Plant Physiol. (1998) 117: 1023-1030

Second Messengers Mediate Increases in Cytosolic Calcium in Tobacco Protoplasts

Igor D. Volotovski, Sergei G. Sokolovsky, Olga V. Molchan, and Marc R. Knight^{1, *}

Institute of Photobiology, Academy of Sciences of Belarus, Academicheskaja Street 27, 220072 Minsk, Belarus (I.D.V., S.G.S., O.V.M.); and Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, United Kingdom (M.R.K.)

	ABSTRACT

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Addition of membrane-permeable cyclic GMP (cGMP) and cyclic AMP (cAMP) were shown to cause elevation of cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) in tobacco (Nicotiana plumbaginofolia) protoplasts. Under the same conditions these cyclic nucleotides were shown to provoke a physiological swelling response in the protoplasts. Nonmembrane-permeable cAMP and cGMP were unable to trigger a detectable [Ca²⁺]_cyt response. Cyclic-nucleotide-mediated elevations in [Ca²⁺]_cyt involved both internal and external Ca²⁺ stores. Both cAMP- and cGMP-mediated [Ca²⁺]_cyt elevations could be inhibited by the Ca²⁺-channel blocker verapamil. Addition of inhibitors of phosphodiesterases (isobutylmethylxanthine and zaprinast) and the adenylate cyclase agonist forskolin to the protoplasts (predicted to elevate in vivo cyclic-nucleotide concentrations) caused elevations in [Ca²⁺]_cyt. Addition of the adenylate cyclase inhibitor 2,5-dideoxyadenosine before forskolin significantly inhibited the forskolin-induced [Ca²⁺]_cyt elevation. Taken together, these data suggest that a potential communication point for cross-talk between signal transduction pathways using cyclic nucleotides in plants is at the level of Ca²⁺ signaling.

	INTRODUCTION

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Environmental and hormonal signals regulate various physiological processes in plants via signal transduction pathways. Essential components of signal transduction pathways include second messengers. Vital roles for the second messengers Ca²⁺, cAMP, cGMP, IP₃, and 1,2-diacylglycerol were first discovered in animal systems (Berridge and Irvine, 1984; Nishizuka, 1984). The last decade has been marked by substantial progress in elucidating the mechanisms of intracellular signaling in plants (Gilroy and Trewavas, 1994). Attempts to draw comparisons with mammalian systems have been successful in showing that Ca²⁺-calmodulin, cGMP (Neuhaus et al., 1993), and phosphoinositide signal mechanisms (Alexandre et al., 1990; Allen at al., 1995) also exist in plants, although cAMP involvement in transduction pathways has remained controversial (Assmann, 1995; Bolwell, 1995).

It is known that a number of chemical and physical stimuli mediate their effects via transient increases in the concentration of intracellular free Ca²⁺ (Gilroy and Trewavas, 1994). Ca²⁺ influx into the cytosol can occur because of the opening of Ca²⁺ channels in plasma membrane, with Ca²⁺ entering the cell down a concentration gradient. Receptor activation, at least in mammalian cells, can trigger the phosphoinositide cascade, which leads to the production of IP₃ and 1,2-diacylglycerol (Berridge and Irvine, 1984). IP₃ can then provoke the release from the internal Ca²⁺ stores of plants cells by opening intracellular Ca²⁺ channels (Alexandre et al., 1990; Gilroy et al., 1990; Allen et al., 1995). Increases in [Ca²⁺]_cyt produced either by influx or release from internal Ca²⁺ stores stimulate the phosphorylation of proteins within the cell (Poovaiah and Reddy, 1990). An increase in [Ca²⁺]_cyt detected by Ca²⁺ microelectrodes, Ca²⁺-sensitive fluorescence probes, and extrinsic and intrinsic aequorin (Cobbold and Rink, 1987; Chae et al., 1990; Knight et al., 1991; Shacklock et al., 1992), followed by the alteration of the activities of intercellular targets, enzymes, genes, pumps, and channels, have all been demonstrated in plants (Fallon et al., 1993; Neuhaus et al., 1993; Bowler et al., 1994a). In some circumstances activated plant cells can display repeated Ca²⁺ spikes or oscillations (McAinsh et al., 1995) and spreading Ca²⁺ waves (Shacklock et al., 1992; Campbell et al., 1996). Such elevations in [Ca²⁺]_cyt have been shown to be involved in several signaling pathways triggered, for example, by light via phytochrome (Neuhaus et al., 1993; Bowler et al., 1994a, 1994b), by phytohormones (Felle, 1988; Gehring et al., 1990), and even by mechanical signals and cold shock (Knight et al., 1991, 1992; Haley et al., 1995; Campbell et al., 1996). The multifunctional role of Ca²⁺ suggests that it participates in many signaling pathways in the plant cell. A simple but effective method for measuring changes in [Ca²⁺]_cyt in whole plants has been developed (Knight et al., 1991). This involves the expression of the Ca²⁺-activated photoprotein aequorin in transgenic plants (Knight et al., 1993; Knight and Knight, 1995). This approach can be adapted for use in protoplasts that provide a superlative system for calibration of the Ca²⁺ signal (Haley et al., 1995; Knight et al., 1996).

