Systemin-induced cytoplasmic Ca2+ increase
Systemin is a polypeptide known to elicit the systemic synthesis of PIs in tomato plants (Pearce et al. 1991). Up to now the early events evoked by systemin at the plasma membrane surface are far from understood. It is supposed that systemin binds to a putative receptor and activates a lipase which leads to the release of JA. However, the steps between the binding of systemin to its receptor and the activation of the octadecanoic pathway are unclear. It has been shown that one of the first events triggered by systemin in Lycopersicon esculentum mesophyll cells is a membrane depolarization and complex effects on H+ fluxes (Moyen & Johannes 1996). Increases of extracellular pH and K+ concentration have also been reported following the addition of systemin to the bathing medium of Lycopersicon peruvianum suspension-cultured cells (Felix & Boller 1995).
In this study, we showed that in Lycopersicon esculentum mesophyll cells, systemin also triggers, within 2 min of its application, an increase in cytoplasmic Ca2+ concentration ([Ca2+]c). The choice of using tomato plants expressing apoaequorin was made to allow us to monitor [Ca2+]c changes non-invasively on mesophyll tissue because the use of protoplasts, which are required for [Ca2+]c measurements with calcium binding fluorescent dyes, has several disadvantages. For example, during protoplast isolation, the systemin receptor could be damaged. Furthermore, the cell wall digesting enzymes release elicitors such as OG which are thought to trigger defence reactions through the same signalling pathway as systemin (Farmer & Ryan 1992) and could, therefore, affect the systemin response. It has also been shown that cell cultures show a less complex response to systemin (Moyen & Johannes 1996) and to hormones such as auxin (Felle, Peters & Palme 1991) than tissues. Moreover, the cultured cell response to systemin differs with the cell line used (Felix & Boller 1995). For our experiments the pieces of mesophyll used did not contain any major vascular bundles and minor ones were usually removed when the lower epidermis was stripped off. Previous data (Moyen & Johannes 1996) have shown that in a few hours the pieces of mesophyll have recovered from the stripping of their lower epidermis and are able to react again to systemin. The number of epidermal cells is small compared with the number of mesophyll cells. Therefore, although we cannot entirely rule out that part of the response to systemin might occur in the upper epidermal cells, it can be assumed that the main effect seen represents the mesophyll cell response. A consequence of not having all the cells reacting to systemin would be an underestimation of the systemin-induced [Ca2+]c change occurring in the responding cells.
Our results show that the systemin-induced [Ca2+]c increase occurred within 1 min and reached a peak after ≈ 2 min. Our data also showed that in many cases oscillations occurred following the initial Ca2+ increase triggered by systemin. These oscillations, which have a particularly high magnitude on the example shown in 2Fig. 2b, can also be seen in many of the other figures but with a lower magnitude. One of the hypotheses for this discrepancy could be that in some recordings most of the individual cell oscillating responses occurred in phase leading to marked overall oscillations, while in other recordings the individual cell oscillating responses were not in phase or had different frequencies, therefore leading to less pronounced oscillations. Another hypothesis could be that only some types of mesophyll cells show oscillating responses. To address this question, individual cell responses would need to be recorded with a different method more sensitive than aequorin-reported calcium changes.
Oscillations of intracellular Ca2+ concentration have already been described in response to diverse stimuli and are thought to be a way of encoding information (Berridge 1990; McAinsh et al. 1995). Previous studies have also shown that some elicitors and hormones can evoke a rise of cytoplasmic Ca2+ in plant cells. Elicitors such as OG have been reported to evoke an increase in cytoplasmic Ca2+ in carrot protoplasts (Messiaen et al. 1993; Messiaen & Van Cutsem 1994) and a decrease of the Ca2+ concentration of the bathing medium of tobacco suspension cells (Mathieu et al. 1991). However, the kinetics of these OG-induced [Ca2+] changes differ strongly from the one evoked by systemin in tomato mesophyll tissue. In carrot protoplasts, the OG-induced [Ca2+]c increase started between 5 and 10 min after OG addition, and reached a concentration of more than 1 μM in 30 min (Messiaen & Van Cutsem 1994). With tobacco suspension cells, the OG-induced decrease of the external [Ca2+] reached a maximum after 50 min (Mathieu et al. 1991). Plant hormones such as abscisic acid (ABA) (Gilroy et al. 1991; Irving et al. 1992; McAinsh et al. 1992) and indol acetic acid (IAA) (Irving et al. 1992) have also been shown to trigger cytosolic [Ca2+] rises in plant cells. As shown here with systemin, ABA and IAA evoked a 1·5–3-fold increase in resting [Ca2+]c (Irving et al. 1992). Therefore, as in the cases of ABA and IAA, the systemin-induced [Ca2+]c change could be expected to be sufficient to trigger other cell responses.
