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     

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.     

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     

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     

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)     

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     

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.     

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.     

Simultaneous Measurements of Cytoplasmic K Concentration and the Plasma Membrane Electrical Parameters in Single Membrane Samples of Chara corallina

The electrophysiological properties of cytoplasm-rich fragments (single membrane samples) prepared from internodal cells of Chara corallina were explored in conjunction with K⁺-sensitive microelectrode and current-voltage (I-V) measurements. This system eliminated the problem of the inaccessible cytoplasmic layer, while preserving many of the electrical characteristics of the intact cells. In 0.1 millimolar external K concentration (K_o⁺), the resting conductance (membrane conductance G_m, 0.85 ± 0.25 Siemens per square meter (±standard error)) of the single membrane samples, was dominated by the proton pump, as suggested by the response of the near-linear I-V characteristic to changes in external pH. Initial cytoplasmic K⁺ activities (a_K+), judged most reliable, gave values of 117 ± 67 millimolar; stable a_K+ values were 77 ± 31 millimolar. Equilibrium potentials for K⁺ (Nernst equilibrium potential) (E_K) calculated, using either of these data sets, were near the mean membrane potential (V_m). On a cell-to-cell basis, however, E_K was generally negative of the V_m, despite an electrogenic contribution from the Chara proton pump. When K_o⁺ was increased to 1.0 millimolar or above, G_m rose (by 8- to 10-fold in 10 millimolar K_o⁺), the steady state I-V characteristics showed a region of negative slope conductance, and V_m followed E_K. These results confirm previous studies which implicated a K_o⁺-induced and voltage-dependent permeability to K⁺ at the Chara plasma membrane. They provide an explanation for transitions between apparent K_o⁺-insensitive and K_o⁺-sensitive (`K⁺ electrode') behavior displayed by the membrane potential, as recorded in many algae and higher plant cells.     

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     

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⁺     

The Vacuolar pH of Chara Braunii

The intravacuolar pH of penultimate branchlet segments of Charabraunii Gm. was measured using intracellular antimony electrodes.This pH was shown to vary somewhat irregularly with extracellularpH and frequently to be so low that the entire vacuolar potentialcould be accounted for by the Nernst potential of hydrogen.     

Quantitative Analysis of Intercellularly-Transported Photoassimilates in Chara corallina

Using a thin-layer chromatographic technique, we have identifiedthe photoassimilates that are transported intercellularly frombranchlets to internodes in Chara corallina. An internode-branchletcomplex having a primary apex was used in these experiments.After feeding 1 mol m³ NaH¹⁴CO₃ to a branchlet for 10 min, the¹⁴C-labelled photoassimilates (¹⁴C-photoassimilates) found inthe sol endoplasm of the branchlet were composed of sucrose,amino acids, malate, and sugarphosphates. The composition ofthe ¹⁴C-photoassimilates transported from the source branchletto the sink internode in 10 min was the same as that in thesol endoplasm of the source branchlet. From the proportion ofeach ¹⁴C-photoassimilate in both the source branchlet and thesink internode, it was deduced that the main photoassimilatesinvolved in the intercellular transport were sucrose and aminoacids. We found previously that polar transport of photoassimilatesoccurs from a branchlet to an internode with an apex. Determinationof the amount of sucrose, amino acids, glucose-6-phosphate,and malate in both branchlet and internode with or without anapex revealed that there were gradients in the concentrationsof sucrose, serine, and glutamic acid between the sol endoplasmof the two cells. The levels were higher in the branchlet andlower in the internode and the gradients decreased when theapex was detached. Therefore, it is concluded that sucrose andthese amino acids are the compounds involved in the polar intercellulartransport. Key words: Chara corallina, intercellular transport, photoassimilates, ¹⁴C     

Calcium-salinity interactions affect ion transport in Chara corallina

Detached internodes of Chara corallina survived in solutions containing 100 mol m^?3 NaCl when the external concentration of Ca²⁺ was greater than 1 mol m^?3. Na⁺ influx was roughly proportional to external Na⁺ up to 100 mol m^?3 NaCl. Na⁺ influx involved two components: a Ca²⁺-insensitive influx which allowed the passage of Na⁺ independently of external Ca²⁺; and a Ca²⁺-inhibitable mechanism where Na⁺ influx was inversely proportional to external Ca²⁺. The Ca²⁺-inhibitable Na⁺ influx was similar to the Ca²⁺-inhibitable K⁺ influx. Mg²⁺ and Ba²⁺ were able to substitute for Ca²⁺ in partially inhibiting Na⁺ influx in the absence of external Ca²⁺. The effect of Ca²⁺ appears specific to Na⁺ and K⁺ influx since the effects of a Ca²⁺-free solution on the influx of some other cations, anions and neutral compounds is small. It is suggested that Na⁺ influx via the Ca²⁺-inhibitable mechanism represents Na⁺ leakage through K⁺ channels and that cell death at high salinity occurs due to a cytotoxic Na⁺ influx via this mechanism.     

The Uptake of Sugars by Chara corallina

The influx of several monosaccharides into Chara corallina wasstudied under varying conditions of temperature, in the presenceof inhibitors, and by the use of competition experiments. Itwas found that the mechanism of transport was stereospecific,and the complex interactions between the sugars were characteristicof a carrier-mediated system. There is reason to believe thatthe carrier involved in this system has a narrower range ofaffinities than is usually attributed to a sugar carrier, forglucose and fructose appear to have different sites of entry,though each can influence the uptake of the other. In additionthe carrier system is situated in the plasmalemma and maintainedby metabolic energy.     

Fracture mechanics of the cell wall of Chara corallina

Previous mechanical studies using algae have concentrated on cell extension and growth using creep-type experiments, but there appears to be no published study of their failure properties. The mechanical strength of single large internode cell walls (up to 2 mm diameter and 100 mm in length) of the charophyte (giant alga) Chara corallina was determined by dissecting cells to give sheets of cell wall, which were then notched and fractured under tension. Tensile tests, using a range of notch sizes, were conducted on cell walls of varying age and maturity to establish their notch sensitivity and to investigate the propagation of cracks in plant cell walls. The thickness and stiffness of the walls increased with age whereas their strength was little affected. The strength of unnotched walls was estimated as 47 ± 13 MPa, comparable to that of some grasses but an order of magnitude higher than that published for model bacterial cellulose composite walls. The strength was notch-sensitive and the critical stress intensity factor K _1c was estimated to be 0.63 ± 0.19 MNm^−3/2, comparable to published values for grasses. Received: 4 April 2000 / Accepted: 21 July 2000