Effects of Ammonia and Methylamine on Cl- Transport and on the pH Changes and Circulating Electric Currents Associated with HCO3- Assimilation in Chara corallina

Low concentrations of ammonia and methylamine greatly increaseCl^– influx into Chara corallina. Both amines have theirmaximum effect at pH 6.5–7.5. The amine stimulation ofCl^– influx is small below about pH 5.5. Above pH 8.5 theremay be inhibition of influx by amines. Concentrations of 10–25µM ammonia are sufficient to cause the maximum stimulationof Cl^– influx; the corresponding methylamine concentrationsare 0.1–0.2 mM. It is concluded that entry of amine cations(NH₄^$ and CH₃NH₃^$), rather than unionized bases (NH₃ and CH₃NH₂),causes Cl^– transport to be increased. Increases in rates of Cl^– transport are not necessarilyaccompanied by effects on HCO₃^$ assimilation and OH^– efflux.Measurements of localized pH differences at the cell surfaceand of circulating electric currents in the bathing solutionshow that these phenomena are only significantly affected byammonia at or above 50 µM and by methylamine at or above1.0 mM. The significance of the effects of amines is assessedin relation to current ideas about transport of Cl^–, HCO₃^–,and OH^–.     

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     

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     

Chloride Transport in Chara corallina and the Electrochemical Potential Difference for Hydrogen Ions

The pH of the cytoplasm of Chara corallina cells has been measuredwith the weak acid 5,5-dimethyloxazolidine-2,4-dione (DM0).Over an external pH range 4·5–9·5 the resultsfit the regression equation pH_cytoplasm=6·28+0·22pH_out. Using measured values of the electric potential difference acrossthe plasmalemma we have calculated the electrochemical potentialdifference across this membrane for H⁺ and Cl^–. Thesedata are used to test the hypothesis that the inward transportof Cl^– is coupled to the inthix of H⁺ or, which comesto the same thing, efflux of OH^–. One-for-one couplingwill not give net Cl^– uptake from solutions with pH greaterthan about 7·2, unless the cytoplasmic Cl^– concentrationis lower than 10 mM, or the pH just outside the membrane islower than that in the bulk solution. It is shown that net Cl^–uptake proceeds from solutions with pH up to 9. The alternative possibility is that Cl^– transport is broughtabout by co-transport of two H⁺ for each Cl^–; this isnot ruled out by the results reported. Such a mechanism mightbe detectable by its electrogenic effect: although such effectshave not been detected, it is shown that they would be smallunder most conditions. Other possible mechanisms are discussed.     

Control of Intracellular pH in Chara corallina during Uptake of Weak Acid

Butyric acid was used to acidify the cytoplasm of cells of Characorallina in order to study the mechanisms that regulate intracellularpH. Butyric acid was found to enter the cell rapidly, predominantlyas the undissociated acid, and to dissociate in the cytoplasmto yield high concentrations of the butyrate anion. A rapidreduction in cytoplasmic pH was followed by partial recovery.The reductions in cytoplasmic pH resulting from butyrate accumulationwere small compared to the proton load calculated from the cytoplasmicbuffering capacity and intracellular dissociation of butyricacid. The cytoplasmic and vacuolar buffering capacities, calculatedfrom titration of cell extracts, were 17.9 and 0.5 mol m^–3per pH unit respectively. It was concluded that pH control in Chara during weak acid accumulationwas mainly due to membrane transport (active efflux) of protons.The factors which might determine the rate and extent of protonefflux, such as the energy supply and the availability of ionsfor charge balance, were examined. Butyrate strongly inhibitedphotosynthesis and caused a slight reduction in the rate ofrespiration. The mechanism of inhibition of photosynthesis isdiscussed in relation to the reported effects of weak acidson isolated chloroplasts. Key words: Cytoplasmic pH, weak acids, Chara     

The Permeability of Ammonia, Methylamine and Ethylamine in the Charophyte Chara corallina (C. australis)

