Plant Physiol. (1999) 121: 273-280

Elevation of the Cytosolic Free [Ca²⁺] Is Indispensable for the Transduction of the Nod Factor Signal in Alfalfa¹

Hubert H. Felle^*, Éva Kondorosi, Ádám Kondorosi, and Michael Schultze

Botanisches Institut I, Justus-Liebig-Universität, Senckenbergstrasse 17, D-35390 Giessen, Germany (H.H.F.); Institut des Sciences Végétales, Centre National de la Recherche Scientifique, Avenue de la Terrasse, F-91198 Gif-sur-Yvette, France (E.K., A.K., M.S.); and Institute of Genetics, Biological Research Center, Hungarian Academy of Sciences, P.O. Box 521, H-6701 Szeged, Hungary (A.K.)

	ABSTRACT

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In root hairs of alfalfa (Medicago sativa), the requirement of Ca²⁺ for Nod factor signaling has been investigated by means of ion-selective microelectrodes. Measured 50 to 100 µm behind the growing tip, 0.1 µM NodRm-IV(C16:2,S) increased the cytosolic free [Ca²⁺] by about 0.2 pCa, while the same concentration of chitotetraose, the nonactive glucosamine backbone, had no effect. We demonstrate that NodRm-IV(C16:2,S) still depolarized the plasma membrane at external Ca²⁺ concentrations below cytosolic values if the free EGTA concentration remained low (0.01 mM). Externally added Sr²⁺ was able to replace Ca²⁺, and to some extent even enhanced the Nod-factor-induced depolarization, whereas with Mg²⁺ it was decreased. This suggests that the Nod factor response is triggered by Ca²⁺ from external stores. The addition of the endomembrane Ca²⁺-ATPase inhibitor 2,5-di(t-butyl)-1,4-benzohydroquinone, which presumably mobilizes Ca²⁺ from Ins(1,4,5)P₃-sensitive stores, mimicked the Nod factor response, i.e. increased the cytosolic free [Ca²⁺], triggered Cl-efflux, depolarized the plasma membrane, and alkalized the root hair space. In all cases a refractory state toward Nod factor perception was produced, indicating a shortcut of Nod factor signal transduction by releasing Ca²⁺ from internal stores. These latter results strongly support the idea that an elevation of cytosolic free [Ca²⁺] is indispensable for the transduction of the Nod factor signal, which is consistent with the role of Ca²⁺ as a second messenger.

	INTRODUCTION

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In legumes, nitrogen fixation takes place in highly specialized root organs (nodules) that result from symbiotic interaction between the host plant and soil bacteria known as rhizobia. During early stages of this association, molecular signals are synthesized by the rhizobia that are essential for initiating morphogenetic responses in the host plant. These signals, Nod factors, are lipochitooligosaccharides that are highly host specific due to their distinct structural modifications in different rhizobial species (Lerouge et al., 1990; Felle et al., 1995; Long, 1996; Schultze and Kondorosi, 1998).

Early events of Nod factor signaling are cytosolic pH changes (Felle et al., 1996) and delayed depolarization of the plasma membrane (Ehrhardt et al., 1992; Felle et al., 1995; Kurkdjian, 1995). Using ion-selective microelectrodes, we have demonstrated recently that the most rapid response to Nod factors is a Ca²⁺ influx from the root hair space into the cell. This triggers a number of events such as the activation of anion channels through which the cells lose Cl rapidly, causing depolarization followed by K⁺ efflux (Felle et al., 1998). The Ca²⁺ channel antagonist nifedipine inhibited the Nod-factor-induced ion fluxes, and the Ca²⁺ ionophore A₂₃₁₈₇ mimicked the Nod factor responses, suggesting a role of Ca²⁺ in Nod signal transduction (Felle et al., 1998). A role of Ca²⁺ in Nod factor signaling has also been suggested by the absence of Ca²⁺ spiking in a nodulation-defective alfalfa mutant (Ehrhardt et al., 1996). Gehring et al. (1997) and DeRuijter et al. (1998) have demonstrated Nod-factor-induced elevation of the cytosolic free [Ca²⁺] in root hair tips. However, since these changes would primarily be connected with tip growth, whereas the experiments in this study were carried out well behind the tip, we did not consider their observations to be relevant for our investigations.

