Actin dynamics mediates the changes of calcium level during the pulvinus movement of Mimosa pudica

Abstract

The bending movement of the pulvinus of Mimosa pudica is caused by a rapid change in volume of the abaxial motor cells, in response to various environmental stimuli. We investigated the relationship between the actin cytoskeleton and changes in the level of calcium during rapid contractile movement of the motor cells that was induced by electrical stimulation. The bending of the pulvinus was retarded by treatments with actin-affecting reagents and calcium channel inhibitors. The actin filaments in the motor cells were fragmented in response to electrical stimulation. Further investigations were performed using protoplasts from the motor cells of M. pudica pulvini. Calcium-channel inhibitors and EGTA had an inhibitory effect on contractile movement of the protoplasts. The level of calcium increased and became concentrated in the tannin vacuole after electrical stimulation. Ruthenium Red inhibited the increase in the level of calcium in the tannin vacuole and the contractile movement of the protoplasts. However, treatment with latrunculin A abolished the inhibitory effect of Ruthenium Red. Phalloidin inhibited the contractile movement and the increase in the level of calcium in the protoplasts. Our study demonstrates that depolymerization of the actin cytoskeleton in pulvinus motor cells in response to electrical signals results in increased levels of calcium.

Introduction

Mimosa pudica is a model for the study of plant nyctinastic movements. M. pudica is very sensitive to environmental stimuli, such as wounding, touching, vibration, temperature stimulus, and change of illumination. In response to these stimuli, M. pudica rapidly bends its petioles downward and closes the leaflets of its doubly compound leaves. The bending of M. pudica petioles is due to the bending movement of the pulvinus, while the bending movement of the pulvinus is caused by the rapid change in volume of the abaxial motor cells of the pulvinus. This rapid change can be accounted for by the shrinkage of tannin and colloidal vacuoles in the motor cells, which is caused by rapid loss of turgor pressure due to the efflux of K⁺ and translocation of water.^1–5

The actin cytoskeleton is reported to be involved in the bending movement of Mimosa petioles. Treatment of the Mimosa motor organ with cytochalasin B (CB) and phalloidin, to interfere with the actin cytoskeleton, alters the ability of the main pulvinus to bend, which suggests that the rearrangement of actin is important for seismonastic movement.⁶ Further observations of the actin cytoskeleton before and after petiole bending have shown that actin filaments undergo fragmentation during bending.^6,7 In addition, actin in the Mimosa pulvinus is heavily tyrosine-phosphorylated. The extent of phosphorylation correlates with the degree of petiole bending.^7,8 However, the role of actin during Mimosa petiole bending remains to be established.

Besides the involvement of the actin cytoskeleton, Ca²⁺ may also play an important role in Mimosa nyctinastic movements.^5,9,10 The calcium level in motor cells increases during Mimosa petiole bending.¹¹ Pharmacological experiments have indicated that the calcium channels in the tannin vacuole membrane are responsible for the release of calcium from the vacuole, and that calcium pumps are involved in calcium scavenging by the tannin vacuole during recovery of the petiole to its original position.¹² Comparison of the bending movement and the responses to calcium-related reagents upon mechanical perturbation or darkness between plants with or without tannin vacuoles has suggested that the tannin vacuoles are an important calcium resource for bending movements.¹³ In addition, the calcium-sensitive potassium current has been studied by using pulvinar protoplasts from M. pudica.¹⁴

Although it has been shown that the actin cytoskeleton and calcium are both involved in the bending movement of M. pudica petioles, further study is needed to determine whether and if so how these two components are related. In the study reported herein, we (i) induced bending of M. pudica petioles by electrical stimulation and (ii) investigated the dynamic changes of the actin cytoskeleton and changes in Ca²⁺ level during movement by using various reagents that are know to interfere with the actin cytoskeleton and Ca²⁺ changes. Our study provides evidence that actin dynamics mediate the changes in Ca²⁺ level during pulvinus movement of M. pudica.

Results

Actin cytoskeleton and calcium are involved in bending of Mimosa pudica petioles that is induced by electrical stimulation.

Bending of the pulvini of M. pudica can be triggered by electrical stimulation. When an electrical stimulus is applied, the petioles of M. pudica bend downward and the leaflets close rapidly (see Suppl. video 1). In the present study, an electrical stimulus was applied to trigger bending of the pulvini of M. pudica. The size of the petiole bending angle was measured to determine whether or not such movement was affected by the treatments of actin-affecting reagents and calcium channel inhibitors.