Ritchie, R. J. 1987. The permeability of ammonia, methylamineand ethylamine in the charophyte Chara corallina (C. australis).—J.exp. Bot. 38: 67–76 The permeabilities of the amines, ammonia (NH₃), methylamine(CH₃NH₂) and ethylamine (CH₃CH₂NH₂) in the giant-celled charophyteChara corallina (C. australis) R.Br. have been measured andcompared. The permeabilities were corrected for uptake fluxesof the amine cations. Based on net uptake rates, the permeabilityof ammonia was 6?4?0?93 µm s^–1 (n = 38). The permeabilitiesof methylamine and ethylamine were measured in net and exchangeflux experiments. The permeabilities of methylamine were notsignificantly different in net and exchange experiments, norto that of ammonia (P_methylamine = 6?0?0?49 µm s^–1(n = 44)). In net flux experiments the apparent permeabilityof ethylamine was slightly greater than that of ammonia andmethylamine (P_{ethylamine, net} = 8?4?1?2 µm s^–1 (n= 40)) but the permeability of ethylamine based on exchangeflux data was significantly higher (P_{ethylamine, exchange} =14?1?2 µm s^–1 (n = 20)). Methylamine can be validlyused as an ammonium analogue in permeability studies in Chara. The plasmalemma of Chara has acid and alkaline bands; littlediffusion of uncharged amines would occur across the acid bands.The actual permeability of amines across the alkaline bandsis probably about twice the values quoted above on a whole cellbasis i.e. the permeability of ammonia across the permeablepart of the plasmalemma is probably about 12 µm s^–1. Key words: Chara, permeability, ammonia, methylamine     

Intercellular Transport and Photosynthetic Differentiation in Chara corallina

Intercellular transport of ¹⁴C-labelled photoassimilates, bothin isolated upper shoots and in isolated internode-branchletcomplexes of Chara corallina, was measured. The isolated uppershoots were composed of a primary apex, two mature internodes,and three branchlet whorls. A 10 min loading of the isolatedupper shoot with H¹⁴CO₃^– resulted in a greater accumulationof ¹⁴C in the apical complex and branchlets than in the internodes,while a subsequent 50 min chase with unlabelled solution inthe light resulted in a greater accumulation of ¹⁴C in internodesthan in other parts of the shoot. In the isolated internode-branchlet complex, when the apex wasnot detached, the amount of ¹⁴C transported from branchletsto internodes was about fives times that transported from internodesto branchlets. Removal of the apex resulted in a decrease intransport from branchlets to internodes and an increase in transportin the opposite direction. In an attempt to explain the mechanism of the polar transportof photosynthetically fixed carbon between branchlets and internodes,photosynthetic activities of both types of cells were investigated.Detached branchlets have higher photosynthetic ¹⁴C-fixationactivities than those of internodes. Chlorophyll contents, measuredin terms of surface area, in internodes and branchlets werealmost identical. The ribulose-l,5-bisphosphate carboxylase(RuBPCase) activity of branchlets was 1.6 times that of internodes,and the rate of ferricyanide-dependent evolution of oxygen inbranchlets was 1.4 times that in internodes. Key words: Chara, internode, branchlet, polar transport, photosynthesis     

Effect of Temperature and External pH on the Cytoplasmic pH of Chara corallina

Cytoplasmic pH (pH_c) in Chara corallina was measured (from [¹⁴C]stribution)as a function of external pH (pH₀)and temperature. With pH₀near 7, pH_c at 25?C is 7.80; pH_cincreases by 0.005 pH units?C^–1 temperature decrease, i.e. pH_c at 5 ?C is 7.90. WithpH? near 5.5, the increase in pH_c with decreasing temperatureis 0.015 units ?C^–1 between 25 and 15?C, but 0.005 units?C^–1 between 15 and 5?C. This implies a more precise regulationof pH_c with variations in pH_o at 5 or 15 ?C compared with 25?C. The observed dp H_c/dT is generally smaller than the –0.017units ?C^–1 needed to maintain a constant H⁺/OH^–1,or a constant fractional ionization of histidine in protein,with variation in temperature. It is closer to that needed tomaintain the fractional ionization of phosphorylated compoundsor of CO₂–HCO₃^– The value of dpH_c/dT has importantimplications for several regulatory aspects of cell metabolism.These include (all as a function of temperature) the rates ofenzyme reactions, the _H+ at the plasmalemma(and hence the energy available for cotransport processes),and the mechanism for pH_c regulation by the control of bidirectionalH⁺ fluxes at the plasmalemma.     

