Habituation of plant cells does not mean insensitivity to plant growth regulators

Summary Fully habituated organogenic and nonorganogenic sugarbeet calluses reacted to application of the synthetic auxin [3-benzo(b) selenienyl] acetic acid by changes in growth and ethylene production. Treatment of fully habituated cells of periwinkle with 2,4-dichlorophenoxyacetic acid led to the decrease of free cytokinin contents (isopentenyl adenine, zeatin riboside, and zeatin) during the late exponential phase of growth. The polyamine contents were also modified and the capacity to biotransform secologanin into ajmalicine was decreased. Treatment of the habituated periwinkle cells with zeatin greatly increased the amount of a polypeptide of 16 kDa; this response was more marked than that displayed by the auxin-dependent line. These data show that hormone-independent calluses and cell suspensions can retain some sensitivity to growth hormones. However, differences of responses were observed between the auxin-dependent lines and the habituated lines.     

Mechanosensitive ion channels in chara: influence of water channel inhibitors, HgCl2 and ZnCl2, on generation of receptor potential

Characean internodal cells generate receptor potential (ΔE _m) in response to mechanical stimuli. Upon a long-lasting stimulus, the cells generated ΔE _m at the moment of both compression and decompression, and the amplitude of ΔE _m at the moment of decompression, (ΔE _m)_E, was larger than that at compression. The long-lasting stimulus caused a membrane deformation (ΔD _m) having two components, a rapid one, (ΔD _m)_rapid, at the moment of compression and a slower one, (ΔD _m)_slow, during the long-lasting compression. We assumed that (ΔD _m)_slow might have some causal relation with the larger ΔE _m at (ΔE _m)_E. We treated internodal cells with either HgCl₂ or ZnCl₂, water channel inhibitors, to decrease (ΔD _m)_slow. Both inhibitors attenuated (ΔD _m)_slow during compression. Cells treated with HgCl₂ generated smaller (ΔE _m)_E compared to nontreated cells. On the other hand, cells treated with ZnCl₂ never attenuated (ΔE _m)_E but, rather, amplified it. Thus, the amplitude of (ΔD _m)_slow did not always show tight correlation with the amplitude of (ΔE _m)_E. Furthermore, when a constant deformation was applied to an internodal cell in a medium with higher or lower osmotic value, a cell having higher turgor always showed a larger (ΔE _m)_E. Thus, we concluded that changes in tension at the membrane may be the most important factor to induce activation of mechanosensitive Ca²⁺ channel.     

Statoliths and microfilaments in plant cells

Microfilaments have been demonstrated in rhizoids of Chara fragilis Desvaux by labelling of actin with rhodamine-conjugated phalloidin. Each rhizoid contains thick microfilament-bundles arranged longitudinally in the basal region. In the subapical and apical regions, much thinner bundles exist which contact the statoliths and encircle them in the form of a dense envelope. In root statocytes from Lepidium sativum L. the presence of an actin network is indicated by the fact that application of cytochalasin B (25 g·ml^-1 for 4 h) results in an approximately threefold increase in the rate of statolith (amyloplast) sedimentation relative to controls. It is concluded that in gravity-perceiving plant cells statoliths may trigger the transduction mechanism via actin filaments.Abbreviation CB cytochalasin B - ER endoplasmic reticulum - MF microfilament     

Mercurial-sensitive water transport in barley roots

An isolated barley root was partitioned into the apical and basal part across the partition wall of the double-chamber osmometer. Transroot water movement was induced by subjecting the apical part to a sorbitol solution, while the basal part with the cut end was in artificial pond water. The rate of transroot osmosis was first low but enhanced by two means, infilitration of roots by pressurization and repetition of osmosis. Both effects acted additively. The radial hydraulic conductivity (Lp_r) was calculated by dividing the initial flow rate with the surface area of the apical part of the root, to which sorbitol was applied, and the osmotic gradient between the apical and basal part of the root. Lp_r which was first 0.02–0.04 pm s⁻¹ Pa⁻¹ increased up to 0.25–0.4 pm s⁻¹ Pa⁻¹ after enhancement. Enhancement is assumed to be caused by an increase of the area of the plasma membrane which is avallable to osmotic water movement. The increased Lp_r is in the same order of magnitude as the hydraulic conductivity (Lp) of epidermal and cortical cells of barley roots obtained by Steudie and Jeschke (1983). HgCl₂, a potent inhibitor of water channels, suppressed Lp_r of non-infiltrated and infiltrated roots down to 17% and 8% of control values, respectively. A high sensitivity of Lp_r to HgCl₂ suggests that water channels constitute the most conductive pathway for osmotic radial water movement in barley roots.     

