Light- and dark-induced action potentials in Physcomitrella patens

Abstract

Glass microelectrodes were inserted into Physcomitrella patens gametophyte leaves and action potentials (APs) were recorded in response to sudden illumination as well as after darkening, i.e., when the dark-induced membrane depolarization crossed a threshold. Application of 5 mM La³⁺ (a calcium channel inhibitor), 10 mM TEA⁺ (a potassium channel inhibitor) and increased free Ca²⁺ resulted in a loss of excitability. Lack of Ca²⁺ in the external medium did not prevent APs from occurring. It was concluded that during light- dark-induced excitation of Physcomitrella patens, APs might rely upon calcium influxes from the intracellular compartments. APs were not blocked by the proton pump inhibitors (DES, DCCD), although the resting potential (RP) diminished significantly.

Introduction

Light signals are the most important environmental factors that regulate growth and development of plants. In addition to light quantity, its quality, polarization, direction and periodicity provides plants with necessary information.¹ Elucidation of the paths of light-triggered information flow might be sometimes confounded by the existence of multifarious and function-overlapping light receptors.^2,3 In the moss Physcomitrella patens for example, both blue and red light induce chloroplast movements by the activation of phototropin and phytochrome receptors, respectively.^4–6 For the same plant, at least four distinct photoreceptor systems (cryptochrome, photoropin and two distinctly located phytochromes) are involved in the light-evoked side branch formation.⁷ In the process of caulonemal filament branching, light-induced membrane depolarization is engaged.^8,9 First, by the use of ion channel blockers, Ermolayeva et al.⁸ elaborated a possible ionic mechanism of the light-induced membrane potential transients in caulonemal filaments. Next, thanks to voltage-clamp experiments, they demonstrated that Ca²⁺, K⁺ and Cl^- currents are activated during the light-dependent membrane depolarization.⁹

The general scheme of plant excitation, consisting of a transient activation of Ca²⁺, Cl^- and K⁺ channels in a sequence,^10–12 resembles the ionic model of the light-induced membrane depolarization in Physcomitrella patens. Light-induced action potentials (APs) are known in excitable plants.^13–16 Moreover, shade-induced APs were also reported in plants.^17,18 Unexcitable cells respond to illumination or darkening with transient potential changes of relatively low amplitudes which depend on light intensity and quality.^19–21 There is considerable evidence that signal transduction of dark-light-dependent membrane potential changes involves changes in [Ca²⁺]_cyt.^9,22–27 The role for Ca²⁺ fluxes as a messenger between the membrane depolarization and a physiological response was con-firmed many times.^28,29 However, the source of Ca²⁺ ions during light-induced depolarization is under examination and the compelling results point that the extracellular as well as the intracellular calcium stores may be involved in the light-triggered signalling, depending on a plant, a photoreceptor system and its location within a cell.^{5,23,25–27,30}

The aim of this study was to examine light-induced membrane potential changes in gametophytes of Physcomitrella patens. The gamtophyte is a dominating stadium of the moss and most of genetic and physiological experiments have been carried out on gametophyte cells. Our data considering electrical phenomena in gametophyte cells of Physcomitrella patens are widely consistent with Ermolayeva et al.,^8,9 who examined caulonemal filaments being a younger stadium of the moss development. We demonstrate that, in gametophytes of Physcomitrella patens, the light-induced membrane depolarizations are APs. APs also appeared when dark-induced depolarization overcame the threshold of excitation. Thus, both illumination and darkening are able to evoke APs in the same cell. Such a phenomenon has never previously been reported in plant cells. We postulate that light- and dark-induced APs, though having different kinetics, share many aspects of the ionic mechanism.

Results

The sudden illumination after an hour-long darkness evoked transient depolarization of a resting potential (RP) in the gametophyte cells of Physcomitrella patens. The depolarization amplitude was proportional to the light intensity but only to the value of approximately 100 µmol photons m^-2s^-1, above which action potentials (AP_light) of relatively constant amplitudes occurred. The subthreshold responses to light-dark versus dark-light transitions were opposite and almost symmetrical, i.e., light induced brief depolarization while darkening induced fast hyperpolarization (Fig. 1 insets). Long-lasting records revealed multiphasic responses. Transient depolarization evoked by illumination was followed by long-lasting hyperpolarization and another depolarization phase. Upon darkening, the transient hyperpolarization was followed by persistent depolarization. Thus, to get reproducible results, we stimulated each thallus every hour with an alternate light-dark-light transition. The light intensity was established at 300 µmol photons m^-2s^-1 (if not stated otherwise).

Figure 1.