Testing the cytosolic free [Ca²⁺] within the root hairs of alfalfa provided evidence that the [Ca²⁺] increased in response to Nod factors; however, due to technical problems, this increase could not be brought into a clear temporal relationship with the Ca²⁺ influx, the Cl efflux, or the depolarization. Taking this difficulty into account, we argued that the changes in cytosolic [Ca²⁺] need not necessarily spread across the entire cell, but could occur essentially in pockets, e.g. close to either vacuolar or endoplasmatic Ca²⁺ pools. Still, it left the question open as to what extent the concentration of cytosolic free Ca²⁺ is part of Nod factor signal transduction. To test this, we manipulated the cytosolic free [Ca²⁺] using an inhibitor of endomembrane Ca²⁺ ATPases, 2,5-di(t-butyl)-1,4-benzohydroquinone (BHQ), for its effect on the transduction of the Nod signal. Here we demonstrate that BHQ at submicromolar concentrations increases the cytosolic free [Ca²⁺] and strongly inhibits the transduction of the Nod signal, thus contributing to the idea that cytosolic Ca²⁺ is an important element in Nod factor signaling.

	MATERIALS AND METHODS

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General Assay Conditions

Ion-Selective Microelectrodes

	RESULTS

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Influence of External [Ca²⁺] on Nod-Factor-Induced Depolarization

View larger version (18K): [in this window] [in a new window]   Figure 1. Maximal depolarizations of alfalfa root hairs in response to 0.1 µM NodRm-IV(C16:2,S), measured in different Ca2+ solutions (pCa) with and without EGTA, as indicated. Points are from single experiments (, ) or mean values (, ±SE); numbers in brackets refer to the number of experiments. Shaded area compares the effect of NodRm-IV(C16:2,S) on membrane depolarization in the presence of 0 (), 0.1 (), and 0.01 () mM EGTA at equal free [Ca2+].

To decrease free Ca²⁺ concentrations to below 1 µM, it was necessary to add the Ca²⁺ chelator EGTA. EGTA/Ca²⁺ solutions were calculated and the final [Ca²⁺] checked in the root hair space with a Ca²⁺-selective microelectrode. As shown in Figure 1, the free EGTA in these solutions played a critical role in the response of the root hairs to the Nod factor. Whereas in the presence of 0.1 mM free EGTA the depolarizations were clearly lower than in solutions without EGTA, the response was equal or even slightly better in the presence of 0.01 mM free EGTA. Solutions completely free of Ca²⁺ (excess EGTA) were not used because the recordings became unstable, probably due to an unspecific increase in membrane conductivity.

Sr²⁺, acting in a manner similar to Ca²⁺ (Bauer et al., 1998), can replace Ca²⁺. When 0.1 mM Sr²⁺ was added to the solution in which only a small response to 0.1 µM NodRm-IV(C16:2,S) was recorded (pCa 6.75, 0.1 mM free EGTA), the Nod factor substantially depolarized the root hairs (Fig. 2a). Decreasing the free EGTA to 0.01 mM stimulated the response to the Nod factor (Fig. 2a, compare tracks 1 and 4). In the presence of 0.1 mM Sr²⁺ the response was even stronger than the control without Sr²⁺ (track 3), whereas Mg²⁺ inhibited the response (track 5). In solutions with no EGTA added, Sr²⁺ generally induced stronger Nod factor responses than Ca²⁺ did at comparable concentrations (Fig. 2b).

View larger version (17K): [in this window] [in a new window]   Figure 2. Membrane potential (Em) response of alfalfa root hairs to NodRm-IV(C16:2,S). a, Root hairs were preincubated in the indicated pCa solutions in the presence of 0.1 mM (traces 1 and 2) or 0.01 mM free EGTA (traces 3, 4, and 5). After the addition of 0.1 mM Sr2+ or Mg2+, respectively, 0.1 µM NodRm-IV(C16:2,S) (NF) was tested. At "//" drawings of curves were interrupted for 2 to 3 min to allow for normalization of Nod factor addition. b, Depolarization response to 0.01 µM NodRm-IV(C16:2,S) either in the presence of Sr2+ (1) or Ca2+ (2) at the indicated concentrations. Kinetics are representative examples of three to five recordings each carried out under equivalent conditions.

