INTRODUCTION

Top Abstract Introduction Procedures Results Discussion References

Ca²⁺ is well established as a second messenger in plant signal transduction pathways, including those initiated by such stimuli as cold shock (1), heat shock, (2), touch (3), anoxia (4, 5), elicitor addition (3, 6-9), pathogen infection (10-12), hormone administration (13-16), oxidative stress (17), far red light (18), drought (19), pollen tube elongation (20, 21), and egg cell fertilization (22) (for recent reviews, see Refs. 23-26). These temporary elevations of cytosolic Ca²⁺ are believed to participate in signal propagation, resulting in activation/inhibition of such downstream effectors as protein kinases, ion channels, phospholipases, oxidases, hydrolases, and/or calmodulin-dependent enzymes (27). As anticipated, blockade of the above-stimulated Ca²⁺ influxes either inhibits or retards most, if not all, of the associated pathways.

It can be hypothesized that plant cells maintain high Ca²⁺ concentrations both in their extracellular milieus and in certain intracellular compartments to enable rapid gating of the cation into the low Ca²⁺ environment of the cytosol (23-29). Indeed, cytosolic Ca²⁺ transients in hormone/cytokine-treated animal cells have been shown to derive from both internal and external Ca²⁺ stores (27). Because Ca²⁺ channels have been identified in both plasma and internal membranes of plant cells (6, 9, 30-38), both Ca²⁺ pools should be considered as possible sources for mediating plant signaling events.

We and others have observed that elicitation of the oxidative burst in cultured plant cells generally accompanies and requires a rise in cytosolic Ca²⁺ (7-10). However, it has not been established if the induction of the oxidative burst requires Ca²⁺ influx from internal or external Ca²⁺ stores or from both in succession or simultaneously. In the case of the mechanically or osmotically stimulated oxidative burst, the cytoplasmic Ca²⁺ increase is distinctly biphasic. Thus, using aequorin-transformed tobacco cells to quantitate Ca²⁺ influx into the cytoplasm of the cell (3), Ca²⁺ peaks have been repeatedly observed ~15 s and 1.5 min after hypotonic stress (8, 39). In addition, other groups have established that both Ca²⁺ entry across the plasma membrane and Ca²⁺ release from internal stores may play roles in plant cell volume/turgor regulation (14, 23). Because this unusual pattern of Ca²⁺ flux offers the opportunity to examine the Ca²⁺ signal required for both the induction of the oxidative burst and the regulation of cellular turgor pressure, we have decided to identify the Ca²⁺ stores responsible for both phases of the Ca²⁺ influx. We report here that the first Ca²⁺ transient arises from outside the cell, whereas the second derives from intracellular stores. We further identify inhibitors that can selectively block the first, second, or both phases of Ca²⁺ flow, and we report observations regarding possible communication between the two pathways responsible for Ca²⁺ influx.

	EXPERIMENTAL PROCEDURES

Top Abstract Introduction Procedures Results Discussion References

Materials-- Ruthenium red, lanthanum chloride, gadolinium chloride, and niflumate were all purchased from Sigma/Aldrich. ⁴⁵Ca in the form of ⁴⁵CaCl₂ salt was obtained from ICN (Costa Mesa, CA), and coelenterazine was from Molecular Probes (Eugene, OR). Other chemicals used were of reagent grade or better and were obtained from major chemical suppliers.

Aequorin-transformed Tobacco Cell Suspension Cultures-- Aequorin-transformed tobacco cell (Nicotiana plumbaginifolia) suspension cultures were established from transformed seedlings and maintained as described previously (8). Two ml of packed filtered cells were transferred to 100 ml of fresh W-38 liquid medium (40) every 7 days and maintained in suspension by continuous shaking. Cells to be used ~12 h later for Ca²⁺ measurements were transferred at twice their regular cell density. Functional aequorin was reconstituted from the apoenzyme by adding 5 µl of coelenterazine dissolved in ethanol (1 µM final) to 3 ml of suspension-cultured cells at the time of transfer. Functional aequorin capable of reproducible Ca²⁺ measurements was found to develop 5 to 15 h after transfer of the cells to fresh medium and the addition of coelenterazine.

Aequorin Luminescence Measurements and [Ca²⁺] Quantitation-- Luminescence measurements were carried out in a digital luminometer (LKB Wallac model 1250, Gaithersburg, MD), as described previously (8). Briefly, 0.5 ml of coelenterazine-treated cells were transferred to the luminometer cuvette and held in suspension by mild stirring during stimulation. Luminescence data was collected 10 times/s. All inhibitors, chelators, and control solvents were added at the times indicated in the figure legends. Plant cells were hypo-osmotically shocked by diluting the suspensions 1:1 with distilled water at the times indicated in the figures. At the end of each experiment, all remaining aequorin was discharged by the addition of 0.5 mM CaCl₂ in 10% Nonidet P-40, and luminescence was continually quantitated until recordings returned to basal levels and further addition of CaCl₂/detergent solution elicited no further response. Luminescence data were then converted by computer directly to intracellular Ca²⁺ concentration using the equation described by Allen et al. (41), [Ca²⁺] = ((L/L_max)^1/3 + [118(L/L_max)^1/3]  1)/(7 × 10⁶  [7 × 10⁶(L/L_max)^1/3]), where L is the luminescence intensity at any time point, and L_max is the integrated luminescence intensity from that point to the end of the luminescence recording.

