Participation of Ca2+ in cessation of cytoplasmic streaming induced by membrane excitation inCharaceae internodal cells

Summary The mechanism of the cessation of cytoplasmic streaming upon membrane excitation inCharaceae internodal cells was investigated.Cell fragments containing only cytoplasm were prepared by collecting the endoplasm at one cell end by centrifugation. In such cell fragments lacking the tonoplast, an action potential induced streaming cessation, indicating that an action potential at the plasmalemma alone is enough to stop the streaming.The active rotation of chloroplasts passively flowing together with the endoplasm also stopped simultaneously with the streaming cessation upon excitation. The time lag or interval between the rotation cessation and the electrical stimulation for inducing the action potential increased with the distance of the chloroplasts from the cortex. The time lag was about 1 second/15 m, suggesting that an agent causing the rotation cessation is diffused throughout the endoplasm.Using internodes whose tonoplast was removed by replacing the cell sap with EGTA-containing solution (tonoplast-free cells,Tazawa et al. 1976), we investigated the streaming rate with respect to the internal Ca²⁺ concentration. The rate was roughly identical to that of normal cells at a Ca²⁺ concentration of less than 10^–7 M. It decreased with an increase in the internal Ca²⁺ concentration and was zero at 1 mM Ca²⁺.The above results, together with the two facts that Ca²⁺ reversibly inhibits chloroplast rotation (Hayama andTazawa, unpublished) and the streaming in tonoplast-free cells does not stop upon excitation (Tazawa et al. 1976), lead us to conclude that a transient increase in the Ca²⁺ concentration in the cytoplasm directly stops the cytoplasmic streaming. Both Ca influxes across the resting and active membranes were roughly proportional to the external Ca²⁺ concentration, which did not affect the rate of streaming recovery. Based on these results, several possibilities for the increase in Ca²⁺ concentration in the cytoplasm causing streaming cessation were discussed.     

Ca2+-activated Cl− channel in plasmalemma ofNitellopsis obtusa

Summary The mechanisms of Cl^–-channel activation in the plasmalemma ofNitellopsis obtusa was studied by measuring both the transient inward current under voltage clamp and Cl^– efflux during the action potential. 9-anthracenecarboxylic acid (A-9-C) at 1.0mm inhibited both the transient inward current and the Cl^– efflux, but did not uncouple the sudden cessation of the cytoplasmic streaming. Since this excitation-cessation coupling is caused by a transient increase in the cytoplasmic Ca²⁺ concentration, these results suggest that A-9-C inhibited not the Ca²⁺ channel but specifically the Cl^– channel. The following results were found between the Ca²⁺-channel activation and the Cl^–-channel activation: (1) The Ca²⁺-channel blocker La³⁺ uncoupled the excitation-cessation coupling and inhibited both the transient inward current and the Cl^– efflux, although the Cl^–-channel blocker A-9-C did not affect the excitation-cessation coupling. (2) The Cl^– efflux was greatly reduced by depletion of Ca²⁺ from the external solution and restored by an increase in the external Ca²⁺ concentration. (3) An increase in the external ionic, strength which increases Ca²⁺ entry (T. Shiina & M. Tazawa,J. Membrane Biol. 96:263–276, 1987) enhanced the Cl^– efflux. (4) Mg²⁺, which cannot pass through the Ca²⁺ channel, reduced both the transient inward current and the Cl^– efflux. (5) Although Sr²⁺ can pass through the plasmalemma Ca²⁺ channel, Cl^–-channel activation by Sr²⁺ was only partial. These findings support the hypothesis that voltage-dependent Ca²⁺-channel activation, which increases the free Ca²⁺ concentration in the cytoplasm, is necessary for the subsequent Cl^–-channel activation.     