It has been reported that treatment of the Mimosa motor organ with cytochalacin B or phalloidin alters the ability of the main pulvinus to bend.^3,7 We therefore applied actin-affecting reagents to determine whether the actin cytoskeleton is also involved in the petiole bending induced by electrical stimulation. The plants of M. pudica were pretreated with actin-disrupting reagent latrunculin A or actin-stabilizing reagent phalloidin. The degree of petiole bending was measured after electrical stimulation. The degree of petiole bending was reduced significantly by pretreatment with latrunculin A or phalloidin (Fig. 1). The petiole bending angle was reduced by latrunculin A in a concentration-dependent manner (Fig. 1A). Pretreatment with 50 µM phalloidin also reduced the petiole bending angle, compared with the controls, after electrical stimulation (Fig. 1B). These results indicate that the actin cytoskeleton is also involved in petiole bending that is induced by electrical stimulation.

Figure 1.

Effects of actin and calcium-related reagents on the bending angle of Mimosa pudica petioles. (A) latrunculin A; (B) phalloidin; (C) LaCl₃; (D) nifedipine; (E) Ruthenium Red.

It has also been reported that Ca²⁺ may play an important role in Mimosa petiole bending.¹² Therefore, the actions of various reagents that interfere with intracellular calcium level were investigated (Fig. 1C–E). When pretreated with LaCl₃, which is a competitive inhibitor of calcium channels, at concentrations of 1, 1.5 and 3 mM, the bending of the petioles was about 43, 26 and 3°, respectively (Fig. 1C). When pretreated with the calcium-channel inhibitor nifedipine at concentrations of 100, 250 and 500 µM, the bending of the petioles was about 39, 18 and 13°, respectively (Fig. 1D). When pretreated with Ruthenium Red, which is an inhibitor of cytoplasmic calcium channels, at concentrations of 50, 150 and 200 µM, the bending of the petioles was about 44, 18 and 2°, respectively (Fig. 1E). All treatments with these calcium-channel inhibitors had an inhibitory effect on petiole bending. Therefore, we conclude that both the actin cytoskeleton and calcium are important for bending of M. pudica petioles that is induced by electrical stimulation.

Actin cytoskeleton in pulvinus motor cells is disrupted after electrical stimulation.

To further elucidate the changes in actin cytoskeleton during petiole bending, the actin cytoskeleton in motor cells was visualized by confocal microscopy. M. pudica petioles were cut from the plants and dipped in solution until they recovered their original position. Petiole bending was then triggered by electrical stimulation. The petioles were quickly fixed before and after petiole bending, and after petiole recovery. The actin filaments in motor cells were then stained with Alexa Flour 488 phalloidin and observed under a confocal microscope (Fig. 2). Before petiole bending, long actin filaments and bundles were observed (Fig. 2A, D and G). After petiole bending, most actin filaments were fragmented, with only a few short and fine filaments remaining (Fig. 2B, E and H). After recovery for 2–4 h, the long actin filaments and bundles reappeared (Fig. 2C, F and I). Counting the numbers of cells with actin filaments that remained intact before and after electrical stimulation, and in recovered cells, indicated that the filaments were depolymerized during petiole bending (Table 1).

Figure 2.

F-actin reorganization in the motor cells of the primary pulvinus. (A–C) Petioles before (A), after (B), and recovered (C) from electrical stimulation. (D–F) F-actin in the motor cells of the primary pulvinus was stained green by Alexa Flour 488 phalloidin. Before electrical stimulation, F-actin was present as long filaments and bundles (D). After stimulation, most of the F-actin was fragmented (E). F-actin was restored after the petiole returned to its original position (F). (G–I) DIC images of motor cells of primary pulvinus before (G), after (H), and recovered (I) from electrical stimulation. Bar, 20 µm (D–I).

Table 1.

The depolymerizaiton of actin filaments in motor cells before and after the bending of Mimosa pudica petioles triggered by electrical stimulation

	The number of cells with actin filaments that remained intact	The percentage of cells out of the total number of cells observed
Before electrical stimulation	111	82.8% (n = 134)
After electrical stimulation	3	2.8% (n = 109)
After the recovery	54	70.1% (n = 77)

n: the total number of cells observed.