Modulation of a plant mitochondrial K+ATP channel and its involvement in cytochrome c release

Pea stem mitochondria, resuspended in a KCl medium (de-energized mitochondria), underwent a swelling, as a consequence of K⁺ entry, that was inhibited by ATP. This inhibition was partially restored by GTP and diazoxide (K⁺ _ATP channel openers). In addition, glyburide and 5-hydroxydecanate (K⁺ _ATP channel blockers) induced an inhibition of the GTP-stimulated swelling. Mitochondrial swelling was inhibited by H₂O₂, but stimulated by NO. The same type of responses was also obtained in succinate-energized mitochondria. When the succinate-dependent transmembrane electrical potential () had reached a steady state, the addition of KCl induced a dissipation that was inhibited by H₂O₂ and stimulated by NO. The latter stimulation was prevented by carboxy-PTIO, a NO scavenger. Phenylarsine oxide (a thiol oxidant) and NEM (a thiol blocker) stimulated the KCl-induced dissipation of , while DTE prevented this effect in both cases. In addition, DTE transiently inhibited the NO-induced dissipation of , but then it caused a more rapid collapse. These results, therefore, show that the plant mitochondrial K⁺ _ATP channel resembles that present in mammalian mitochondria and that it appears to be modulated by dithiol–disulfide interconversion, NO and H₂O₂. The aperture of this channel was linked to the partial rupture of the outer membrane. The latter effect led to a release of cytochrome c, thus suggesting that this release may be involved in the manifestation of programmed cell death.     

Effects of D2O on permeation and gating in the Ca2+-activated potassium channel fromChara

We studied the effects of H₂O/D₂O substitution on the permeation and gating of the large conductance Ca²⁺-activated K⁺ channels inChara gymnophylla droplet membrane using the patchclamp technique. The selectivity sequence of the channel was: K⁺>Rb⁺≫Li⁺, Na⁺, Cs⁺ and Cl⁻. The conductance of this channel in symmetric 100mm KCl was found to be 130 pS. The single channel conductance was decreased by 15% in D₂O as compared to H₂O. The blockade of channel conductance by cytosolic Ca²⁺ weakened in D₂O as a result of a decrease in zero voltage Ca²⁺ binding affinity by a factor of 1.4. Voltage-dependent channel gating was affected by D₂O primarily due to the change in Ca²⁺ binding to the channel during the activation step. The Hill coefficient for Ca²⁺ binding was 3 in D₂O and around 1 in H₂O. The values of the Ca²⁺ binding constant in the open channel conformation were 0.6 and 6 μm in H₂O and D₂O, respectively, while the binding in the closed conformation was much less affected by D₂O. The H₂O/D₂O substitution did not produce a significant change in the slope of channel voltage dependence but caused a shift as large as 60 mV with 1mm internal Ca²⁺.     

Water permeability of plant cuticles: The effect of temperature on diffusion of water

The effect of temperature on water permeability of plant cuticles (astomatous Citrus leaf cuticles) has been investigated. The Arrhenius plot (logarithm of the permeability coefficient vs. 1/temperature) has two linear portions that intersect at 44° C. Evidence is presented to show that this intersection represents the solid/liquid phase transition of cuticular lipids. As the Arrhenius plot has only one phase transition in the temperature range of 5 to 80° C, it appears that all soluble cuticular lipids in the cuticle are present as a homogeneous mixture rather than as individual layers differing in composition. This view is supported by electron spin resonance evidence showing homogenous distribution of spin label fatty acids. The original distribution of soluble cuticular lipids is irreversibly altered by heating cuticular membranes above the transition temperature. This is accompanied by an irreversible increase in water peremeability, demonstrating the importance of the structure of cuticular lipids with regard to cuticular permeability.Abbreviations CM cuticular membranes - MX polymer matrix - SCL soluble cuticular lipids - MES morpholinoethane sulphonic acid - J flux - ESR electron spin resonance - THO tritiated water     

The long-wavelength limit of plant photosynthesis

It is a common knowledge that the photosynthesis efficiency drops rapidly under the long-wavelength light excitation above 680 nm. We discovered that in sunflower leaves attached to the plant the initial fall is replaced by an unexpected increase at much longer wavelengths, so that a detectable O(2) evolution is remained till 780 nm. The quantum yield of O(2) evolution at the local maximum at 745 nm reaches almost 20% of the yield at 650 nm. We conclude that extreme long-wavelength chlorophylls may be present in the intact photosystem II antenna system, similarly to photosystem I.     