Studies on cessation of cytoplasmic streaming under K+-induced depolarization inNitella axilliformis

Internodal cells ofNitella axilliformis had a membrane potential of about−120mV and showed active cytoplasmic streaming with a rate of about 90 μm/sec in artificial pond water (APW) at 25C. When APW was replaced with 50 mM KCl solution, the membrane potential depolarized accompanying an action potential, and the cytoplasmic streaming stopped. Soon after this quick cessation, the streaming started again, but its velocity remained very low for at least 60 min. Removal of KCl from the external medium led to repolarization of the membrane and accelerated recovery of the streaming. The change in the concentration of free Ca²⁺ in the cytoplasm ([Ca²⁺]_c) was monitored by light emission from aequorin which had previously been injected into the cytoplasm. Upon application of KCl to the external medium, the light emission, i.e., [Ca²⁺]_c, quickly increased. It then decreased exponentially and reached the original low level within 100 sec. The cause of the long-lasting inhibition of cytoplasmic streaming observed even when [Ca²⁺]_c had returned to its low resting level is discussed based on the mechanism proposed for action potential-induced cessation of cytoplasmic streaming; inactivation of myosin by Ca²⁺-dependent phosphorylation or formation of cross bridge between actin filaments and myosin.     

The mechanism of nitrate transport across the tonoplast of barley root cells

Nitrate-selective microelectrodes were used to measure not only nitrate activity in the cytoplasm and vacuole of barley (Hordeum vulgare L.) root cells, but also the tonoplast electrical membrane potential. For epidermal cells, the mean cytoplasmic and vacuolar pNO₃ (-log₁₀ [NO₃]) values were 2.3±0.04 (n=19) and 1.41±0.03 (n=35), respectively, while for cortical cells, the mean cytoplasmic and vacuolar nitrate values were 2.58±0.18 (n=4) and 1.17±0.06 (n=13), respectively. These results indicate that the accumulation of nitrate in the vacuole must be an active process. Proton-selective microelectrodes were used to measure the proton gradient across the tonoplast to assess the possibility that nitrate transport into the vacuole is mediated by an H⁺/NO ₃ ^– antiport mechanism. For epidermal cells, the mean cytoplasmic and vacuolar pH values were 7.12±0.06 (n=10) and 4.93±0.11 (n=22), respectively, while for cortical cells, the mean cytoplasmic and vacuolar pH values were 7.24±0.07 (n=3) and 5.09±0.17 (n=7), respectively. Calculations of the energetics for this mechanism indicate that the observed gradient of nitrate across the tonoplast of both epidermal and cortical cells could be achieved by an H⁺/NO ₃ ^– antiport with a 11 stoichiometry.Abbreviations and Symbols G/F free-energy change for H⁺/NO ₃ ^– antiport - F Faraday constant - pH_c cytoplasmic pH - pH_v vacuolar pH - p[NO₃]_c log₁₀ (cytoplasmic [NO ₃ ^– ]) - P[NO₃]_v -log₁₀ (vacuolar [NO₃]) We wish to thank Dr. K. Moore for assistance with statistical analysis.     

Role of calcium ion in the excitability and electrogenic pump activity of theChara corallina membrane: I. Effects of La3+, verapamil,EGTA, W-7, and TFP on the action potential

Summary The cytoplasmic streaming of the normal internodal cell of giant algaChara stops transiently at about the peak of action potential. Application of La³⁺ or verapamil (a calcium channel blocker) or removal of external Ca²⁺ by EGTA caused a partial depolarization of the resting potential, partial decrease of the membrane conductance and a marked decrease of the amplitude of action potential. Under these conditions, the conductance at the peak of action potential reduced markedly and the streaming of cytoplasm did not cease during action potential (excitation-cessation (EC) uncoupling). The effects of Ca²⁺ channel blockers could not be removed by addition of CaCl₂ to the external medium. In contrast, the effect of EGTA on the excitability could be removed to a greater extent and the cytoplasmic streaming ceased at about the peak of action potential by the addition of Ca²⁺ externally. Application of calmodulin antagonists W-7 or TFP caused similar effects on the action potential and on the cytoplasmic streaming.     