These observations showed that the actin filaments in pulvini motor cells were fragmented during petiole bending that was induced by electrical stimulation, which was consistent with the effect seen when M. pudica pulvini were stimulated by touching with an ice-cold spatula.⁷

Actin cytoskeleton and calcium are involved in contraction of pulvinus motor cell protoplasts that is induced by electrical stimulation.

The bending of M. pudica petioles is due to a rapid change in volume of the abaxial motor cells of the pulvinus. Therefore, we prepared the protoplasts from motor cells of M. pudica pulvini. The motor cell protoplasts contracted rapidly in response to the electrical stimulation (see Suppl. video 2), which indicated that it was feasible to use the protoplasts in further experiments.

To ascertain whether the actin cytoskeleton was also involved in the contractile movements of the motor cell protoplasts, various actin- and calcium-related reagents were applied and the percentages of protoplasts that showed contractile movement in response to electrical stimulation were calculated (Table 2). Without treatment, ∼84.6% of protoplasts showed contractile movement in response to electrical stimulation. No obvious changes were observed when the protoplasts were pretreated with 2 µM latrunculin A, compared with the controls (Table 2). However, treatment with 100 µM phalloidin completely inhibited the contractile movement (Table 2). Therefore, actin depolymerization plays an important role in the contractile movement of the protoplasts.

Table 2.

The contractile movement of the motor cell protoplasts and changes in the cytosolic level of calcium that was induced by electrical stimulus under various treatments

Reagents of treatments	Contractile movement (%)	Increase of [Ca2+]cyt (%)	Increase of [Ca2+] in the tannin vacuoles (%)
2 µM Latrunculin A	80.3% (53/66)	88.6% (31/35)	82% (9/11)
1 µM phalloidin	0% (0/55)	0% (0/30)	0% (0/8)
10 mM EGTA	5% (2/40)	0% (0/30)	0% (0/6)
200 µM Ruthenium Red	6.4% (3/47)	0% (0/30)	0% (0/6)
2 µM Latrunculin A +
200 µM Ruthenium Red	86.3% (44/51)	90% (40/44)	78% (7/9)
2 µM Latrunculin A +
10 mM EGTA	11.4% (5/44)	0% (0/30)	0% (0/9)
1 µM phalloidin +
200 µM Ruthenium Red	0% (0/33)	0% (0/30)	0% (0/7)
Without treatment	84.6% (55/65)	-	70% (7/10)

The data are presented as percentages (the number of cells with contractile movement or increase of [Ca²⁺]/the total number of cells observed).

In addition, when 10 mM EGTA was added to remove free calcium, the percentage of motor cell protoplasts that showed contractile movement in response to electrical stimulation was reduced to 5% (Table 2). When pretreated with 200 µM Ruthenium Red, which is an inhibitor of cytoplasmic calcium channels, the percentage of motor cell protoplasts that showed contractile movement was reduced to 6.4% (Table 2). These observations suggest that both extracellular and intracellular calcium mediates the contractile movement of the motor cell protoplasts.

Furthermore, latrunculin A had a rescue effect on treatment with Ruthenium Red. About 86.3% of protoplasts showed contractile movement in response to electrical stimulation when pretreated with 2 µM latrunculin A and 200 µM Ruthenium Red (Table 2). However, no significant effect was observed if latrunculin A was added to medium that contained EGTA (Table 2). The contractile movement of the protoplasts was also completely inhibited when 1 µM phalloidin was added together with 200 µM Ruthenium Red (Table 2).

These observations indicate that both the actin cytoskeleton and calcium are involved in the contractile movement of motor cell protoplasts in response to electrical stimulation and suggest that actin depolymerization mediates the change in cytoplasmic calcium levels.

Changes in cytosolic free calcium level during contractile movement depends on actin depolymerization.

In the experiments described above, we demonstrated that both the actin cytoskeleton and calcium are important for the bending of M. pudica petioles and the contractile movement of the motor cell protoplasts. It remains to be determined how these two components are related during such movements.