Induction of chondrogenic cells from dermal fibroblast culture by defined factors does not involve a pluripotent state

There is a significant need for cell sources for cartilage regenerative medicine. It has been reported that the combined transduction of two reprogramming factors (c-Myc and Klf4) and one chondrogenic factor (SOX9) directly induces chondrogenic cells from mouse dermal fibroblast (MDF) culture. To gain insights into the process by which cellular characteristics are altered by transduction of c-Myc, Klf4 and SOX9, we examined marker gene expression in the MDF culture at various time points after transduction. The expression of fibroblast-markers was reduced first, followed by an increase in the expression of a chondrocyte-marker. We detected no expression of pluripotent markers at any time point examined. To determine whether or not induced chondrogenic cells go through a pluripotent state after transduction, we analyzed MDFs prepared from Nanog-GFP transgenic mice by monitoring expression of the GFP-labeled pluripotent marker Nanog-GFP in the MDF culture, using time-lapse microscopic observation. Whole-well time-lapse observation revealed that none of the induced chondrogenic cells displayed GFP fluorescence during induction. These results indicate that cells do not undergo a pluripotent state during direct induction of chondrogenic cells from fibroblast culture by transduction of c-Myc, Klf4 and SOX9.     

Response of plants to CO2 under water limited conditions

The influence of inefeased atmospheric CO₂ on the interaction between plant growth and water use is proving to be one of the most profound impacts of the anthropogenic Greenhouse Effect. This paper illustrates the interaction between CO₂ and water in plant growth at a range of scales. Most published work has concentrated on water use efficiency, especially at shorter time scales, and has shown large increases of leaf water use efficiency with increased CO₂. However, the magnitude of the effect is variable, and does not consistently agree with predictions from simple leaf gas exchange considerations. The longer the time scales considered, the less the information and the more the uncertainty in the response to CO₂, because of the additional factors that have to be considered, such as changes in leaf area, respiration of non-photosynthetic tissues and soil evaporation. The need for more detailed studies of the interactions between plant evaporation, water supply, water status and growth is stressed, as increased CO₂ can affect all of these either directly, or indirectly through feedbacks with leaf gas exchange, carbon partitioning, leaf growth, canopy development and root growth.     

Voltage-gated calcium and Ca2+-activated chloride channels and Ca2+ transients: voltage-clamp studies of perfused and intact cells of Chara

The voltage-clamp technique was used to study Ca²⁺ and Cl^– transient currents in the plasmalemma of tonoplast-free and intact Chara corallina cells. In tonoplast-free cells [perfused medium with ethylene glycol bis(2-aminoethyl ether)tetraacetic acid] long-term inward and outward currents through Ca channels consisted of two components: with and without time-dependent inactivation. The voltage dependence of the Ca channel activation ratio was found to be sigmoid-shaped, with about –140-mV activation threshold, reaching a plateau at V>50 mV. As the voltage increased, the characteristic activation time decreased from approximately 10³ ms in the threshold region to approximately 10 ms in the positive region. The positive pulse-activated channels can then be completely deactivated, which is recorded by the Ca²⁺ tail currents, at below-threshold negative voltages with millisecond-range time constants. This tail current is used for fast and brief Ca²⁺ injection into tonoplast-free and intact cells, to activate the chloride channels by Ca²⁺ . When cells are perfused with EDTA-containing medium in the presence of excess Mg²⁺, this method of injection allows the free submembrane Ca²⁺ concentration, [Ca²⁺]_c, to be raised rapidly to several tens of micromoles per liter. Then a chloride component is recorded in the inward tail current, with the amplitude proportional to . When Ca²⁺ is thus injected into an intact cell, it induces an inward current in the voltage-clamped plasmalemma, having activation–inactivation kinetics qualitatively resembling that in EDTA-perfused cells, but a considerably higher amplitude and duration (approximately 10 A m^–2 and _inact~0.5 s at –200 mV). Analysis of our data and theoretical considerations indicate that the [Ca²⁺]_c rise during cell excitation is caused mainly by Ca²⁺ entry through plasmalemma Ca channels rather than by Ca²⁺ release from intracellular stores.     

