K+, NO,- Activities in Liverwort Conocephalum conicum Action

Plant Physiol. (1994) 106: 1073-1084

Cytoplasmic Ca2+, K+, CI', and NO,- Activities in the Liverwort Conocephalum conicum 1. at Rest and during

Action Potentials'

Kazimierz Trebacz', Wilhelm Simonis, and Cerald Schonknecht*

Julius-von-Sachs-lnstitut für Biowissenschaften der Universitat Würzburg, Lehrstuhl Botanik I, Mittlerer Dallenbergweg 64, 97082 Würzburg, Germany

lntracellular Caz+, K+, CI-, and NOs- activities were measured with ion-selective microeledrodes in the liverwort Conocephalum conicum 1. at rest, during dark/light changes, and in the couese of adion potentials triggered by light or electrical stimuli. l h e average free cytosolic Caz+ concentration was 231 f 65 nM. We did not observe any light-dependent changes of the free cytosolic Caz+ concentration as long as no adion potential was triggered. During adion potentials, on average a 2-fold increase of the free cytoplasmic Caz+ concentration was recorded. lntracellular K+ adivity was 76 f 10 mM. It did not depend on K+ concentration changes in the bath solution between 0.1 and 10 mM. The average equilibrium potential for K+ in the standard medium containing 1 mM K+ was -110 mV, which differed significantly from the resting poten- tia1 of -151 f 2 mV. During adion potentials, either a slight decrease or no changes in intracellular K+ activity were recorded. The average CI- adivity was 7.4 f 0.2 mM in the cytoplasm and 43.5 f 7 mM in the vacuole. l h e adivities of NO,- were 0.63 I: 0.05 mM in the cytoplasm and 3.0 f 0.3 mM in the vacuole. For both anions the vacuolar activity was 5 to 6 times higher than the cytoplasmic activity. After the light was switched off both the CI- and the NOs- adivity showed either no change or a slight increase. lllumination caused a gradual return to previous values or no change. During action potentials a slight decrease of intracellular CI- activity was recorded. It was concluded that in Conocephalum, as in characean cells, chloride channels are involved in the depolarization phase of the action potentials. We discuss a model for the ion fluxes during an action potential in Conocephalum.

APs in plants play an important role in intracellular signaling and in the regulation of different physiological proc- esses (Davies, 1987; Dziubifiska et al., 1989; Wildon et al., 1992; Fromm and Spanswick, 1993). To understand the function of APs in plant physiology, their ion mechanism has to be known in detail. Data conceming the sequence of ion fluxes during APs in plants were obtained primarily on the highly specialized giant characean cells (Beilby, 1984). According to the present model, APs in these plants are

~

This work was supported by the Deutsche Forschungsgemein- schaft (SFB 176, TP B6) and an Alexander-von-Humboldt Fellowship (K.T.). ' Permanent address: Department of Plant Physiology, Mana

Curie Sklodowska University, Akademicka 19, PL-20-033 Lublin, Poland.

* Corresponding author; fax 49-931-71446. 1073

initiated by an elevation of the free Ca2+ concentration in the cytoplasm due to opening of voltage-dependent Ca2+ channels in the plasma membrane and in the tonoplast (William- son and Ashley, 1982; Kikuyama and Tazawa, 1983). This causes activation of Ca2+-dependent anion channels, and massive efflux of chloride depolarizes the plasma membrane (Gaffey and Mullins, 1958; Lunevsky et al., 1983; Okihara et al., 1991). The depolarization leads to opening of K+ channels (outward, delayed rectifiers), and as a consequence the K+ efflux repolarizes the plasma membrane again to the resting potential (Oda, 1976; Kikuyama et al., 1984), which is established near the equilibrium potential for K+.

There is mostly indirect evidence confirming functioning of individual elements of that model in excitable higher plants such as Aldrovanda vesiculosa (Iijima and Sibaoka, 1985), Dionaea muscipula (Hodick and Sievers, 1988), Mimosa pudica (Abe, 1981; Samejima and Sibaoka, 1982), and Salix viminalis (Fromm and Spanswick, 1993). A complete investigation of the ion fluxes during APs in higher plants is still lacking. It is not clear whether the scheme proposed for characean cells can be adopted without changes to terrestrial higher plants. There seems to be a general agreement as to the participation of Caz+ in the excitation (Iijima and Sibaoka, 1985; Hodick and Sievers, 1988). A C1- efflux as a consequence of an increased cytoplasmic free Ca2+ concentration was not recorded in a11 investigations (Minorsky and Spanswick, 1989) even in characean algae (Kikuyama et al., 1984). In some plants or in some circumstances Ca2+ influx alone may depolarize the membrane during APs, whereas in other cases Ca2+ mainly plays the role of an anion channel activator (Beilby, 1984). It remains an open question to what extent there are also ion fluxes across intemal membranes during APs.

The exact knowledge of intracellular ion activities is important for the understanding of membrane transport (San- ders, 1990), metabolism, nutrition, and cellular signaling. Although the intracellular activities for cations (H+, K+, Na', and Ca2+) are well established (Wyn Jones et al., 1979; Tyerman, 1992) and time-dependent changes for H+ and

Abbreviations: AP, action potential; E, membrane potential; E,=,, equilibrium potential for C1-; Een, potential reflecting the activity for the respective ion; EK, equilibrium potential for K+; pCa, negative logarithm of the free Ca'+ concentration; TEA, tetraethyl- ammoniumchloride.

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Ca2+ can be measured with different techniques (Guem et al., 1991; Felle, 1993; Graziana et al., 1993), little is known about the intracellular activities for anions (Cl-, Nos-) (Wyn Jones et al., 1979; Tyerman, 1992) and their time-dependent Ichanges (Thaler et al., 1992).

The liverwort Conocephalum conicum L., the organism stud- ied here, can be regarded as an intermediary form between algae and higher plants. It is a multicellular plant but without conducting bundles. Conocephalum generates APs singly or in series consisting of several dozens of excitations, sponta- neous or evoked by light, and electrical stimuli, or they can be generated by wounding (Dziubinska et al., 1983; Trpbacz and Zawadzki, 1985). These APs are quite long lasting, from about 30 s to over 1 min, and therefore it is possible to apply ion-selective microelectrodes to measure changes in ion activities accompanying them. It was demonstrated (Dziubiliska et al., 1989) that in Conocephalum the rate of respiration begins to increase several seconds after the stimulation and transiently reaches twice the resting level. This effect is observed regardless of whether damaging stimuli (incision of i i thallus edge) or nondamaging electrical stimuli are applied to trigger APs. No change in the rate of respiration is observed when no AP is evoked by the respective stimuli either in unexcitable plants or when excitability is blocked by TEA (Dziubiliska et al., 1989). Since it was reported that Ca2+ activates respiration in animal cells (Fein and Tsacopoulos, 1988; Brustovetsky et al., 1993), a change in cytosolic Ca2+ might also be the reason for the increased rate of the respiration caused by APs in Conocephalum (Dziubinska et al., 1989). The transmembrane potential of Conocephalum cells at rest contains a large electrogenic component (Trgbacz et al., 1989), which raises the question about the nature of the repolarizing ion.