In order to observe the changes in calcium level in the motor cell protoplasts, the calcium dye Fura-2 was employed to indicate the level of cytosolic-free calcium in the protoplasts. No obvious fluorescence was detected before the staining of Fura-2 (see Suppl. Fig. 1). After incubation with 5 µM Fura-2, ∼80% of the protoplasts retained contractile movement in response to electrical stimulation, which indicates that Fura-2 has no obvious effect on contractile movement. Then, the changes in cytosolic calcium level were monitored by fluorescence microscopy and recorded with a CCD camera.

When calcium was removed by the addition of 10 mM EGTA to the medium, both contractile movement and changes in [Ca²⁺]_cyt were blocked (Fig. 3A and B, and Table 2). Pretreatment of the protoplasts with 200 µM Ruthenium Red caused no obvious change in [Ca²⁺]_cyt, and the contractile movement of protoplasts was blocked after electrical stimulation (Fig. 3C and D, and Table 2). Treatment with 2 µM latrunculin A without electrical stimulation caused a dramatic increase in [Ca²⁺]_cyt, especially in the tannin vacuole (Fig. 3E and F). However, after the addition of 200 µM Ruthenium Red with 2 µM latrunculin A, the calcium level in the cytosol was increased, but not in the tannin vacuole (Fig. 3G and H). Furthermore, the addition of 10 mM EGTA to the medium that contained 2 µM latrunculin A had an inhibitory effect on the increase in [Ca²⁺]_cyt that was induced by latrunculin A (Fig. 3I and J). The change in [Ca²⁺]_cyt was completely inhibited when protoplasts were treated with 1 µM phalloidin (Fig. 3K and L, and Table 2). These observations suggest that polymerization of the actin cytoskeleton is important for the changes in [Ca²⁺]_cyt and that, in turn, the change in the level of cytosolic-free calcium mediates the contractile movement of motor cell protoplasts.

Figure 3.

Change in calcium level in motor cell protoplasts in response to electrical stimulation in the presence of calcium and actin-affecting reagents. The protoplasts were stained with 5 µM Fura-2-AM and the change in calcium level was monitored by fluorescence microscopy and recorded with a CCD camera. In the presence of 10 mM EGTA, there was no change in the level of calcium before (A) and after (B) electrical stimulation. Ruthenium Red (200 µM) also prevented any change in the level of calcium before (C) and after (D) electrical stimulation. Latrunculin A (2 µM) markedly increased the level of calcium in the cytosol and the tannin vacuole, when compared to before (E) and after (F) the treatment of latrunculin A. However, in the presence of both latrunculin A and Ruthenium Red, the calcium in the cytosol still obviously increased, but no change was observed in the tannin vacuole, when compared to before (G) and after (H) the treatment. By contrast, in the presence of both latrunculin A and 10 mM EGTA, there were no changes in the level of calcium either in the cytosol or tannin vacuole before (I) and after (J) electrical stimulation. Nevertheless, treatment with phalloidin to stabilize F-actin blocked completely any changes in the level of calcium, before (K) and after (L) electrical stimulation. Bar, 20 µm (A–L).

The results from latrunculin A treatment showed that the level of free calcium in tannin vacuoles was significantly higher than that in the cytosol (Fig. 3E and F). When an electrical stimulus was applied, the cytosolic Ca²⁺ was concentrated into the tannin vacuoles (see Suppl. video 3). It would be of interest to determine whether the movement of free calcium into tannin vacuoles is important for the contractile movement of motor cell protoplasts. When the protoplasts were pretreated with 2 µM latrunculin A, the free calcium in the cytosol increased and was concentrated in the tannin vacuoles (Fig. 4). Treatment with 200 µM Ruthenium Red inhibited the increase in Ca²⁺ level in the tannin vacuoles (Fig. 3C and D), even in the presence of 2 µM latrunculin A (Fig. 3G and H). After treatment with 200 µM Ruthenium Red followed by 2 µM latrunculin A, electrical stimulation induced an increase in calcium level in the tannin vacuoles (Fig. 4) and the contractile movement of motor cell protoplasts was restored (Table 2). These observations indicate that the movement of free calcium from the cytosol to tannin vacuoles is important for the contractile movement of motor cell protoplasts.

Figure 4.