Involvement of Ca2 + and flowing endoplasm in recovery of cytoplasmic streaming after K+-induced cessation

Summary When K⁺ of high concentration (50 mM) was applied toNitella cells, the cytoplasmic streaming stopped instantly as in the case of electrical stimulation. Recovery of the streaming after chemical stimulation was much slower than after electrical stimulation. When the endoplasm content was modified by centrifugation, streaming recovery was accelerated in the centrifugal cell fragments rich in endoplasm and deccelerated in those poor in it. The recovery was also accelerated either by permeabilizing the plasmalemma in the presence of EGTA in the external solution or by removing the tonoplast by vacuolar perfusion with the EGTA-containing medium. We concluded that the streaming was recovered due to decrease of the cytoplasmic Ca²⁺ concentration, which seems to be accelerated by sequestering of Ca²⁺ by endoplasmic components. The slow recovery of the streaming after KCl-stimulated cessation is assumed to be caused by continuous influx of Ca^{2 +} during the prolonged membrane depolarization.Abbreviations ATP adenosine 5-triphosphoric acid - EGTA ethyleneglycol-bis-(-aminoethyl ether)N,N-tetraacetic acid - PIPES piperazine-N,N-bis(2-ethanesulfonic acid)     

Membrane control of cytoplasmic streaming in characean cells

When a characean cell generates an action potential, cytoplasmic streaming transiently stops and then recovers gradually. Calcium ion is one of the most important factors mediating between membrane excitation and cessation of cytoplasmic streaming. When an internode ofNitella flexilis is subjected to transcellular osmosis, both membrane depolarization and cessation of streaming take place at the endoosmotic part of the cell. It was also found that Ca²⁺ plays a key role in mediating between osmosis induced hydration of the cytoplasm and the cessation of cytoplasmic streaming. The present article reviews how Ca²⁺ acts as a second messenger in intracellular signal transduction in controlling the cytoplasmic streaming.     

Cessation of cytoplasmic streaming follows an increase of cytoplasmic Ca2+ during action potential inNitella

Summary Taking advantage of prolonged action potential under low temperature, we studied temporal relationship among the action potential, increase of cytoplasmic Ca²⁺ concentration and cessation of cytoplasmic streaming inNitella. The Ca²⁺ concentration began to increase at a very early stage of the action potential and the cessation of streaming followed that increase.Abbreviations APW artificial pond water     

Head and stalk structures of soybean vacuolar membranes

Highly purified tonoplast fractions isolated by preparative free-flow electrophoresis from hypocotyls of etiolated soybean (Glycine max L. (Merr.)) were examined by negative-staining electron microscopy, and many but not all vesicles were found to exhibit head and stalk structures resembling the 9-nm stalked F₁ ATPase particles reported previously for Neurospora (Bowman et al., 1989, J. Biol. Chem. 264, 15606–15612). The structures show distinguishing characteristics similar to those for Neurospora. These include a cleft in the particle not exhibited by mitochondrial F₁ ATPase and a tendency to disappear from the membrane when treated with nitrate plus Mg^–2+-ATP-containing solutions. The position of the stalked ATPase structures, indicates that some of the tonoplast vesicles were oriented cytoplasmic side out whereas others were oriented cytoplasmic side in.The technical assistance of Keri Safranski and Dorothy Werderitsh is gratefully acknowledged. Electron-microscope facilities were provided through the Purdue University Electron Microscope Laboratory under the direction of Professor Charles Bracker. One of the authors (G.F.E.S.) was supported by the Bundesministerium für Forschung und Technologie.     

Fluxes of 86Rb+ in “isolated” guard cells of Vicia faba L.

