Effect of Sodium, Potassium, Calcium, Magnesium and Tetraethylammonium on the Transient Voltage Response to a Galvanostatic Step and of the Temperature on the Steady Membrane Conductance of Chara corallina: a Further Evidence for the Involvement of Potassium Channels in the Fast Time Variant Conductance

Homble F. 1985. Effect of sodium, potassium, calcium, magnesiumand tetraethylammonium on the transient voltage response toa galvanostatic step and of the temperature on the steady membraneconductance of Chara corallina: A further evidence for the involvementof potassium in the fast time variant conductance.—J.exp. Bot. 36: 1603–1611. Potassium channels of Chara corallina have an activation energyof 36±1 kJ mol^–1 and 50±2 kJ mol^–1at temperatures higher and lower than 15°C respectively.The fast time variant conductance property of potassium channelsis insensitive to sodium and magnesium ions and is depressedby the presence of calcium, potassium and tetraethylammoniumions. It is suggested that in Chara two different kinds of potassiumchannels exist, each kind being distinguished by their kineticsand their response to calcium and magnesium ions. Key words: —Chara corallina, membrane conductance, potassium channels, temperature     

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     

Recording of the potassium efflux during a single action potential in Chara corallina

The potassium efflux from cells of Chara corallina was recordedsimultaneously with an action potential using a flame photometerand a perfusion system. The potassium efflux increased instantaneouslywith the action potential and lasted for 12–76 sec. Thenet potassium efflux during an action potential averaged 308picomoles/cm₂ impulse. (Received December 21, 1974; )     

Effect of Ca2+ on Tonoplast Potential of Permeabilized Characeae Cells

Using permeabilized characean cells in which the ionic conditionsat the cytoplasmic side of the tonoplast are easily controlled,effects of Ca²⁺ ion on tonoplast potential were examined. Whenthe cell was treated with 1 µM Ca²⁺, the tonoplast potential(E_M became positive in a complicated manner in Chara corallinawhile it simply became negative in Nitella axilliformis. Whenthe cell was treated with 9-antracenecarboxylic acid, a Cl^–-channelinhibitor, E_m became more negative and the response of E_m toCa²⁺ was significantly suppressed. It is suggested that Ca²⁺activates Cl^–-channel at a low concentration and inactivatesat a higher one in C. corallina while it simply inactivate Cl^–-channelin N. axilliformis. ¹Present address: Biological Laboratory, The University of theAir, Wakaba 2-11, Wakaba, 260 Japan. (Received August 22, 1988; Accepted December 26, 1988)     

The Apparent Induction of Nitrate Uptake by Chara corallina Cells following Pretreatment with or without Nitrate and Chlorate

Nitrate provision has been found to regulate the capacity forChara corallina cells to take up nitrate. When nitrate was suppliedto N sufficient cells maximum nitrate uptake was reached after8 h. Prolonged treatment of the cells in the absence of N alsoresulted in the apparent ability of these cells to take up nitrate.Chlorate was found to substitute partially for nitrate in the‘induction’ step. The effects on nitrate reductionwere separated from those on nitrate uptake by experiments usingtungstate. Tungstate pretreatment had no effect on NO^–₃uptake ‘induced’ by N starvation, but inhibitedNO^–₃ uptake associated with NO^–₃ pretreatment. Chloridepretreatment similarly had no effect on NO^–₃ uptake ‘induced’by N deprivation, but inhibited NO^–₃ uptake followingNO^–₃ pretreatment. The data suggest that there are atleast two mechanisms responsible for the ‘induction’of nitrate uptake by Chara cells, one associated with NO^–₃reduction and ‘induced’ by CIO^–₃ or NO^–₃and one associated with N deprivation. Key words: Nitrate, Chlorate, Chara corallina, Induction     

Water channels in Chara corallina

Water relations parameters ofChara corallina inter-nodes weremeasured using the single cell pressure probe. The effect ofmercurials, which are recognized as non-specific water channelinhibitors, was examined. HgCl₂ concentrations greater than5 mmol m^–3 were found to inhibit hydraulic conductivity{Lp) close to 90%, whereas pCMPS was found to have no effecton Lp. The activation energy of water flow was increased significantlyfrom 21.0 kJ mol^–1 to 45.6 kJ mol^–1, following theapplication of HgCl₂. These results are in accordance with evidencefor Hg²⁺sensitive water channels in the plasma membrane of charophytes(Henzler and Steudle, 1995; Tazawa et al., 1996). The metaboliceffects must, however, be considered in view of the rapid inhibitionof respiration and the depolarization of the membrane potentialwith HgCl₂ concentrations lower than those found to affect Lp.It was possible to measure simultaneously water relations andmembrane PD, in order to examine the contribution of potassiumchannels to Lp. Cells were induced into a K⁺ permeable state.The K⁺ channels, assumed to be open, were subsequently blockedby various blockers. No significant difference in Lp was foundfor any of these treatments. Finally, the permeability of C.corallina membranes to ethanol was examined. HgCl₂ was foundto cause a decrease in reflection coefficient, coinciding witha decrease in Lp, but there was no change in the ethanol permeabilitycoefficient. This has been interpreted in terms of both thefrictional model and composite model of non-electrolyte membranetransport. Key words: Water channels, Chara, hydraulic, conductivity, membrane transport models, reflection coefficient     