The data presented in this paper show that increases in intracellular concentrations of the second messengers cGMP and cAMP (Tepper et al., 1995) provoke increases in [Ca²⁺]_cyt in plant cells. This suggests that a potential communication point for cross-talk between signal transduction pathways using these second messengers (Bowler et al., 1994b) is the release of intracellular and extracellular Ca²⁺ stores.

	MATERIALS AND METHODS

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Plant Material and Protoplast Isolation

2

1

1

Aequorin Reconstitution and [Ca²⁺]_cyt Monitoring

Protoplast Swelling

	RESULTS

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All in vivo [Ca²⁺]_cyt measurement experiments were repeated at least three times. The traces presented were taken from the replicates and represent luminescence over time. The size of the luminescence peak and the luminescence kinetics are governed not only by the rate and concentration of [Ca²⁺]_cyt, but also by the absolute amount of aequorin present in the cells. Higher or lower amounts of aequorin will give larger or smaller peaks in response to the same changes in Ca²⁺. If the consumption of aequorin becomes significant, then a smaller-than-expected peak is obtained. All of these factors are taken into account when the peak [Ca²⁺]_cyt values are calibrated. Thus, these calibrated [Ca²⁺]_cyt values may be compared between treatments in the same experiment. For the reasons outlined above, however, no qualitative comparisons can be made between experiments or treatments based on magnitude and kinetics of luminescence alone. Addition of 2 mM extracellular CaCl₂ to the protoplasts (Fig. 1, A and B) provoked increases of [Ca²⁺]_cyt of about 0.2 to 0.3 µM, indicating successful in vivo reconstitution of aequorin. Regardless of whether this extracellular Ca²⁺ was added before or after second messengers, its addition produced maximum [Ca²⁺]_cyt values that were quite similar (data not shown).

View larger version (18K): [in this window] [in a new window]   Figure 1. Influence of db-cGMP (A) and db-cAMP (B) on Ca2+-dependent chemiluminescence of aequorin in protoplasts isolated from transgenic tobacco leaves (traces from chart recorder). Applications of stimuli are shown by arrows. Aliquots (200 µL) of cyclic mononucleotides diluted in buffer to a final concentration of 10 µM, were added to the suspension (0.5 mL) of protoplasts (2 × 105 cells mL1) in 0.5 M mannitol, 20 mM Hepes, pH 7.0, 2 mM MgCl2, and 0.1 mM EGTA. Before the addition of cyclic mononucleotides, CaCl2 solution (200 µL) was added to give a final CaCl2 concentration of 1 mM (shown by arrows). C, Peak [Ca2+]cyt values after the addition of cyclic mononucleotides and Ca2+ to protoplasts were calculated after chemiluminescence discharge under the action of 0.1% Triton X-100/100 mM CaCl2, as described previously (Cobbold and Rink, 1987; Knight and Knight, 1995). Experiments were performed seven times; the error bars on histograms indicate SD.

