of about50 mol m^–3 H⁺pH_c-unit. The modes of proton re-entry and the effects of active and passiveproton transport on cellular pH control are critically discussed.It is suggested that the proton leak, consisting of H⁺-cotransport(e.g. H⁺/Cl^–) rather than H⁺-uniport, is no threat topH_c. The proton export pump, although itself reacting to changesin pH_c, influences pH_c only to a minor extent. It is concludedthat buffer capacity and membrane transport play moderate rolesin pH_c control in Sinapis, while the interlocked H⁺-producingand -consuming reactions of cellular metabolism are the mainregulating factors. This makes pH control in Sinapis quite differentfrom bacterial and animal cells. Key words: Cytoplasmic pH, double-barrelled pH micro-electrode, pH control, proton transport, Sinapis     

Intracellular pH homeostasis in cultured human placental syncytiotrophoblast cells: recovery from acidification

Resting or basal intracellular pH (pH_i) measured in cultured human syncytiotrophoblast cells was 7.26 ± 0.04 (without HCO₃^–) or 7.24 ± 0.03 (with HCO₃^–). Ion substitution and inhibitor experiments were performed to determine whether common H⁺-transporting species were operating to maintain basal pH_i. Removal of extracellular Na⁺ or Cl^– or addition of amiloride or dihydro-4,4'-diisothiocyanatostilbene-2,2'-disulfonate (H₂DIDS) had no effect. Acidification with the K⁺/H⁺ exchanger nigericin reduced pH_i to 6.25 ± 0.15 (without HCO₃^–) or 6.53 ± 0.10 (with HCO₃^–). In the presence of extracellular Na⁺, recovery to basal pH_i was prompt and occurred at similar rates in the absence and presence of HCO₃^–. Ion substitution and inhibition experiments were also used to identify the species mediating the return to basal pH_i after acidification. Recovery was inhibited by removal of Na⁺ or addition of amiloride, whereas removal of Cl^– and addition of H₂DIDS were ineffective. Addition of the Na⁺/H⁺ exchanger monensin to cells that had returned to basal pH_i elicited a further increase in pH_i to 7.48 ± 0.07. Analysis of recovery data showed that there was a progressive decrease in pH per minute as pH_i approached the basal level, despite the continued presence of a driving force for H⁺ extrusion. These data show that in cultured syncytial cells, in the absence of perturbation, basal pH_i is preserved despite the absence of active, mediated pH maintenance. They also demonstrate that an Na⁺/H⁺ antiporter acts to defend the cells against acidification and that it is the sole transporter necessary for recovery from an intracellular acid load. sodium/hydrogen antiporter; pH regulation; fluorescence; 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein     

Short-term Measurements of the Cytoplasmic pH of Chara corallina Derived from the Intracellular Equilibration of 5,5-Dimethyloxazolidine-2,4-Dione (DMO)

Smith, F. A. 1986. Short-term measurements of the cytoplasmicpH of Chara corallina derived from the intracellular equilibrationof 5,5-dimethyloxazolidine-2,4-dione (DMO).—J. exp. Bot.37: 1733–1745. Measurements of the time-course of influx of ¹⁴C-labelled 5,5-dimethyloxazolidine-2,4-dione(DMO) into the cytoplasm and vacuole of internodal cells ofChara corallina, and of efflux of DMO into non-radioactive solutions,have shown that exchange of DMO across the tonoplast is veryrapid compared with exchange across the plasma membrane. Thishas made possible calculations of cytoplasmic pH from distributionof DMO between cytoplasm and vacuole over short periods (5 or10 min) even when intracellular DMO is not at flux equilibriumwith external DMO. Using this new method, estimates have beenmade of the rates and magnitude of: (i) acidification of thecytoplasm caused by acidic growth regulators (IAA and NAA) andby metabolic inhibitors (azide, DNP, CCCP and DCMU), and (ii)alkalinization caused by uptake of ammonium and methylammoniumions. The potential application of the method to future studiesof membrane transport in charophyte cells is assessed. Key words: Charophyles, cytoplasmic pH.     

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)     

The Hyperpolarization of the Chara Membrane at High pH: Effects of External Potassium, Internal pH, and DCCD

At high external pH, the Chara membrane is known to switch toa new state in which the membrane potential is highly negative;it has been characterized as a passive diffusion potential forH⁺ (or 0H^–). DCCD is shown to inhibit the increased conductanceand the highly negative membrane potential associated with thisstate. DCCD also inhibits the plasmalemma H⁺-ATPase, as wellas cytoplasmic streaming. The alkaline state of the membraneis shown to involve a decreased permeability to K⁺; this enhancesthe selectivity for H⁺ (or OH^–) which results from theincreased permeability to H⁺ (or OH^–). Altering the cytoplasmicpH affects the membrane potential at both neutral and alkalinepH. It can also affect the ability of the cell to make the transitionto the alkaline state.     

