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Volume 405, Issue 3 p. 390-393
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Repetitive Ca2+ spikes in a unicellular green alga

C.S. Bauer

C.S. Bauer

Lehrstuhl Botanik I, Universität Würzburg, Mittlerer Dallenbergweg 64, D-97082 Würzburg, Germany

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C. Plieth

C. Plieth

Institut für Angewandte Physik, Universität Kiel, Olshausenstr. 40, D-24098 Kiel, Germany

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U.-P. Hansen

U.-P. Hansen

Institut für Angewandte Physik, Universität Kiel, Olshausenstr. 40, D-24098 Kiel, Germany

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B. Sattelmacher

B. Sattelmacher

Institut für Pflanzenernährung und Bodenkunde, Universität Kiel, Olshausenstr. 40, D-24098 Kiel, Germany

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W. Simonis

W. Simonis

Lehrstuhl Botanik I, Universität Würzburg, Mittlerer Dallenbergweg 64, D-97082 Würzburg, Germany

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G. Schönknecht

Corresponding Author

G. Schönknecht

Lehrstuhl Botanik I, Universität Würzburg, Mittlerer Dallenbergweg 64, D-97082 Würzburg, Germany

Corresponding author. Fax: (49) (931) 888 6158; E-mail: [email protected]Search for more papers by this author
First published: 07 November 1997
Citations: 26

Abstract

Cytosolic Ca2+ activity ([Ca2+]cy) and membrane potential were measured simultaneously in the unicellular green alga Eremosphaera viridis. Steady state [Ca2+]cy was about 160 nM. A ‘light-off’ stimulus induced a transient elevation of [Ca2+]cy ([Ca2+]cy spike) in parallel with a transient hyperpolarization of the plasma membrane. Caffeine and Sr2+, known to release Ca2+ from intracellular stores in animal cells, induced repetitive [Ca2+]cy spikes in Eremosphaera which were always accompanied by parallel repetitive transient hyperpolarizations. These transient hyperpolarizations could be used as an indicator for [Ca2+]cy spikes. Repetitive [Ca2+]cy spikes in Eremosphaera were similar to repetitive [Ca2+]cy spikes in excitable animal cells. The mechanisms underlying these [Ca2+]cy oscillations seem to be comparable in animal and plant cells.

1 Introduction

Transient elevations of [Ca2+]cy play a central role in intracellular signal transduction in plant and animal cells [1, 2]. Repetitive transient elevations of [Ca2+]cy, so-called [Ca2+]cy oscillations, are well established in animal cells [3-5]. In plant cells, repetitive increases of [Ca2+]cy, e.g. as induced by phytohormones [6, 7], were strongly damped and ceased after a few repetitions [6, 7]. Only recently stable [Ca2+]cy oscillations were reported for plant cells [8, 9]. The shape and frequency of transient repetitive [Ca2+]cy elevations vary considerably [4]. A regular spiking pattern for [Ca2+]cy elevations was observed in excitable animal cells and recently in a highly specialized plant cell [10]. The physiological significance of Ca2+ oscillations is a matter of debate [3-5], but the potential to encode and transduce information in the pattern of an oscillation is obvious.

Very little is known about the occurrence and mechanisms of repetitive transient elevations of [Ca2+]cy in plant cells. The unicellular green alga Eremosphaera viridis responds to a ‘light-off’ stimulus by a transient hyperpolarization of the plasma membrane [11]. The ‘light-off’ signal is received inside the chloroplast [11]. The hyperpolarization is caused by the transient opening of K+ channels. It is not known how this signal is transduced to the K+ channels of the plasma membrane. Different chemical effectors like Ba2+, caffeine, and Ins-P3, known to release Ca2+ from intracellular stores in animal cells [5, 12], induce single or repetitive transient hyperpolarizations in Eremosphaera [13-15]. This indicates that transient elevations of [Ca2+]cy may be the origin of the transient hyperpolarizations. Here the occurrence of repetitive [Ca2+]cy spikes in an algal cell is shown.

2 Materials and methods

The coccal green alga Eremosphaera viridis de Bary (algal culture collection Göttingen LB 228-1, Germany) was cultured and prepared for measurements according to Köhler et al. [11]. Measurements were performed in artificial pond water (APW, 0.1 mM KNO3, MgCl2, CaCl2, 2.0 mM MES adjusted to pH 5.6 with NaOH). For measurements at a low external concentration of divalent cations, the medium contained 0.1 mM KNO3, 0.1 mM EGTA, 2.0 mM MES/NaOH pH 5.6. At pH 5.6 the buffering capacity of EGTA for divalent cations is reduced and the free Ca2+ concentration as measured with Ca2+ selective microelectrodes was in the range of 300 nM. Sr2+, Gd3+, and La3+ were added as chloride salts. Caffeine was directly added to APW. The fluorescent Ca2+ sensitive dye fura-2 dextran (M r=10 ,000; Molecular Probes, Leiden, Netherlands) was injected mechanically into the cytosol of the alga [16, 17]. Afterwards a microelectrode containing 3 M KCl was impaled to record the membrane potential. Parallel to the membrane potential the Ca2+-dependent fura-2 dextran fluorescence [Ca2+]cy was measured by fluorescence ratio imaging using a CCD camera [16, 17]. For in vitro calibrations standard calibration solutions (C3722, Molecular Probes) containing 1.0 mM Mg2+ were used [16, 17].