The relationship between carbon and water transport in single cells ofChara corallina

Summary The hydraulic resistance of the plasma membrane was measured on single internodal cells ofChara 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.Dedicated to our teacher, Professor Masashi Tazawa, on the occasion of his 65th birthday     

Vitamin B12 transport in Escherichia coli K12 does not require the btuE gene of the btuCED operon

Summary Transport of vitamin B₁₂ across the cytoplamic membrane ofEscherichia coli requires the products ofbtuC andbtuD, two genes in thebtuCED operon. The role ofbtuE, the central gene of this operon, was examined. Deletions withinbtuE were constructed by removal of internal restriction fragments and were crossed onto the chromosome by allelic replacement. In-frame deletions that removed 20% or 82% of thebtuE coding region did not affect expression of the distalbtuD gene. These nonpolar deletions had little effect on vitamin B₁₂ binding (whole cells or periplasmic fraction) and transport. They did not affect the utilization of vitamin B₁₂ or other cobalamins for methionine biosynthesis, even in strains with decreased outer membrane transport of vitamin B₁₂. ThebtuE mutations did not impair adenosyl-cobalamin dependent catabolism of ethanolamine or repression ofbtuB expression. Thus, despite its genetic location in the transport operon, thebtuE product plays no essential role in vitamin B₁₂ transport.     

Sodium efflux from perfused giant algal cells

Internodal cells of the giant alga Chara corallina were perfused internally to replace the native cytoplasm, tonoplast and vacuole with artificial cytoplasm. Sodium efflux from perfused cells, measured by including ²²Na in the perfusion media, was increased by increasing the internal sodium concentration and by decreasing the external pH, and was inhibited by external application of the renal diuretic amiloride. The sodium efflux was markedly ATP-dependent, with a 50-fold decrease in efflux observed after perfusion with media lacking ATP. Efflux in the presence of ATP was reduced by 33% by inclusion of 10 M N,N-dicyclohexylcarbodiimide in the perfusion medium. The membrane potential of the perfused cells approximated that of intact cells from the same culture. It is suggested that sodium efflux in perfused Chara cells proceeds via a secondary antiporter with protons, regulated by ATP in a catalytic role and with the proton motive force acting as the energy source.Abbreviations DCCD N,N-dicyclohexylcarbodiimide - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - Mes 2-(N-morpholino)ethanesulphonic acid - Mops 3(N-morpholino)propanesulphonic acid - Taps tris(hydroxymethyl)methylaminopropanesulphonic acid     

Effects of flooding and drought on stomatal activity, transpiration, photosynthesis, water potential and water channel activity in strawberry stolons and leaves

Transpiration, xylem water potential and water channel activity were studied in developing stolons and leaves of strawberry (Fragaria × ananassa Duch.) subjected to drought or flooding, together with morphological studies of their stomata and other surface structures. Stolons had 0.12 stomata mm^–2 and a transpiration rate of 0.6 mmol H₂O m^–2 s^–1, while the leaves had 300 stomata mm^–2 and a transpiration rate of 5.6 mmol H₂O m^–2 s^–1. Midday water potentials of stolons were always less negative than in leaves enabling nutrient ion and water transport via or to the strawberry stolons. Drought stress, but not flooding, decreased stolon and leaf water potential from –0.7 to –1 MPa and from –1 to –2 MPa, respectively, with a concomitant reduction in stomatal conductance from 75 to 30 mmol H₂O m^–2 s^–1. However, leaf water potentials remained unchanged after flooding. Similarly, membrane vesicles derived from stolons of flooded strawberry plants showed no change in water channel activity. In these stolons, turgor may be preserved by maintaining root pressure, an electrochemical and ion gradient and xylem differentiation, assuming water channels remain open. By contrast, water channel activity was reduced in stolons of drought stressed strawberry plants. In every case, the effect of flooding on water relations of strawberry stolons and leaves was less pronounced than that of drought which cannot be explained by increased ABA. Stomatal closure under drought could be attributed to increased delivery of ABA from roots to the leaves. However, stomata closed more rapidly in leaves of flooded strawberry despite ABA delivery from the roots in the xylem to the leaves being strongly depressed. This stomatal closure under flooding may be due to release of stress ethylene. In the relative absence of stomata from the stolons, cellular (apoplastic) water transport in strawberry stolons was primarily driven by water channel activity with a gradient from the tip of the stolon to the base, concomitant with xylem differentiation and decreased water transport potential from the stolon tip to its base. Reduced water potential in the stolons under drought are discussed with respect to reduced putative water channel activity.     