The effects of db-cGMP and db-cAMP on [Ca²⁺]_cyt levels of tobacco protoplasts were tested. As can be seen in Figure 1, A and B, the addition of either of these second messengers at 10 µM provoked large transient increases in [Ca²⁺]_cyt. db-cGMP caused a transient [Ca²⁺]_cyt elevation of around 0.8 µM, lasting for approximately 40 s (Fig. 1, A and C). db-cAMP caused a transient [Ca²⁺]_cyt elevation of around 0.7 µM, lasting for approximately 100 s (Fig. 1C). Figure 2, C and D, demonstrates the effect of 8-Br-cGMP and 8-Br-cAMP. These experiments were performed on protoplasts suspended in Ca²⁺-deficient medium to compare the results obtained in Figure 1 (in medium containing Ca²⁺) and hence gauge the involvement of external/internal Ca²⁺ stores in the [Ca²⁺]_cyt responses to cAMP/cGMP. Both types of cyclic-nucleotide derivatives caused similar transient [Ca²⁺]_cyt elevations, unequivocally indicating that the mononucleotide parts of the molecules were responsible. The transients also lasted about 40 and 100 s, respectively. Both db-cGMP and db-cAMP were able to produce a [Ca²⁺]_cyt elevation in Ca²⁺-free medium (Fig. 2, A and B), strongly suggesting Ca²⁺ release from internal stores of ion. There was substantial variability in the calibrate cyclic-nucleotide-mediated [Ca²⁺]_cyt peak values between experiments performed under the same conditions (compare Figs. 2 and 3). Also, the apparent relative potency of the cAMP and cGMP varied (compare Figs. 2 and 3). This is most likely because of physiological differences between protoplasts from different preparations leading to differences in signaling. Practically, this means that the relative quantitative contributions of intracellular/extracellular stores cannot be gauged. Qualitatively, it is clear from our results that both types of Ca²⁺ stores are involved, although the 8-Br- derivatives were less potent than the db- derivatives at concentrations that mediated a [Ca²⁺]_cyt increase (Fig. 2). This difference might be attributable to nuances in uptake efficiencies of the two derivative forms of cAMP/cGMP.

View larger version (21K): [in this window] [in a new window]   Figure 2. Influence of db-cGMP (A), db-cAMP (B), 8-Br-cGMP (C), and 8-Br-cAMP (D) on Ca2+-dependent chemiluminescence of aequorin in protoplasts isolated from transgenic tobacco leaves and suspended in Ca2+-deficient medium (traces from chart recorder). The conditions of the experiments were the same as in Figure 1. E, Peak [Ca2+]cyt values after the addition of cyclic mononucleotides and Ca2+ to protoplasts were calculated after chemiluminescence discharge under the action of 0.1% Triton X-100/100 mM CaCl2 as described previously (Cobbold and Rink, 1987; Knight and Knight, 1995). Experiments were performed seven times; the error bars on histograms indicate SD.

View larger version (12K): [in this window] [in a new window]   Figure 3. Dose dependence of [Ca2+]cyt elevation in protoplasts to db-cGMP () and db-cAMP () concentration without any external Ca2+.

Figure 3 shows a dose-response curve for cyclic-nucleotide concentration plotted against peak [Ca²⁺]_cyt values as a result of intracellular Ca²⁺ release (this experiment was performed in the absence of external Ca²⁺). The concentration dependence reached a plateau somewhere between 1 and 10 µM for both cyclic mononucleotides (Fig. 3). The ability of cyclic nucleotides to cause [Ca²⁺]_cyt elevations was further demonstrated by the use of membrane-permeable modulators of anabolic and catabolic pathways of cyclic-mononucleotide metabolism. [Ca²⁺]_cyt elevations were observed with the addition of the cyclic-mononucleotide phosphodiesterase inhibitor IBMX (Fig. 4A), the cGMP phosphodiesterase inhibitor zaprinast (Fig. 4B), and the adenylate cyclase activator forskolin (Fig. 4C). When the adenylate cyclase inhibitor DDOA was added before forskolin, the forskolin-mediated [Ca²⁺]_cyt increase was inhibited (Fig. 4D). It seems that Ca²⁺-sensitive chemiluminescence of cytosolic aequorin can be induced by the changes in the endogenous cyclic-mononucleotide concentrations, stimulating their formation or inhibiting their hydrolysis.