Calcium movement in response to electrical stimulation. The protoplasts were stained with 5 µM Fura-2-AM and the change in calcium level was monitored by fluorescence microscopy and recorded with a CCD camera. Without the treatment of reagents, the cytosolic Ca²⁺ was concentrated into the tannin vacuoles. Pretreated the protoplast with 2 µM latrunculin A for 30 min, the cytosolic Ca²⁺ was remarkably decreased in response to the electrical stimulation. Pretreated the protoplast with 200 µM Ruthenium Red for 10 min to block the increase of calcium level in the tannin vacuole (see Fig. 3C and D), additional pretreatment with 2 µM latrunculin A for 30 min, however, still resulted in the movement of cytosolic Ca²⁺ into the tannin vacuole. BES: before electrical stimulation; AES: after electrical stimulation. Bar, 20 µm.

In addition, the fluorescence intensity (total calcium concentration) seems decreased after the electrical stimulation (Fig. 4). Therefore, calcium might also move out of the cells after the stimulation. On the whole, the movements of free calcium into tannin vacuoles and out of the cells are both involved.

Discussion

Actin cytoskeleton may interact with calcium channels in Mimosa pudica pulvinus bending.

Our results about the actin cytoskeleton are similar to those obtained from touching the pulvinus with an ice-cold spatula⁷ or icy water,⁶ which indicates that the actin cytoskeleton is also involved in petiole bending that is induced by electrical stimulation.

Our results show that stabilizing F-actin arrests both the increase in cytosolic calcium and the contractile movement of motor cell protoplasts, while disruption of F-actin restores the increase in calcium and the contractile movement that are inhibited by Ruthenium Red. Therefore, there is interaction between the actin cytoskeleton and changes in calcium level. Calcium channels may interact with actin. For example, the osmo-sensitive and stretch-activated calcium-permeable channels in Vicia faba guard cells are regulated by actin dynamics.¹⁵ In addition, some membrane components may contribute to the interaction between the actin cytoskeleton and calcium. For example, annexin may have some calcium channel activity and in vitro analysis has shown that annexin from Mimosa bundles actin filaments in a calcium-dependent manner.¹⁶

Our present study and reports from other groups^6,7 show that reagents that disrupt or stabilize the actin cytoskeleton reduce the bending ability of the main pulvinus. However, our experiments using motor cell protoplasts showed that treatment with latrunculin A to depolymerize actin filaments had no effect on the contractile movement of the protoplasts. How can this apparent contradiction be resolved?

The actin cytoskeleton might play multiple roles in the bending of the pulvinus in M. pudica. Two processes for pulvinus bending should be considered. First, the stimuli (including electrical stimulation) trigger the transmission of signals from the stimulation site to the pulvinus motor cells. For example, the action potential is considered an important signal. Second, the contractile movement of the motor cells is induced by the signal. Our experiments using motor cell protoplasts indicate that depolymerization of the actin cytoskeleton is necessary for the contractile movement of the motor cells. Given that there is no transmission process for the contractile movement of motor cell protoplasts that is induced by electrical stimulation, we hypothesize that actin-disrupting reagents hinder the transmission process, which results in a decrease in petiole bending.

Movement of calcium between the cytosol and tannin vacuoles during contractile movement of the motor cell protoplasts.

Although it is reported that the tannin vacuoles change their size in response to external stimuli,¹² our observations showed that change in colloidal main vacuole was evident but no apparent change in tannin vacuole were observed after the electrical stimulation (see Suppl. video 2). This contradiction could be due to different materials used in the observations. We used the protoplasts for the observations. Nevertheless, we propose that the size change of colloidal main vacuoles might be the major reason for the rapid change in volume of the abaxial motor cells.

It has been proposed that tannin vacuoles may be involved in calcium storage, and release Ca²⁺ upon mechanical perturbation or the onset of darkness.¹⁷ We showed that the level of Ca²⁺ in tannin vacuoles increased after the application of electrical stimulation. Ruthenium Red or EGTA inhibited both the increase in [Ca²⁺]_cyt upon electrical stimulation and the contractile movement of motor cell protoplasts. These observations suggest that the release of calcium from and its concentration in the tannin vacuoles may be involved in the contractile movement of motor cell protoplasts.