Surface potentials near the mouth of the large-conductance K+ channel from Chara australis: A new method of testing for diffusion-limited ion flow

The kinetics of single K⁺ channels were derived for patch-clamp recordings of membrane patches excised from cytoplasmic drops from the plant, Chara australis R. Br. Specifically, the tilt effect model of MacKinnon, Latorre and Miller (1989. Biochemistry28:8092–8099) has been used to measure the electrostatic potential (surface PD) and fixed charge at the entrances of the channel. The surface PD is derived from the difference between the trans-pore potential difference (PD) and that between the two bulk phases. The trans-pore PD is probed using three voltage-dependent properties of the channel. These are (1) the association and dissociation rates of Ca²⁺ binding to the channel, from both the cytoplasmic and vacuolar solutions. These were determined from the mean blocked and unblocked durations of the channel in the presence of either 20 mmol liter^–1 vacuolar or 1 mmol liter^–1 cytoplasmic Ca²⁺; (2) the closing rate of the channel's intrinsic gating process. This was determined from the mean channel open time in the absence of vacuolar Ca²⁺ at membrane PDs more negative than –100 mV; and (3) the effect of Mg²⁺ on channel conductance when added to solutions initially containing 3 mmol liter^–1 KCl.The voltage dependence of properties 1 and 2 shifts along the voltage axis according to the ionic strength of the bathing media, consistent with the presence of negative charge in the channel vestibules. Furthermore, the magnitude of this shift depends on the current in a manner consistent with diffusion-limited ion flow in the channel (i.e., the rate of ion diffusion in the external electrolyte limits the channel conductance). Mg²⁺ on either side of the membrane alters channel conductance in a voltage-dependent way. A novel feature of the Mg²⁺ effect is that it reverses, from a block to an enhancement, when the membrane PD is more negative than –70 mV. This reversal only appears in solutions of low ionic strength. The attenuating effect is due to voltage-dependent binding of Mg²⁺ within the pore, which presumably plugs the channel. The enhancing effect is due to screening by Mg²⁺ of surface potentials arising from diffusion-limited flow of K⁺.     

Flooding-induced soil and plant ethylene accumulation and water status response of field-grown tobacco

Summary Ethylene (C₂H₄) accumulation in flooded soil was related to oxygen (O₂), redox potential (Eh), and flooding rate. The water status response of tobacco (Nicotiana, tabacum L.) to these conditions was evaluated from stem diameter, relative water content, leaf water potential, and C₂H₄ content of leaf tissue. Treatments were: flooded with either 0,5, or 15 cm of water per day for 6 days. By the third day, O₂ in the soil decreased to less than 9% in treatments flooded with 5 or 15 cm of water. When O₂ in the soil air was less than 9% and redox potential (Eh) was less than +150 mv, most of the soil air samples contained some C₂H₄ and 16% contained more than 6 ppm. Very little C₂H₄ was present in soil air when O₂ exceeded 9%. Tobacco leaf C₂H₄ peaked 3 days after flooding and then declined to the preflooding level a day later, one day ahead of the rapid increase in soil C₂H₄. Wilting developed progressively beginning with the rise of C₂H₄ in the soil; leaf water potential, stem diameter, and relative leaf water content all were decreased. Soil-and plant-produced C₂H₄ are suggested as factors in reducing root permeability and increasing resistance to water uptake by tobacco.Contribution of the USDA-SEA/AR, in cooperation with the South Carolina Experiment Station.     