Steady-state ⁸⁶Rb⁺ fluxes and contents were measured in stomatal guard cells of Vicia faba L., using isolated epidermal strips in which all cells other than the guard cells had been killed by a brief ultrasound treatment. Flux experiments were carried out for a range of stomatal apertures, achieved by incubation in light or dark, or on solutions containing different concentrations of RbCl (1–30 mM). At pH 5.5 the efflux curve of ⁸⁶Rb⁺ could be fitted to the sum of two exponential terms and it was possible to calculate compartmental contents and fluxes. The data indicate that steady-state stomatal opening is achieved by regulation of both influx and efflux. Salt accumulation was not always adequate to contribute the observed osmotic requirement for opening, and this may indicate that some other solute accounts for the discrepancy.Abbreviations and Symbols A amplitude - k rate constant - Mes 2-(N-morpholino)-ethanesulphonic acid - Q_T, Q_C, Q_V total, cytoplasmic, vacuolar chemical content - Q^* tracer content - Ø_P, Ø_l plasmalemma, tonoplast fluxes This work was supported by a Research Studentship from the Science and Engineering Research Council. I thank Professor E.A.C. MacRobbie for much helpful discussion and advice.     

Proton flows across the plasma membrane in microperforated characean internodes: tonoplast injury and involvement of cytoplasmic streaming

Microperforation of characean cell wall with a glass micropipette in the absence of the tonoplast impalement was found to cause rapid alkalinization of the apoplast by 2–3 pH units, which may rigidify the cell wall structure, thus protecting the cell from further injury. A similar but a deeper insertion of a microneedle, associated with piercing the tonoplast and with an action potential generation, led to a considerable delay in the apoplast alkalinization without affecting the amplitude of the eventual increase in pH. The retardation by the mechanically elicited action potential of the incision-mediated pH transients in the apoplast contrasted sharply to the enhancement of these pH transients by the action potential triggered electrically before the microperforation. Hence, the delay of the apoplast alkalinization was not related to basic ionic mechanisms of plant action potentials. Measurements of the vacuolar pH after mechanical elicitation of an action potential indicate that the tonoplast piercing was accompanied by leakage of protons from the vacuole into the cytoplasm, which may strongly acidify the cytoplasm around the wounded area, thus collapsing the driving force for H⁺ influx from the medium into the cytoplasm. The lag period preceding the onset of external alkalinization was found linearly related to the duration of temporal cessation of cytoplasmic streaming. The results suggest that the delayed alkalinization of the apoplast in the region of tonoplast wounding reflects the localized recovery of the proton motive force across the plasmalemma during replacement of the acidic cytoplasm with fresh portions of unimpaired cytoplasm upon restoration of cytoplasmic streaming.     

ATP,pH and Mg2+ modulate a cation current in Beta vulgaris vacuoles: A possible shunt conductance for the vacuolar H+-ATPase

Macroscopic instantaneous and time-dependent currents have been measured in the vacuolar membrane of Beta vulgaris using a patch clamp configuration analogous to whole cell mode. At low cytosolic Ca²⁺ and in the absence of Mg²⁺, only an instantaneous current was observed. This current is carried predominantly by cations (P_KP_Cl 71, p_nap_cl 41 and arginine is also conducted). The instantaneous current can be activated by ATP^4– (e.g., ATP-activated mean K⁺ current density was –20 mA.m^–2 at a membrane voltage of –20 mV) and by increasing cytosolic pH and Mg²⁺ (raising Mg²⁺ from 0 to 0.4 mm induced a mean current density increase of –7 mA.m^–2 at –20 mV). Such current can be activated by simultaneous addition of putative in vivo concentrations of ATP^4–/MgATP/Mg _free ²⁺ (in the presence of bafilomycin to inhibit the vacuolar ATPase) and further modulated by cytosolic pH. With vacuolar K⁺ concentration greater than that of the cytosol, activation of the instantaneous current would mediate vacuolar K⁺ release over the range of physiological membrane voltage. It is argued that the ATP^4–-activated current, in addition to acting as a K⁺ mobilization pathway, could provide a counter-ion (shunt) conductance, allowing the two electrogenic H⁺ pumps which reside in the vacuolar membrane to acidify the vacuolar lumen.A separate time-dependent current, which was not observed at low Ca²⁺ concentrations (less than 500 nm) could also be elicited by addition of Mg²⁺ at the cytoplasmic membrane face. This current was stimulated by increasing cytoplasmic pH.The authors are grateful to the BBSRC for financial support (Grant PG87/529) and to the Royal Society (University Research Fellowship to J.M.D.). We thank C. Abbott, K. Partridge and J. Robinson for plant cultivation; A. Amtmann, A. Bertl, D. Gradmann and G. Thiel for helpful discussion.     