Plasmalemma Chloride Transport in Chara corallina: Inhibition by 4,4'-Diisothiocyano-2,2'-Disulfonic Acid Stilbene

Chloride transport, presumably via a Cl⁻-2H⁺ co-transport system, was investigated in Chara corallina. At pH 6.5, the control influx (3.1 picomoles per centimeter² per second) was stimulated 4-fold by an 18-hour Cl⁻ starvation. The stimulated influx was inhibited to 4.7 picomoles per centimeter² per second after a 60-minute pre-exposure to 0.5 millimolar 4,4′-diisothiocyano-2,2′-disulfonic acid stilbene (DIDS). This compares with a nonsignificant inhibition of the control under similar conditions. At 2 millimolar DIDS, both stimulated and control influx were inhibited to values of 1.1 and 2.2 picomoles per centimeter² per second, respectively; in all cases, DIDS inhibition was reversible. Over the pH range 4.8 to 8.5, the control and DIDS-inhibited influx showed only slight pH sensitivity; in contrast, the stimulated flux was strongly pH dependent (pH 6.5 optimum). Inasmuch as changes in pH alter membrane potential, N-ethylmaleimide was used to depolarize the membrane; this had no effect on Cl⁻ influx. A transient depolarization of the membrane (about 20 millivolts) was observed on restoration of Cl⁻ to starved cells. The membrane also depolarized transiently when starved cells were exposed to 0.5 millimolar DIDS, but the depolarization associated with Cl⁻ restoration was inhibited by a 40-minute pretreatment with DIDS. Exposure of control cells to DIDS caused only a small hyperpolarization (about 7 millivolts). DIDS may have blocked Cl⁻ influx by inhibiting the putative plasmalemma H⁺-translocating ATPase. Histochemical studies on intact cells revealed no observable effect of DIDS on plasmalemma ATPase activity. However, DIDS application after fixation resulted in complete inhibition of ATPase activity.

The differential sensitivity of the stimulated and control flux to inhibition by DIDS may reflect an alteration of transport upon stimulation, but could also result from differences in pretreatment. The stimulated cells were pretreated with DIDS in the absence of Cl⁻, in contrast to the presence of Cl⁻ during pretreatment of controls. The differential effect could result from competition between Cl⁻ and DIDS for a common binding site. Our histochemical ATPase results indicate that Cl⁻ transport and membrane ATPase are separate systems, and the latter is only inhibited by DIDS from the inside of the cell.

     

Intracellular measurement of ammonium in Chara corallina using ion-selective microelectrodes

The study of ammonium (NH₄ ⁺) transport across plant cell membranes requires accurate measurement of NH₄ ⁺ gradients across subcellular gradients. We have developed an ammonium-selective microelectrode based on the ionophore nonactin. This electrode can detect NH₄ ⁺ activities (a_NH4) in vivo in the millimolar range in the presence of cytosolic levels of potassium, the main interfering ion. The electrode was used to measure intracellular a_NH4 in internodal cells of the giant alga Chara corallina. Results from cells incubated in media supplemented with 1 mM NH₄ ⁺ produced two populations, with means of 7.3 and 30.8 mM, respectively. HPLC analysis of vacuolar sap suggests the higher population represents vacuolar impalements, and the lower population can thus be assumed to be cytosolic. These results suggest a four-fold accumulation of NH₄ ⁺ in the vacuolar compartment of Chara. This revised version was published online in June 2006 with corrections to the Cover Date.     