View larger version (28K): [in this window] [in a new window]   Figure 4. Effect of IBMX (A), zaprinast (B), forskolin (C), and DDOA plus forskolin (D) on [Ca2+]cyt in tobacco protoplasts as measured by aequorin chemiluminescence. The compounds were added to the suspension of tobacco protoplasts at the indicated concentrations. The composition of the buffer with no Ca2+ was as described for Figure 1. E, The histograms show the peak [Ca2+]cyt values obtained for each treatment. The error bars on the histograms indicate SD.

Figure 5 shows the correlation between the concentration of forskolin added and the [Ca²⁺]_cyt responses obtained. The most pronounced effect on [Ca²⁺]_cyt was observed at 20 µM, a concentration that is consistent with the concentration range of optimal activity of forskolin in animal cells (Siamon et al., 1981). Higher concentrations of forskolin seem to have inhibitory effects (Fig. 5), most likely because of cell toxicity at these concentrations.

View larger version (9K): [in this window] [in a new window]   Figure 5. Dose dependence of [Ca2+]cyt elevation in tobacco protoplasts to forskolin concentration under the same conditions as described for Figure 1.

As described previously (Haley et al., 1995), additions to protoplasts in solution may result in the so-called "touch" effect, a consequence of mechanical induction of Ca²⁺ release from intracellular Ca²⁺ stores. To minimize the touch effect, Ca²⁺ solution was added to protoplast suspensions very carefully. This was verified by testing the effect of adding membrane-impermeable cAMP and cGMP to protoplasts as well as by the addition of buffer alone, including ethanol (as a pharmaceutical diluent), as controls (Fig. 6). The addition of cAMP, cGMP, or buffer alone immediately produced very rapid and small transients of cytosolic free Ca²⁺ attributable to the touch response. As can be seen in Figures 1 and 2, a rapid transient is also seen immediately upon addition of db-cGMP or db-cAMP, which can be attributed to the same phenomenon. This is clearly separate in time and magnitude from the very large and prolonged responses seen with these membrane-permeable second messengers.

View larger version (10K): [in this window] [in a new window]   Figure 6. Effects of buffer alone and water-soluble cAMP and cGMP on [Ca2+]cyt levels in tobacco protoplasts. Media for cAMP, cGMP, and buffer plus ethanol (200-µL solutions) were the same as for Figure 1. Peak [Ca2+]cyt values after the addition of cyclic mononucleotides and Ca2+ to protoplasts were calculated after chemiluminescence discharge under the action of 0.1% Triton X-100/100 mM CaCl2 as described previously (Cobbold and Rink, 1987; Knight and Knight, 1995).

Figure 7 shows the effect of the Ca²⁺-channel blocker verapamil on cyclic-nucleotide-mediated [Ca²⁺]_cyt increases. Verapamil inhibited the effects of db-cAMP and db-cGMP, producing lower elevations of [Ca²⁺]_cyt. The inhibiting action of verapamil on db-cAMP/db-GMP-induced [Ca²⁺]_cyt elevation was more pronounced for cGMP than for cAMP (Fig. 7C).

View larger version (18K): [in this window] [in a new window]   Figure 7. Inhibition of db-cGMP-induced (A) and db-cAMP-induced (B) [Ca2+]cyt elevation by verapamil in Ca2+-deficient medium. C, Percentage of inhibition by verapamil. Experiments were performed three times; the error bars on histograms indicate SD.

To understand the signaling role of Ca²⁺ in plants in relation to the involvement of cyclic nucleotides as the messengers, the physiological action of protoplast swelling was investigated. Figure 8 shows that the time course of protoplast swelling was caused by db-cAMP and db-cGMP. According to our results, a 15-min incubation was sufficient for the curves to reach a plateau corresponding to about a 10% and 15% increase in volume for db-cGMP and db-cAMP, respectively.