Here we report measurements of intracellular Ca2+, K+, C1-, and NO3- activities in Conocephalum with ion-selective microelectrodes at rest, during dark/light changes, and in the course of APs. On this basis we calculate the gradients and driving forces for these ions and discuss the ion fluxes during APs in plants.

MATERIALS AND METHODS

Plant Material

The liverwort Conocephalum conicum L. was grown in a greenhouse at moderate illumination, in the temperature range 20 to 26OC at high humidity. Every 2 to 3 weeks young parts of the thalli were put into pots with freshly prepared soil. Ten- to 14-d-old thalli were used in the experiments. Two to 4 h before the experiments were started, the thalli were rinsed with water to remove soil, blotted on tissue paper, and mounted horizontally in the experimental chamber in the standard experimental medium containing 1 mM KCI, 0.1 mM CaC12, 2 mM Tris/Mes, pH 7.0. Two sharpened silver wires 0. 1 mm in diameter were inserted into the thallus 5 to 10 mm from each other to serve as stimulation electrodes later in the experiment (2 to 4 h). Electrodes and General Electrophysiology

Micropipettes were prepared from borosilicate glass capil- Iaries 1.5 mm in outer diameter (Hilgenberg, Malsfeld, Ger- many) with filament, or without filament for N03--selective

microelectrodes. Micropipettes were pulled eitlier with a L/M-3P-A puller (List Medical, Darmstadt, Germmy) or with a Getra micropipette puller (Munich, Germany). Tip diameters were in the range of 0.2 pm (Thaler et al., 1992). The potential electrodes were filled with 3 M KCl for Ca2+-selective-, 1 M NaCl for K+-selective-, and 0.5 M K2S#O4 plus 10 m~ KCl for C1-- and N03--selective microelectrodes. The capillaries destined to become ion-selective microelectrodes were silanized according to Chao et al. (1988). The silanized micropipettes were filled with reference solution from their blunt ends. The reference solution for Ca2+-selec:tive microelectrodes (Ammann et al., 1987a) contained 10 m~ EGTA, 6.28 mM CaCl,, 44.9 mM KOH, 55.1 mM KCl, and Mops, pH 7.4 (pCa 7.0; 100 mM K+ as interfering ion). K+-selective microelectrodes contained 1000 mM KCI, 10 mM NaCI, 2 m~ Hepes/NaOH, pH 7.2. Cl--sensitive microelectrodes were filled with 100 mM KC1, 10 mM Tris/H2S04, pH 7.4 (Thaler et al., 1992), and N03--selective microelectrodes contained 100 mM NaN03, 100 mM KCl (Miller and Zlien, 1991). Remaining air in the shank was pushed away by means of a 10-cm3 syringe.

Two methods of filling the ion-selective cocktail into the tip were applied (Felle, 1988). A small portion of the cocktail was placed near the filament from the blunt end by means of another glass pipette with a broken tip. The drop of cocktail moved spontaneously along the filament toward the tip within about 1 to 2 min. In the other method, the cocktail was sucked up into the tip of the microelectrode by applying negative pressure from its other end. The cockt#dl for Ca2+- selective microelectrodes (Ammann et al., l987a) was composed of 5% (w/w) Caz+ ionophore I1 (ETH 129, N,N,N’,N’-tetra-cyclohexyl-3-oxapentanediamid e), 94% (w/ w) 2-nitrophenyl-octyl ether (o-NPOE), and 1% (w/w) so- dium tetraphenylborate; 86% (w/w) of this solution was added to 14% (w/w) polyvinylchloride and dissolved in two volumes of tetrahydrofuran (a11 components frorn Fluka AG, Buchs, Switzerland). For K+-selective microelectrodes (Am- mann et al., 198%) the cocktail contained 5% (w/w) K+ ionophore I (valinomycin), 93% (w/w) 1,2-dime:hyl-3-nitro- benzene, and 2% (w/w) potassium tetralcis(4-chloro- pheny1)borate (a11 from Fluka). The cocktail for (-I--sensitive microelectrodes was composed as described by Kondo et al. (1989). For N03--selective microelectrodes the cocktail (Miller and Zhen, 1991) contained (w/w): 28% polyvinylchloride (high mo1 wt polymer); 1% methyltriphenyl phos- phonium bromide; 65% 2-nitrophenyl octyl ethc.r; 6% meth- yltridodecylammonium nitrate, dissolved in 3 volumes of tetrahydrofuran (a11 from Fluka). CI--sensitivc? microelectrodes were preincubated in 100 mM KCl solution for at least 3 h before use.

The Ca2+-selective microelectrodes were calibrated according to Ammann et al. (1987a) and the K+-selective microelectrodes were calibrated with solutions containing 1, 10, 50, 100, or 1000 mM KCI. Ten millimolar NaCl and 2 mM Hepes was added to each solution and the pH was adjusted to 7.2 with NaOH. Calibration solutions for Cl--sensitive microelectrodes were prepared according to Thaler cat al. (1992) and for N03--selective microelectrodes according to Miller and Zhen (1991). The selectivity coefficients wclre obtained by the fixed interference method (Vaughan-Jones and Aickin,

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lon Activities in Conocephalum 1075

1987). The ion-selective microelectrodes were calibrated before and after each experiment. Data obtained by the recalibration were the basis of the calculations.

The electrodes were connected to the inputs of a high- impedance ( 1015 Q ) differential amplifier (FD 223, World Precision Instruments, New Haven, CT). The amplifier sub- tracts the voltage of the potential electrode ( E ) from the voltage of the ion-selective electrode ( E + Eio,). The three traces representing E , E + Eion, and E,,, were fed to a pen recorder (model314, Kontron, Munich, Germany) and E and Eion parallel to a digital storage oscilloscope (HM 205-3, Hameg, Frankfurt, Germany). The traces presented in the figures were copied by a scanner from original records. For the sake of clarity in the figures, only the voltage trace ( E ) and the trace for the ion activity (Ei,,,) are shown. An Ag/ AgCl reference electrode had contact with the plant via a salt bridge and the flowing medium. The stimulation electrodes were connected through a switch to a regulated DC current source.

