HCO(3) Influx across the Plasmalemma of Chara corallina: Physiological and Biophysical Influence of 10 mm K

The effect of 10 mm K⁺ on the HCO₃⁻ influx in Chara corallina has been used to distinguish a Ca²⁺-dependent membrane integrity site from the HCO₃⁻ transport site which is also Ca²⁺-dependent (Lucas and Dainty, Plant Physiology 1977 60: 862-867).     

Sulfhydryl Group Involvement in Plasmalemma Transport of HCO(3) and OH in Chara corallina

The effect of the sulfhydryl reagents (—SH) p-chloromercuribenzene-sulfonic acid (PCMBS), N-ethylmaleimide (NEM), and inorganic mercury on H¹⁴CO₃⁻ assimilation in Chara corallina is reported. Commercial grade PCMBS caused severe inhibition of H¹⁴CO₃⁻ assimilation. Results obtained using purified PCMBS (stock solution passed through a chelating resin) indicated that inhibition observed using unpurified PCMBS was due predominantly to the presence of inorganic mercury (as a contaminant). The inhibitory role of inorganic mercury was verified using HgCl₂. This chemical caused a dramatic inhibition of H¹⁴CO₃⁻ assimilation, while it had little effect on cellular ¹⁴CO₂ fixation. Reversal of the Hg²⁺ inhibition of H¹⁴CO₃⁻ assimilation (in presence of 1.0 millimolar dithioerythritol) was extremely slow, requiring 2 to 3 hours for the reestablishment of control rates. This slow recovery may reflect de novo synthesis of transport proteins.     

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

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

Influence of Turgor Pressure Manipulation on Plasmalemma Transport of HCO(3) and OH in Chara corallina

A modified version of the osmotic shock technique was used to investigate HCO₃⁻ and OH⁻ transport in the alga Chara corallina. Cell turgor was brought close to zero and then restored. When turgor was reduced to near the plasmolytic point using an osmoticum, little effect was observed on H¹⁴CO₃⁻ assimilation and OH⁻ transport. However, when turgor was recovered in these cells, there was a large reduction in HCO₃⁻ and OH⁻ transport activity. In contrast, when cells were air-dried to zero turgor, and rewetted to restore turgor, no significant effect on OH⁻ transport was observed.     

Plasmalemma Transport of HCO-3 and OH- in Chara corallina: Inhibitory Effect of Ammonium Sulphate

The influence of (NH₄)₂SO₄ on ¹⁴C assimilation and cyclosisin internodal cells of Chara corallina was investigated. Severeinhibition of ¹⁴C assimilation was found at pH values above7·0, this inhibition being correlated with the exogenouslevel of NH3 rather than NH⁺₄. Cyclosis was also affected athigher concentrations of (NH₄)₂SO₄. This effect was similarlycorrelated with exogenous levels of NH₃. ¹⁴C assimilation was inhibited non-competitively by (NH₄)₂SO₄,the apparent Km being increased from 0·55 to 1·5mM. The results suggest that the site(s) of inhibition is locatedat the plasmalemma, rather than at the chloroplasts. (Evidencein support of in vivo uncoupling of photophosphorylation, bylow concentrations of (NH₄)₂SO₄, was not obtained). Significant perturbation of the OH^– efflux pattern wasobserved as the level of (NH₄)₂SO₄ was increased. Induced migrationof efflux sites indicates that NH₃ may interfere with the cellularmechanism that controls OH^– transport. Using a cell-segmentisolating chamber it was shown that (NH₄)₂SO₄ inhibited OH^–efflux rather than HCO^–₃ transport. This inhibitory effectwas readily reversible. These data are discussed in terms of a possible relationshipbetween the observe NH₄)²SO₄ stimulation of ³⁶Cl^– influxand the effect of this compound on ¹⁴C assimilation.     

Effect of Sulfhydryl Reagents on the Biophysical Properties of the Plasmalemma of Chara corallina

The administration of the sulfhydryl reagent N-ethyl-maleimide (NEM) to internodal cells of Chara corallina caused alterations in the biophysical properties of the plasmalemma, as measured with electrophysiological and radioactive tracer techniques. The membrane potential depolarized to, or near, the calculated Nernst potential for potassium (E_K) after 30 seconds' exposure to 0.1 millimolar NEM. During this time, the ATP level did not decrease below the control value, and the specific membrane resistance did not increase; only upon further exposure to NEM did the resistance approach the value observed in the dark. In the depolarized state, the membrane potential responded to changes in the external potassium concentration in the manner of a K⁺-electrode, but it retained it's relative insensitivity to external sodium.     

Plasmalemma transport of OH- in Chara corallina: dynamics of activation and deactivation.

The light-mediated, time-dependent rise in the pH value at the center of an alkaline band was analyzed using the methods of numerical analysis. From this analysis an expression of the time-dependent build-up of OH- efflux was obtained for these bands. This information can now be employed to determine whether the light-activated transport of OH- and HCO3- influences the electrical properties of the plasmalemma. The dark-induced deactivation of OH- transport was also characterized, revealing a transition from efflux to a transient influx phase during deactivation. Numerical analysis of the steady-state OH- diffusion pattern, established along the surface of an alkaline band, revealed that the OH- efflux width was wider than previously envisaged. It was also found the OH- sink regions exist on either side of the efflux zone. These, and other characteristics revealed by the numerical analysis, enabled us to extend the OH- transport model proposed by Lucas (J. Exp. Bot. 1975, 26:347).     