Intercellular Transport and Subcellular Distribution of Photoassimilates in Chara corallina

The mechanism of polar transport of ¹⁴C-photoassimilates betweenChara corallina branchlets and internodes was investigated usingan intracellular perfusion procedure. When the apex remained,more ¹⁴C-photoassimilates were transported from branchlet tointernode than in the opposite direction. When the apex wasdetached, this polarity disappeared. To study the polar transportmechanism, we examined the subcellular distribution of ¹⁴C-photoassimilatesin the three main intracellular compartments, the cortical chloroplastlayer, streaming sol endoplasm and vacuole. When the internode or branchlet of an internode-branchiet complexwas exposed to ¹⁴C-bicarbonate for 10 min, 70 to 80% of thetotal fixed carbon was found in the sol endoplasm. In the subsequent3 h chase, the ¹⁴C-photoassimilates in the sol endoplasm ofthe source cell decreased greatly to 20 to 40% of the totallabelled substances. When the apex remained, the branchlet showed higher photosyntheticactivity than the internode, which resulted in a concentrationgradient of 13·5 mol m^–3 in the sol endoplasm frombranchlet to internode after photosynthesis for 10 mm. In the3 h chase period, this gradient decreased to about 1·0mol m^–3. In the other three cases, involving transportfrom branchlet to internode when the apex was detached, or transportfrom internode with or without apex to branchlet, the intercellulargradient of ¹⁴C after 10 mm photosynthesis was about 70 molm^–3 in the sol endoplasm. Thus, when the apex remains,the greater gradient of photoassimilates between the sol endoplasmof branchlet and internode seems to be one of the reasons forpolar intercellular transport. Key words: Chara corallina, polar transport, ¹⁴C photoassimilates     

Measurements of the Cytoplasmic pH of Chara corallina using Double-barrelled pH Micro-electrodes

A range of polymeric compounds was examined for their suitabilityas pressure-stabilizing agents in liquid membrane pH micro-electrodesfor intracellular use in plant cells. Of the compounds tested,mixtures of liquid proton sensor and nitrocellulose were foundto be superior to epoxy resins, polyvinylchloride and ethylcellulose. The electrical resistance of silicone rubber mixtureswas too high for micro-electrodes with tip diameters of 1.0µm. Double-barrelled micro-electrodes containing nitrocellulosemaintained excellent pH sensitivity for up to 1.0 impalementsof charophyte cells. Measurements of cytoplasmic pH were madein both internodal and whorl cells of Chora corallina over arange of experimental conditions. The response of cytoplasmicpH to rapid changes in external pH or illumination occurredover several minutes. The advantages of the use of double-barrelledpH micro-electrodes over other methods of intracellular pH measurementsuch as the distribution of weak acids (DMO), ³¹P-NMR and single-barrelledmicro-electrodes is discussed. Key words: pH micro-electrodes, cytoplasmic pH, charophytes     

The Effect of Darkness on Active and Passive Transport in Chara corallina

In Chara corallina, the membrane potential may stay much morenegative than the equilibrium potential for potassium in thedark, indicating that the proton pump is operative. The highproton conductance which occurs at high external pH, as indicatedby a high membrane conductance and a membrane potential nearthe equilibrium potential for protons, is not seen in the darkat pH 11. This effect is likely to be related to inhibitionof photosynthesis since DCMU has the same effect. The effectis similar but not identical to the effect of a decreased internalpH. Key words: Light, dark, membrane potential, Chara     