3 Results

[Ca2+]cy and the membrane potential of Eremosphaera viridis were measured simultaneously in the same algal cell. At steady state the free running membrane potential (E) was −85±11 mV and [Ca2+]cy was 163±42 nM (n=50). A ‘light-off’ stimulus induced a transient hyperpolarization of the plasma membrane in 75% of the measurements. It had a duration of 53.1±10.2 s. As shown in Fig. 1 this hyperpolarization was accompanied by a transient elevation of [Ca2+]cy. The [Ca2+]cy increase started a few seconds after ‘light-off’. Extending the time scale (Fig. 1, right) demonstrates that the rise in [Ca2+]cy precedes the hyperpolarization. Besides this [Ca2+]cy spike, we did not observe any statistically significant light-dependent changes in [Ca2+]cy.

figure image
A transient change of [Ca2+]cy (bottom) and the membrane potential (E, top) after a ‘light-off’ stimulus (black bar at the bottom). Left, normal time scale; right, the same signals immediately after light off on a 20-fold extended time scale (the dashed lines indicate the baselines and the last sampling interval before hyperpolarization, respectively). Sampling interval was 1.5 s.

Caffeine applied externally in the millimolar range is known to release Ca2+ from internal Ca2+ stores in animal cells, and [Ca2+]cy oscillations induced by caffeine were repeatedly reported [18, 19]. Fig. 2 shows the effect of 20 mM caffeine on [Ca2+]cy and on membrane potential in Eremosphaera. Caffeine induced a [Ca2+]cy spike and a simultaneous transient hyperpolarization in five out of six experiments. The transient changes of [Ca2+]cy and membrane potential had a duration of 67.4±12.6 s. Steady state [Ca2+]cy was not changed by the perfusion of caffeine. The probability of inducing a hyperpolarization by caffeine was concentration dependent. It decreased from 81% at 20 mM (n=16) to 53% at 10 mM (n=17) and to 33% at 1 mM (n=18). When divalent cations were omitted from the external medium (0.1 mM EGTA, no divalent cations added) the addition of 20 mM caffeine induced fast repetitive [Ca2+]cy spikes and parallel repetitive transient hyperpolarizations (Fig. 3 ) in 50% of the measurements (n=20). In 20% of the measurements, only one or two [Ca2+]cy spikes accompanied by transient hyperpolarizations were observed. The caffeine-induced [Ca2+]cy spikes and hyperpolarizations had a frequency of 1.0±0.4 per minute and a duration of up to 60 min. The repetitive [Ca2+]cy spikes resulted in a significant increase of the [Ca2+]cy baseline (249±43 nM; n=10) compared to 160 nM under control conditions. The addition of 100 μM GdCl3, a well-known Ca2+ channel blocker, reversibly inhibited the [Ca2+]cy spikes and the transient hyperpolarizations (Fig. 4 ). Beside 100 μM GdCl3, the addition of 100 μM LaCl3 (n=6) or 10 μM verapamil (n=7) also reversibly blocked caffeine-induced oscillations (not shown).

figure image
A transient change of [Ca2+]cy and the membrane potential (E) induced by addition of 20 mM caffeine to the external medium (bar on top gives perfusion protocol). Sampling interval was 3 s.
figure image
Repetitive [Ca2+]cy spikes and membrane potential (E) oscillations induced by addition of 20 mM caffeine at a low external concentration of divalent cations (0.1 mM KNO3, 0.1 mM EGTA, 2.0 mM MES/NaOH pH 5.6) Sampling interval was 1.5 s.
figure image
The repetitive [Ca2+]cy spikes and potential (E) oscillations induced by 20 mM caffeine at a low external concentration of divalent cations were reversibly inhibited by 100 μM GdCl3 applied for 5 min and after wash-out for another 7 min (upper bars give perfusion protocol). Sampling interval was 3 s.