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     

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

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

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

     

Plasmalemma Transport of OH in Chara corallina: III. FURTHER STUDIES ON TRANSPORT SUBSTRATE AND DIRECTIONALITY

The identity of the plasmalemma-transported species that develops the alkaline bands of Chara corallina was investigated. The effect of fusicoccin on the rate of HCO₃⁻ assimilation, and on the time-dependent alkaline band pH buildup following low pH flushing, was found to be small, with no stimulatory effect. Computer simulation of the flushing experiments showed that in the experimental situation the alkaline band transport system was slowed down, rather than speeded up, by low pH flushing. A detailed theoretical examination of the maximum rate of proton production from water showed that measured alkaline band fluxes are too large to be explicable in terms of an H⁺ influx system. The experimental and theoretical results indicate that the plasmalemma transport of OH⁻ ions is responsible for the measured negative external electric potential and alkalinity flux which are associated with the alkaline band phenomenon. Consequently, HCO₃⁻ influx across the characean plasmalemma must be charge-balanced by the efflux of OH⁻ ions.     

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)     

Activation of Divalent Cation Influx into S. cerevisiae Cells by Hypotonic Downshift

Subjecting Saccharomyces cerevisiae cells to a hypotonic downshift by transferring cells from YPD medium containing 0.8 m sorbitol to YPD medium without sorbitol induces a transient rapid influx of Ca²⁺ and other divalent cations into the cell. For cells grown in YPD at 37°C, this hypotonic downshift increases Ca²⁺ accumulation 6.7-fold. Hypotonic downshift-induced Ca²⁺ accumulation and steady-state Ca²⁺ accumulation in isotonic YPD medium are differentially affected by dodecylamine and Mg²⁺. The Ca²⁺-influx pathway responsible for hypotonic-induced Ca²⁺ influx may account for about 10–35% of Ca²⁺ accumulation by cells growing in YPD. Ca²⁺ influx is not required for cells to survive a hypotonic downshift. Hypotonic downshift greatly reduces the ability of S. cerevisiae cells to survive a 5-min exposure to 10 mm Cd²⁺ suggesting that mutants resistant to acute Cd²⁺ exposure may help identify genes required for hypotonic downshift-induced divalent cation influx. Received: 14 January 1997/Revised: 20 June 1997     

Plasmalemma Transport of OH- in Chara corallina: II. FURTHER ANALYSIS OF THE DIFFUSION SYSTEM ASSOCIATED WITH- OH EFFLUX

The dynamic and steady state aspects of the pH and electricpotential () profiles, that develop in the experimental mediumin association with the photosynthetic assimilation of exogenousHCO^–₃ by internodal cells of Chara corallina, were investigated.A theoretical treatment is presented which explains the originof the phenomenon. This theory was tested by comparing thepH and values generated by a numerical analysis model (whichsimulated the experimental system) against experimental data.Verification of our model indicates that the steady state ionicfluxes, associated with HCO^–3 assimilation (HCO^–₃,OH^–, and CO^23–, are not significantly influencedby the electric potential gradients. The main driving forcecausing the observed fluxes is the diffusion gradient associatedwith the respective ion. By simultaneous measurement of and pH, at the centre of analkaline band, a direct correlation was established betweenlight-activation and dark-deactivation of the OH^– transportsystem and the light-mediated changes in at the cell surface.In addition, under steady state conditions, an almost perfectcorrelation was observed between alkaline band pH centres andthe negative electric potential maxima. These data offer strongsupport for the hypothesis that OH^–efflux, in this system,is an electrogenic process. Based on our present analysis, the profile along the cell indicatesthat, in terms of the spatial aspect of HCO3^– transport,the rate of HCO^–₃ influx varies quite dramatically alongthe length of an internodal cell. This aspect is discussed interms of the cellular integration of OH^– and HCO^–₃transport in this species.     

Control of Passive Permeability in the Chara Plasmalemma

Conductance to K⁺ alters as a function of membrane potential(_m). Conductance to H⁺ (or OH^–) changes with externalpH (pH^o) This conductance change can be modulated by alteringcytoplasmic pH or external K⁺ concentration, both of which alsoalter _m. We suggest a role for H⁺ conductance in regulatingcytoplasmic pH above pH^o 7.0.     

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⁺     

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     

Immunolocalisation of the cytoskeleton to plasmodesmata of Chara corallina

The macromolecular structure of plasmodesmata in the giant celled freshwater alga, Chara corallina, was examined using antibodies against cytoskeletal elements. The large internodal cells of Chara are separated by a nodal complex of smaller cells which are interconnected by plasmodesmata. Putative plasmodesmata-associated proteins can be identified by a comparison of proteins extracted from preparations of clean walls of nodal complexes and those extracted from the external walls of internodal cells which have no plasmodesmata. Actin and tubulin were identified in the protein extracts of nodal walls and the cytoplasm of nodes and internodes but not in the extracts of internodal external walls. Immunogold labelling confirmed the localisation of actin and myosin to plasmodesmata of Chara.     

Energy-linked Adenosine Diphosphate Accumulation by Corn Mitochondria: II. Phosphate and Divalent Cation Requirement

The requirement for phosphate and Mg²⁺ in energy-linked [³H] ADP accumulation by corn mitochondria has been studied. Arsenate will fully substitute for phosphate; sulfate partially substitutes; acetate, bicarbonate, and pyrophosphate are ineffective. Phosphate is also taken up by the mitochondria, but the ADP/Pi ratio varies widely with experimental treatments. ADP does not exchange with endogenous labeled phosphate, although Pi/³²Pi exchange occurs.     

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