Effects of osmotic concentrations and IAA on the rapid shortening of the cortex in the main pulvinus ofMinosa pudica

In 0.8 M mannitol solution, no rapid shortening was recorded, while normal action potential was recorded. This result suggests that motive force of rapid shortening is an elastic stretch of the cell wall caused by the turgor pressure, and that osmotic concentration of motor cell is 0.6–0.7 M. IAA increased the rapid shortening, recovery rate and Cl⁻-efflux. The magnitudes of rapid shortening in IAA were 3–10 times as large as those in APW. The mean and maximum values of the rapid shortening in IAA were 87.0±2.2μm and 207 μm, respectively, or 1.6 and 3.9% of the whole length of motor tissue. When the large rapid shortening occurred, the large recovery rate was observed. These results suggest that both mechanisms, expulsion and re-entry of cell sap, are enhanced by IAA treatment. IAA-induced hyperpolarization was observed with a short time lag, which suggests that IAA enhances the electrogenic ion pump.     

The shortening and action potential of the cortex in the main pulvinus ofMimosa pudica

The shortening and action potential of the upper and lower cortices of the main pulvinus ofMimosa pudica were recorded simultaneously. The shortening and action potential were observed only in the lower cortex. The extensibility and the excitability of the cortex are discussed.     

IAA-induced opening of excisedMimosa pulvinules

Movement ofMimosa pudica L. pulvinules was investigated by using excised ones which were placed on a moist filter paper. The pulvinules excised in the morning opened at the addition of IAA (10⁻⁷ M to 10⁻⁴M) in the dark. The lag period for the onset of the opening was about 15 min. Na-acetate buffer (pH 4) also induced the opening of pulvinules in the dark, and the buffer-induced opening was inhibited by the uncouplers of oxidative phosphorylation. Na-MES and Na-citrate buffers (pH 4) did not induce the opening. Pulvinules taken from closed leaves in the evening were less responsive to IAA than those taken from open leaves in the morning. The pulvinules taken in the evening slightly opened with incandescent light (4000 lux), but those preincubated with IAA (10⁻⁷M and 10⁻⁶M) opened distinctly upon the illumination.     

Ultrastructural features of the starch sheath cells of the primary pulvinus after gravistimulation of the sensitive plant (Mimosa pudica L.)

Summary InMimosa pudica the primary and secondary motor organs (pulvini) of fully grown leaves are capable of graviresponse. These organs possess sedimentable amyloplasts in their starch sheath cells.In the primary pulvinus these cells are characterized by a structural polarity induced by the localization of nucleus at their (morphologically) apical part and the localization of amyloplasts at their (physically) basal part. These cells also display structural peculiarities including plasmodesmatal disposition, little development of the endoplasmic reticulum and an absence of vacuolar tannins; moreover, the sedimentation of the amyloplasts, induced by gravistimulation, is accompanied by the variation of localization of the cytoplasm, vacuole and mitochondria and by structural modifications of the nucleus and endoplasmic reticulum.     

Chloride ion efflux during an action potential in the main pulvinus ofMimosa pudica

The shortening, action potential and Cl⁻-efflux of the excised lower half cortex in the main pulvinus ofMimosa pudica were simultaneously recorded. The mean values±(S.E.) for Cl⁻-efflux and shortening were 183±18 picomoles/mg fresh weight/impulse and 87.0±2.2 μm, respectively.     

Energy coupling for membrane hyperpolarization in Lemna: Evidence against an ATP-fueled electrogenic pump as the exclusive mechanism