The Nod Factor Response in the Presence of High and Low External [Ca²⁺]

View larger version (26K): [in this window] [in a new window]   Figure 3. Effect of 0.1 µM NodRm-IV(C16:2,S), NodRm-IV(C16:0,S), and chitotetraose ([GlcNAc]4) on cytosolic free [Ca2+] (pCa) and membrane potential (Em) of alfalfa root hairs. Kinetics are representative examples of 21 (NodRm-IV[C16:2,S]), three (NodRm-IV[C16:0,S]), and five ([GlcNAc]4) carried out under equivalent conditions.

As shown in Figure 4a, the addition of 20 mM Ca²⁺ to the standard solution (0.1 mM Ca²⁺) increased the cytosolic [Ca²⁺] by 0.2 to 0.3 pCa, whereas 0.1 µM NodRm-IV(C16:2,S) added thereafter only evoked a minor increase in cytosolic [Ca²⁺] and a small depolarization. Changing the external [Ca²⁺] from 10 mM to a solution with no extra Ca²⁺ added (1-2 µM free Ca²⁺) decreased the cytosolic [Ca²⁺] just slightly below the level measured in the presence of 0.1 mM Ca²⁺. Lowering the external [Ca²⁺] to pCa 7.44 (36 nM Ca²⁺; free EGTA 0.01 mM) did not change the cytosolic free [Ca²⁺] any further. Subsequent addition of 0.1 µM NodRm-IV(C16:2,S), however, caused a rapid increase in cytosolic free [Ca²⁺] in the presence of 0.01 mM EGTA.

View larger version (21K): [in this window] [in a new window]   Figure 4. Effect of external [Ca2+] on cytosolic free [Ca2+] (pCaC) and membrane potential (Em) of alfalfa root hairs before and after the addition of 0.1 µM NodRm-IV(C16:2,S). Shown is the Nod factor response in the presence of high extracellular [Ca2+] (a) and in the presence of low extracellular [Ca2+] (b). "Ca2+-free" refers to the addition of a solution with no extra Ca2+ added (i.e. 1-2 µM free Ca2+, as measured with a Ca2+-selective microelectrode within the root hair space). pCa 7.44 refers to a solution with 0.01 mM free EGTA. The initial transients on the Ca2+ traces arise from the different response times of the two electrodes. Kinetics are representative examples of four recordings each carried out under equivalent conditions.

BHQ Mimicks Nod Factor Action But Induces a Refractory State to Nod Factor Perception

View larger version (18K): [in this window] [in a new window]   Figure 5. Effect of BHQ at the indicated concentrations and subsequently added 0.1 µM NodRm-IV(C16:2,S) on cytosolic free [Ca2+] (pCaC) (a) and cytosolic [Cl] (pClC) of M. sativa root hairs (b). At "//" drawings of curves are interrupted for 2 to 3 min to allow for normalization of Nod factor addition. The initial transients on the Ca2+ or Cl traces arise from the different response times of the two electrodes. External [Ca2+] was 0.1 mM. Kinetics are representative examples of four recordings each carried out under equivalent conditions.

As shown in Figure 6, BHQ at concentrations above 1 µM transiently depolarized the root hairs, whereas submicromolar concentrations did not. After a pre-incubation with BHQ, the subsequent addition of 0.1 µM NodRm-IV(C16:2,S) in the presence of BHQ resulted in a concentration-dependent reduced depolarization.

View larger version (22K): [in this window] [in a new window]   Figure 6. Effect of BHQ at the indicated concentrations on the membrane potential (Em) of alfalfa root hairs before and after the addition of 0.1 µM NodRm-IV(C16:2,S). At "//" drawings of curves were interrupted for 2 to 3 min to allow for normalization of Nod factor addition. External [Ca2+] was 0.1 mM. Kinetics are representative examples of three to five recordings carried out under equivalent conditions. Inset, Dose-response relationship of maximal Nod factor- () and BHQ-induced depolarizations () are plotted against the BHQ concentration during the experiment.