JA and bestatin are ineffective on [Ca2+]c
JA and bestatin are known to induce some of the cell responses also evoked by systemin, in particular PI synthesis (Farmer & Ryan 1990; Schaller et al. 1995). More recently it has been shown that, like systemin, JA and bestatin also induce aspartic protease synthesis [Schaller & Ryan 1996; see also Schaller & Ryan (1995)] and wound-inducible polyphenol oxidase synthesis (Constabel et al. 1995). Our data show that under our experimental conditions, unlike systemin, JA and bestatin had no effect on the [Ca2+]c of tomato mesophyll cells. JA is one of the end products of the octadecanoid pathway by which the systemin signal is transduced within the plant (Farmer & Ryan 1992). Our results therefore indicate that the systemin-induced [Ca2+]c increase is a step occurring upstream of JA biosynthesis.
Bestatin is an inhibitor of aminopeptidase which induces the same group of defence genes that are activated by systemin (Schaller & Ryan 1995, 1996; Schaller et al. 1995). The absence of a bestatin effect on [Ca2+]c agrees with the conclusion of Schaller et al. (1995) that bestatin appears to be exerting its effect close to the transcriptional control of the defence gene, i.e. late in the signalling pathway.
Ca2+ mobilization from different cellular pools upon systemin addition
The systemin-induced [Ca2+]c increase is inhibited partially by compounds such as ruthenium red, LiCl and neomycin, which are three compounds thought to interfere with the release of calcium from intracellular compartments such as the vacuole. Neomycin and LiCl both affect IP3-mediated Ca2+ release (Berridge 1984; Frankling-Tong et al. 1996), whereas ruthenium red is known to block the cADPR-mediated Ca2+ release from vacuoles (Allen, Muir & Sanders 1995). The marked inhibition of the systemin-induced Ca2+ increase by these compounds argues for the involvement of a Ca2+ release from intracellular compartments which might be part of the systemin signalling pathway.
The present data also show that the systemin-induced [Ca2+]c increase was slightly but not significantly inhibited by organic blockers of voltage-gated Ca2+ channels (nifedipine, verapamil and diltiazem), and not inhibited by Cl– channel blockers (DIDS, A9C, niflumic acid) or by K+ channel blockers (TEA, CsCl; data not shown). By contrast, LaCl3, CdCl2 and GdCl3 which are known to be very potent calcium channel blockers (Tsien et al. 1987; Hess 1988) abolished almost completely the [Ca2+]c increase triggered by systemin. In our study these three channel blockers were only applied at 1 mM for 10 min before systemin was added. Considering that mesophyll tissues and not protoplasts were used as an experimental system, the short incubation time and low inhibitor concentration suggest that these antagonists exert their effect mainly on the plasma membrane and the possibility that these ions enter the cells and have an additional effect on Ca2+ mobilization from internal stores is unlikely. It should also be kept in mind that no changes in the resting [Ca2+]c have been observed during the inhibitor treatments (data not shown) as could be expected if the calcium antagonists were entering the cell and displacing Ca2+ from its intracellular binding sites. The stronger effect triggered by inorganic Ca2+ channel blockers compared with organic blockers could be attributed to the blockade of more types of channels by these cations. Indeed it has been shown in animal cells, that Cd2+ and La3+ block at least two types of voltage-activated Ca2+ channels (L- and N-types), whereas organic antagonists such as nifedipine and verapamil only block the L-type (Tsien et al. 1987; Hess 1988). It should be acknowledged that like most inhibitors, calcium channel blockers are not perfectly specific and have been shown to block some other channel types such as K+ channels in hypocotyls and root cells of Arabidopsis (Lewis & Spalding 1998). However, it should be kept in mind that even if these blockers block channels that are not specific Ca2+ channels, this would not affect our conclusions because we have measured their effect on the aequorin-reported intracellular [Ca2+]. An argument in support of that point comes from the fact that blockers of K+ channels and of Cl– channels tested in this study showed no effect on the systemin-induced [Ca2+] changes.
Thus, taken together these results suggest that some of the calcium mobilized by systemin comes from the apoplast through a non-selective Ca2+ permeable channel or simultaneously through several types of channels. It has recently been shown in parsley protoplasts that a Ca2+ permeable channel sensitive to LaCl3 is activated upon addition of a polypeptide elicitor (Zimmermann et al. 1997). Activation of non-selective Ca2+ permeable channels by abscisic acid has also been reported (Schroeder & Hagiwara 1990).