The vacuolar electrical potential of Lemna paucicostata 6746 has an active component of about-130 mV. This hyperpolarization above the diffusion potential was maintained when dicyclohexyl carbodiimide (DCCD) or arsenate (0.1 mM or 5 mM final concentrations, respectively) were added in the light or after the plants had been kept in darkness for 1 h. The ATP level was reduced to 11±3% by DCCD and to 56±6% by arsenate under conditions identical to those during the potential measurements. In this report, it is discussed whether these results could be interpreted in terms of a putative electrogenic ATPase in the plasma membrane of Lemna. Rb⁺-influx in illuminated plants was 12.5% or 52% of the control when ATP generation was inhibited by DCCD or arsenate. This finding is regarded as justifying the assumption that the availability of ATP at plasmalemma-located transport sites is drastically decreased by these inhibitors.A passive proton-permeability in the cell membrane was induced with different concentrations of carbonyl cyanide m-chlorophenyl hydrazone (CCCP). The potential decrease, caused by the current through this shunt, was not affected by DCCD. It therefore seems less conceivable that the cell membrane remains hyperpolarized because of an increase of membrane resistance concomitant to the inhibition of the pump.The significance of respiratory processes for membrane hyperpolarization is displayed by the depolarizing action of anoxia or KCN. As ATP was found to be non-limiting under these conditions, the inhibition of the electrogenic pump is regarded as being in discord with the concept of an electrogenic ATPase, which is solely responsible for membrane hyperpolarization.Abbreviations CCCP carbonyl cyanide m-chlorophenyl hydrazone - DCCD N, N-dicyclohexyl carbodiimide - DES diethylstilbestro - DNP 2,4-dinitrophenol - POPOP 1,4-bis (2-(5-phenyloxazolyl))-benzene - PPO 2,5-diphenyloxazole     

Absence of plasma membrane H+-ATPase in plasmodesmata located in pit-fields of the young reactive pulvinus ofMimosa pudica L.

Summary Immunocytochemical techniques were employed to study the spatial distribution of the plasma membrane H⁺-ATPase within various cell types of the young reactive primary pulvinus ofMimosa pudica L. These cells were interconnected by large numbers of plasmodesmata, being concentrated within pit-fields. Although we could routinely detect evidence of the H⁺-ATPase along the plasma membrane, immunolabelling was rarely, if ever, observed along the plasma membranes of the plasmodesmata. This finding is discussed with respect to the likely specialized supramolecular structure of the plasmodesma.Abbreviations SEL size exclusion limit of plasmodesmata     

Phytochrome-mediated changes in the membrane potential of subepidermal cells of Lemna paucicostata 6746

Light-stimulated transmembrane potential changes have been measured continuously after implantation of microelectrodes into subepidermal cells of the short-day plant Lemna paucicostata 6746. Irradiation for 5 min with white or red light caused a transient hyperpolarization. These potential changes could be suppressed with 10^-6 M DCMU. Irradiation of DCMU-inhibited plants with far-red light for 5 min hyperpolarized the membrane potential, which thereafter was not changed by further far-red application. Consecutive red light irradiation for 5 min depolarized the membrane potential. The red/far-red reversibility of the potential changes (which could be repeated several times with a single plant) suggests the participation of phytochrome.Abbreviations EDTA ethylenediaminetetraacetate - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - P_r, (P_fr) red- (far-red-) absorbing form of phytochrome     

Photogeneration of membrane potential hyperpolarization and depolarization in non-excitable cells

We monitored femtosecond laser induced membrane potential changes in non-excitable cells using patchclamp analysis. Membrane potential hyperpolarization of HeLa cells was evoked by 780 nm, 80 fs laser pulses focused in the cellular cytoplasm at average powers of 30–60 mW. Simultaneous detection of intracellular Ca²⁺ concentration and membrane potential revealed coincident photogeneration of Ca²⁺ waves and membrane potential hyperpolarization. By using non-excitable cells, the cell dynamics are slow enough that we can calculate the membrane potential using the steady-state approximation for ion gradients and permeabilities, as formulated in the GHK equations. The calculations predict hyperpolarization that matches the experimental measurements and indicates that the cellular response to laser irradiation is biological, and occurs via laser triggered Ca²⁺ which acts on Ca²⁺ activated K⁺ channels, causing hyperpolarization. Furthermore, by irradiating the cellular plasma membrane, we observed membrane potential depolarization in combination with a drop in membrane resistance that was consistent with a transient laser-induced membrane perforation. These results entail the first quantitative analysis of location-dependent laser-induced membrane potential modification and will help to clarify cellular biological responses under exposure to high intensity ultrashort laser pulses.     