Illumination was provided by a cold light source equipped with light-conducting fiber (KL 1500 Schott, Mainz, Ger- many). The photon fluence rate at the plant surface was kept at 29 pE m-’ s-’ or 50 PE m-‘ s-’ as measured with a quantum meter (LI-189, Li-Cor, Lincoln, NE).

lon-Selective Measurement

After the ion-selective microelectrode was calibrated, the two microelectrodes, an ion-selective and a potential one, were inserted perpendicularly into the tissue near to the midrib. The two microelectrodes were located as close to each other as possible (less than 0.1 mm) under dissection micro- scope (Leitz, Wetzlar, Germany) observation. The electrodes were inserted 50 to 100 pm deep in the thallus, in the second layer of cells, which are relatively large (100-300 pm). There- fore, the two microelectrodes were located in the same cell or in neighboring cells. The magnitude of the electric potential difference as well as the slope and direction of AP records were good indicators of proper microelectrode insertion into the cell. The distance between the stimulating electrodes and the site of microelectrodes (ion-selective and potential) insertion was 7 to 10 mm.

Even after addition of polyvinylchloride and with the small tip diameters, the ion-selective microeleckodes used had only a limited pressure resistance. Therefore, the cell turgor had to be reduced by the addition of sorbitol (Thaler et al., 1992). Thirty to 60 min before the microelectrodes were inserted, 250 to 320 mM sorbitol, depending on the actual water potential of cells, was added to the medium perfused through the measuring chamber at a rate of approximately 50 mL h-’. Sorbitol in this concentration range influenced neither the resting potential nor the APs. To exclude the possible artifact (Felle and Bertl, 1986a) that the turgor pushed back the ion- selective cocktail inside the cell, the resistance of ion-selective microelectrodes was measured before the impalement and every 10 min during the experiments. Because the resistance of the ion-selective electrodes (>10 GQ) is severa1 orders of magnitude larger than the membrane resistance and the resistance of the potential electrode (both in the MQ range), the electrical resistance must not change during impalement;

when the cocktail was pushed back the resistance decreased strongly. Only measurements with stable resistances (ion- selective microelectrode) and stable potentials, .which were followed by a successful recalibration (see “Results”), were evaluated. Anion chromatography was performed as described by Schroppel-Meier and Kaiser (1988).

RESULTS

Measurements of the Membrane Potential and the Cytoplasmic lon Activity in the Thallus of Conocephalum

The transmembrane potential in cells of Conocephalum differs significantly among different thalli, but it is constant within one thallus. This is an important prerequisite for measuring ion activities with an ion-selective microelectrode and a separate potential electrode in a tissue where the electrodes might be located in different cells. Because the ion activity is calculated from the difference between the two electrodes (the potential electrode measuring E and the ion- selective one measuring E + Eion), the E sensed by the two electrodes has to be identical. With a rate of AP transmission of 10 to 30 cm min-’ (in the standard medium) and a distance between the two microelectrodes of 0.1 mm, the lag time between the electrodes is smaller than 100 ms, which is below the time resolution of the measuring system. So, even during the rapid potential changes of APs no significant differences (in E ) for the two measuring electrodes are to be expected.

In Figure 1, traces from two potential electrodes (both without ion-selective cocktail) inserted into neighboring cells and the difference of both signals are shown. Neither during the relatively large light-dependent potential changes nor during the electrically triggered AP were there significant deviations between the potentials measured by the two electrodes. Rapid potential changes (light-dependent or APs) did not cause deviations between the two electrodes larger than 2 mV in any of our control experiments ( n = 11). This allows us to cany out measurements of ion activities with an ion- selective microelectrode inserted into one cell and a potential microelectrode not necessarily inserted in the same cell but into one of the neighboring cells.

Measurements of the Free Cytoplasmic Ca2+ Concentration in Cells of Conocephalum

After fabrication, the Ca’+-selective microelectrodes had a resistance between 40 and 100 GQ. Their response time to a 10-fold change in the free Ca2+ concentration was usually 5 to 10 s. The slope of microelectrodes was nearly Nemstian between pCa 5 and 7 and decreased to less than 20 mV in most microelectrodes between pCa 7 and 8 (Fig. 2, a and b). After impalement it took up to 15 min before a stable poten- tia1 was registered by the Ca’+-selective microelectrode. The response rate of Ca2+-selective microelectrodes allowed measurement of not only the resting leve1 of the free cytosolic Ca2+ concentration, but also its changes during the relatively long-lasting APs. However, this took place in only approximately 50% of the experiments. In the remaining cases, after impalement the resistance of the microelectrodes grew to over 1000 GR, resulting in a drift of the potential and large

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51 O

-50

-1 O0

-1 50

-200

!I 5 min

I - I

Figure 1. Parallel potential measurements in two neighboring cells both impaled with a potential microelectrode filled with 3 M KCI (top and bottom), and the difference between the two potentials (middle, note the 5-times expanded voltage axis for the difference trace). The white and black bars indicate illumination and darkening of the plant, respectively. The downward pointing arrow indicates the moment of electrical stimulation to trigger an AP.

offsets during APs. The reason for this was probably clogging of the microelectrode tips. These experiments were rejected. Only measurements with stable potentials and resistances that were followed by a calibration with slopes as given in Figure 2b were evaluated. These measurements showed an average cytoplasmic free Ca2+ concentration in Conocephalum of 231 k 65 nM in six different thalli at an externa1 Ca2+ concentration of 0.1 mM.

Although there were some differences in the free Ca2+ concentration for cbfferent thalli (from 100 up to 310 nM), the free Ca2+ concentration measured in a single thallus was constant within 0.1 pCa (see Figs. 3 and 4). In none of the experiments did the free Ca2+ concentration exceed the sub- micromolar range, which indicates that the tip of Ca2+- selective microelectrodes was always located in the cytoplasm rather than in the vacuole. We think that Ca2+-selective microelectrodes were sometimes inserted into the vacuole (as were other ion-selective microelectrodes) but that they were probably excluded from the vacuole (see below) before a stable potential, and thus a reliable free Ca2+ concentration, could be measured. No significant change in the cytoplasmic free Ca2+ concentration was observed ( n = 12) after light was tumed off (Figs. 3 and 4). Turning the light on evoked a transient depolarization (approximately 10 mV) called the generator potential, which could trigger an AP. In cases where the generator potential was too small to evoke an AP,

there was no detectable change in the free Ca2+ coixentration after illumination (n = 7) (Fig. 3).

Figure 4 shows an example of a parallel measiirement of the free cytoplasmic Ca2+ concentration and trans membrane potential changes. The first two APs were triggered electrically (downward pointing arrows), AP number 1 in light and number 2 in darkness. The third AP was evokr?d by light (note the lack of an artifact from electrical stimulation). One can observe a significant increase in the free cytoplasmic Ca2+ concentration during the APs that was prolonged for several seconds after the repolarization. The increase of the free cytoplasmic Ca2+ concentration during an AP did not depend on the stimulus (light or electric pulse) used to trigger the AP. The average leve1 of the free cytoplasmic Ca2+ concentration during 11 APs recorded in six different thalli was 477 f 114 nhi. The greatest value detected was 1458 nivr. It should be stressed that the increase in the free cytophsmic Ca2+ activity recorded during the AP cannot be an artifact resulting from the lower time resolution of 'the Ca2+-selective microelectrode in comparison with the potential microel ectrode. At depolarization cation-selective microelectrodes show an apparent decrease in ion activity (and an increase at hyper- polarization) when potential changes are too fast for them to follow. The fast transients during rapid depolarization and repolarization phases of the APs were caused by such a difference in time resolution of the two types of microelectrodes.