The observation that the systemin-induced Ca2+ increase occurred even when the bathing pH buffered medium of the mesophyll pieces contained EGTA does not rule out the possibility that part of the Ca2+ involved comes from the extracellular space. Indeed, the measurement of free [Ca2+] in the bathing medium of mesophyll tissues (by spectrophotometry using Arsenazo III) showed that in our experimental conditions, after a 30 min incubation with 1 mM EGTA, or after a 30 min or 60 min incubation with 10 mM EGTA the free [Ca2+] reached several micromolar (data not shown). Therefore, it can be assumed that during an incubation with EGTA, the chelation of the external calcium maintains a calcium gradient between the bathing medium and the tissues, and leads to a release of calcium from the tissues. The tissue released enough Ca2+ to get an external Ca2+ concentration near the plasma membrane which was low but sufficient to allow Ca2+ influx through a high-affinity permeable Ca2+ channel similar to the one recently described by Pineros & Tester (1997).
From the data obtained with Ca2+ channel blockers we could suggest that the systemin-induced Ca2+ mobilization occurred through a non-selective Ca2+ permeable channel or simultaneously through several types of channels. The results with EGTA would be consistent with the hypothesis that the [Ca2+] increase results from Ca2+ fluxes through several types of calcium permeable channels, one of which must be a high-affinity Ca2+ channel. The contribution of several types of Ca2+ permeable channels (and of several cellular Ca2+ pools) could also explain why the inhibition of the systemin-induced [Ca2+] changes seen in the presence of the various Ca2+ antagonists tested was never complete.
If we were to suppose that the systemin-induced calcium changes are one of the first steps of the signalling pathway leading to important defence reactions following wounding, it would not seem unreasonable for a cell to have several ways of raising its intracellular [Ca2+] as a safe mechanism to trigger vital defence processes.
This influx of external Ca2+ might then induce the release of Ca2+ from intracellular stores. Some evidence for a Ca2+-induced Ca2+ release via slowly activated vacuolar (SV) channels has been shown in plant vacuoles (Ward & Schroeder 1994; Allen & Sanders 1995). In the ABA-induced Ca2+ increase observed in guard cells, it has also been postulated that both influx of external calcium and release of calcium from intracellular stores occurred, as Ca2+ channel blockers could only partially inhibit the calcium increase triggered by ABA (McAinsh, Brownlee & Hetherington 1991; McAinsh et al. 1995). A model describing the mobilization of Ca2+ from two different pools, one IP3-sensitive and one IP3-insensitive has been proposed to explain the initiation of Ca2+ oscillations (Berridge 1990) and could account for the oscillations triggered by systemin in tomato mesophyll cells.
It is likely that the systemin-induced changes in [Ca2+]c play an important role in the signalling pathway. This is suggested by recent findings of Fisahn (1997) which implicate that elevation of [Ca2+]c is involved in the induction of wound-inducible Pin II genes. This was concluded from simultaneous recordings of membrane potential and of aequorin-emitted luminescence changes performed to correlate the electrical response and the calcium component of the long distance signal mediating the systemic induction of Pin II gene expression.
The present report does not intend to elucidate the effects of [Ca2+]c changes on the induction of wound-inducible genes. However, the direct target of the systemin-induced [Ca2+]c elevations and the physiological significance of this systemin-induced calcium increase will need to be identified in future studies keeping in mind that local changes in [Ca2+]c (e.g. in the vicinity of the plasma membrane and tonoplast) are likely to be much higher than the average values obtained from a large number of mesophyll cells.
Taken together our data suggest that systemin triggered an influx of extracellular Ca2+ simultaneously through several types of channels such as non-specific Ca2+ permeable channels and a high-affinity Ca2+ channel, and a release of Ca2+ from intracellular stores sensitive to blockers of IP3- and cADPR-mediated Ca2+ release.
Our findings of a systemin effect on tomato mesophyll cell membrane potential (Moyen & Johannes 1996) and on [Ca2+]c could also contribute to the understanding of the nature of the long distance signal involved in wounding response. Besides chemical signalling via the mobile polypeptide systemin (Bergey, Hoi & Ryan 1996), there is also evidence for electrical (Wildon et al. 1992; Stankovic & Davies 1996) and hydraulic signalling (Malone, Alarcon & Palumbo 1994). From our results, it can be expected that systemin propagation across mesophyll tissue leads to an electrical (depolarizing) wave and a calcium wave. Therefore, it is possible that in cells containing voltage-gated and Ca2+-activated channels action potential could be triggered. In this way it could be envisaged that a combination of chemical and electrical signalling is involved.
The kinetics of the systemin-induced [Ca2+] changes and depolarization (Moyen & Johannes 1996) are quite similar and could suggest a close link between these two parameters. So far it is difficult to speculate whether the depolarization is caused by calcium influx, or rapidly follows as a consequence of the changes in [Ca2+] (e.g. by Ca2+ activation of anion channels). Furthermore, other systemin-induced ion fluxes – such as H+ (Moyen & Johannes 1996) and K+ (Felix & Boller 1995) – have been shown to occur early following the systemin application and are, therefore, also very likely to contribute to the systemin-induced depolarization. The relationship between calcium uptake and membrane depolarization is an issue that will need to be specifically addressed in future experiments.