The membrane potential in whole cells of Methanobacterium thermoautotrophicum

of whole cells of Methanobacterium thermoautotrophicum was estimated under varying conditions using an electrode sensitive to the lipophilic cation tetraphenylphosphonium chloride (TPP⁺). Since was found to be extremely sensitive to air, a special reaction vessel was developed to maintain strict anaerobiosis. The cells took up TPP⁺ under energization by H₂ and CO₂ thus allowing to calculate the from the distribution of TPP⁺ inside and outside the cells. The unspecific uptake of deenergized cells was around 10% of the total uptake of energized cells. TPP⁺ itself slightly diminished the , but had no effect on the formation of methane. Typical values of were in the range of-150 to-200 mV. showed a quantitative dependence on both the electron donor H₂ and the electron acceptor CO₂. NaCl stimulated the extent of the , whereas KCl slightly diminished it. Valinomycin resulted in a linear decline of , whereas the methane production rate was only slightly affected. In contrast, monensin reduced both methanogenesis and .Abbreviations pmf proton motive force - membrane potential - TPP⁺ tetraphenylphosphonium (chloride salt) - TPMP⁺ triphenylmethylphosphonium (chloride salt, if not otherwise indicated) - d.w. dry weight - t _d doubling time - PVC polyvinylchloride     

Determination of the membrane potential of vacuoles isolated from red-beet storage tissue

The membrane potential of isolated vacuoles of red beet (Beta vulgaris L.) was estimated using several methods. The quenching of the fluorescence of the cyanine dyes 3,3-diethylthiodicarbocyanine iodide (DiS-C₂–(5)) and 3,3-dipropylthiodicarbocyanine iodide (DiS-C₃–(5)) in vacuoles indicated a transmembrane potential difference, negative inside at low external potassium concentrations. The was found to be-55 mV with two other methods, the distribution of ²⁰⁴T1⁺ in the presence of valinomycin and the distribution of the lipophilic cation triphenylmethylphosphonium. Uncouplers reduced this value to-35 mV. High external potassium concentrations, comparable to cytosolic values, abolished the membrane potential almost completely. The addition of 1 mM Tris-Mg²⁺-ATP markedly hyperpolarized the membrane to-75 mV. This effect was prevented by inhibitors of the ATPase activity located in isolated vacuole membranes.Abbreviations ANS aminonaphthalene sulfonate - DiS-C₂–(5) 3,3-diethylthiodicarbocyanine iodide - DiS-C₃–(5) 3,3-dipropylthiodicarbocyanine iodide - EDAC 1-ethyl-3-C-3dimethylaminopropylcarbodiimide - FCCP carbonylcyanide-p-trifluoromethoxyphenylhydrazone - MES morpholinoethylsulfonic acid - TPP⁺ tetraphenylphoshonium - TPMP triphenylmethylphosphonium - Tris tris(hydroxymethyl)aminomethane     

Calcium-sensitivity of the plasmalemmal delayed rectifier potassium current suggests that calcium influx in pulvinar protoplasts from Mimosa pudica L. can be revealed by hyperpolarization

Isolated protoplasts from pulvinar motor cells of Mimosa pudica were studied using conventional whole-cell patch clamp techniques. With internal solutions weakly buffered for Ca²⁺ (0.2 mm EGTA), a run-down of the outward delayed rectifier K⁺ current was induced by hyperpolarizing the holding potential, and this effect was strongly promoted by high external Ca²⁺ concentrations. This rundown could be reversed by coming back to less hyperpolarized holding potentials or by lowering the external [Ca²⁺]. Such rundown was absent when pipette internal solutions strongly buffered (10 mm EGTA) for Ca²⁺ were used. Ionomycin induced run-down of the K⁺ current with internal solutions containing 0.2 mm but not 10 mm EGTA. The hyperpolarization-associated rundown was reversibly blocked by Gd³⁺ and La³⁺.We thank Christophe Untereiner and Denis Wagner for expert technical assistance in facilitating the experiments and data acquisition and analysis.     

