Internodal Cells Of Chara Corallina Research Articles

Cl? and K+ channel currents during the action potential in Chara. simultaneous recording of membrane voltage and patch currents

Patch currents in the cell attached-mode and action potentials (AP) have been recorded simultaneously in internodal cells of Chara corallina. The action potentials are closely correlated with transient patch currents. With pipettes containing either 50 mM CaCl2 or 100 mM KCl plus 1 or 5 mM CaCl2, these transients measured up to 100 to 200 pA per patch at zero mV. Transients had a mean duration (time during which the current was > or = half maximum peak amplitude) of about 1 sec, a maximum slope for current rising of about 400 pA sec-1 and a maximum rate of about 100 pA sec-1 for current decay, with no obvious effect of external Ca2+ on either of these parameters. In well-resolved recordings of current transients triggered by an action potential (AP), activities of two types of Cl--conducting channels (15 and 38 pS) have been identified. Since activity of these channels was only observed during action potentials but not upon positive voltage steps, these channels are not directly voltage gated but point to a cytoplasmic gating factor which accumulates during excitation and propagates from excited areas to the patch. A K(+)-conducting channel (40 pS) could be identified as well during an AP, when 100 mM KCl was in the pipette solution. The activity of this channel relaxed at the end of the APs with a time constant of about 3 sec. Stimulated activity of this channel is understood to cause the repolarization overshoot during the final phase of the action potential, whereas the transient activation of the Cl- channels determines the fast voltage changes of the action potential.

The relationship between carbon and water transport in single cells of Chara corallina.

The hydraulic resistance of the plasma membrane was measured on single internodal cells of Chara corallina using the method of transcellular osmosis. The hydraulic resistance of the plasma membrane of high CO2-grown cells was significantly higher than the hydraulic resistance of the plasma membrane in low CO2-grown cells. Therefore we tested the possibility that the "bicarbonate transport system", postulated to be present in low CO2-grown cells, serves as a water channel that lowers the hydraulic resistance of the plasma membrane. We were unable to find any correlation between agents that inhibited the "bicarbonate transport system" and agents that increased the hydraulic resistance of low CO2-grown cells. We did, however, find a correlation between the permeability of the cell to water and CO2. We propose that the reduced hydraulic resistance of the plasma membrane of the low CO2-grown cells is a function of a change in either the structural properties of the lipid bilayer or the activity of a CO2 transport protein so that under conditions of reduced inorganic carbon, the plasma membrane becomes more permeable to CO2, and consequently to other small molecules, including H2O, methanol and ethanol.

Magnetic detection of a single action potential in Chara corallina internodal cells

The electrical activity that occurs in plants has not yet been detected magnetically. Magnetic detection of electrical activity in some animal as well as in human cells and organs, on the other hand, is an established research method. Our experiments demonstrate the propagation of a single action potential in the internodal cell of the green algae Chara corallina, measured magnetically. The propagation velocity and the intracellular current were determined.

Characterization of the Ca2+-Dependent Cl− Efflux in Perfused Chara Cells

The mechanism of the Ca2+-dependent Cl− efflux was studied in tonoplast-free cells, in which the intracellular chemical composition can be freely controlled. Tonoplast-free cells were prepared by perfusing the cell interior of internodal cells of Chara corallina with a medium that contained EGTA. The Ca2+-induced Cl− efflux was measured together with the membrane potential during continuous intracellular perfusion. The dependencies of Cl− efflux and the membrane potential on the intracellular Ca2+ or Cl− concentrations were analyzed. When perfusion was started with medium that contained Ca2+ ions, Cl− efflux and membrane depolarization were induced. The amount of Cl− efflux varied considerably among individual cells. The rate of efflux decreased exponentially but a residual efflux remained detectable. The Cl− efflux was induced at concentrations of Ca2+ ions above 1 μM and reached a maximum at 1 mM. By contrast, the membrane depolarization reached a maximum at about 10 μM Ca2+. The rate of Cl− efflux increased linearly with logarithmic increases in the intracellular Cl− concentrations. These findings suggest that more than two kinds of Ca2+-dependent Cl− channel might be present in the plasma membrane. Addition of ATP or its removal from the perfusion medium did not affect the Ca2+-dependent Cl− efflux. Calmodulin antagonists slightly inhibited the Ca2+-dependent Cl− efflux.

