Effects of Divalent Cations on the Excitability and on the Cytoplasmic Streaming of Chara corallina

The plasmalemma of Chara corallina remained excitable, whenit was treated with 1 to 100 µM of TFP. However, excitationcessation (EC) uncoupling, i.e. no cessation of cytoplasmicstreaming during an action potential, was observed in a concentrationrange of TFP between 30 to 100 µM. The percentage of occurrenceof the EC-uncoupling increased with the concentration of TFP.The EC-uncoupling effect of TFP could be removed by externalperfusion with 0.1 min or higher concentration of Ca²⁺ but notwith Mg²⁺, Ba²⁺, Sr²⁺ or Pb²⁺. These results suggest that excitationof the plasmalemma and EC-coupling is regulated via calmodulinor calmodulin-like system. (Received December 15, 1986; Accepted April 4, 1987)     

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.     

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     

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     

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     

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     

Measurement of the Cytoplasmic and Vacuolar Buffer Capacities in Chara corallina

The cytoplasm and the vacuole were isolated from internodal cells of Chara corallina by using the intracellular perfusion technique, and their buffer capacities (β_i) were determined from the titration curves. The pH of the isolated vacuolar sap was 5.19 ± 0.029 (mean ± standard error). At this pH, β_i was minimal and amounted to 0.933 ± 0.11 millimoles H⁺/pH unit/liter vacuolar sap. The pH of isolated cytoplasm was 7.22 ± 0.028. β_i was minimal in this pH region and amounted to 14.2 ± 0.80 millimoles H⁺/pH unit/liter cytoplasm. When 1% (volume/volume) Triton X-100 was added to the cytoplasmic solution to permeabilize the subcellular organelles, the cytoplasmic pH increased to 7.32 ± 0.026, where β_i was 20.35 ± 2.66 millimoles H⁺/pH unit/liter cytoplasm. This shows that alkaline subcellular compartments exist in the cytoplasm and also that the cytoplasmic pH before adding Triton X-100 may represent the cytosolic pH. These data indicate that the pH values of the cytoplasm and the vacuole are regulated at the values where the β_i values are minimal. This suggests that ATP- and inorganic pyrophosphate-dependent H⁺ pumps in the plasma membrane and the tonoplast could efficiently regulate the pH of both cytoplasm and vacuole in Chara internodal cells.     

Cytoplasmic streaming in Chara corallina studied by laser light scattering.

An apparatus is described by means of which the power versus frequency spectrum of the photomultiplier current can be obtained for laser light scattered by streaming cytoplasm in the algal cell Chara corallina. A Doppler peak is noted in the spectrum which is abolished when cytoplasmic streaming is arrested by electrical stimulation. For 5 cells of Chara, this simple laser-Doppler velocimeter gave streaming velocities (46-7 mum s-1, S.D. +/- 4-8 at 20 degrees C) similar to those obtained for the same cells using the light microscope (44-3 mum s-1, S.D. +/- 5-3 at 20 degrees C). A narrow distribution of streaming velocities is indicated. The technique described provides a rapid, quantitative assay of the in vivo rheological properties of cytoplasm.     

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 Lanthanum on Calcium and Magnesium Contents and Cytoplasmic Streaming of Internodal Cells of Chara corallina

Biological and environmental effects of lanthanide series of elements have received much attention recently due to their wide applications. In this study, effects of La³⁺ treatments on calcium and magnesium concentrations as well as cytoplasmic streaming of internodal cells of Chara corallina were investigated. At all treatment concentrations (10, 100, and 1,000 μM), La³⁺ significantly decreased calcium concentrations in the cell-wall fractions after 5-h treatments. Calcium concentrations in the cell contents and magnesium concentrations in the cell-wall fractions were reduced by 100 and 1,000 μM La³⁺ treatments. However, cytoplasmic streaming as an indicator of [Ca²⁺]_cyt was only inhibited at the highest La³⁺ concentration (1,000 μM). The results suggest that La³⁺ may affect cellular calcium homeostasis by actions other than as a simple Ca²⁺ antagonist. La³⁺ could partially compensate for calcium deficiency at certain concentrations.     

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     

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.     

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.     

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     

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     

Plasmalemma Voltage Noise in Chara corallina

Voltage noise analysis is applied to plasmalemma ion transport in Chara corallina. There is a component in the noise power spectrum that is probably associated with current fluctuations within passive transport channels, and another component that may be associated either with fluctuations in the number of open channels, or with active transport. The data allow the calculation of time constants that may be attributable to molecular level events in these transport processes.     