High-frequency oscillations and circadian rhythm of the membrane potential in Spinach leaves

The microelectrode technique was used to follow oscillations in membrane potential in mesophyll cells of spinach (Spinacia oleracea L.) during exposure do different photoperiodic conditions. Both high-frequency oscillations and circadian variations were observed. The circadian rhythm was imposed on the period of high-frequency oscillation during short days as well as in continuous light: The free-running period was 25.2 h. The average period of high-frequency oscillation increased from 7.64 min in the dark to 19.95 min in the light within several minutes after dark to light transition. This period length coincides with the established period length for oscillations in the redox potential in the chloroplast suspensions of spinach.Abbreviations CL continuous light - SD short day - MP membrane potential     

Electrophysiological properties ofDictyostelium derived from membrane potential measurements with microelectrodes

Summary Electrical membrane properties of the cellular slime moldDictyostelium discoideum were investigated with the use of intracellular microelectrodes. The rapid potential transients (1 msec) upon microelectrode penetration of normal cells had a negative-going peak-shaped time course. This indicates that penetration of a cell with a microelectrode causes a rapid depolarization, which can just be recorded by the microelectrode itself. Therefore, the initial (negative) peak potential transient valueE _p (–19 mV) should be used as an indicator of the resting membrane potentialE _m ofD. discoideum before impalement, rather than the subsequent semistationary depolarized valueE _n (–5 mV). Using enlarged cells such as giant mutant cells (E _p=–39 mV) and electrofused normal cells (E _p=–30 mV) improved the reliability ofE _p as an indicator ofE _m. From the data we concluded thatE _m ofD. discoideum cells bathed in (mm) 40 NaCl, 5 KCl and 1 CaCl₂ is at least –50 mV. This potential was shown to be dependent on extracellular potassium. The average input resistanceR _i of the impaled cells was 56 M for normalD. discoideum. However, our analysis indicates that the membrane resistance of these cells before impalement is >1 G. Specific membrane capacitance was 1–3 pF/cm². Long-term recording of the membrane potential showed the existence of a transient hyperpolarization following the rapid impalement transient. This hyperpolarization was associated with an increase inR _i of the impaled cell. It was followed by a depolarization, which was associated with a decrease inR _i. The depolarization time was dependent on the filling of the microelectrode. The present characterization of the electrical membrane properties ofDictyostelium cells is a first step in a membrane electrophysiological analysis of signal transduction in cellular slime molds.     

Short-term effects of plant hormones on membrane potential and membrane permeability of dwarf maize coleoptile cells (Zea mays L. d 1) in comparison with growth responses

The membrane potential difference of dwarf maize coleoptile cells is increased by both 10^-5moll^-1 gibberellic acid (GA₃) and indoleacetic acid (IAA) a few minutes after application. A final level is reached after 10–20 min. The membrane permeability ratio P _Na:P _K is altered by both hormones during the first 15 min after application, indicating a rapid effect on the membrane. Elongation growth of coleoptile segments, however, is only stimulated by IAA. The auxin-induced growth as well as the auxin effect on membrane permeability depends on the calcium ion concentration of the medium. It is concluded that IAA acts via a proton extrusion pump that is electrically balanced by a potassium ion uptake, driven by the electromotive force of the pump. The mode of action of GA₃ on elongation growth is assumed to involve a process that depends on the physiologic state of the tissue and/or metabolic energy.Abbreviations IAA indoleacetic acid - GA₃ gibberellic acid - FC fusicoccin - PD electric potential difference between the vacuole and the external medium     

Effects of membrane potential on calcium fluxes of Pelvetia eggs

⁴⁵Ca²⁺ fluxes across the plasma membrane of zygotes of the fucoid alga, Pelvetia fastagiata (J. Ag.) De Toni, were studied in artificial sea waters of various potassium concentrations. Except for two cases, hyperpolarization of the cell membrane (with low [K⁺]) increases, and depolarization (with high [K⁺]) decreases the influx of Ca²⁺ over the range of [K⁺] studied (1–100 mM). The fractional increases of influx during hyperpolarization are close to the fractional increases in membrane potential but the decreases during depolarization are much smaller than those in membrane potential. In two anomalous cases, the influxes of ⁴⁵Ca²⁺ at a potassium concentration of 30 mM were about 20% higher than the control value instead of being 10% lower.The effluxes of ⁴⁵Ca²⁺ are increased by both hyperpolarization and by depolarization. On balance (and excepting the two anomalous cases) the net result of hyperpolarization should be to increase and that of depolarization to decrease intracellular [Ca²⁺].     