Tonoplast Anion Channel Activity Modulation by pH in Chara corallina

The patch-clamp technique was used to investigate regulation of anion channel activity in the tonoplast of Chara corallina in response to changing proton and calcium concentrations on both sides of the membrane. These channels are known to be Ca²⁺-dependent, with conductances in the range of 37 to 48 pS at pH 7.4. By using low pH at the vacuolar side (either pH_vac 5.3 or 6.0) and a cytosolic pH (pH_cyt) varying in a range of 4.3 to 9.0, anion channel activity and single-channel conductance could be reversibly modulated. In addition, Ca²⁺-sensitivity of the channels was markedly influenced by pH changes. At pH_cyt values of 7.2 and 7.4 the half-maximal concentration (EC ₅₀) for calcium activation was 100–200 μm, whereas an EC ₅₀ of about 5 μm was found at a pH_cyt of 6.0. This suggests an improved binding of Ca²⁺ ions to the channel protein at more acidic cytoplasm. At low pH_cyt, anion channel activity and mean open times were voltage-dependent. At pipette potentials (V _p) of +100 mV, channel activity was approximately 15-fold higher than activity at negative pipette potentials and the mean open time of the channel increased. In contrast, at pH_cyt 7.2, anion channel activity and the opening behavior seemed to be independent of the applied V _p. The kinetics of the channel could be further controlled by the Ca²⁺ concentration at the cytosolic membrane side: the mean open time significantly increased in the presence of a high cytosolic Ca²⁺ concentration. These results show that tonoplast anion channels are maintained in a highly active state in a narrow pH range, below the resting pH_cyt. A putative physiological role of the pH-dependent modulation of these anion channels is discussed. Received: 14 March 2001/Revised: 16 July 2001     

Regulation of the Cytoplasmic pH of Chara corallina: Response to Changes in External pH

Effects of external pH (pH_o) on the cytoplasmic pH (pH_c) ofChara corallina have been measured with the weak acid 5, 5-dimethyloxazolidine-2,4-dione (DMO) following standardized pretreatment of cells insolutions at pHo 4.5, 6.3 and 8.3. Irrespective of pH_c duringpretreatment, pH_o responded to pHo during the experimental periodsof 150–180 min or (in one experiment) 90–110 min.There were increases or decreases of about 0.5 in pHo when cellswere transferred from pH_o 4.5 to 8.3 or vice versa. In the darkpH_c was 0.2–0.3 units lower than the corresponding valuein the light. The results are discussed in relation to the factorsinvolved in the regulation of pH_c in C. corallina, which maybegin to break down below about pH_o4.5, as indicated by relativelylarge decreases in pH_c at low pH_o. Key words: Chara corallina, Cytoplasmic pH, External pH, DMO     

Hydroxyl- and Bicarbonate-Associated Transport Processes in Chara corallina: Studies on the Light-Dark Regulation Mechanism

Lucas, W. J. and Ogata, K. 1985. Hydroxyl– and bicarbonate–associatedtransport processes in Chara corallintr. Studies on the light–darkregulation mechanism.—J. exp. Bot. 36: 1947–1958.Experiments were undertaken on the fresh water alga Chara corallinato investigate the nature of the coupling between the chloroplastsand the light–dependent OH^– and -associated plasmalemma transport systems. Electrophysiologicalexperiments, in which DCMU was employed, revealed that thischemical could elicit a hyperpolarization of the membrane potentialthat was identical to that normally obtained by turning offthe light. This DCMU–induced hyperpolarization was obtainedunder control () and phosphate–decoupled conditions (). Measurements of the extracellular electric potentialswhich are associated with the acidic () and alkaline (OH^–) regions, indicated that, in the presenceof control ()or phosphate–decoupled conditions, normal profiles were established under air, oxygenor nitrogen environments. These results indicate that the generationof the control signal(s) is related to events associated withchloroplast electron transport, rather than to changes in theflow or levels of carbon intermediates within the reductivepentose phosphate or photorespiratory cycles. Although the levelof oxygen was found to have no effect on the light–inducedactivation of the OH regions, we found that in pure oxygen thedark–induced inactivation of the OH^– efflux systemwas delayed, and that partial transport function could be maintainedin the dark. The possible involvement of changes in either theratio of oxidized to reduced ferridoxin or NADP^? to NADPH, aspart of this light–mediated control signal, is discussed. Key words: Chara corallina, Plasma membrance transport, OH^– and , regulation     