Measurements of the lntracellular K+ Activity wi th K'-Selective Microelectrodes

The K+-selective microelectrodes had a lower resistance, between 15 and 30 GQ, and an almost Nemstian slope (Fig. 2, c and d). They responded to a 10-fold change of the K+ concentration within 3 to 5 s . The response time W(IS probably limited by the exchange of the solutions.

The average intracellular K+ activity in C. conicum im- mersed in the standard medium containing 1 rnM K+ was 76 f 10 mM (n = 8). The results could not be divided into two separate groups. This means that either the tips of K+- selective microelectrodes were located in a11 expximents in the cytoplasm or there was no significant difference in K+ activity between the *cytoplasm and the vacuole. An increase of K+ concentration in the extemal medium frorn 0.1 to 10 m~ had no significant influence on the intracellular K+ activity (Table I). The plasma membrane was depolaiized by 11 and 14 mV after the transitions from 0.1 to 1 antl from 1 to 10 mM K+ in the medium, respectively. The iequilibrium potential for K+ came close to the resting potential at 0.1 mM K+ in the extemal medium, but it differed significantly by 41 and 82 mV when the medium contained 1 and 10 m~ K+ even after several hours of perfusion. No chariges of the intracellular K+ activity were recorded after tuming light off or on in cases where no AP was evoked by illumination. The amplitudes and the shapes of APs showed alrriost no de- pendente on the extemal K+ concentration. Duriiig APs, the intracellular K+ activity underwent either a slight decrease or no changes were recorded. In Figure 5, an example is shown of APs accompanied by a slight (<8 mM) decrease in the cytoplasmic K+ activity. Again, the observed activity changes

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did not depend on whether the APs were triggered electrically (AP number 1, downward pointing arrow) or by light (AP number 2).

Measurements of intracellular CI- and NOs- Activities with lon-Selective Microelectrodes

The C1--sensitive microelectrodes (ETH 500 based) had a Nemstian slope in solutions containing 1 mM or more C1-, but the slope was significantly smaller (20 mV) between 0.1 and 1 mM C1- (Fig. 2, e and f). The microelectrodes had a high resistance, between 150 and 200 GQ, and therefore responded relatively slowly to C1- activity changes. They needed 5 to 20 s to reach 90% of the voltage after a 10-fold change in C1- activity. The C1--sensitive cocktail used by us, like most known anion sensors, had a higher selectivity toward nitrate over C1-. Thus, it was important to know exactly the selectivity coefficient of these microelectrodes. We used the fixed interference method (Vaughan-Jones and Aickin, 1987) to obtain it. Figure 2e presents original traces from one of the C1--sensitive microelectrodes calibrated without and with 1.3 mM NO3- as interfering ion. The average characteristics for five such microelectrodes are given in Figure 2f. The selectivity coefficient Kcl calculated on the basis of these curves was 3.83 f 0.9, which means that C1-- sensitive microelectrodes were approximately 4 times more selective for NO3- than for C1-. The interference of NO3- with C1--sensitive microelectrodes might lead to a gross over-

W ::h O

-25 5 6 7 0

-100 -200- -1 50 - tkl '";

-100 -1 -j\ 50

-200 o 1 2 3

-log ([Ca2']/M) -log ([K']/M)

> '1 i2 E - 1 ~ ~ 1 -1 50

Figure 3. Transmembrane potential (top trace) and cytoplasmic free Caz+ concentration (bottom trace) in the thallus of C. conicum in the absence of APs. A subthreshold electric stimulus (downward pointing arrow) was followed by a light/dark and a dark/light change (black and white bars) without induction of an AP. Even at the enlarged pCa axis (cf. Fig. 4) there were no detectable changes in the free cytoplasmic Caz+ concentration (note expanded time axis compared to the other figures).

estimation of the intracellular C1- content if the intracellular Nos- activity is not known.

To solve this problem we applied N03--selective microelectrodes recently developed by Miller and Zhen (1991), which are much more selective toward Nos- over C1- than the C1--sensitive microelectrodes described above. These mi-

100- 7GZ e

-50-

-100-

f l O 0 l

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-100 1 2 3 4

-lOg ([CI-]/M)

'Oo- 9 0.1 +78mMCI-

20

-100- - -50- 100 100

5 min

1001 h

O

-100 -50 50K 1 2 3 4

Figure 2. Calibration data of ion-selective microelectrodes. a, Recalibration trace of the Caz+-selective microelectrode used in the experiment presented in Figure 4. The numbers 5, 6, 7, and 8 denote pCa of calibration solutions prepared according to Ammann et al. (1 987a). b, Averaged calibration curves of Caz+-selective microelectrodes: A, first calibration (dashed line); O, recalibration (solid line) resulting from measurements as shown in a. For recalibrations SE values are given. Average slope of nine measurements was 33 mV between pCa 5 and 6,28 mV between pCa 6 and 7, and 16 mV between pCa 7 and 8. c, Calibration traces of K+-selective microelectrode used in the experiment presented in Figure 5. Reference and potential electrodes were filled with 1 M NaCI. 1, 10, 50, 100, and 1000 denote concentrations of K+ in calibration solutions in mM. Left, Calibration; right, recalibration. d, Averaged calibration curves of nine K+-selective microelectrodes (as in b). The abscissa was corrected for K+ activity according to Ammann (1986). e, Original calibration traces of a CI--sensitive microelectrode in solutions containing 0.1, 1, 10, and 100 mM (concentration) KCI (left trace) and in the same solutions with addition of 1.3 m M NO,- (right trace). f, Average calibration curves of five CI--sensitive microelectrodes: O, in solutions without N o s - (solid line); O, with addition of 1.3 m M NO3- as interfering ion (dashed line) resulting from measurements as shown in e. g, Original traces obtained during calibration of a NO,--selective microelectrode in solutions containing 0.1, 1, 10, 20, and 100 mM (activity) NO3- (left trace) and in the same solutions with addition of 78 mM CI- (activity) (right trace). h, Average calibration curves of five N03--selective microelectrodes: O, in solutions without CI- (solid line); O, with addition of 78 m M CI- as interfering ion (dashed line).