Simulation of the light-induced oscillations of the membrane potential in Potamogeton leaf cells

Summary An attempt has been made to simulate the light-induced oscillations of the membrane potential of Potamogeton lucens leaf cells in relation to the apoplastic pH changes. Previously it was demonstrated that the membrane potential of these cells can be described in terms of proton movements only. It is hypothesized that the membrane potential is determined by an electrogenic H⁺-ATPase with a variable H⁺/ATP stoichiometry. The stoichiometry shifts from a value of two in the dark to a value of one in the light. Moreover, this H⁺ pump shows the characteristics of either a pump or a passive H⁺ conductance: the mode of operation of the H⁺ translocator is considered to be regulated by the external pH. The pump conductance is assumed to be dominant at low or neutral pH, while the passive H⁺ conductance becomes more significant at alkaline pH. The pH dependence of the transport characteristic is expressed by protonation reactions in the plasma membrane. The proposed model can account for most features of the light-induced oscillations but not for the absolute level of the membrane potential.This research was supported by the Foundation of Biophysics, part of the Dutch Organization for Scientific Research (NWO) ECOTRANS publication No. 34.     

A theory for the membrane potential of living cells

We give an explicit formula for the membrane potential of cells in terms of the intracellular and extracellular ionic concentrations, and derive equations for the ionic currents that flow through channels, exchangers and electrogenic pumps. We demonstrate that the work done by the pumps equals the change in potential energy of the cell, plus the energy lost in downhill ionic fluxes through the channels and exchangers. The theory is illustrated in a simple model of spontaneously active cells in the cardiac pacemaker. The model predicts the experimentally observed intracellular ionic concentration of potassium, calcium and sodium. Likewise, the shapes of the simulated action potential and five membrane currents are in good agreement with experiment. We do not see any drift in the values of the concentrations in a long time simulation, and we obtain the same asymptotic values when starting from the full equilibrium situation with equal intracellular and extracellular ionic concentrations. Received: 9 December 1998 / Revised version: 30 August 1999 / Accepted: 15 October 1999     

Electrical characteristics of stomatal guard cells: The ionic basis of the membrane potential and the consequence of potassium chlorides leakage from microelectrodes

The membrane electrical characteristics of stomatal guard cells in epidermal strips from Vicia faba L. and Commelina communis L. were explored using conventional electrophysiological methods, but with double-barrelled microelectrodes containing dilute electrolyte solutions. When electrodes were filled with the customary 1–3 M KCl solutions, membrane potentials and resistances were low, typically decaying over 2–5 min to near-30 mV and <0.2 k·cm² in cells bathed in 0.1 mM KCl and 1 mM Ca²⁺, pH 7.4. By contrast, cells impaled with electrodes containing 50 or 200 mM K⁺-acetate gave values of-182±7 mV and 16±2 k·cm² (input resistances 0.8–3.1 G, n=54). Potentials as high as (-) 282 mV (inside negative) were recorded, and impalement were held for up to 2 h without appreciable decline in either membrane parameter. Comparison of results obtained with several electrolytes indicated that Cl^- leakage from the microelectrode was primarily responsible for the decline in potential and resistance recorded with the molar KCl electrolytes. Guard cells loaded with salt from the electrodes also acquired marked potential and conductance responses to external Ca²⁺, which are tentatively ascribed to a K⁺ conductance (channel) at the guard cell plasma membrane.Measurements using dilute K⁺-acetate-filled electrodes revealed, in the guard cells, electrical properties common to plant and fungal cell membranes. The cells showed a high selectivity for K⁺ over Na⁺ (permeability ratio P_Na/P_K=0.006) and a near-Nernstian potential response to external pH over the range 4.5–7.4 (apparent P_H/P_K=500–600). Little response to external Ca²⁺ was observed, and the cells were virtually insensitive to CO₂. These results are discussed in the context of primary, charge-carrying transport at the guard cell plasma membrane, and with reference to possible mechanisms for K⁺ transport during stomatal movements. They discount previous notions of Ca²⁺-and CO₂-mediated transport control. It is argued, also, that passive (diffusional) mechanisms are unlikely to contribute to K⁺ uptake during stomatal opening, despite membrane potentials which, under certain, well-defined conditions, lie negative of the potassium equilibrium potential likely prevailing.Abbreviations and symbols EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - Mes 2-(N-morpholino) propanesulfornic acid - E equilibrium potential - G_m membrane conductance - R_in input resistance - V_m membrane potential