Beside caffeine, Sr2+ is an established effector for [Ca2+]cy oscillations in animal cells [20]. In Eremosphaera the addition of 1.0 mM Sr2+ to the external medium induced repetitive [Ca2+]cy spikes and parallel repetitive transient hyperpolarizations of the membrane potential (Fig. 5 ) in nearly all experiments (95%, n=27). Neither the probability of inducing oscillations by Sr2+ nor their duration or frequency were influenced by the external concentration of Ca2+ (or Mg2+) in the range between 1 mM EGTA (no Ca2+ or Mg2+ added) and 1 mM Ca2+ or 10 mM Mg2+. The Sr2+-induced [Ca2+]cy spikes and hyperpolarizations had a frequency of 0.5±0.2 per minute and a very long lifetime. Durations of more than 2 h were frequently observed. The baseline of the repetitive [Ca2+]cy spikes (168±43 nM) was identical with the steady state [Ca2+]cy under control conditions (160 nM). Compared to caffeine, the repetitive transient changes induced by Sr2+ displayed an about twofold lower frequency. The duration of a single [Ca2+]cy spike during Sr2+-induced repetitive changes was comparable (35±8 s) to caffeine-induced repetitive changes (30±2 s). The higher frequencies were mainly caused by shorter intervals between the single caffeine-induced [Ca2+]cy spikes compared to Sr2+-induced spikes.

figure image
Repetitive [Ca2+]cy spikes and membrane potential (E) oscillations induced by addition of 1 mM SrCl2. Sampling interval was 3 s.

4 Discussion

The experiments show the suitability of the unicellular green alga Eremosphaera (∅=150 μm) for simultaneous measurements of membrane potential and determination of cytosolic Ca2+ activity ([Ca2+]cy) by means of the fluorescent dye fura-2 dextran. The measured steady state value of [Ca2+]cy of 163±42 nM coincides well with the value of 164 nM obtained with Ca2+-selective microelectrodes in the same species [21]. It is also in the range reported for other plant cells [22]. Therefore, the in vitro calibration seems to be reliable under the conditions of these experiments.

The benefit of the greater temporal resolution of fluorescent dyes over Ca2+-selective microelectrodes is demonstrated by the fact that the ‘light-off’-induced [Ca2+]cy spike (Fig. 1) was not resolved with electrodes [21]but with the experiments here. The permanent light-dependent changes of [Ca2+]cy reported for Nitellopsis [23]were observed neither in Eremosphaera (Fig. 1) [21]nor in the liverwort Conocephalum [24]. The origin of the light-dependent changes of [Ca2+]cy in plant cells seems to be a release of Ca2+ from the chloroplasts. In characean cells, the same time constant was found for light-induced changes in chlorophyll fluorescence and [Ca2+]cy-induced changes in plasmalemma resistance [25]. Isolated chloroplasts were shown to release Ca2+ upon darkening [26]. Dark-induced Ca2+ release from chloroplasts may be involved in the [Ca2+]cy spike observed in Eremosphaera upon darkening. Other internal Ca2+ stores as the vacuole or the endoplasmic reticulum may contribute as well.

It had been shown earlier that an artificial increase of [Ca2+]cy by Ca2+ ionophores or Ca2+ injection causes a hyperpolarization in Eremosphaera [27, 15]. One major issue of our investigations was the demonstration of the close relationship between hyperpolarizations of the plasma membrane and changes in [Ca2+]cy. This was found in all experiments, we never observed a transient hyperpolarization in the absence of a [Ca2+]cy spike. Especially during the application of blockers like Gd3+ (Fig. 4), the perfect synchronization of the [Ca2+]cy spikes and the repetitive transient hyperpolarizations became obvious. Within our time resolution (1.5 or 3 s per sampling interval) the [Ca2+]cy increase started at the same time as the hyperpolarization or a few sampling intervals in advance (Fig. 1). Obviously, the transient hyperpolarization of the plasma membrane is caused by the [Ca2+]cy spikes, probably via the opening of Ca2+-activated K+ channels in the plasma membrane. The hyperpolarization can be used as a qualitative indicator for transient elevations of [Ca2+]cy in Eremosphaera.

Little is known about the effect of caffeine or Sr2+ in plant cells [14, 27]. In animal cells, the involvement of a release of Ca2+ from internal stores is known to occur when caffeine and Sr2+ enter the cells [3, 28, 29]. Thus, it is suggested that a similar mechanism applies to the [Ca2+]cy oscillations in Eremosphaera which resemble the rhythmic spiking pattern commonly found in excitable animal cells [4]. The rapidly reversible blockage of oscillations by Gd3+ indicates that some Ca2+ flux across the plasma membrane is necessary for sustained oscillations. If some basic mechanisms of [Ca2+]cy oscillations were developed before plant and animal cells diverged, Eremosphaera seems to be an excellent candidate for the study of these common mechanisms.

Acknowledgements

This work was financially supported by the Deutsche Forschungsgemeinschaft (SFB 176, TP B11).