Treatment with latrunculin A increased the level of Ca²⁺ in both the cytosol and tannin vacuoles, but the addition of Ruthenium Red inhibited the increase in the Ca²⁺ level in tannin vacuoles but not in the cytosol. Therefore, the calcium channels in tannin vacuoles may be involved in the increase in the level of Ca²⁺ that is induced by latrunculin A. However, upon electrical stimulation, both the increase in Ca²⁺ in tannin vacuoles and the contractile movement of the protoplasts were restored by the addition of latrunculin A and Ruthenium Red. This observation indicates that the increase in Ca²⁺ level in tannin vacuoles upon electrical stimulation cannot be inhibited by Ruthenium Red. Therefore, calcium may move into tannin vacuoles via means other than calcium channels after electrical stimulation. For instance, the calcium pumps might be involved in calcium scavenging by tannin vacuoles.¹² This hypothesis is more likely to be correct if calcium moves into tannin vacuoles against its concentration gradient.

Nevertheless, treatment with phalloidin demonstrated that actin depolymerization mediated the increase in [Ca²⁺]_cyt and the contractile movement of the motor cell protoplasts. In addition, our observations of the actin cytoskeleton in motor cells showed that actin filaments are fragmented upon electrical stimulation. Furthermore, treatment with latrunculin A dramatically increased the [Ca²⁺]_cyt in motor cell protoplasts. We showed that actin depolymerization mediated the increase in [Ca²⁺]_cyt, and in turn, the contractile movement of the motor cell protoplasts upon electrical stimulation.

In conclusion, we propose that, during the contractile movement of motor cell protoplasts, the release of calcium from tannin vacuoles that is induced by electrical stimulation may proceed via calcium channels whose activity is regulated by actin dynamics, while the free cytosolic calcium returns to tannin vacuoles via calcium pumps.

Materials and Methods

Plants and protoplasts.

The seeds of Mimosa pudica L. were sterilized with 0.1% hypochlorite. Then they were soaked in warm water (30°C) for 24 h. Then they were germinated on Murashigeskoog media medium (MS). After growing for 1 week, the seedlings were transplanted into pots in a growth room, where they were grown with a 16 h light (7:00–23:00)/8 h dark cycle. During the 16 h light period, they were irradiated with light of 800–1500 µmol photons m⁻² s⁻¹ at 23°C. The plants were watered every two days. Two-month-old plants were used for the experiments.

To prepare motor cell protoplasts, pulvini were excised from the petioles of 6–10 primary leaves by using a razor blade. The pulvinal slices were placed in incubation buffer (0.5 M sorbitol, 1 mM CaCl₂, 2 mM KCl, 2 mM MgCl₂, 1% w/v Bovine serum albumin (BSA), and 25 mM MES, pH 5.6) that contained 2% w/v Cellulase Onozuka R-10 (Yakult, Japan) and 0.5% w/v Macerozyme Onozuka R-10 (Yakult), at 21°C in the dark for 2–3 h. The protoplasts were selected under a micromanipulator (BX50WI; Olympus, Japan). The selected protoplasts were sucked into a glass microneedle and transferred into a small incubation chamber on a glass slide before observation. The incubation chamber was made from a ring ∼1 cm in diameter and ∼2 mm in height, cut from the 1-mL tip, and sealed onto a glass slide with nail polish. The small chamber was filled with paraffin oil. A drop of 5 µL varying solution according to the experimental need was injected into the oil to prevent it from evaporating.

Measurements of petiole bending and protoplast contractile movement.

Electrical stimulation was used to induce petiole bending and protoplast contraction. Petiole bending was measured by following the procedure described by Fleurat-Lessard et al., (1988). Briefly, a primary leaf with petiole and stem ∼3 cm in length was excised from a Mimosa pudica L. plant. The excised branch was then set vertically in an Eppendorf tube with its tip dipped in distilled water. Electrical stimulation was applied with an electrical stimulator (JL-B; JiaLong, China). The positive electrode was positioned ∼3 cm from the base of the petiole, and the negative electrode at ∼2 cm from the base. A 9-V electrical pulse of 0.5 s duration was used to trigger petiole bending. The course of the petiole bending was recorded with a digital video camera (Cybershot DSC-S85; Sony, Japan). The bending angles were then measured on the computer with E-Ruler software (WonderWebware, USA). In each experiment, at least four petioles were measured. For each petiole, the bending movements were induced repetitively four times by the electrical stimulus at 1-h intervals. The bending angles were measured and calculated. Each experiment was repeated at least 10 times.