Current-voltage relationships of a sodium-sensitive potassium channel in the tonoplast ofChara corallina

Summary The membrane of mechanically prepared vesicles ofChara corallina has been investigated by patch-clamp techniques. This membrane consists of tonoplast as demonstrated by the measurement of ATP-driven currents directed into the vesicles as well as by the ATP-dependent accumulation of neutral red. Addition of 1mm ATP to the bath medium induced a membrane current of about 3.2 mA·m^–2 creating a voltage across the tonoplast of about –7 mV (cytoplasmic side negative). On excised tonoplast patches, currents through single K⁺-selective channels have been investigated under various ionic conditions. The open-channel currents saturate at large voltage displacements from the equilibrium voltage for K⁺ with limiting currents of about +15 and –30 pA, respectively, as measured in symmetric 250mm KCl solutions. The channel is virtually impermeable to Na⁺ and Cl^–. However, addition of Na⁺ decreases the K⁺ currents. TheI–V relationships of the open channel as measured at various K⁺ concentrations with or without Na⁺ added are described by a 6-state model, the 12 parameters of which are determined to fit the experimental data.     

Involvement of photosynthesis in the action of temperature on plasmalemma transport inNitella

Summary At membrane potentials different fromE _K, the temperature effect on membrane potential ofNitella consists of two components. One of them changes its sign atE _K, the other one does not. This leads to the assignment of these components to changes in the K⁺ channel and in the H⁺ pump, respectively. It is shown that the fast time constant (3 to 30 sec) of the temperature effect on the H⁺ pump measured as a change in membrane potential and that of the temperature effect on the K⁺ channel measured as a change in resistance (having about twice the value of that of the pump) are sensitive to light intensity. Both time constants measured inNitella become smaller if light intensity increases from 0 to 15 Wm^–2. This supports the suggestion of Fisahn and Hansen (J. Exp. Bot. 37:440–460, 1986) that temperature acts on plasmalemma transport via photosynthesis via the same mechanism as light does.     

Ca2+ ion reversibly inhibits the cytoplasmic streaming ofNitella

Summary When Ca²⁺, K⁺ or Cl^– was injected iontophoretically into the cytoplasm of intactNitella cell, only Ca²⁺ reversibly inhibited the cytoplasmic streaming. However, when an extremely large current was used, the cytoplasmic streaming was reversibly inhibited irrespective of the ion species. This inhibition may be due to a transient increase of free Ca²⁺.     

Electrical tolerance (breakdown) of theChara corallina plasmalemma: I. Necessity of Ca2+

Summary The relationship between the external Ca²⁺ concentrations [Ca²⁺]₀ and the electrical tolerance (breakdown) in theChara plasmalemma was investigated. When the membrane potential was negative beyond –350–400 mV (breakdown potential, BP), a marked inward current was observed, which corresponds to the so-called punch-through (H.G.L. Coster,Biophys. J. 5:669–686, 1965). The electrical tolerance of theChara plasmalemma depended highly on [Ca²⁺]₀. Increasing [Ca²⁺]₀ caused a more negative and decreasing it caused a more positive shift of BP. BP was at about –700 mV in 200 M La³⁺ solution. [Mg²⁺]₀ depressed the membrane electrical tolerance which was supposed to be due to competition with Ca²⁺ at the Ca²⁺ binding site of the membrane. Such a depressive effect of Mg²⁺ was almost masked when the [Ca²⁺]₀/[Mg²⁺]₀ ratio was roughly beyond 2.     