Membrane potential changes of Physcomitrella patens gametophyte cells after illumination and darkening (representative traces). White light of 300 µmol photons m^-2s^-1 was applied. The cells were kept in the standard solution containing 1 mM KCl, 1 mM CaCl₂, 50 mM sorbitol (pH 7). Grey bars indicate darkness periods. Insets, responses to subthreshold illumination (100 µmol photons m^-2s^-1) and darkening after subthreshold light stimulus (60 µmol photons m^-2s^-1).

Once the threshold was crossed, action potentials were generated both in response to illumination, AP_light, and darkening, AP_dark. The amplitudes of AP_light and AP_dark reached approximately a constant value (Fig. 1). The AP_light were triggered by white, red and blue light as well (not shown). Apart from light intensity, the duration of darkness also mattered for AP_light (Fig. 1). The average minimal duration of darkness was 13 min for white light of 300 µmol photons m^-2s^-1. APs after darkening (AP_dark) appeared only if the duration of illumination was longer than 20 min and light intensity preceding darkening was higher than 60 µmol photons m^-2s^-1. Existence of refractory periods once more indicates that light- and dark-induced membrane potential changes are indeed APs. The peak values for AP_light and AP_dark did not significantly differ between each other for the same leaf (Fig. 1). The profound difference between AP_light and AP_dark was related to their duration—AP_dark lasted several times longer (Fig. 1 and Table 1). Upon darkening, the threshold of excitation was exceeded at a higher potential as compared to AP_light. This was marked by a sudden increase in the depolarization rate of AP_dark.

Table 1.

The influence of respective treatments on APs

	APlight [mV]	t1/2 light [min]	APdark [mV]	t1/2 dark [min]
Standard white light	88 ± 4	3.1 ± 0.2	95 ± 3	22.8 ± 2.5a
	(n = 25)		(n = 23)
Ca2+-free	113 ± 5b	2.1 ± 0.1b	118 ± 4b	12.9 ± 2.0ab
	(n = 8)		(n = 16)
0.05 mM DES	38 ± 4b	9.1 ± 2.7	45 ± 3b	15.2 ± 2.7
	(n = 18)		(n = 14)
0.05 mM DCCD	77 ± 10	8.9 ± 4.6	61 ± 6b	10.3 ± 3.0a
	(n = 4)		(n = 3)

Parameters characterizing potential changes: AP, the average AP amplitude ± SE; t_1/2, the average AP duration given as the half-time of AP i.e., time measured in the half of the amplitude ± SE.

^aStatistically significant difference between light and dark conditions (p < 0.01).

^bStatistically significant difference between respective treatments and the standard (p < 0.01).

The ionic mechanism of AP in plants is based upon subsequent fluxes of Ca²⁺, Cl^- and K⁺ ions. Thus, we used different inhibitors of the respective ion channels engaged in membrane excitation to elaborate a comprehensive model for the APs in Physcomitrella gametophyte cells. Since the important role of the proton pump in restoring RP after excitation is well established,^14,33 we also used H⁺-ATPase inhibitors. The effects of all the compounds used on AP amplitudes and durations are summarized in Table 1.

La³⁺ ions are known to block a variety of Ca²⁺ channel types. When added at 5 mM concentration to the standard medium, they caused a total inhibition of APs—those evoked by light as well as by darkness (Fig. 2A). The membrane potential changes that remained upon La³⁺ treatment resembled the responses to subthreshold stimuli. To further test the role of Ca²⁺ fluxes in AP generation we submersed the moss in the standard medium deprived of Ca²⁺ overnight. Such a treatment had no hampering effect on APs occurrence and appearance. Decrease of AP amplitudes would be expected if external Ca²⁺ was the only ion participating in cell depolarization. Instead of decrease, AP amplitudes even grew, although slightly but significantly for statistics (Table 1). In order to examine the effect of increased external Ca²⁺, we used standard medium enriched with 10 mM CaCl₂. The excess of calcium ions was comparable to lanthanum action (Fig. 2B). The plant lost its excitability and generated only slow responses to alternate light-dark, dark-light transition.

Figure 2.

Responses of Physcomitrella patens cells to illumination and darkening treated with (A) 5 mM LaCl₃ added to the standard medium and (B) high Ca²⁺ concentration (10 mM) obtained 10 min (continuous line) and 9 h (dashed line) after the treatment.

Addition of 10 mM TEA⁺, potassium channel inhibitor, to the standard medium caused a total loss of excitability, too (Fig. 3). In the plants treated with TEA⁺, the RP did not fluctuate so strongly as in untreated plants (Fig. 3). The responses to illumination and darkening resembled these to subthreshold stimuli in the control plants.

Figure 3.

The effect of 10 mM TEA⁺ treatment of Physcomitrella cells on the membrane potential changes evoked by light and darkness.