Effects of salinity and turgor on calcium influx in Chara

ABSTRACTMeasurements were made of the influx of 45Ca into internodal cells of Chara corallina in solutions containing high concentrations of NaCl. Increasing salinity in the range 4–100mol m−3 NaCl resulted in a doubling of Ca2+ influx at the plasmalemma. A time‐course of Ca2+ influx in 50 mol m−3 NaCl, 0.5mol m−3 CaCl2 showed that while influx at the plasmalemma increased only 1.5‐fold, influx to the vacuole increased by up to 15‐fold. This was interpreted as being due to inhibition of active Ca2+ efflux from the cell. The stimulation of Ca2+ influx by increasing salinity appeared to be principally a response to reduced turgor since similar stimulations were obtained when turgor was reduced by NaCl, Na2SO4 or mannitol. When cells were plasmolysed Ca2+ influx increased by 10–20‐fold. The increased permeability was relatively specific for Ca2+ and was inhibitable by La3+. Survival of cells in high salt conditions was increased by 30 mmol m−3 La3+, which inhibited Ca2+ influx. Paradoxically, survival can also be extended by increasing external Ca2+ which leads to a higher influx. Therefore, it seems unlikely that the ameliorative effect of Ca2+ on the sensitivity of plants to high NaCl is mediated by Ca2+ entry across the plasmalemma. It seems more likely that the principal role of Ca2+ under these conditions is exerted externally through the control of membrane voltage and permeability.

Cortical Microtubule Organization and Internodal Cell Maturation in Chara corallina

SummaryThe arrangement of cortical microtubules, as documented by immunofluorescence and immunogold‐silver enhancement, changes during the growth and maturation of giant internodal cells of Chara corallina, a process taking approximately 45 days. Transverse microtubules are found throughout growth along with a subset of distinctly non‐transverse microtubules. During the second half of the growing period, when relative growth rates are diminishing, these non‐transverse microtubules become more abundant but a few days prior to growth cessation, they are mostly absent. At about the time of growth cessation the microtubules, while retaining their locally parallel alignment, begin to show increasing deviation from the transverse axis. Eventually, a mosaic of locally parallel yet variably oriented fields of microtubules forms. Many days after growth stops, microtubules become shorter and less numerous and lose parallel alignment, leading to the formation of a random MT pattern.

Regulation of Calcium Influx in Chara

Measurements were made of (45)Ca influx into isolated internodal cells of Chara corallina and also into internodal cells of intact plants. (45)Ca influx was closely related to growth. In rapidly expanding internodal cells, the influx was approximately 1.4 nmol m(-2) s(-1) compared to the influx in mature cells from slow-growing cultures of 0.2 nmol m(-2) s(-1). Isolated internodal cells had influxes in the range 0.2 to 0.7 nmol m(-2) s(-1), but this increased to approximately 2 nmol m(-2) s(-1) in high calcium solutions and to 4 nmol m(-2) s(-1) in high potassium solutions. No significant effects on calcium influx were observed for changes in external pH or for treatments that changed internal pH, except that NH(4) was slightly inhibitory. Severe metabolic inhibition by carbonylcyanide-m-chlorophenyl-hydrazone stimulated influx, whereas dicyclohexylcarbodiimide had no effect and darkness inhibited influx. La(3+) also inhibited influx, but the organic channel blockers nifedipine and bepridil stimulated influx. Verapamil had no effect. The results are generally consistent with voltage regulation of calcium channels as in animal cells.

The contribution of the extracellular matrix to gravisensing in characean cells.