Tonoplast action potential inNitella in relation to vacuolar chloride concentration

Summary The action potential ofNitella internode was studied in relation to K⁺ and Cl^– concentrations in the vacuole. When the vacuole ofNitella pulchella was filled with an artificial solution with extremely low Cl^– concentration, a diphasic action potential (DAP) was observed. The first phase consists of a rapid depolarization followed by a relatively rapid repolarization, and the second one consists of a strong hyperpolarization followed by a gradual return to the resting potential.When the cell was stimulated immediately after the generation ofDAP, a monophasic action potential which resembles an action potential of the natural cell was observed, indicating that theDAP consists of two components with different refractory periods. The refractory period of the component responsible for the depolarizing phase is shorter than that of a component responsible for the hyperpolarizing phase. Measuring the plasmalemma potential and vacuolar potential separately, it was demonstrated that the hyperpolarizing component ofDAP originates from the tonoplast.The action potential of the tonoplast, in contrast with that of the plasmalemma, could be generated independently of concentration of K⁺ in the vacuole. Since the maximum amplitude of hyperpolarization decreased significantly by increasing Cl^– concentration of the vacuole, it is concluded that the tonoplast is very sensitive to Cl^– during excitation.     

Physiological characteristics and regulation mechanisms of the H+ pumps in the plasma membrane and tonoplast of characean cells

The relationship between the physiological characteristics and changes in the activities of H⁺ pumps of the plasma membrane and tonoplast of characean cells is discussed. The large size of the characean internodal cells allows us to perform various experimental operations. The intracellular perfusion technique developed by Tazawaet al. (1976) is a powerful tool for analyzing the characteristics and control mechanisms of the H⁺ pumps (Tazawa and Shimmen 1987, Tazawaet al. 1987, Shimmenet al. 1994) Respiration-dependent changes in the activity of the plasma membrane H⁺ pump are explained by changes in the supply of energy substrate. Photosynthesis-dependent changes in activities of both the plasma membrane and the tonoplast H⁺ pumps are explained in terms of changes in the level of inorganic phosphate in the cytoplasm. Cytoplasmic and vacuolar pHs are suggested to be controlling factors forin vivo activities of the H⁺ pumps. Furthermore, the membrane potential and various ions are considered to bein vivo factors that regulate the activities of the H⁺ pumps. Recipient of the Botanical Society Award of Young Scientists, 1993.     

Possible involvement of protein phosphorylation/dephosphorylation in the modulation of Ca2+ channel in tonoplast-free cells ofNitellopsis

Summary The regulation of voltage-dependent Ca²⁺ channels by protein phosphorylation and dephosphorylation was studied using tonoplast-free cells ofNitellopsis. Since the Ca²⁺-channel activation has a dominant role in the membrane excitation of tonoplast-free cells (T. Shiina and M. Tazawa,J. Membrane Biol. 96:263–276, 1987), it seems to be reasonable to assume that any change of the membrane excitability reflects a modulation of the Ca²⁺ channel. When agents that enhance phosphoprotein dephosphorylation (protein kinase, inhibitor, phosphoprotein phosphatase-1, -2A) were introduced to the intracellular surface of the plasmalemma (twice-perfused tonoplast-free cells), the membrane potential depolarized and the membrane resistance decreased under current-clamp experiments. By contrast, when cells were challenged with agents that enhance protein phosphorylation (phosphoprotein phosphatase inhibitor-1, -naphthylphosphate), the membrane potential hyperpolarized, and the membrane resistance increased. When phosphoprotein phosphatase-1 or -2A was perfused, the current-voltage (I–V) curve which was obtained under ramp voltage-clamp condition exhibited the so-called N-shaped characteristic, indicating an acceleration of the Ca²⁺-channel activation. This effect was suppressed by the addition of phosphoprotein phosphatase inhibitors. ATP--S, which is assumed to stimulate protein phosphorylation, decreased the inward current in theI–V curve. The dependence of the Ca²⁺-channel activation on intracellular ATP was different between the once-perfused and twice-perfused cells. In once-perfused cells, the membrane excitability was reduced by low intracellular ATP concentration. By contrast, in twice-perfused cells, excitability was enhanced by ATP.