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

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

Salinity-induced Malate Accumulation in Chara

Ion absorption by Chara corallina from solutions containingpredominantly KC1 or RbCl at up to 100 mol m^–3 resultedin accumulation of salts and turgor regulation. Turgor regulationdid not occur in solutions containing Na⁺ or Li⁺salts. Duringion absorption from various salts of K⁺ and Rb⁺ vacuolar cationconcentration exceeded Cl^– concentration. This differencewas shown to be balanced by the synthesis and accumulation ofmalate. Vacuolar malate concentration reached 48 mol m₃^–,with accumulation occurring at rates of up to 0.45 mol m^–3h^–1. Malate accumulation was inhibited by low externalpH and was dependent upon external HCO₃^– concentration.The synthesis of malic acid and its subsequent dissociationimposed a severe acid load on the cell. Biophysical regulationof cellular pH was achieved by a H⁺efflux at a rate of about40 nmol m^–2 s^–1from the cell. The results presentedargue against cytoplasmic Cl^–, HCO₃^– or pH regulatingmalate accumulation in Chara and it is suggested that malatetransport across the tonoplast may regulate malate accumulation. Key words: Malate, Chara corallina, pH regulation, salinity     

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     

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     

Cl- Fluxes during Osmoregulation in Chara

Measurements of Cl^– influx in cells of Chara corallinashow that control of this flux contributes to the ability ofthis cell to regulate its osmotic pressure. Transcellular osmosiswas used to generate cell fragments with abnormally high ₁,(H-cells), and with abnormally low ₁, (L-cells). Plasmalemmainflux (_oc) was very high in L-cells, and markedly reduced inH-cells. Influx was not affected by the presence of sucrosein the pond water and the consequent reduction in turgor. InH-cells the chloride flux from cytoplasm to vacuole (_cv) wasalso strongly inhibited. It is suggested that control of Cl^–fluxes at both plasmalemma and tonoplast is involved in osmoregulationin these cells. Key words: Chara corallina, osmoregulation, Cl^– flux     

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     

Ionic Currents across the Plasmalemma of Chara inflata Cells: III. WATER-RELATIONS PARAMETERS AND THEIR CORRELATION WITH MEMBRANE ELECTRICAL PROPERTIES

The water-relations parameters of Chara inflata cells were determineddirectly using the micro pressure probe technique. The turgorpressure of cells in artificial pond water (₀ = 0.06 MPa) wasabout 0.65 MPa and the half-time (T_1/2) for water exchange wasabout 6.5 s. The calculated values of the hydraulic conductivity(L_P) were in the range 1–2 ? 10^–6m s^–1 (MPa)^–1.The volumetric elastic modulus () was 32.8 MPa for turgor rangingfrom 0.77 to 0.82 MPa. Large changes in the water-relations parameters and the electricalproperties of the membrane occurred when the turgor was decreasedto low values. These changes included: (i) a decrease in theT_1/2 for water exchange, (ii) an increase in L_P and (iii) depolarizationof the membrane potential difference (V_m). The micro pressure probe, which enabled the turgor pressureof the cell to be altered, was used in combination with thevoltage-clamp technique to determine the relationship betweenK⁺ and Cl^– conductances of the plasmalemma and the cellturgor. The K⁺ conductance increased reversibly as the turgorwas reduced in the range 0 to 0.6 MPa and the Cl^– -conductanceincreased as the turgor was reduced in the range 0.1 to 0.5MPa. It is suggested that these pressure-dependent K⁺ and Cl^–conductances may have a dual role in electrical events and thenon-electrical responses such as changes in the cell volume. Key words: Chara inflata, membrane conductances, ion channels, water-relations parameters     

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)     

Studies on Mechano-Perception in Characeae: Effects of External Ca2+ and Cl-

The effects of modification of extracellular concentrationsof Ca²⁺ and C^– on mechano-perception were studied in internodalcells of Chara corallina. Cells were stimulated by droppinga piece of glass tubing on them, and the resulting receptorpotentials and action potentials were analyzed. When the Ca²⁺concentration was extremely lowered by adding EGTA, the amplitudesof both receptor potentials and action potentials were attenuated,suggesting the involvement of Ca²⁺ channels. However, the possibilityremained that attenuation of the amplitude of the receptor potentialwas caused by modification of membrane characteristics by extremelowering of [Ca²⁺]_o. When the plasma membrane was depolarizedto about 0 mV by adding 100 mM KC1, responses in the negativedirection were induced upon mechanical stimulation. When theplasma membrane was depolarized by adding 50 mM K²SO⁴, responsesin the positive direction were induced. Thus, Cl^– channelsmay be involved in responses induced by mechanical stimulationunder K+-induced depolarization. (Received January 16, 1996; Accepted March 25, 1997)     