View larger version (11K): [in this window] [in a new window]   Figure 8. Time course of tobacco protoplast swelling after treatment with db-cGMP and db-cAMP in complete darkness at 22°C, expressed as percent volume change (V,%). Protoplasts were suspended in the media described for Figure 1. Cyclic mononucleotides were added at a concentration of 10 µM and incubation times varied as shown. The volume changes were determined as described in ``Materials and Methods''. The percent volume change is relative to the control without cyclic mononucleotides. Each point represents the mean of at least three separate experiments; the error bars indicate SE.

	DISCUSSION

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The data presented in this paper show that second messengers such as cGMP and cAMP can provoke [Ca²⁺]_cyt elevations in tobacco protoplasts. In the case of cAMP and cGMP, the targets of action are likely to be intracellular, because even though membrane-permeable analogs at concentrations as low as 0.1 µM provoked a large [Ca²⁺]_cyt response (Fig. 1, A and B), membrane-impermeable cAMP and cGMP did not produce any significant effect (Fig. 6). An intracellular site of action is also supported by the fact that a significant [Ca²⁺]_cyt response was induced by db-cGMP and db-cAMP in the absence of external Ca²⁺. Protoplasts in EGTA (zero external Ca²⁺) still showed [Ca²⁺]_cyt responses to these second messengers, implicating the release of Ca²⁺ from internal stores as a mechanism for this response. Although variability was seen between protoplast preparations, cyclic-nucleotide-mediated [Ca²⁺]_cyt increases were generally higher in the presence of external Ca²⁺. This suggests that cyclic mononucleotides may also trigger changes in the Ca²⁺ permeability of the plasma membrane and thus cause a Ca²⁺ influx. The results obtained with verapamil (Fig. 7) indicate that a significant proportion of the [Ca²⁺]_cyt responses occurs via the activation of verapamil-sensitive Ca²⁺ channels.

Similar [Ca²⁺]_cyt elevations were also mediated by the addition of 8-Br-cAMP and 8-Br-cGMP, which also penetrate membranes very easily (Fig. 2, C and D). The [Ca²⁺]_cyt responses stimulated by these two types of derivative of membrane-permeable cyclic nucleotides were relatively large, much greater in magnitude, and much more prolonged than the touch [Ca²⁺]_cyt response caused by the mechanical stimulation resulting from the addition of solutions to the protoplasts (Fig. 6).

[Ca²⁺]_cyt elevation in protoplast cytoplasm was also induced by manipulating endogenous cyclic-mononucleotide levels. The addition of various modulators controlling cyclic-nucleotide metabolism to protoplasts produced significant changes in [Ca²⁺]_cyt. As can be seen in Figures 4 and 5, compounds that should increase endogenous cyclic-nucleotide concentration (forskolin, IBMX, and zaprinast) produced increases in [Ca²⁺]_cyt. The interesting result that is consistent with the above explanation was obtained when DDOA was used before forskolin. Forskolin stimulates adenylate cyclase within the cell and, consequently, increases the concentration of endogenous cAMP. It seems likely that DDOA competitively inhibits the adenylate cyclase and prevents its stimulation by forskolin (Fig. 4, C and D).

In addition to [Ca²⁺]_cyt elevation induced by db-cAMP and db-cGMP, the physiological response of protoplast swelling was observed at the same concentrations of cyclic mononucleotides (Fig. 8). These data are consistent with those obtained by other authors (Kim et al., 1986; Bossen et al., 1988; Gilroy and Trewavas, 1994). It has also been shown that protoplast swelling is under the control of red light absorbed by phytochrome (Bossen et al., 1988; Chung et al., 1988; Shacklock et al., 1992). It has been shown that the release of caged Ca²⁺ can cause protoplast swelling in the absence of light (Shacklock et al., 1992). Therefore, it is possible that cyclic-nucleotide-induced increases in [Ca²⁺]_cyt mediate protoplast swelling. Furthermore, it is possible that the phenomenon described in this paper is relevant to the photophysiology of phytochrome action, i.e. signal transmission from the photoreceptor to target elements within the cell (Barnes et al., 1997), where cyclic mononucleotides have been shown to be downstream signaling molecules (Bowler at al., 1994b).