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Figure 4. Transmembrane potential (top trace) and cytoplasmic free Ca2+ concentration (bottom trace) in Conocephalum during APs. All traces shown here were obtained during a single measurement on one thallus lasting 28 min. APs 1 and 2 were evoked by electrical stimuli: 1 in light, 2 in darkness; AP 3 was triggered by light on. The fast transients in t h e bottom traces during fast voltage changes are artifacts caused by different time resolutions of the ion-selective and the potential microelectrode. Other denotations are as in Figure 1 .

croelectrodes had a low resistance, from 5 to 20 GQ, and responded within 2 to 3 s to 10-fold changes in NO3- activities. They showed a slight shift from a Nernstian slope between 0.1 and 1 mM NO3- (Fig. 2, g and h). The average slope in this range of activities was 36 mV. The selectivity coefficient K N 0 3 obtained by the fixed interference method was 0.023 f 0.004, which means that N03--selective microelectrodes were approximately 43 times more selective for NO3- than for C1-. Figure 2g shows an example of calibration traces of NO,--selective microelectrodes obtained without and with 78 mM C1- as interfering anion in the calibration solutions. The respective calibration curves are presented in Figure 2h.

The apparent intracellular C1- activity measured with C1-- sensitive microelectrodes, (Cl), is the sum of the real C1- activity, [Cl], and the real NO3- activity, [NO3], multiplied by the selectivity coefficient Kcl:

(Cl) = [Cl] + 3.83 X [NO,]

-50

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-200 1 1 1 ' 5 min

Figure 5. Transmembrane potential (top traces) and c!/toplasmic K+ activity (bottom traces) in Conocephalum during APs obtained during a single measurement. AP 1 was triggered by electrical stimulation and AP 2 by light on. Other denotations are as in Figure 1 .

Similarly, the intracellular nitrate activity measured with N03--selective microelectrodes, (Nos), was grea ter than the real one by the factor KN03 X [Cl]:

(NO,) = [NO,] + 0.023 x [Cl]

Solving this equation system, we obtained the following formulas for the calculation of intracellular Cl-. and NO3- activities:

[NO3] = 1.1 X (NO3) - 0.025 X (C1;i

[Cl] = 1.1 X (CI) - 4.2 x (NO,)

The lntracellular CI- and NO3- Activities in Cells of Conocephalum

Applying the above calculations, we obtained NO different groups of values for both the C1- and the NO,'- activity. It seems reasonable to postulate that the lower values were measured in the cytoplasm and the higher values were measured in the vacuole (Table 11). During single measurements, transitions from high to low values (but never reverse

Table 1. 7he cytoplasmic K+ activity of C. conicum, the EK calculated from cytoplasmic and external K+ activities, the resting potential, and the AP amplitudes are compared at external media containing O. 1 , I , and 10 mM KCl

A constant ion strength of the medium was adjusted with NaCI.

Amplitude of AP Cytoplasmic K+ Calculated

Resting Potential Externa1 K+

Concentration Activitv EK

mM mM mV mV mV

o. 1 108 f 2 ( n = 22) 1 7 6 + l O ( n = 8 ) -110f3 -151 f 2 ( n = 2 1 ) 1 0 4 f 2 ( n = 2 1 )

103 * 2 (n = 22)

73 f 9 (n = 8)

78 f 10 ( n = 9)

- 1 6 6 f 3

-55 f 3

-162 f 2 (n = 22)

-137 t 2 (n = 22) 10

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Table II . The intracellular Cl- and NO3- activities in C. conicum The cytoplasmic and vacuolar activities resulted from measurements with ion-selective microelec-

trodes. The whole cell activities were calculated from t h e data obtained with ion-selective microelectrodes (ISM) on the basis of 20% cytoplasm and 80% vacuole (v/v). Additionally, the whole cell concentrations were determined bv anion chromatoaraphv (AC) of the cell sap of whole thalli.

Whole Whole Cell (AC) Cytoplasm (activity) Vacuole (activity) (ISM) (concentration) (activity)

[CI-l/mM 7.4 f 0.2 (n = 15) 43.5 f 7 (n = 4) 36.3 38.3 f 4 ( n = 4) 2.2 f 0.9 ( n = 3) [ N o , - ] / m ~ 0.63 f 0.05 ( n = 16) 3.0 f 0.3 ( n = 5) 2.5

changes) were frequently observed (see, for instance, Fig. 7b) that were probably caused by the exclusion of the microelectrode tip by the tonoplast from the vacuole (Thaler et al., 1992). On the other hand, the membrane potential (obtained by potential microelectrodes) was quite constant. We never recorded a sudden potential decrease, which would be expected if the potential electrode were excluded from a vacuole with a positive potential inside. This indicates that the poten- tia1 difference across the tonoplast in Conocephalum is negli- gible. In approximately 50% of the experiments we succeeded in measuring C1- and NO3- activities on the same thallus. In the remaining cases C1- and NO3- activities were measured on different thalli and the average activities of the interfering ion from a11 measurements was taken for calculation. The average cytoplasmic and vacuolar activities for a11 thallus investigated (Table 11) were 7.4 f 0.2 mM ( n = 15) and 43.5 f 7 mM ( n = 4) for C1-, and 0.63 f 0.05 mM ( n = 16) and 3.0 * 0.3 mM ( n = 5) for NO3-, respectively. The average cytoplasmic C1- and Nos- activities calculated on the basis of the results obtained on the same thalli equalled 7.2 + 0.3 mM ( n = 7) and 0.53 k 0.09 mM ( n = 8), respectively, which is not significantly different from the C1- and NO3- activities that were obtained on different thalli. The ion-selective microelectrodes were only occasionally located in the vacuole, so that in none of experiments were vacuolar C1- and N03- activities obtained on the same thallus.

The cell sap from complete thalli of Conocephalum was analyzed by anion chromatography. The concentrations ob-

> W 5 -100

n / I

tained were 38.3 f 4 mM ( n = 4) for C1- and 2.2 f 0.9 mM (n = 3) for NO3- (Table 11). One has to point out that the conditions of plant culture must be well controlled. Cultivat- ing plants in a soil containing nitrate fertilizers causes accumulation of NO3- and a decrease of C1- contents (data not shown).

Figure 6 shows an example of parallel measurement of the transmembrane potential and the cytoplasmic C1- activity. After tuming off the light we observed either no change of the cytoplasmic C1- activity or a slow increase by 1 to 2 mM. Reillumination caused a gradual retum to the previous value or no change in the C1- activity. Similarly, only slight or no changes of the intracellular NO3- activity were recorded after transitions between darkness and light (Fig. 7).

In 30% of our experiments the dynamics of C1--sensitive microelectrodes allowed us to measure changes in the C1- activity during APs. In these cases a small decrease, 1.5 k 0.7 mM ( n = 12), of C1- activity was recorded. An example of C1- activity changes during an AP is presented in Figure 6. The transients accompanying fast depolarization and repolarization phases of the AP were caused by the longer response time of the C1--sensitive microelectrode in comparison with the potential one. In most experiments (70%), the inertia of C1--sensitive microelectrodes was too high. They could not resolve the rapid potential changes during the AP. This was manifested by large offsets in the differential signal, which lasted almost as long as APs. Results of these experiments were only taken into account for calculations of the

Figure 6. Original traces of transmembrane potential changes (upper trace) and the cytoplasmic CI- activity (lower trace). An action potential was triggered electrically. Other denotations are as in Figure 1 .