Intracellular pH Regulation in the Giant-Celled Marine Alga Chaetomorpha darwinii

The vacuolar pH (pH_v) and the cytoplasmic pH (pH_c) of the marinegiant-celled green alga Chaetomorpha darwinii were measuredby pH microelectrode techniques on extracted vacuolar sap, andby the [^I4C]DMO distribution method respectively. Equilibrationof DMO occurred with a half-time of about 2 h, with an apparentP_DMO of 3.6 x 10^–5 cm s^–1, but the vacuolar concentrationof free, undissociated DMO was always less than the externalconcentration. The explanation offered for freshwater giant-celledalgae of net DMO^– leakage across the plasmalemma cannotapply to Chaetomorpha darwinii, since electrically-driven DMO^–exit from the cytoplasm should be similar across the plasmalemmaand the tonoplast in these cells with large, vacuole-positivepotential differences across the tonoplast. pHc was accordinglycomputed assuming either tonoplast or plasmalemma equilibrationof DMO, with correction for DMO metabolism within the cell.pH_c was 8.0–8.3 in the light in artificial seawater (pH_oabout 8.0), was some 0.5 units lower in the dark, and was slightlylower with an external pH of 7. Vacuolar pH was 6.5–6.9,without consistent effects of illumination or of external pHof 7 rather than 8. While µ_H+ at the tonoplast was similarto that in giant-celled freshwater algae (although with a greatercontribution from relative to pH), µ_H+ at the plasmalemmawas less than 8 kJ mol^–1, i.e. less than one-third ofthe value in freshwater green algae. µ_Na+ was some 13kJ mol^–1 at the plasmalemma. The possibility that theprimary active transport process at the plasmalemma of Chaetomorphadarwinii (and certain other marine algae) is Na⁺ efflux ratherthan H⁺ efflux is discussed.     

Automated Recording of Lettuce Root Elongation as Affected by Auxin and Acid pH in A New Rhizometer with Minimum Mechanical Contact to Roots

The elongation of many lettuce roots was separately recordedat the same time by a new rhizometer with minimum mechanicalcontact to root tips. The apparatus was operated by a microcomputersystem interfaced with field-effect transistor circuits. Elongationresponses of intact seedling roots (ISR), decotylized seedlingroots (DSR) and excised apical roots (EXR) to different concentrationsof H⁺ and IAA were compared. A pH 4-induced acid growth wasobserved only in DER and EXR, whereas a pH 3-induced one wasobserved in all roots. Duration of pH 3-induced elongation wasshorter in ISR than in DSR and EXR. Growth curves suggested that ISR is more susceptible to acid-injurythan EXR. The maximum acid growth was obtained at pH 2.5 inEXR which is comparable to thick roots of maize [Edwards andScott (1974) Planta 119: 27]. Indole-3-acetic acid showed growthinhibition at concentrations higher than 10^–8 M whichis comparable to other reports on thick roots. It is concludedthat fine lettuce roots have the same magnitude of sensitivityto external concentration of H⁺ and IAA as thick roots. It isalso suggested that root excision or decotylization makes rootsless susceptible to acid-injury, resulting in greater acid-growthof EXR. Further characteristics and application of the new rhizometerare discussed. (Received March 19, 1986; Accepted September 8, 1986)     

Comparison of the Effects of Ammonia and Methylamine on Chloride Transport and Intracellular pH in Chara corallina

Ammonium and methylammonium ions greatly increase the rate ofCl^– transport in Chara corallian. This effect is dependenton the pH of the bathing solution. The amine-stimulated Cl^–influx is small at pH 5·5, increases to a maximum atpH 6·5–7·5, and decreases again as the pHis raised to 8·5. Increased Cl^– influx is accompaniedby an increase in cytoplasmic pH, as calculated from the distributionof DMO. When the external pH lies between 5·5 and 7·3,cytoplasmic pH in the absence of amine is 7·65–7·70,with an increase of 0·15–0·25 in the presenceof amine. As external pH is increased above 7·3, cytoplasmicpH also increases, with progessively less effect of amine. Although the relationship between Cl^– influx and cytoplasmicpH is not simple, the results provide evidence in accord withthe hypothesis that Cl^– transport in Chara involves H⁺—Cl^–symport, or the equivalent OH^–—Cl^– antiport.The possible role of cytoplasmic pH as a factor involved inthe regulation of membrane transport in Chara is discussed.     