External alkalinization is a common phenomenon observed after the application of Nod factors and elicitors. Although its exact nature has not yet been elucidated, alkalinization is a good parameter with which to test the action of both symbiotic and defense agents (Felix et al., 1993; Nürnberger et al., 1994; Felle et al., 1998). As shown in Figure 7, 10 µM BHQ caused a transient alkalinization in the root hair space. Compared with the Nod-factor-induced alkalinization, it reached the peak faster and turned into an acidification at the end; 0.1 µM NodRm-IV(C16:2,S) added subsequently in the presence of BHQ only slightly alkalized the root hair space at that time. In contrast, when these agents were added in inverse order, BHQ alkalized the root hair space in the regular manner, indicating that the mechanisms leading to alkalinization came from different sources.

View larger version (18K): [in this window] [in a new window]   Figure 7. Effect of 10 µM BHQ and 0.1 µM NodRm-IV(C16:2,S) on the pH of the alfalfa root hair space. External [Ca2+] was 0.1 mM. Results are representative of five equivalent tests each.

	DISCUSSION

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In this paper we demonstrate that external and cytosolic Ca²⁺ are important factors in Nod-factor-induced depolarization. It was interesting, however, to observe that substantial responses to NodRm-IV(C16:2,S) occurred even at external [Ca²⁺] far below the cytosolic value if EGTA did not hamper the response. According to Equation 1, this is not surprising. The electrochemical driving force for Ca²⁺ (µ_Ca/F) into the cell is given by:

(1)

where Em is the membrane potential and pCa_C and pCa_O are the negative logarithms of the free [Ca²⁺] of the cytosol (c) and the outside (o), respectively. Inserting the membrane potential and the transmembrane [Ca²⁺] gradient, Equation 1 shows that even if the external [Ca²⁺] is less than the cytosolic [Ca²⁺], the inwardly directed driving force for Ca²⁺ is still sufficiently negative to drive Ca²⁺ into the cell. Unfortunately, because of the remaining free EGTA, it was not possible to exactly pinpoint the minimal external Ca²⁺ concentration required for a response to Nod factors. In the presence of 0.1 mM free EGTA, the cells ceased to respond around 10 nM (pCa 8.0). Extrapolating from the measuring points obtained in the presence of 0.01 mM EGTA, even lower [Ca²⁺] would be sufficient to support responsiveness.

It is intriguing that the Nod factor response is apparently insensitive over a wide range of [Ca²⁺]. This is because the measuring parameter, depolarization, is a secondary response apparently induced by elevated cytosolic [Ca²⁺]. It is not yet clear how much Ca²⁺ is actually needed to trigger the response, but it appears sufficient even at concentrations below 100 nM (Fig. 1). As demonstrated in Figure 2, Sr²⁺ could replace Ca²⁺ in triggering the Nod factor response, whereas Mg²⁺ could not, supporting the notion that external Ca²⁺ and its influx into the cytosol is essential for the Nod-factor-induced depolarization. This is in line with our previous observation that nifedipine, presumably through blocking much of the Ca²⁺ influx, prevent downstream Nod factor effects (Felle et al., 1998) such as Cl efflux and subsequent depolarization (data not shown). The inhibitory effect of Mg²⁺ on the Nod factor response could be explained by obstructing Ca²⁺ from freely entering the respective channels.

We also present evidence that cytosolic [Ca²⁺] is an element of the Nod factor signal chain. BHQ obviously increased cytosolic free [Ca²⁺], transiently decreased cytosolic [Cl] (Fig. 5), alkalized the root hair space (Fig. 7), transiently depolarized the plasma membrane, and inhibited the Nod signal (Fig. 6). BHQ mimicks the Nod factor response presumably just by increasing cytosolic [Ca²⁺], which was observed using the Ca²⁺ ionophore A₂₃₁₈₇ (Felle et al., 1998). Although both BHQ and Nod factors obviously trigger similar events through increasing cytosolic free [Ca²⁺], the data in Figure 6 clearly show that it matters which mechanism leads to the elevation of cytosolic [Ca²⁺]. With Nod factors, Ca²⁺ channels at the plasma membrane are activated, which may require the involvement of G-proteins (Pingret et al., 1998), whereas with BHQ, such a step was not necessary.