In order to observe the contractile movements of the protoplasts, selected protoplasts were transferred with a glass microneedle into an observation chamber with electrodes on a slide (Fig. 5) and adhered to the slide with polylysine. The slide was then placed under an Olympus microscope (BX51; Olympus, Japan) equipped with a CCD camera (CoolSNAP HQ; Olympus, Japan) immediately before observation. The contractile movements of the protoplasts were trigged by a 9-V electrical pulse of 0.5 s duration.

Figure 5.

Schematic view of the observation chamber. The double-sided adhesive tape was stuck on a glass slide. Two Ag-AgCl electrodes were placed on the adhesive tape. Then a cover slide was put on the electrodes and stuck by the adhesive tap. The sample was injected into the chamber for further treatments and observation.

Observation of F-actin.

The petioles with the stem were excised from the plants and inserted into MSB buffer (50 mM Piperazine-N',N'-bis(2-morpholino) Ethane-sulfonic fluoride (PIPES), pH 6.9, 5 mM EGTA, pH 8.0, 5 mM MgSO₄ and 0.01% Triton X-100) in Eppendorf tubes. The petioles were then placed in a growth chamber for 30 min until they recovered their original position. Electrical stimulation was then applied. The petioles were fixed before and after electrical stimulation, and after 2–4 h of recovery in their original position.

To fix the cells, MSB buffer was gently replaced by fixative (8% paraformaldehyde, pH 6.9 with MSB buffer). Fixation was carried out for 20 min with negative-pressure by a vacuum pump. After fixation, the pulvini were excised from the petioles into very thin slices by using a razor blade. The pulvinal slices were incubated with 50 nM Alexa Flour 488 phalloidin for 10 min in darkness. The samples were washed three times with MSB buffer and observed under a confocal microscope (LSM 510 META; Zeiss, Jena, Germany), using Zeiss x40 oil objectives (Plan-NEOFLUAR, NA 1.3).

Measurement of cytosolic free calcium.

The level of cytosolic free calcium was measured according to the method of Grabov and Blatt (1998). The protoplasts were placed in the incubation chamber with incubation buffer that contained 5 µM Fura-2-AM (Invitrogen, Carlsbad, CA, USA), diluted from 1 mM stock in Dimethey sulfoxide (DMSO) with incubation buffer, at 4°C for 1 h in darkness. Then they were transferred to the observation chamber. Then, the level of cytosolic free calcium was measured and recorded with an optical microscope (BX51; Olympus) that was equipped with a CCD camera (Cool SNAP HQ; Olympus) before and after electrical stimulation. The dye Fura-2 was excited at 340 nm and 380 nm, and MetaFlour software (Moclecular Devices, Sunnyvale, CA, USA) was using to control the image acquisition. Fluorescent images were acquired with a 20x objective (Olympus Plan, NA 0.50) and CCD camera, with a time scale of 10 s at intervals of 540 ms. Photometric and image analyses were carried out using MetaFlour Analyst software.

Pharmacological treatments.

Latrunculin A and phalloidin were employed to investigate the role of actin filaments in petiole bending and protoplast contraction. Nifedipine, Ruthenium Red, and LaCl₃ were used to establish whether calcium channels were involved. Latrunculin A (500 µM; Sigma, St Louis, MO, USA) dissolved in DMSO, 1 mM phalloidin (Molecular Probes, Eugene, OR, USA) in methanol, 1 M nifedipine (Sigma, USA) in DMSO, 1 mM Ruthenium Red (Merck, Darmstadt, Germany) and 10 mM LaCl₃ (Merck, Gemany) in distilled water, 1 M EGTA were made up as stock solutions, kept at 4°C, and diluted with incubation buffer to varying concentrations before the experiments. For treatment of excised branches, the incubation buffer in the Eppendorf tubes was replaced by the treatment solutions. The electrical stimulus was applied after 60 min.

Protoplasts were incubated with the reagents in the incubation chamber for 15–30 min, then transferred to the observation chamber for further observation. To determine calcium levels, protoplasts were first incubated with Fura-2 as described above, then treated with the reagents according to the experimental need. The protoplasts were then transferred to the observation chamber for further observation.

For combination treatment with Ruthenium Red and latrunculin A, protoplasts were treated with Fura-2 as described above, then incubated with 200 µM Ruthenium Red for 10 min in the incubation chamber. Then 2 µM latrunculin A was added and the protoplasts were incubated for a further 30 min. The protoplasts were then transferred to the observation chamber for further observation.