A key area that needs to be understood concerns the mechanism by which these second messengers bring about Ca²⁺ release from intracellular Ca²⁺ stores into cytoplasm. It could be indirect via effects on protein kinases (Stone and Walker, 1995), or possibly by direct action on ion channels (Ward et al., 1995). The first possibility is very important because the activities of many plant cell proteins are modulated by phosphorylation (Ranjeva and Boudet, 1987). The activities of cGMP- and cAMP-dependent protein kinases have been detected in animals (Krebs, 1985). There is also some evidence for cyclic-mononucleotide-regulated kinases in plant cells (Komatsu and Hirano, 1993). However, unlike the situation in mammalian cells, the involvement of cAMP still needs to be unequivocally elucidated in plants (Bolwell, 1995). Generally, the problem is that the low levels of cAMP present in plant cells means that cAMP-dependent processes that are detected experimentally are viewed with suspicion. However, analogs and modulators of cAMP metabolism have been shown to have physiological effects in plant cells (Assmann, 1995). The data presented here suggest the possibility of a role for cAMP in the control of Ca²⁺ release from intracellular stores in plant cells. The second potential targets for cyclic mononucleotides, at least in animals, are ion channels, which can be directly controlled by cyclic mononucleotides, e.g. in visual and olfactory cells in animals (Fesenko et al., 1985; Delgado et al., 1991). Cyclic mononucleotides in these cases have been shown to be bound directly to specific binding sites located at the components of so-called cGMP- and cAMP-dependent ion channels.

Environmental, chemical, and hormonal stimuli that are capable of inducing changes in [Ca²⁺]_cyt are characterized by individual Ca²⁺ signatures with different amplitudes, kinetics, and spatial distribution, which, it has been suggested, allow cells to distinguish between stimuli (Gilroy and Trewavas, 1994; Haley et al., 1995; Campbell et al., 1996). Thus, stimulus-induced changes in [Ca²⁺]_cyt may be transient, sustained, or oscillatory (Knight et al., 1991; McAinsh et al., 1995; Campbell et al., 1996). Typically, these [Ca²⁺]_cyt responses can last from a few seconds to several hours. It is generally accepted that changes in [Ca²⁺]_cyt are closely associated with transduction of the biological action of auxin, ABA, cytokinins, gibberellic acid, fungal elicitors, and light (Gilroy and Trevawas, 1994; Bush, 1995). The involvement of second messengers other than Ca²⁺ in these pathways has not been demonstrated, although cGMP involvement in phytochrome-mediated chs gene expression has been shown (Bowler et al., 1994a, 1994b). cGMP was shown to substitute for the action of red light in the phytochrome-dependent regulatory control of anthocyanin synthesis (Neuhaus et al., 1993; Bowler et al., 1994a). It is clearly possible that cyclic nucleotides (Bolwell, 1995) and metabolites from phosphoinositide cycles are involved in many other plant signal transduction pathways (Alexandre et al., 1991; Allen et al., 1995). The major challenge for the future will be to determine whether cyclic-nucleotide-mediated [Ca²⁺]_cyt increases are actually used in bona fide signal transduction pathways in plants.

	FOOTNOTES

   Received January 5, 1998; accepted April 17, 1998.

	ABBREVIATIONS

Abbreviations: 8-Br-cAMP, 8-brom-cyclic AMP. 8-Br-cGMP, 8-brom-cyclic GMP. [Ca²⁺]_cyt, cytosolic Ca²⁺ concentration. db-cAMP, 2-O-dibutyryl AMP. db-cGMP, 2-O-dibutyryl GMP. DDOA, 2,5-dideoxyadenosine. IBMX, 3-isobutyl-1-methylxanthine. IP₃, inositol 1,4,5-trisphosphate.

	ACKNOWLEDGMENTS

We thank Prof. A.A. Milutin (Sakcharov's College, Minsk), who donated coelentrazine, and Dr. V.B. Gavrilov (Institute of Photobiology, Academy of Sciences of Belarus, Minsk) for the use of the chemiluminometer.

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