-1 50 1 - ,

5 min

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Figure 7. Examples of parallel measurements of the transmembrane potential (upper traces)

traces). a, The NO,--selective microelectrode was located in the cytoplasm. b, The NO,-- selective microelectrode was inserted into the vacuole and was spontaneously excluded into the cytoplasm. Traces in a and b were obtained on two different thalli. APs were triggered electrically. Other denotations are as in Figure 1.

and the intracellular NO,- activity (lower -50

> Lu

-100

-1 50

Plant Physiol. Vol. 106, 1994

a

I =I 2.2 -

om 2.6- I I

$ 5 min z - 3.0-

3.4- 7 A .

average steady-state C1- activity. Almost no change in NOs- activity was recorded during APs, no matter whether the ion- selective microelectrode was located in the cytoplasm or in the vacuole (Fig. 7).

DISCUSSION

In this paper we describe the measurement of cytosolic Ca2+, K+, C1-, and NO3- activities in intact thalli of the liverwort C. conicum by ion-selective microelectrodes. This technique allows the registration of changes of the ion activity of a single cell within an intact Chl-containing tissue, while simultaneously, the E is monitored. This is hardly possible with other methods using photoproteins, fluorescent dyes (Read et al., 1993), or classical isotope techniques. Instead of double-barreled microelectrodes, which are much harder to handle (Felle, 1988), we used two separate electrodes, one ion-selective and one potential. This method is applicable only in cases in which the system is under good control (no differences in membrane potential of neighboring cells) (Felle, 1988), as was the case here (Fig. 1; see also Trgbacz, 1992). It might not be applicable to other plants, especially to those with complex tissues built up from different cell types. In our case the impalement of two neighboring cells might even have the advantage of causing less damage to the cells compared with the impalement of one cell on two microelectrodes. Additionally, the method was insensitive to distor- tions that might be caused by electrolyte leakage from the potential electrode.

The Cytoplasmic Free Caz+ Concentration in C. conicum

The concentration of free Ca2+ under steady-state conditions in the cytoplasm of the liverwort C. conicum cells (231 & 65 nM, n = 6) was similar to those recorded in the liverwort Riccia fluitans (127 & 33 nM) (Felle, 1988) and in other plant species (Bush, 1993). We did not investigate to what extent the cytoplasmic free Ca2+ concentration varied during changes of the extemal Ca2+ concentration, but it was shown by Felle (1988, 1991) that the cytoplasmic free Ca2+ concentration is tightly regulated in the liverwort R. fluitans

and in other plant cells (Felle et al., 1992). When the light- dependent potential changes in Conocephalum were not large (and thus the artifacts caused by the different time resolution of the electrodes were relatively small; see Figs. 3 and 4), the resolution of Ca2+-selective microelectrodes for related activity changes was well below 0.1 pCa. Within thc,se limits we did not detect significant changes in the free cytoplasmic Ca2+ concentration after tuming light on and off, as long as no AP was induced. This is in contrast to Miller and Sanders (1987), who measured long-lasting and relatively large light- dependent changes in the free cytoplasmic Ca2'+ concentration in Nitellopsis.

The Relationship between the Cytoplasmic Frec! Ca*+ Concentration and the AP

The results presented here show a significant increase of the free Ca2+ concentration during the AP. This increase was on average 2-fold (from 231 f 65 nM to 477 k '114 nM), and an up to 6-fold increase was registered. Williamson and Ashley (1982) estimated that in Chara a 30-fold and in Nitella a 42-fold increase of the free Caz+ concentration in the cytoplasm occurred during an AP. The 10-fold lower increase measured in our case may be caused by the different plant species or by different methods; Williamson and Ashley (1982) used aequotin. In principle, it is also possible that the increase in the free cytoplasmic Ca2+ concentration in Cono- cephalum during the AP was underestimated if there was a very rapid and transient increase to which Ca2+-selective microelectrodes responded too slowly to sense. The relatively high variability of the free CaZ+ concentration chmges during APs measured by us may be caused by the different location of microelectrode tips within an inhomogeneou s cytoplasm possessing local Ca2+ gradients (Gilroy et al., 1991). The increase of the free Ca2+ concentration lasted only a few seconds longer than the AP did. This is similar in Conoce- phalum and in intact characean cells (Williamson and Ashley, 1982) but at variance with the measurements by Kikuyama and Tazawa (1983) in tonoplast-free preparations, in which the free Ca2+ concentration remained high severa1 minutes after the repolarization.

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In Conocephalum, as in other species, the role of Ca2+ during the AP may be rather complex (Beilby, 1984). A depolarizing Ca2+ influx across the plasmalemma seems to be essential, since different Ca2+ channel inhibitors such as La3+, Mn2+, and verapamil entirely cancel the excitability of Conocephalum (Trebacz et al., 1989). But the observed increase in the free cytoplasmic Ca2+ concentration might also result from a Ca2+ efflux from interna1 stores into the cytoplasm (Forster, 1990; Bush, 1993). The increased Ca2+ activity might be the trigger that opens ion channels in the plasmalemma to further depolarize the membrane. These could be Caz+-dependent C1- channels, as in characean cells (Lunevsky et al., 1983; Shiina and Tazawa, 1987; Okihara et al., 1991) (see below), or Ca2+-dependent K+ channels, as in Eremosphaeva (Thaler et al., 1989).

Maintenance of Ca2+ activity on a low level is an element of the strategy developed by cells to respond to different stimuli whose consequence is an increase in the cytoplasmic Ca2+ activity (Bush, 1993). The increase of the free Ca2+ concentration in the cytoplasm of Conocephalum during the AP seems sufficient to evoke physiological effects such as the observed enhancement of the rate of respiration accompanying the AP (Dziubinska et al., 1989). It is worth mentioning that the increase in the free cytoplasmic CaZ+ concentration was clearly related to the AP itself and not to the stimuli used to trigger it; this is also the case for the described increase in the rate of respiration (Dziubinska et al., 1989). It was pre- viously demonstrated using antimony-filled H+-selective microelectrodes that in Conocephalum the pH changes in the course of APs did not exceed 0.05 pH unit and thus seem to be too small to influence the activity of respiratory enzymes (Trgbacz, 1992).

The Cytoplasmic K+ Activity and Its Relation to the Transmembrane Potential

The intracellular K+ activity in the intact thallus of the liverwort C. conicum (76 f 10 mM, n = 8) was in the same range as in other plant species (Leigh and Wyn Jones, 1984; Maathuis and Sanders, 1993). It was slightly lower than in the thallus of the liverwort R. fluitans (100-130 mM) (Felle and Bertl, 1986b; Felle, 1988), where, in contrast to our measurements, light-dependent changes of the cytoplasmic K+ activity were registered (Felle and Bertl, 1986b). In Cono- cephalum the cytoplasmic K+ activity did not significantly change at a 10-fold increase or decrease of the extemal K+ concentration (Table I).