Physiological Control of Chloride Transport in Chara corallina: II. THE ROLE OF CHLORIDE AS A VACUOLAR OSMOTICUM

The extent to which Cl⁻ is replaceable as the major anionic constituent of the vacuole of Chara corallina was investigated. It was found that external Cl⁻ is not essential in order for nongrowing cells to increase internal osmotic pressure. After growth of cells in low (9 micromolar) Cl⁻, the vacuolar Cl⁻ concentration is one-half that of cells grown at normal external Cl⁻ concentration (850 micromolar). In contrast, both internal osmotic pressure and total concentration of the major cations, K⁺ and Na⁺, in the same cells were found to be only slightly sensitive to the external Cl⁻ concentration. Thus, it is proposed that, at limiting external Cl⁻ concentration, the cell is able to transport or synthesize another anion for vacuolar use rather than utilize a neutral solute.     

Biophysical and Biochemical Regulation of Cytoplasmic pH in Chara corallina during Acid Loads

The contribution of membrane transport to regulation of cytoplasmicpH in Chara corallina has been measured during proton-loadingby uptake of butyric acid. In the short-term (i.e. up to 20min) uptake of butyric acid is not affected by removal of externalK⁺, Na⁺ or Cl^– but over longer periods uptake is decreased(by 20–50% in different experiments) in the absence ofexternal Na⁺ or, sometimes, K⁺. Influxes of both Na⁺ and K⁺increase temporarily after addition of butyrate, Na⁺ immediatelyand K⁺ after a lag. Effects on Cl^– influx are small butCl^– efflux increases enormously after a short lag. Anapproximate comparison of internal butyrate with changes inthe concentration of K⁺, Na⁺, and Cl^– suggests that initially(i.e. for a few min) cytoplasmic pH is determined by bufferingand possibly by some decarboxylation of organic acids (biochemicalpH regulation), and that biophysical pH regulation involvingefflux of H⁺ balanced by influxes of K⁺, Na⁺ and especiallyefflux of Cl^– progressively becomes dominant. When butyric acid is washed out of the cells, cytoplasmic pHis restored completely or partially (depending on the butyrateconcentration used) and this is independent of the presenceor absence of external Cl^–. Where Cl^– is present,its influx is relatively small. It is suggested that cytoplasmicpH is then controlled biochemically, involving the synthesisof an (unidentified) organic acid and the accumulation of acidicanions in place of butyurate lost from the cell. During thesecond application of butyrate, net Cl^– efflux is small:it is suggested that control of cytoplasmic pH then involvesdecarboxylation of the organic acid anions. The questions of the source of Cl^– lost from the cell(cytoplasm or vacuole) and of possible cytoplasmic swellingassociated with the accumulation of butyrate are discussed. Key words: Chara corallina, butyric acid, cytoplasmic pH, membrane transport     

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.     

Plasmalemma Transport of HCO3- and OH- in Chara corallina: Non-antiporter Systems

An experimental system was designed to test the obligate couplingbetween HCO₃^– and OH^– fluxes (i. e. a ‘Mitchell-type’antiporter) proposed by Lucas and Smith (1973). The resultsof these experiments demonstrated categorically that the OH^–efflux process can function in the absence of exogenous HCO₃^–at the actual OH^– efflux site. Hence, the obligate couplinghypothesis is invalid. It is proposed that HCO₃^– and OH^–are transported across the plasmalemma ‘independently’,on quite distinct carriers. It is possible, therefore, thatthese fluxes contribute towards determining the electrical propertiesof this membrane when the bathing solution pH value is 6.5.It was also found that HCO₃^– can be transported acrossthe dark segment of a partly illuminated cell. The observedrates were always much less than those obtained in the illuminatedcell segment. The significance of this result is discussed.