⁴⁵Ca Influx Experiments-- As above, 3 ml of suspension-cultured tobacco cells transferred at twice the density of continuously cultured cells were used for determination of ⁴⁵Ca influx as described by Atkinson et al. (10). Briefly, 0.1 µCi of ⁴⁵Ca was added to 3 ml of cells just before hypo- or iso-osmotic treatment. Inhibitors were added at the times indicated in the figure legends. ⁴⁵Ca-treated cells were then incubated with gentle shaking for 7 min before suction filtration through Whatman filter paper at 20 mm of Hg and immediate rinsing with 3 ml of normo-osmotic buffer containing 10 mM CaCl₂, 5 mM MES,¹ and 160 mM sucrose (pH 5.6) to displace any externally bound ⁴⁵Ca. Filtered and rinsed cells were then resuspended in 3 ml of rinse buffer and shaken for 20 min before suction filtering and rinsing again with 3 ml of rinse buffer. Finally, the cells were scraped into pre-weighed scintillation vials containing 3 ml of ICN Ecolite scintillation liquid. After counting the radioactivity in a Packard 1600CA liquid scintillation analyzer (Meriden, CT) and weighing the vials, the radioactivity/mg of filtered cells was calculated. Data are presented as detailed in the figure legend.

	RESULTS

Top Abstract Introduction Procedures Results Discussion References

The Biphasic Ca²⁺ Flux in Tobacco Induced by Hypo-osmotic Shock Is the Result of the Activation of Specific Ion Channels-- It was conceivable that the previously observed hypo-osmotic shock generated Ca²⁺ transients (8, 39) were not due to regulated ion channels but rather to a membrane disturbance resulting in nonspecific Ca²⁺ leaks. To evaluate this possibility, we tested the effectiveness of several broad-spectrum Ca²⁺ channel inhibitors on the induced cytosolic Ca²⁺ fluxes. Ruthenium red and the trivalent lanthanides gadolinium and lanthanum are known to block many types of Ca²⁺ channels in both plasma and sarcoendoplasmic reticular membranes of animal cells (42-44), and all three have also been used for characterization of Ca²⁺ channels and Ca²⁺-dependent processes in plant cells (5, 8, 30-34, 45, 46). As can be seen in Fig. 1, each of these inhibitors is effective at blocking both phases of Ca²⁺ flow into hypo-osmotically stimulated tobacco cells. Inhibition of Ca²⁺ influx by ruthenium red at 50 µM (Fig. 1A) is consistent with the pharmacologic data of both Allen et al. (30) and Pineros and Tester (34), who observed blockade of cyclic-ADP-ribose-activated tonoplast Ca²⁺ channels and voltage-gated plasma membrane Ca²⁺ channels, respectively, at similar concentrations. Although this effect of ruthenium red has been previously published (8), we present these data here to allow direct comparisons of the inhibitory effects of Ca²⁺ channel blockers on these Ca²⁺ fluxes. Lanthanum and gadolinium ions, which required a 10-min equilibration to achieve maximal channel inhibition, also displayed concentration dependences similar to those observed by others (5, 9, 17). At 10 mM concentration, both lanthanides inhibited greater than 80% total Ca²⁺ influx, whereas at 1 mM they primarily suppressed influx during the first Ca²⁺ peak (Figs. 1, B and C).

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Effect of ruthenium red, La3+, and Gd3+ on the hypo-osmotic shock induced Ca2+ fluxes in aequorin-transformed tobacco cells. Coelenterazine-treated aequorin-transformed tobacco cells (0.5 ml) were transferred to a luminometer cuvette after 15 min of treatment with the following inhibitors: A, ruthenium red (RR); B, LaCl3; and C, GdCl3. Final concentrations of inhibitors are indicated for each trace. Traces marked control represent data from cells not treated with any inhibitor. Luminescence was monitored, and [Ca2+]cyt was calculated as outlined under "Experimental Procedures." The cells were hypo-osmotically shocked in the cuvette by the addition of an equal volume of distilled water at the times indicated by the arrow. The data shown were collected on the same day from the same batch of cells and are representative of three independent experiments conducted on separate days with different batches of cells.