The resting potential in Conocephalum in the standard medium containing 1 mM K+ ranged from -140 to -170 mV in different thalli; occasionally values up to -210 mV were measured. On the other hand, the mean value of EK was only -110 mV under this condition (Table I). This indicates that the transmembrane potential is to a great extent established by an electrogenic pump (Trgbacz et al., 1989), comparable to results in the liverwort R. fluitans (Felle and Bentrup, 1976). One has to point out that at rest there exists a driving force for K+ directed toward the cytoplasm. Thus, there must be a

mechanism that keeps intracellular K+ concentration at a physiological level.

A possible consequence of a less-negative E K in comparison to the resting potential might be that in an early depolarization phase of the AP, K+ channels that carry K+-influx toward the cell (inward rectifying) may open, and this could contribute to an initial depolarization. The lack of a corresponding, transient increase in the cytoplasmic K+ activity in the early depolarization phase of the AP might be caused by the fact that it was too small to be detected by the K+-selective microelectrode. During the repolarization phase of the AP at potentials less negative than E K , outward-rectifying K+ channels may open, which would cause a K+ efflux and a return to E K . A slight decrease of intracellular K+ activity during APs was frequently recorded. The complete repolarization to the resting potential is probably established by an electrogenic pump. The amplitudes and the shapes of APs showed no dependence on the externa1 K+ concentrations tested (O. 1-10 mM). Direct characterization of K+ channels in this plant by voltage-clamp or patch-clamp measurements should help to clarify this picture.

The lntracellular CI- and NO3- Activities and Their Relationship to the AP

Most, if not all, anion sensors used for fabrication of ion- selective microelectrodes have higher selectivity for NO3- than for C1- (Ammann, 1986). Direct measurements of intracellular C1- activity with C1--sensitive microelectrodes give reliable values only when the NO3- activity is negligibly low, as in Eremosphaera uiridis (Thaler et al., 1992). Here we used a method that allows the measurement of intracellular C1- and NO3- activities when significant amounts of both anions are present inside the cells. The purpose of this method is to measure ion activities in the same plant with two different anion-sensitive microelectrodes whose selectivity coefficients toward the respective anions are different and well determined. We obtained cytoplasmic and vacuolar anion activities of 7.4 f 0.2 mM ( n = 15) and 43.5 f 7 m~ ( n = 4) for C1-, and 0.63 k 0.05 mM ( n = 16) and 3.0 f 0.3 m~ (n = 5) for NO,-, respectively (Table 11). Taking into account that the cytoplasm in Conocephalum cells constitutes about 20% of the cell volume, as estimated by centrifugation, one can calculate an activity for C1- and NOs- of 36.3 f 7 mM and 2.5 f 0.3 m ~ , respectively, for the whole cells. This is comparable to the ion activities calculated for the whole thallus from anion chromatographic analysis: 28.3 f 3 mM ( n = 4) and 1.6 f 0.6 m~ ( n = 3), respectively. The concentrations obtained by anion chromatography (given in Table 11) were converted into activities with an activity coefficient of 0.74 (Ammann, 1986).

Our data (Table 11) show relatively low cytoplasmic C1- and NOs- activities. Very divergent data conceming the cytoplasmic C1- concentration of glycophytes ranging from 1 to 60 mM (and up to 500 mM for halophytes) have been published (Wyn Jones et al., 1979; Sanders, 1984). More recent data (Schroppel-Meier and Kaiser, 1988; Thaler et al., 1992) indicate that the cytoplasmic C1- activity is probably 4 0 mM, at least in glycophytes. Compartmental analyses with thalli of the liverwort R. fluitans give 11 and 30 m~ for

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1082 Trebacz et al. Plant Physiol. Vol 106, 1994

Table 111. The intracellular ion activities in C. ronicum (the pH data are unpublished) [ NOa- l /m~ [ K + l / m ~ [Ca” l / n~ [ C l - ] / m ~ PH

Cytoplasm 7.16 k 0.05 76 k 10 231 k 65 7.4 f 0.2 0.63 ? 0.05 Vacuole 5.83 k 0.23 (about 76) 43.5 f 7 3.0 k 0.3

the cytoplasmic and vacuolar C1- concentration, respectively (Felle and Bentrup, 1976). The cytoplasmic NOs- activity in Conocephalum was at the lower end of recently published values (Miller and Zhen, 1991; King et al., 1992). Our data point to a 6- and 5-fold concentration gradient for C1- and NO3- across the tonoplast, respectively. We have good indi- cations that the electrical potential across the tonoplast is negligibly small. This would mean a net driving force for both anions from the vacuole into the cytoplasm and thus an active accumulation of these anions in the vacuole.

We measured no or very slight changes in the anion activities after light/dark transitions. It was recently reported (Thaler et al., 1992) that in Eremosphaera darkening resulted in an increase of C1- activity. Reillumination caused a return to the light value. It was postulated that changes in the cytoplasmic C1- activity reflected ion fluxes compensating for light-induced pH changes (Thaler et al., 1992). In Conoce- phalum such pH changes are much smaller than in Eremo- sphaera (Trpbacz, 1992), and C1- fluxes accompanying them might be too small to be detected with ion-selective microelectrodes, especially on the higher background (7.4 compared to 2.2 mM) of C1- activity.

Our results indicate that in the standard experimental medium the Ecl (across the plasmalemma) is +50 mV. At about -150 mV E there is a large driving force for C1- out of the cell, and opening of C1- channels would result in C1- efflux. We frequently measured a small decrease of the cytoplasmic C1- activity during APs. C1- channels probably opened during the AP in Conocephalum, allowing C1- efflux and giving rise to a depolarization.