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     

Nitrate Transport into Chara corallina Cells using 36ClO-3 as an Analogue for Nitrate: I. INTERACTION BETWEEN 36ClO-3 AND NO-3 AND CHARACTERIZATION OF 36CIO-3/NO-3 INFLUX

The use of chlorate as an analogue for NO^–₃ during nitrateuptake into Chara corallina cells has been investigated. NO^–₃inhibits ³⁶C1O^–₃ influx into Chara over the concentrationrange 0–1000 mmol m^–3. Lineweaver-Burke plots ofthe data are characteristic of competitive inhibition by NO^–3in the low concentration range (0–300 mmol m^–3 ClO^–₃)and apparent K_INO3 is 140 mmol m^–3 which is of a similarorder of magnitude as apparent K_mCIO3- 180 mmol m^–3. Athigher substrate concentrations the inhibition by NO^–₃was not characteristic of competitive or uncompetitive inhibition. ³⁶C1O^–₃/NO^–₃ influx was dependent on K⁺ and Ca²⁺in the external medium and inhibited by FCCP. NO^–₃ pretreatmentor N starvation increased subsequent ³⁶C1O^–₃/NO^–₃influx into Chara. A comparison between rates of net NO^–₃uptake and ³⁶C1O^–₃/NO^–₃ influx supported the previoushypothesis that NO^–₃ efflux is an important componentin the determination of overall uptake rates. Key words: Nitrate, Chara, ³⁶CIO^–₃     

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.     

Effects of Temperature and Water Potential on Germination, Radicle Elongation and Emergence of Mungbean

Controlled environment experiments were performed to determinethe effects of temperature and water potential on germination,radicle elongation and emergence of mungbean (Vigna radiata(L.) Wilczek cv. IPB-M79-17-79). The effects of a range of constant temperatures (15–45°C) and water potentials (0 to –2.2 MPa) on germinationand radicle elongation rates were studied using an osmoticumtechnique, in which seeds were held against a semi-permeablemembrane sac containing a polyethylene glycol solution. Linearrelationships were established between median germination time(Gt₅₀) and water potential at different temperatures, and betweenreciprocal Gt₅₀ (germination rate) and temperature at differentwater potentials. Germination occurred at potentials as lowas –2.2 MPa at favourable temperatures (30–40 °C),but was fastest at 40 °C when water was not limiting, withan estimated base temperature (T_b) of about 10 °C. Subsequentradicle elongation, however, was restricted to a slightly narrowertemperature range and was fastest at 35 °C. The conceptof thermal time was used to develop an equation to model thecombined effects of water potential and temperature on germination.Predictions made using this model were compared with the actualgermination obtained in a related series of experiments in columnsof soil. Some differences observed suggested the additionalimportance of the seed/soil/water contact zone in influencingseed germination in soil. Seedling emergence appeared to reflectfurther the radicle elongation results by occurring within anarrower range of temperatures and water potentials than germination.Emergence had an estimated T_b of 12.6 °C and was fastestat 35 °C. A soil matric potential of not less than about–0.5 MPa at sowing was required to obtain 50% or moreseedling emergence. Key words: Germination, temperature, water potential     

Inhibition of Electrogenesis by Aluminum in Characean Cells

The effects of aluminum (AI) on electrogenesis at the plasmamembrane were examined in internodal cells of Chara corallina.After treating cells with 0.1 mM A1C1₃ (pH 4.5), we measuredboth the membrane potential and the membrane resistance in artificialpond water (pH 5.6). Electrogenesis at the membrane was significantlyinhibited by the treatment with A1C1₃ A decrease in the pumpcurrent of the electrogenic proton pump and/or a decrease inthe electrical resistance (an increase in conductance) of thepassive diffusion channel were considered to be responsiblefor the inhibition of electrogenesis. CaCI₃ had a partial amelioratingeffect. Both malic acid and citric acid were very effectivein reversing the effects of A1C1₃. In addition, these organicacids restored electrogenesis in cells that had been treatedwith A1CI₃. It is suggested that Al affects electrogenesis fromthe exterior of the membrane, at least during the initial stagesof treatment (4-24 h). ¹Present Address: Graduate School of Agriculture, Kyoto University,Sakyo-ku, Kyoto, 606-01 Japan