Whereas BHQ is poorly characterized in plant cells, it is known from mammalian cells that BHQ is a potent inhibitor of endomembrane Ca²⁺ ATPases of the E1E2-type, and it has been proposed that it mobilizes Ca²⁺ from Ins(1,4,5)P₃-sensitive stores. Although we could clearly demonstrate that BHQ induced an increase in cytosolic [Ca²⁺], we had no way to tell whether this came from Ins(1,4,5)P₃-sensitive or other stores. However, for the questions raised here such considerations would not be important, because we used BHQ only as a convenient tool with which to manipulate cytosolic free [Ca²⁺] and to test the effect this change would have on the Nod factor response.

The question of whether Nod factors drain intracellular Ca²⁺ stores has been addressed by Pingret et al. (1998) by applying the ryanodine receptor antagonist ruthenium red. Originally characterized in animal studies (see Galione, 1994), there is convincing evidence that a ryanodine receptor also exists in the endomembranes of plants (Knight et al., 1992; Allen et al., 1995). Pingret et al. (1998) observed that ruthenium red blocked the epidermal Nod factor response in alfalfa, and inferred that the transduction of the Nod factor signal required the mobilization of intracellular Ca²⁺ stores. Although they did not directly show that Nod factors increased cytosolic [Ca²⁺] and that ruthenium red prevented it, we demonstrate in this paper that Nod factors do cause an increase of the cytosolic [Ca²⁺] of alfalfa (Figs. 3-5), and this increase is indispensable for the activation of downstream events such as the activation of anion channels (Fig. 5b). On the other hand, using EGTA (to reduce external [Ca²⁺]) and La³⁺ (to block Ca²⁺-influx), Pingret et al. (1998) demonstrated that the investigated Nod factor response was also dependenton external Ca²⁺, an observation that corresponds with our observations.

The finding that BHQ at micromolar concentrations inhibits the Nod-factor-induced depolarization (and external alkalinization) is in agreement with the idea that events that require elevated cytosolic [Ca²⁺] are refractory to another Nod factor stimulus when triggered previously through increased cytosolic [Ca²⁺]. Since we have good indications now that the main cause for the Nod-factor-induced depolarization is a Ca²⁺-triggered stimulation of the Cl efflux (Felle et al., 1998), previously elevated cytosolic [Ca²⁺] at the site of action would largely prevent such a stimulation, because BHQ, through increasing cytosolic Ca²⁺, would have already activated the anion channels. This idea is supported by the finding that BHQ itself caused Cl efflux (Fig. 5b) and transient depolarization (Fig. 6), as well as by the observation that in the presence of 10 or 20 mM external Ca²⁺, the Nod factor response was strongly decreased, probably also due to the increased cytosolic [Ca²⁺] under these conditions (Figs. 1 and 4a). In this context it is interesting that BHQ induced a refractory state for Nod factor action, but not vice versa (Fig. 7). The reason for this could be that the Nod-factor-induced increase in cytosolic [Ca²⁺] is slightly transient or, more likely, that the suppression of the Nod factor response through increased cytosolic Ca²⁺ does not occur at the anion channel but at a more upstream part of the respective transduction chain.

Regardless of these considerations, an elevation of cytosolic Ca²⁺ appears to be a prerequisite for the initiation of a cascade of events at the plasma membrane of alfalfa root hairs, leading to rapid Cl efflux and depolarization. Since the experiments with Sr²⁺ and nifedipine strongly indicate that this Ca²⁺ comes from the outside, these responses must be regarded as different from the Ca²⁺ spikes observed by Erhardt et al. (1996). Their delayed Nod-factor-induced Ca²⁺ signals very likely originate from intracellular stores around the nucleus. Both responses obviously target different stages within the framework of a complex Nod factor action. Whereas the spikes, by propagating along the root hair, may carry information from the nucleus to other parts of the cell, the more stationary Ca²⁺ changes presented in this paper, by triggering channel activities at the plasma membrane, may pave the way for the rhizobia to enter the cells. This could be initiated by localized alterations in the ion concentrations or transmembrane electrochemical ion gradients, including pH changes at the rhizobial docking site of the root hair. Both responses, however, underline the role of Ca²⁺ as a second messenger in Nod factor signaling.

	FOOTNOTES

   Received March 29, 1999; accepted May 27, 1999.

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