A Model for the lon Fluxes during an AP

Table I11 summarizes the intracellular ion activities in Con- ocephalum known so far. Together with the extemal ion

concentrations of the standard experimental medium (pH 7.0, 1 mM K+, 0.1 mM Ca2+, 1.2 mM C1-, and no h03- added) and the E (about -150 mV), this gives the framework of ion gradients and driving forces one needs to unders.and APs in Conocephalum. On the basis of previous studies (Trpbacz et al., 1989) and the data presented here, we suggest a basic model for the ion fluxes across the plasma ml.mbrane of Conocephalum in the steady state and during AR; (Fig. 8). In the steady state the transmembrane potential is established by the electrogenic H+ pump extruding H+ out of the cytoplasm (Trpbacz et al., 1989), comparable to what happens in the liverwort R. fluitans (Felle and Bentrup, 1976). At the large negative resting potential most physiologically important ions are kept far away from their electrochemical equilibrium (Table 111). Strong driving forces promote Ca2+ influx and anion, especially C1-, efflux. We demonstrated that the free cytoplasmic Ca2+ concentration increased significantly and the cytoplasmic C1- activity decreased duIing an AP. This points to Ca2+ influx and C1- efflux during ihe depolarization phase of APs in Conocephalum. Probably Ca2+ and anion channels are opened during the first ph,ise of APs, causing the depolarization and the respective iori fluxes. C1- seems to play a straightforward part as a depolarizing efflux, the role of Ca2+ is probably more complex (Beilby, 1984). Ca2+ influx across the plasmalemma would depolarize the membrane, yet Ca2+ might also be necessary to open C1- chan els of the plasma membrane, as in characean cells (Lunevsky et al., 1983; Shiina and Tazawa, 1987. Okihara et al., 1991). Peak depolarization during APs in Ccnocephalum never reaches, at least in the standard medium, ihe Ecl. This is possibly because of a rapid inactivation of aniim channels and/or the opening of other ion channels. We measured a slight decrease of the cytoplasmic K+ activity during APs, which suggests a K+ efflux. The opening of K+ channels at potentials more positive than the EK would cause a K+ efflux

Figure 8. A scheme illustrating the sequence IN OUT IN OUT of ion fluxes across the plasma membrane of Conocephalum in the steady state and during APs. T h e transient depolarization of the E during an AP is shown at the bottom in an expanded time scale (compared to the other figures). In steady state the transmembrane potential is established by an electrogenic p u m p that extrudes H+ from t h e cytoplasm at

2 Ca2+4 x CI-

B H +

t h e expense of ATP. Most ion channels remain closed in steady state. In t h e depolarization phase of APs, Ca2+ influx and CI- efflux take place. K+ efflux and t h e activity of the H+ p u m p cause the repolarization of the transmembrane potential; t h e complete return to resting poten- tia1 is governed by H+ p u m p activity.

IN OUT

1

1 min

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and a membrane repolarization before Ecl is reached. This K+ efflux can repolarize the membrane to EK, but the resting potential more negative than EK can be achieved only by an electrogenic ion pump (cf. Table III), probably a H+ pump (Trsbacz et al., 1989). Meanwhile, the membrane conductance for K+ has to decrease again to prevent a K+ influx. This ion mechanism for the repolarization phase of APs is probably common for a11 plant species whose resting potential has a large “metabolic” component.

With the above model and the data given in Table 111, some quantitative considerations become meaningful. The microelectrodes were inserted into the cells of the second layer of the thallus of Conocephalum, which have a length of about 150 to 200 pm and a diameter of about 80 pm. This gives a surface of about 6 X 104 pm2 and a volume of about 106 pm3. In these cells the cytoplasm constitutes about 20% of the cell volume, i.e. a cytoplasmic volume of 2 x 105 pm3, or 0.2 nL. Taking the well-established value of 1 pF/cm2 for the specific capacitance of biological membranes (Cole, 1970), one cal- culates that for a depolarization of 100 mV only about 6 X 10-l6 mo1 ions have to cross a membrane with a surface of 6 X 104 pm2. This is equivalent to a concentration change of about 3 p~ for a cytoplasmic volume of 0.2 nL. Therefore, the flow of ions needed to cause the observed depolarization had an insignificant effect on cytosolic free concentration and thus would not have been detected by our measurements. This also holds for Ca2+, since about 99.9% of the total cytosolic Ca2+ are bound and not free (Carafoli, 1987). It seems to be characteristic for plant APs (in contrast to animal APs) that the ion fluxes are much larger than required for loading the membrane capacitance; this is also the case for different giant alga1 cells (Gaffey and Mullins, 1958; Kiku- yama et al., 1984; Mummert and Gradmann, 1991) and probably for higher plants as well (Abe, 1981; Fromm and Spanswick, 1993). It has even been suggested that the origin of electrical excitation in plants is more a matter of osmoregulation and that AP in plants could well be an epiphenom- enon of the osmotic events (Mummert and Gradmann, 1991; Gradmann et al., 1993). However, there are different reports in which the role of APs for intercellular signaling in plants was established (Dziubiríska et al., 1989; Wildon et al., 1992; Fromm and Eschrich, 1993). The large ion currents occumng during an AP have to flow in parallel, and the K+ efflux probably electrically balances most (99.9%) of the Ca2+ influx and the C1- efflux. The arrows in Figure 8 may be interpreted as the very small portion of ion fluxes not balanced by counter ions and causing the transient depolarization.

It is worth mentioning that the activity changes we measured during APs were transient for a11 ions. In a11 cases the activity before and after an AP did not differ significantly. On the other hand, for Characeae it is well established that during an AP there is a net uptake of Ca2+ and a net release of K+ and C1- from the cells (Gaffey and Mullins, 1958; Oda, 1976; Kikuyama et al., 1984). The data available suppose that this is also the case for higher plants (Samejima and Sibaoka, 1980; Abe, 1981; Fromm and Spanswick, 1993). Net ion fluxes from the cell to the extemal medium without corresponding permanent activity changes in the cytoplasm point to intemal organelles, probably mainly the vacuole, as source for the ions involved. In tonoplast-free Characeae cells no

net C1- efflux could be detected (Kikuyama et al., 1984), and the free Ca2+ concentration remained increased severa1 minutes after the repolarization (Kikuyama and Tazawa, 1983). So there are probably also ion fluxes across the tonoplast during APs in plants, an efflux of anions from the vacuole into the cytoplasm driven by the electrochemical gradient (Table 111) and electrically compensated by a parallel K+ efflux.

The rapid retum of the free cytoplasmic Ca2+ concentration to the low resting leve1 during the repolarization phase (Fig. 4) could be achieved by the plasma membrane CaZ+-ATPase (Felle et al., 1992) or by an active uptake of Ca2+ into the vacuole or another intemal Ca2+ store (Chanson, 1993; Pfeiffer and Hager, 1993). Therefore, the transient changes in the cytoplasmic ion activities reported here probably indicate the net efflux of K+ and C1- from the vacuole to the extemal medium accompanied by an opposite Ca2+ uptake. As the measured activity changes are by orders of magnitude larger than necessary for the depolarization of the plasma membrane, the APs, besides their role in signaling (Dziubiií- ska et al., 1989; Wildon et al., 1992; Fromm and Eschrich, 1993), likely contribute to osmoregulation (Mummert and Gradmann, 1991; Gradmann et al., 1993).

ACKNOWLEDCMENTS

We thank Dr. W.M. Kaiser, Wiirzburg, Germany, for analyses by anion chromatography and B. Bethmann and M. Thaler for helpful discussion and advice.

Received March 14, 1994; accepted June 6, 1994. Copyright Clearance Center: 0032-0889/94/l06/1073/12.

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K+, NO,- Activities in Liverwort Conocephalum conicum Action

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