The cell-extracellular matrix junction, which includes the cell wall and the outer surface of the plasma membrane, may be an essential region for the perception of gravity by the internodal cells of Chara corallina. Typically, when an internodal cell is oriented vertically, the downwardly directed cytoplasmic stream travels at a velocity that is 10% faster than that of the upwardly directed stream. However when the cells are treated with impermeant hydrolytic enzymes that partially digest cellulose or hemicellulose, the cells lose their ability to respond to gravity even though streaming continues. By contrast, enzymes that digest pectins have no effect on the gravity-induced polarity of cytoplasmic streaming. Furthermore, gravisensing is sensitive to protease treatment; Proteinase K, thermolysin and collagenase but not trypsin, alpha-chymotrypsin or carboxypeptidase B, inhibit gravisensing. These findings indicate that proteins in the cell-extracellular matrix junction may be required for gravisensing. Moreover, the tetrapeptide Arg-Gly-Asp-Ser (RGDS) inhibits gravisensing in a concentration-dependent manner, indicating that the gravireceptor may be an integrin-like protein. The macromolecules necessary for gravisensing have been localized to the cell ends. As a consequence of the exoplasmic site of action of the enzymes and the tetrapeptides, we interpret the results to mean that they are acting on the gravireceptor, although we cannot eliminate the possibility that they are acting on the signal transduction chain. On the whole, our observations indicate that the cell-extracellular matrix junction is a sine qua non for graviperception in statolith-free Chara internodal cells and we suggest that the gravireceptor is located in this region.

Transformation of Protoplasmic Droplets of Chara by Ion Injection

Summary Protoplasmic droplets were isolated from internodal cells of Chara corallina . When ions were injectediontophoretically into droplets having a diameter of 100-200 μm, their diameter increased, suggesting that the droplets were flattened. The flattening was completely reversible. When the duration of current injection was fixed at 1 sec, the current intensity of more than 0.2 μA was necessary to cause a significant increase in droplet diameter. No specificity of ions was observed among the salts used for ion injection (KC1, CaCl}in2}, Ca(NO 3 ) 2 , NaNO 3 , MES, Tris). Cytochalasin E which stopped the rotation of chloroplasts contained in the droplet did not interfere with the flattening, evidence that the actin cytoskeleton is not involved in this phenomenon. In a few cases the droplet diameter changed rhythmically with the amplitude of several to 10 mV withoutcurrent application. The positive and negative shift of the membrane potential corresponded precisely to swelling and shrinking of the droplet, respectively. It is assumed that changes in membrane properties associated with the surface tension are responsiblefor transformation of droplets.

Influence of protein synthesis inhibitors on the extracellular current pattern and electrophysiological properties of internodal cells of Chara corallina

Internodal cells of Chara corallina exhibit a spatially controlled pattern of alternating acid and alkaline regions along their surface, corresponding to regions of outward and inward current density, respectively. Incubation of isolated cells in either 25 μg∙mL−1 cycloheximide or 50 μM 2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide resulted in a 70–90% reduction in current density and a 50% decrease in the magnitude of the dark-induced membrane hyperpolarization. When cells were supplied with dissolved inorganic carbon, in the form of either CO2 or HCO3−, 25 μg∙mL−1 cycloheximide caused a minimal and 30% reduction in photosynthetic O2 evolution, respectively. In vivo protein synthesis, measured by [3H]-leucine incorporation, showed an 11% decrease in the presence of cycloheximide, while chloramphenicol caused a 91.5% inhibition. It is interesting to note that although exposure to chloramphenicol elicited only a slight decrease in current density, the dark-induced membrane hyperpolarization was inhibited by 50%. All treatments failed to perturb the regions on the cell surface, of zero current, giving stable band location, even though the current density was reduced. The effect of these protein synthesis inhibitors is discussed in terms of the cellular control mechanism involved in regulating the supply of inorganic carbon for photosynthesis. Key words: Chara corallina, extracellular currents, cycloheximide, protein synthesis inhibitors, proton transport, membrane potential.