RP obviously depended on H⁺-ATPase status since DES and DCCD were able to depolarize membrane potential in gametophyte cells. DES is a potent inhibitor of plasmalemmal proton pump, while DCCD affects both P-type and V-ATPase.³⁴ However, DES and DCCD did not inhibit Physcomitrella patens excitability. The plants examined did not lose their excitability even when the RP depolarization exceeded 50 mV, though decreased RP resulted in lowering of AP amplitudes (Fig. 4 and Table 1). The depolarization was the cause of lowering the threshold of excitation. The responses to illumination/darkening consisted of two to four consecutive APs. The second AP_light in DES or DCCD treated cells lasted much longer than the first one and its time course was nearly identical to that of AP_dark. The responses to illumination and darkening remained profound in spite of the proton pump inhibition.

Figure 4.

Light- and dark-induced membrane potential changes in Physcomitrella gametophyte cells treated with (A) 0.05 mM DCCD and (B) 0.05 mM DES.

Discussion

The moss Physcomitrella patens is a model plant in photobiology.^4,6,7 Despite being a simple organism, Physcomitrella exhibits responses to gravity, light and growth substances similar to those involved in angiosperms.³⁵ Physcomitrella patens is also a model system in plant functional genomics. It is the only plant known so far that shows a high rate of homologous recombination in its nuclear DNA.³⁶ It is thus possible to analyze the definite gene function by targeted knockouts, especially that most of Physcomitrella genes exist as single copies. Only recently the moss has attracted attention of plant electrophysiologists, too.^8,9,37

Ermolayeva and co-workers^8,9 demonstrated a phytochrome-mediated membrane potential depolarization in caulonemal filaments of the moss. Illumination is an important morphogenetic factor inducing side branch formation. Although threshold-type dependence between the light intensity and the depolarization magnitude as well as the ion mechanism of responses to light pointed to action potentials, the authors were reluctant to use the term: action potential.^8,9 The responses to darkening were not considered in detail.^8,9

We did our measurements on the haploid gametophyte, a dominating phase in mosses. We demonstrated, for the first time in plants, that gametophyte cells of P. patens generate APs both in response to illumination and after darkening. Whether such APs are generated by caulonemal filaments remains to be examined. Both AP_light and AP_dark fulfil the all-or-none law exhibiting constant amplitudes above a certain threshold. Moreover, AP_light and AP_dark are generated only if the refractory period is exceeded. Thus, two of three criteria of action potential have been fulfilled. Propagation is the third criterion in addition to all-or-none characteristics and existence of refractory periods. However, it was hard to demonstrate propagation in such small objects as leaves of Physcomitrella gametophytes, especially that light/dark stimuli act on the whole leaf at the same time.

The ion mechanism of APs generated in plants was elaborated using giant internodal Charophyta cells.^10,12,38 Basic aspects of that mechanism were confirmed to operate in the liverwort Conocephalum conicum closely related to Physcomitrella patens.^{14,30,32,39,40} According to this scheme, AP is initiated by Ca²⁺ influx into the cytoplasm. It activates Ca²⁺-dependent Cl^- channels and Cl^- efflux causes membrane depolarization. Repolarization occurs after the opening of outward-rectifying K⁺ channels and activation of the electrogenic proton pump. The source of Ca²⁺ ions entering the cytoplasm in an early phase of the AP is still a matter of debate. Either external⁴¹ or internal stores⁴² are postulated as being responsible for Ca²⁺ fluxes. Here, we show that lanthanum, a rather unspecific Ca²⁺ channel blocker, completely inhibits APs in Physcomitrella cells. Excess of Ca²⁺ (10 mM) evoked a comparable effect to that of La³⁺ consistent with the inhibition of Ca²⁺ channels by a steep Ca²⁺ gradient. This undoubtedly points to a role of calcium ions in AP generation. On the other hand, long lasting incubation of P. patens cells in a medium without Ca²⁺ slightly enlarged AP amplitudes (Table 1). If external Ca²⁺ was solely responsible for the depolarization phase of the AP, and thus for its amplitude, then, according to the Nernst law, AP amplitude should substantially decrease upon removal of Ca²⁺ from the external medium. It was not the case. This effect may be interpreted by still incomplete removal of Ca²⁺ from the cell walls or by participation of intracellular stores in AP generation. Increase in cytosolic Ca²⁺ is regarded as an activator of anion channels and increased permeability for anions, mainly Cl^-, determine the amplitude of APs.¹⁰ TEA⁺, K⁺ channel inhibitor, caused inhibition of APs in P. patens cells. A similar effect was observed in Conocephalum³⁹ and in Physcomitrella caulonemal filaments.⁸ Membrane potential in P. patens cells is negative to E_K and one may expect that K⁺—in addition to Ca²⁺—fluxes may cause the initial depolarization. Under TEA⁺ treatment, responses to illumination and darkening have the same multiphasic character as in untreated plants subjected to subthreshold stimuli indicating rather a minor role of K⁺ channels in light-induced membrane potential changes. On the other hand, K⁺ channels can be responsible for the differences in AP_dark duration in comparison to AP_light. It was previously demonstrated that Chara cells exhibit a high K⁺ conductance in darkness.¹⁵ This was confirmed in P. patens. Increasing K⁺ concentration from 1 to 100 mM in darkness caused depolarization by 77 mV—close to the Nernstian shift, whereas in light the depolarization was much lower, 30 mV (not shown). Thus, the high K⁺ conductance in darkness is probably responsible for the AP_dark plateau.