Modeling of hysteresis in pH pattern formation along the cell membrane of algae Chara corallina

It is known that illumination of the algae Chara corallina results in the formation along the membrane of regions with inhomogeneous distribution of pH. It was shown that, in a particular range of illumination intensities, two states with different pH distribution are realized at one and the same value of light intensity: an entirely homogeneous state and completely formed structures (pattern). The transition from the homogeneous state to the pattern formation takes place at one value of light intensity, and the back transition, at another light intensity, i.e., the hysteresis is observed. This phenomenon was studied by mathematical modeling. The mechanism of hysteresis is discussed.     

Influence of Cytoplasmic Streaming and Turgor Pressure Gradient on the Transnodal Transport of Rubidium and Electrical Conductance in Chara corallina

In order to study the transnodal transport of Rb⁺ in internodalcells of Chara corallina, a low-temperature loading system wasestablished to separate the loading process from the transportprocess. Tandem cells, consisting of internode-node-internode,were isolated from algal plants. Treatment of a single internodewith 100 mM RbCl at 5°C for 30 min caused an accumulationof 43 mM Rb⁺ in the cytoplasm of this cell (= source cell),but no Rb⁺ was found in the other internode (= sink cell) ofthe tandem cells. In 40 min after a return to 25°C, about12% of the Rb⁺ loaded in the source cell was transported intothe sink cell. The apparent transnodal permeability of Rb⁺ wascalculated to be 4.6 x 10^–7 m.s^–1. Under the assumptionthat the total cross-sectional area of plasmodesmata occupies10% of the nodal area, the diffusion coefficient of RbCl throughplasmodesmata was calculated to be 2.3 x 10^–11 m².s^–1which is about 1% of the free diffusion coefficient in water(2 x 10^–9m².s^–1). The transnodal transport of Rb⁺ was intimately correlated withthe rate of cytoplasmic streaming. The rate of streaming inboth the source and sink cells was varied either by treatingthe cells with cytochalasin B (CB) or by lowering the temperature.The transport rate correlated with the streaming rate irrespectiveof the method used. Since the level of ATP was not influencedby CB or low temperature, the transnodal transport is assumedto be the result of passive diffusion process through plasmodesmata. A turgor pressure gradient across the node decreased both thenodal electrical conductance and the transnodal transport ofRb⁺. By contrast, the exposure of both internodal cells to asolution of sorbitol had no effect on either of them. A turgorpressure gradient of 240 mOsm decreased the transport of Rb⁺in the first hour to 3% of the control, while it decreased thenodal conductance to about 50%. The increase in the electricalresistance occurred on the junction side between the node andthe internode that was treated with sorbitol. Cytochalasin Ehad no effect on the nodal electrical resistance. It is assumedthat plasmodesmata are equipped with a valve-like mechanismwhich is sensitive to the gradient of turgor pressure acrossthe node and is not regulated by an actomyosin system. (Received February 15, 1989; Accepted April 20, 1989)     

Calcium influx at the plasmalemma of Chara corallina

Influx of ⁴⁵Ca into internodal cells of Chara corallina has been measured, using short uptake times, and a wash in ice-cold La³⁺-containing pondwater after the labelling period to overcome the difficulty of distinguishing extracellular tracer from that in the cell. Over 5–15 min the uptake was linear with time, through the origin. The basal influx from 0.1 mM Ca²⁺ externally was 0.25–0.5 pmol·cm^-2·s^-1, but some batches of cells showed higher fluxes. The influx was markedly stimulated by depolarisation in pondwater containing 20 mM K⁺. In cells in which the control flux was less than about 0.5 pmol·cm^-2·s^-1 there was no effect of 50 M nifedipine. In cells in which the control flux was greater than about 0.5 pmol·cm^-2·s^-1 (whether by natural variability, pretreatment, or by depolarisation in 20 mM K⁺), the flux was reduced by 50 M nifedipine to a value in the range 0.25–0.59 pmol·cm^-2·s^-1. It is suggested that two types of Ca-channel are probably involved, both opening on depolarisation, but only one sensitive to nifedipine. The flux was inhibited by 10 M BAY K 8644, which in animal cells more commonly opens Ca-channels. The apparent influx measured over long uptake times was much reduced, and the kinetics indicated filling a pool of apparent size about 1.45 nmol·cm^-2 with a halftime of about 38 min, probably representing cytoplasmic stores. It is argued that in spite of the very small pool of (free+bound) cytoplasmic Ca²⁺ the measured influx is a reasonable estimate of the influx at the plasmalemma.Abbreviations 0.4K-APW6 artificial pondwater, pH 6, containing 0.4 mM KCl - 20 K-APW6 artificial pondwater, pH 6, containing 20 mM KCl - Ca_o external Ca²⁺