Measurement of intracellular nitrate concentrations in Chara using nitrate-selective microelectrodes

Nitrate-selective microelectrodes have been made using a quaternary ammonium sensor, methyl-tridodecylammonium nitrate, in a Polyvinylchloride matrix. These electrodes showed a log-linear response from 0.1 to 100 mol · m(-3) nitrate with a typical slope of 55.6 mV per decade change in nitrate concentration. The only physiologically significant interfering anion was chloride but the lower limit of nitrate detection was 0.5 mol · m(-3) in the presence of 100 mol · m(-3) chloride which means this interference will not be important in most physiological situations. These microelectrodes were used to measure nitrate concentrations in internodal cells of Chara corallina cultured under low nitrate and nitrate-replete conditions for 6 to 30 weeks. Cells maintained in low nitrate only showed measurements which were less than the detection limit of the electrodes, while cells grown under nitrate-replete conditions showed two populations of measurements having means of 1.6 and 6.2 mol · m(-3). Chemical analysis of the high-nitrate cells indicated that they contained a mean nitrate concentration of 5.9 mol · m(-3). As vacuolar nitrate concentration would dominate this whole-cell measurement, it is concluded that the higher concentration measured with the electrodes represents vacuolar nitrate concentration and the lower value represents the cytoplasmic concentration. This intracellular distribution of nitrate could only be achieved passively if the electrical potential difference across the tonoplast is between +25 and + 35 mV.

Subcellular Distribution of Free Amino Acids in Relation to Protein Synthesis in Cells of Chara corallina

AbstractThe influence of protein‐synthesis inhibitors on the subcellular distribution of free amino acids was studied in internodal cells of Chara corallina. Use of an intracellular perfusion technique allowed separate measurements of amino acids in the vacuole, in the flowing sol endoplasm and in the gel layer. The sol endoplasm predominantly represents the cytosol, while the gel layer is occupied, for the most part, by chloroplasts.When cells were treated with 0.5 mM chloramphenicol (CRP) in the dark, both the total concentration of amino acids and the subcellular distribution were almost the same as in cells without treatment. In the light, however, the subcellular distribution changed dramatically, although the total concentration of amino acids was unchanged. The vacuolar concentration of amino acids was 3 times greater in CRP‐treated cells than in the control. The concentrations of amino acids in the sol endoplasm and in the gel layer were only half of those in the control. Amino acid permeability of the chloroplast envelope, measured using the perfused internodal cells, slightly increased after the CRP treatment in the light.Time‐dependent changes in concentrations of amino acids in the CRP‐treated cells were also measured in the light. The total concentration of amino acids in the cytoplasm gradually decreased, while that in the vacuole increased commensurately. The concentration and/or subcellular distribution of alanine, glutamine, glutamate and glycine changed dramatically. The concentration of alanine increased considerably both in the vacuole and in the cytoplasm. The cytoplasmic concentration of glutamine increased transiently within 1 −2 h after treatment with CRP. The cytoplasmic concentrations of glutamate and glycine decreased. Although the concentrations of some amino acids changed so markedly both in the vacuole and cytoplasm, only small differences in the activities of glutamic‐pyruvic transaminase, glutamic‐oxaloacetic transaminase and glutamine synthetase were detected between the control and the CRP‐treated cells.

Inversion of extracellular current and axial voltage profile inChara andNitella

Reducing the pH of the bathing solution from 8.2 to pH 6 can induce an inversion of the extracellular current pattern that develops at the surface of Chara corallina internodal cells. A similar result can be obtained on some cells by changing the medium to a pH value of 10. In noninverting Chara cells the currents were strongly reduced when the pH value of the medium was changed between 3 and 11. Simultaneous measurements of the Chara transmembrane potential in the acid and alkaline regions revealed that a light-induced electrical potential gradient of approximately 24 mV was present in the axial (or longitudinal) direction. Correlated to the external current pattern inversion was an inversion of this internal longitudinal voltage gradient. Reillumination of Nitella cells, after a period of darkness, often resulted in a complete inversion of the extracellular current pattern. These results are discussed in terms of spatial and temporal control of membrane transport processes, and in particular the control of current loops that pass through these cells.