The cells of P. patens exhibit a very low membrane potential located far below the equilibrium potential of basic ions. This points out to the existence of a large electrogenic component of the membrane potential. Indeed, after application of proton pump inhibitors (DES and DCCD) significant depolarization of the membrane potential took place, although the membrane potential upon light on and off occasionally reached a value of -200 mV which is far below the electrochemical equilibrium potential for any ion present in the medium. This may indicate operation of other than H⁺ATPase electrogenic ion pump in the plasma membrane of Physcomitrella patens. It was recently demonstrated that P. patens cells express Na⁺-ATPase.⁴³ Simultaneously with the depolarization, the threshold of excitation was lowered and not only single but also series of APs consisting of 2–4 peaks were generated. Upon the proton pump inhibitors treatment AP_light became broader being almost identical as AP_dark. This indicates that the duration of AP plateau may depend on activity of H⁺ATPase, too. Vanishing series of APs were recorded in Conocephalum conicum in response to proton pump inhibitors.⁴⁰ Conocephalum, however, in contrast to Physcomitrella lost its excitability after approximately one hour of H⁺ATPase inhibitor application. P. patens cells remained excitable up to six hours of DES treatment. The difference between these two Bryophyta species may be a result of a shift in the activation potential of ion channels responsible for AP generation to more positive values. Our results confirmed the validity of electrophysiological measurements on intact cells, which are necessary but sometimes missing steps to a more detailed study on the molecular level.

Materials and Methods

Physcomitrella patens was grown on solidified Knop medium as described by Reski and Abel.³¹ Upon cultivation the plants developed into leafy gametophytes. The gametophyte cultures were maintained by sub-culturing them monthly.

Electrophysiological experiments were performed as previously described (ref. ³²). The transmembrane potential was measured with glass microelectrode (Hilgenberg, Malsfeld, Germany) filled up with 3 M KCl. Ag/AgCl wires of the microelectrode and the reference electrode made of Teflon pipe with porous plug filled with 100 mM KCl were connected to FD 223 amplifier (World Precision Instruments, Sarasota, FL, USA). The output signal was digitalized and stored on a PC hard drive. The sampling frequency was 2 Hz. For microelectrode experiments, the whole gametophyte was immobilized with a strip of Parafilm and immersed into the standard solution containing 1 mM KCl, 1 mM CaCl₂, 50 mM sorbitol (pH 7). Electrical micromanipulator DC3001 (World Precision Instruments, Sarasota, Fl, USA) served to microelectrode impalement into cells. The site of impalement controlled by a microscope (SZ11, Olympus, Japan) was as close to the tip of the reference electrode as possible (less than 0.2 mm). Sudden drop in potential difference was an indication of successful insertion of the microelectrode into a cell. Precise localization of the micro-electrode tip (cytosol or vacuole) was not examined. The gametophyte was kept in the standard solution in darkness overnight at room temperature (22°C) before the recording started. Action potentials were triggered by sudden changes in illumination conditions. The changes (light-on, light-off) were executed every hour. The plants were illuminated with white light of 300 µmol photons m^-2s^-1 from a halogen lamp (HL-EKE 21V/150W, Poland).

The very first APs in the standard solution were treated as a control for further proceedings when the external solution was changed for:

• standard supplemented with 5 mM LaCl₃;

• standard supplemented with excess of calcium (10 mM CaCl₂);

• standard deprived of Ca²⁺;

• supplemented with 10 mM tetraethyl ammonium chloride (TEACl);

• supplemented with 0.05 mM diethystilbestrol (DES);

• supplemented with 0.05 mM 1,3-dicyclohexylcarbodiimide (DCCD)

In order to establish whether or not the treatment had a significant effect on APs, T-test was applied. The values of the parameters are given as mean ± SE.

Abbreviations

AP

action potential

DCCD

1,3-dicyclohexylcarbodiimide

DES

diethystilbestrol

RP

resting potential

t1/2

half-time of action potential

TEA⁺

tetraethyl ammonium ion