Contribution of L-type Ca2+ channels to early afterdepolarizations induced by I Kr and I Ks channel suppression in guinea pig ventricular myocytes

Early afterdepolarizations (EADs) induced by suppression of cardiac delayed rectifier I (Kr) and/or I (Ks) channels cause fatal ventricular tachyarrhythmias. In guinea pig ventricular myocytes, partial block of one of the channels with complete block of the other reproducibly induced EADs. Complete block of both I (Kr) and I (Ks) channels depolarized the take-off potential and reduced the amplitude of EADs, which in some cases were not clearly separated from the preceding action potentials. A selective L-type Ca(2+) (I (Ca,L)) channel blocker, nifedipine, effectively suppressed EADs at submicromolar concentrations. As examined with the action potential-clamp method, I (Ca,L) channels mediated inward currents with a spike and dome shape during action potentials. I (Ca,L) currents decayed mainly due to inactivation in phase 2 and deactivation in phase 3 repolarization. When EADs were induced by complete block of I (Kr) channels with partial block of I (Ks) channels, repolarization of the action potential prior to EAD take-off failed to increase I (K1) currents and thus failed to completely deactivate I (Ca,L) channels, which reactivated and mediated inward currents during EADs. When both I (Kr) and I (Ks) channels were completely blocked, I (Ca,L) channels were not deactivated and mediated sustained inward currents until the end of EADs. Under this condition, the recovery and reactivation of I (Ca,L) channels were absent before EADs. Therefore, an essential mechanism underlying EADs caused by suppression of the delayed rectifiers is the failure to completely deactivate I (Ca,L) channels.     

Mitochondrial uncoupler FCCP activates proton conductance but does not block store-operated Ca current in liver cells

Uncouplers of mitochondrial oxidative phosphorylation, including carbonilcyanide p-triflouromethoxyphenylhydrazone (FCCP) and carbonilcyanide m-cholorophenylhydrazone (CCCP), are widely used in experimental research to investigate the role of mitochondria in cellular function. Unfortunately, it is very difficult to interpret the results obtained in intact cells using FCCP and CCCP, as these agents not only inhibit mitochondrial potential, but may also affect membrane potential and cell volume. Here we show by whole-cell patch clamping that in primary rat hepatocytes and H4IIE liver cells, FCCP induced large proton currents across the plasma membrane, but did not activate any other observable conductance. In intact hepatocytes FCCP inhibits thapsigargin-activated store-operated Ca²⁺ entry, but in patch clamping under the conditions of strong Ca²⁺ buffering it has no effect on store-operated Ca²⁺ current (I_SOC). These results indicate that there is no direct connection between mitochondria and activation of I_SOC in liver cells and support the notion of indirect regulation of I_SOC by mitochondrial Ca²⁺ buffering.     

Demonstration and characterization of Ca2+ channel in tonoplast-free cells ofNitellopsis obtusa

Summary The presence of a Ca²⁺ channel in the plasmalemma of tonoplast-freeNitellopsis obtusa cells was demonstrated and its characteristics were studied using current- and voltage-clamp techniques. A long-lasting inward membrane current (I _m ), recorded using a step voltage clamp, consisted of a single component without time-dependent inactivation. Increasing either [Ca²⁺] _o or [Cl^–] _o 1) enhanced the maximum amplitude of inwardI _m ((I _m ) _p ) and 2) shifted the peak voltage ((V _m ) _p ) at(I _m ) _p to more positive values under ramp-shaped voltage clamping and 3) depolarized the peak value of action potentials. This behavior is consistent with predictions based on the Nernst equation for Ca²⁺ but not for Cl^–. DIDS (4,4-diisothiocyano-2,2-disulfonic acid stilbene) did not suppress(I _m ) _p in tonoplast-free cells, in contrast with its effect on normal cells. La³⁺ and nifedipine blocked(I _m ) _p irreversibly. On the other hand, Ca²⁺ channel agonist, BAY K 8644 irreversibly enhanced(I _m ) _p . Both Sr²⁺ influx and K⁺ efflux increased upon excitation. The charge carried by Sr²⁺ influx was compensated for by K⁺ efflux. It is concluded that only the Ca²⁺ channel is activated during plasmalemma excitation in tonoplast-free cells. In terms of the magnitude of(I _m ) _p , Sr²⁺ could replace Ca²⁺, but Mn²⁺, Mg²⁺ and Ba²⁺ could not. External pH affected(I _m ) _p and the membrane conductance (g _m ) at(I _m ) _p ((g _m ) _p ). Increasing the external ionic strength caused increases in both(I _m ) _p and(g _m ) _p , and shifted(V _m ) _p to positive values. At the same time, Sr²⁺ influx increased. Thus Ca²⁺ channel activation seems to be enhanced by increasing external ionic strength. The possible involvement of surface potential is discussed.