Cytoplasm-enriched fragments ofChara: structure and electrophysiology

Cytoplasm-enriched fragments prepared from internodal cells ofChara corallina by centrifugation contain membrane bound vesicles ranging in size from a few μm to hundreds of μm. If the fragments are incubated in artificial pond water (APW) of pH0 above ∼ 6.5, neutral red stains the inside of many vesicles bright crimson, suggesting the presence of inward proton-pumping. In APW of pH0 below 6 crimson vesicles are found less frequently. Under such conditions most vesicles remain unstained inside and some develop indistinct pink halos. After a few days most fragments form a central vacuole, which stains red, regardless of the pH0. The cytoplasmic layer still contains vesicles after vacuole formation. In order to identify the membrane bounding the vesicles various fluorescent probes were applied either by injection into the fragment or directly onto the vesicles released into artificial cytoplasm. Lucifer yellow or 6 COOH-F move readily across the tonoplast in intact cells, but did not enter any vesicles. On the other hand, the fluorescent cationic stain DIOC, which is used to highlight mitochondria and especially endoplasmic reticulum, stained the vesicle membrane. Numerous elliptical or kidney shaped nuclei in the flowing cytoplasm were highlighted with DAPI. In some fragments the nuclei formed large agreggates sometimes filling the width of the fragment. Patch-clamping the vesicles in artificial cytoplasm showed the presence of several kinds of channels, some displaying similar behaviour to the K+ channels observed in cytoplasmic droplets. Analogous to the plasmalemma of intact cells, the fragments without vacuoles displayed electrophysiological states dominated by either K+ conduction, H+ (or OH−) conduction or the proton pump. On the other hand, excitation transients in fragments were of low amplitude or absent altogether. Detailed comparisons of data from fragments and intact cells are shown. The effect of vacuole formation on fragment electrophysiology was also explored.

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 (beta(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, beta(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. beta(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 beta(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 beta(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.

Calcium influx at the plasmalemma of Chara corallina

Influx of (45)Ca into internodal cells of Chara corallina has been measured, using short uptake times, and a wash in ice-cold La(3+)-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 Ca(2+) 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 μM 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 μM 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 μM 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 Ca(2+) the measured influx is a reasonable estimate of the influx at the plasmalemma.

Effect of dinitrophenol on membrane potential, membrane resistance and chlorophyll fluorescence of Chara corallina internodal cells at various pH values

The effect of DNP on the electrophysiological and fluorescent light emission properties of internodal cells of Chara corallina was investigated at various external pHs. Whatever the external pH, DNP acts first at the plasmalemma, where it is able to increase the permeability to some ions (at least that to protons). This effect is related to both initial depolarization of the membrane and a decrease of the membrane resistance. At acid pH, the membrane permeability of DNP is high enough and its cytoplasmic concentration may be high enough so that it is able to act in chloroplasts (and probably in mitochondria), thus lowering the cytoplasmic ATP concentration. In consequence, the relative chlorophyll fluorescence emission decreases, cyclosis stops, the membrane depolarizes again and membrane resistance falls: the cell dies. At alkaline pH the membrane permeability of DNP is low and it affects neither the cytoplasmic streaming nor the chlorophyll fluorescence emission. Moreover, after the initial changes the resting membrane potential and resistance are steady.

Increase in electrical resistance of plasmodesmata of Chara induced by an applied pressure gradient across nodes

By insertion of microelectrodes into the vacuoles of two adjacent internodal cells of Chara corallina, the changes of the potential differences across the plasma membranes and node were noted when a current of 0.40 μA was passed through the cells bathed in artificial pond water. When the solution bathing one cell was changed to artificial pond water + 102 mol m−3 mannitol, a significant increase was detected both in the membrane potential difference response and in the nodal potential difference response. This indicates that the lowering of turgor on one cell by 0.24 MPa increased the electrical resistance of the membrane of that cell and also the resistance of the plasmodesmata connecting the two internodal cells via the node. This is evidence in support of valving in plasmodesmata when a pressure difference is induced across their ends.

Membrane impedance of internodal cells of Chara corallina obtained by analysis of low level transients

The relaxation of the membrane operational impedance of Chara cells has been investigated using a transient technique. This relaxation changes with the amplitude the sign of the electrical stimulation. A model which accounts for the time- and voltage-dependent channels conductance of the membrane is proposed. It describes both the time course of the transient electrical response and the relaxation of the operational impedance of the membrane of these plant cells.

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.