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     

Pseudo-Inductive Behaviour of the Membrane Potential of Chara corallina under Galvanostatic Conditions: A TIME-VARIANT CONDUCTANCE PROPERTY OF POTASSIUM CHANNELS

The membrane potential of Chara corallina Klein ex Willd, emR.D.W. displays an oscillatory behaviour in response to an appliedcurrent step. The relative amplitude and the frequency of oscillationof the overshoot increase with the strength of the current. Increasing temperature from 10 °C to 30 °C decreasesboth the relative overshoot amplitude and the static membraneresistance. The activation energy calculated from the Arrheniusplot of the frequency of the overshoot has a value of 36.1±2.3kJ mol_–1. Raising the external pH from 5.0 to 6.0 decreases the relativeamplitude of the overshoot and increases the steady state membraneresistance. Treating the cells for 30 s with 0.1 mol m_–3 N-ethylmaleimideinduces a rapid fall in both static membrane resistance andovershoot. These results are interpreted in terms of changes in potassiumchannels conductance. Key words: Chara corallina, Membrane potential, Potassium channels conductance     

Reversible closing of water channels in Chara internodes provides evidence for a composite transport model of the plasma membrane

Treatment of internodal cells of Chara corallina with the waterchannel blocker HgCl₂ caused a decrease of the hydraulic conductivityof the membrane (Lp) by a factor of three to four. In the presenceof (practically) non-permeating solutes such as sugars or salts,the osmotic responses were similar to those found in controls,i.e. reflection coefficients (     

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     

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     

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)     

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     

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     

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     

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     

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)     

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     

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     

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     

Hydraulic and osmotic properties of spruce roots

Hydraulic and osmotic properties of roots of 2-year-old Norwayspruce seedlings (Plcea abiea (L.) Karst) were investigatedusing different techniques (steady flow, pressure probe, andstop flow technique). Root pressures were measured using theroot pressure probe. Compared to roots of herbaceous plantsor deciduous trees, excised root systems of spruce did not developappreciable root pressure (-0.001 to 0.004 MPa or -10 to 40cm of water column). When hydrostatic pressure gradients wereused to drive water flows across the roots, hydraulic conductivities(Lp_r) were determined in two types of experiments: (i) rootpressure relaxations (using the root pressure probe) and (ii)steady flow experiments (pneumatic pressures applied to theroot system or xylem or partial vacuum applied to the xylem).Root Lp_r ranged between 0.2 and 810^–8m s^–1 MPa^–1(on average) depending on the conditions. In steady flow experiments,Lpr depended on the pressure applied (or on the flow acrossthe roots) and equalled (0.190.12) to (1.21.7)10^–8m s^–1 MPa^–1 at pressures between 0.2 and 0.4 MPaand (1.51.3)10^–8 m s^–1 MPa^–1 at appliedpressures between 0.8 and 1.0 MPa. When pressures or vacuumwere applied to the xylem, Lp_r values were similar. The hydraulicconductivity measured during pressure relaxations (transientwater flows) was similar to that obtained at high pressures(and water flows). Although there was a considerable scatterin the data, there was a tendency of the hydraulic conductivityof the roots to decrease with increasing size of the root system.When osmotic gradients were used to drive water flows, Lp_r valuesobtained with the root pressure probe were much smaller thanthose measured in the presence of hydrostatic gradients. Onaverage, a root Lp_r=0.01710^–8 was found for osmotic andLp_r=6.410^–8 m s^–1 MPa^–1 in correspondinghydrostatic experiments, i.e. the two values differed by a factorwhich was as large as 380. The same hydraulic conductivity asthat obtained in osmotic experiments using the pressure probewas obtained by the 'stop flow techniquel. In this technique,the suction created by an osmoticum applied to the root wasbalanced by a vacuum applied to the xylem. Lp_r values of rootsystems did not change significantly when measured for up to5 d. In osmotic experiments with different solutes (Na₂S0₄,K₂S0₄, Ca(NO₃)₂, mannitol), no passive uptake of solutes couldbe detected, i.e. the solute permeability was very low whichwas different from earlier findings on roots of herbs. Reflectioncoefficients of spruce roots (O were low for solutes for whichplant cell membranes exhibit values of virtually unity (     

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     

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.     

Water channels in plants: do basic concepts of water transport change?

A review and re-examination of literature data shows that highlyselective water channels (aquaporins) have marked effects onthe overall transport properties of the plasma membrane of plantcells. The application of the channel blocker HgCl₂ (50 µM)or of high external concentrations of permeating solutes reducedthe water permeability (hydraulic conductivity, Lp) of Charainternodes down to 25% of the control. In treated cells, reflectioncoefficients (     

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^–.     

Mechanisms Controlling Changes in Water Movement Through the Roots of Helianthus annuus L. During Continuous Exposure to Oxygen Deficiency

The effects of oxygen deficiency on suction-induced water flux(Jv) through detached roots of hydroponically-grown sunflowerwere investigated over a period of up to 6 d. Jv was reducedby the time the oxygen partial pressure in the solution, spargedwith oxygen-free N₂, had fallen below 2 kPa (air – 20.6kPa). This reduction resulted from a decline in the hydraulicconductivity of the radial pathway for water movement (Lp),although up to 46% of the decline was attributable to changesin the osmotic component of the driving force (). Lp (mm s^–1MPa^–1), for aerobic roots was 2.23 ? 10^–5 and foranaerobic roots during the initial 16 h, 1.21 ? 10^–5. At 22 h after the onset of anaerobic treatments, Jv and Lp increased,with Lp values becoming 3 times greater than those measuredbetween 4 h and 16 h of treatment and 1.4 times greater thanin aerated roots. However, Lp increased a further 15-fold whenroots were killed by immersion in boiling water for 2 min. Duringand up to 6 d of anaerobic treatment, some roots retained Lpvalues similar to those at 22 h, while others displayed characteristicstypical of dead roots. At no time was there any indication ofreduced axial conductivity due to xylem vessel blockage. The results are discussed in terms of possible energy sourcesfor the maintenance of root integrity and their importance toplant survival during long periods of severe oxygen shortage. Key words: Anaerobiosis, oxygen deficiency, hydraulic conductivity, osmotic potential, water