Intracellular pH shifts in cultured kidney (A6) cells: effects on apical Na+ transport

We report, for the epithelialNa⁺ channel (ENaC) in A6 cells,the modulation by cell pH (pH_c)of the transepithelial Na⁺ current(I_Na), thecurrent through the individual Na⁺channel (i), the openNa⁺ channel density(N_o), and thekinetic parameters of the relationship betweenI_Na and theapical Na⁺ concentration. Thei andN_o were evaluatedfrom the Lorentzian I_Na noise inducedby the apical Na⁺ channel blocker6-chloro-3,5-diaminopyrazine-2-carboxamide.pH_c shifts were induced, understrict and volume-controlled experimental conditions, byapical/basolateral NH₄Cl pulses orbasolateral arrest of theNa⁺/H⁺exchanger (Na⁺ removal; block byethylisopropylamiloride) and were measured with the pH-sensitive probe2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Thechanges in pH_c were positivelycorrelated to changes inI_Na and theapically dominated transepithelial conductance. The sole pH_c-sensitive parameter underlyingI_Na wasN_o. Only thesaturation value of theI_Na kinetics wassubject to changes in pH_c.pH_c-dependent changes inN_o may be causedby influencingP_o, the ENaC openprobability, or/and the total channel number,N_T = N_o/P_o.

     

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     

Flexible Coupling of Phosphate Uptake in Dunaliella acidophila at Extremely Low pH Values

The acidophilic alga Dunaliella acidophila exhibits optimalgrowth at pH 1. We have investigated the regulation of phosphateuptake by this alga using tracer techniques and by performingintracellular phosphate measurements under different growthconditions including phosphate limitation. In batch culturewith 2·2 mol m^–3 phosphate in the medium the uptakeof phosphate at micromolar phosphate concentrations followeda linear time dependence in the range of minutes and rates werein the range of 1 µmol phosphate mg^–1 chl h^–1,only. However, under discontinuous phosphate-limited growthconditions, tracer influx revealed a biphasic pattern at micromolarphosphate concentrations: An initial burst phase resulted ina 10⁴-fold internal phosphate accumulation and levelled offafter about 10 s. A double reciprocal plot of the initial influxrates obtained for phosphate-limited and unlimited algae exhibitedMichaelis-Menten kinetics. Phosphate limitation caused a significantactivation of the maximum velocity of uptake, yielding V_maxup to 1 mmol mg^–1 chl h^–1 as compared to valuesin the order of 50 µmol phosphate mg^–1 chl h^–1for the second phase (this magnitude is also representativefor non-limited batch cultures). Concomitantly the Michaelisconstant was altered from 4 mmol m^–3 to 0·7 mmolm^–3. The rapid uptake of phosphate was inhibited by arsenateand FCCP and was not stimulated by Na⁺. The pH dependence oftracer accumulation and measurements of the intracellular phosphatepool under different growth conditions indicate that at lowpH and low external phosphate concentrations the high protongradient present under these conditions is utilized for a H₃PO₄uptake or a H⁺/H₂PO^–4 cotransport. However, when the externalphosphate concentration was increased to levels sufficientlyhigh for transport to be driven by the positive membrane potential(10 mol m^–3 phosphate), the pH dependence of phosphateuptake was more complex, but could be explained by the uptakeof H₃PO₄ or a H⁺/H₂PO^–₄-cotransport at low pH and a differenttype H₂PO^–4-transport (with unknown type of ion coupling)at high pH-values. It is suggested that this flexible couplingof phosphate transport is of essential importance for the acidresistance of Dunaliella acidophila. Key words: Acid resistance, Dunaliella acidophila, phosphate cotransport, phosphate limitation, plasma membrane, sodium     

Changes in the Cytoplasmic and Vacuolar pH in Intact Cells of Mung Bean Root-Tips under High-NaCl Stress at Different External Concentrations of Ca2+ Ions

The cytoplasmic pH and the vacuolar pH in root-tip cells ofintact mung bean seedlings under high-NaCl stress were measuredby in vivo ³¹P-nuclear magnetic resonance (³¹P-NMR) spectroscopy.When roots were incubated with high levels (100 mM) of NaClat the control external concentration (0.5 mM) of Ca²⁺ ions,the vacuolar pH increased rapidly from 5.6 to 6.2 within 3 h,while the cytoplasmic pH only decreased by a mere 0.1 pH uniteven after a 24-h incubation under high-NaCl conditions. Theincrease in vacuolar pH induced by the high-NaCl stress wasdiminished by an increase in the external concentration of Ca²⁺ions from 0.5 mM to 5 mM. The intracellular concentration ofNa⁺ ions in the root-tip cells increased dramatically upon perfusionof the root cells with 100 mM NaCl, and high external levelsof Ca²⁺ ions also suppressed the in flow of Na⁺ ions into thecells. The vacuolar alkalization observed in salt-stressed rootsmay be related to the inhibition of an H⁺-translocating pyrophosphatasein the tonoplast, caused by the increase in the cytoplasmicconcentration of Na⁺ ions. It is suggested that, although thevacuolar pH increased markedly under salt stress, the cytoplasmicpH was tightly regulated by some unidentified mechanisms, suchas stimulation of the H⁺-translocating ATPase of the plasmalemma,in roots of mung bean under salt stress. (Received April 18, 1992; Accepted July 6, 1992)