The First Pulse of Cytosolic Ca²⁺ Derives from External Ca²⁺ Pools-- To begin to identify the cellular sources of the two pulses of cytosolic Ca²⁺, we first performed manipulations that would specifically modify the flux of external Ca²⁺ across the plasma membrane. EGTA, a membrane-impermeable selective chelator of Ca²⁺ ions, was added to the extracellular medium at a concentration of 1.5 mM, approximately the concentration of free Ca²⁺ in the medium. As shown in Fig. 2A, chelation of extracellular Ca²⁺ with EGTA exerts an inhibitory effect selectively on the first Ca²⁺ spike generated by hypo-osmotic shock, suggesting that this Ca²⁺ transient, but not the second, derives from an external source. Inhibition of the first peak was also seen using 1.5 mM 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) in place of EGTA, indicating that the nature of the Ca²⁺-chelating agent is unimportant to the inhibition (data not shown). In contrast to Ca²⁺ chelation, the addition of extra CaCl₂ to achieve a final medium concentration approximately 2.7 and 4.3 times that normally present in the cell culture was seen to elevate the amount of Ca²⁺ entering during the first phase of the Ca²⁺ influx (Fig. 2B). Furthermore, supplementation of the growth medium with excess Mg²⁺, which can either compete with Ca²⁺ as a channel substrate or antagonize Ca²⁺ entry as a weak channel blocker (31, 34), also reduced the magnitude of the first Ca²⁺ peak (Fig. 2C). Mn⁺² and Fe²⁺ were similarly found to inhibit the first, but not alter the second, of the two Ca²⁺ peaks (data not shown). Inhibition of the first phase of Ca²⁺ influx was also observed after replacement of the chloride with the sulfate salts of Mg²⁺ and Fe²⁺ (data not shown), indicating that this initial phase of Ca²⁺ entry into the cytosol is not controlled by the nature of the counter ion. Based on the selective suppression of the first Ca²⁺ transient by either EGTA or nonsubstrate cations and the selective enhancement of this peak by elevated extracellular Ca²⁺, we propose that the first peak of Ca²⁺ influx during osmotic shock originates from an apoplastic pool.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Effect of extracellular Ca2+ modulators on Ca2+ fluxes. Aequorin luminescence recordings of cytosolic Ca2+ concentration were taken as outlined under "Experimental Procedures." A, cells were diluted 1:1 at the arrow with either 3 mM EGTA dissolved in distilled water (final concentration, 1.5 mM) or distilled water (control). B, cells were diluted 1:1 with either 10 mM CaCl2 dissolved in distilled water (final concentration, 5 mM), 5 mM CaCl2 in distilled water (2.5 mM), or distilled water (control) at the time indicated by the arrow. C, cells were diluted with either 20 mM MgCl2 in distilled water (final concentration, 10 mM), 4 mM MgCl2 in distilled water (2 mM), or distilled water (control). Data presented are representative of three independent experiments. Although Mg2+ is an established modulator of the affinity of aequorin for Ca2+ ions (67), since both L and Ltotal (and thus Lmax) are measured in the presence of elevated Mg2+, no artifact in the measurement or calculation of Ca2+ concentration should result. Additionally, since Mg2+ does not itself stimulate aequorin to luminesce (67), the calculations of intracellular Ca2+ concentration in the presence of Mg2+ should be accurate.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Effect of Ca2+ modulators on 45Ca2+ influx. Aequorin-transformed tobacco cells (3 ml) were exposed to 0.1 µM Ci 45Ca according to the protocol outlined under "Experimental Procedures" and subjected to the following treatments: control, hypo-osmotic shock induced by 1:1 dilution with distilled water; La3+, 10 mM LaCl3 was added and followed immediately by a 1:1 dilution with distilled water; Mg2+, 10 mM MgCl2 was added and followed immediately by a 1:1 dilution with distilled water; and niflumate, 500 µM niflumate dissolved in Me2SO was added 10 min before 45Ca exposure and dilution with distilled water. Data are presented as relative nuclide uptake/mg of cells and corrected for basal 45Ca binding/uptake, measured in iso-osmotically treated control cells exposed to vehicle alone (1% water or 0.1% Me2SO). Error bars represent mean (S.D. for three independent experiments. Iso-osmotically treated samples gave 45Ca uptake readings of ~20% of the hypo-osmotically treated samples, presumably due to 45Ca binding in the cell wall. 45Ca uptake was greater in control cells pretreated with 0.1% Me2SO than in control cells pretreated with 1% water. This difference in basal 45Ca uptake appears to be due to a transient leak of 45Ca due to Me2SO at the time of its addition. Because uptake in vehicle-treated control cells was invariably subtracted, this perturbation should not influence the results shown.

The Second Peak of Ca²⁺ Derives from Intracellular Ca²⁺ Stores-- Although the results shown in Fig. 2 also argue that the second peak of cytosolic Ca²⁺ must stem from opening an internal store, we set out to confirm this hypothesis with selective modulators of internal Ca²⁺ release. For this purpose, caffeine, a Ca²⁺ channel regulator believed to activate Ca²⁺ release from cyclic-ADP-ribose and ryanodine-sensitive stores in many eukaryotic systems (4, 47, 48), was added to the tobacco suspensions, and the osmotically induced Ca²⁺ transients were again examined. As anticipated, the addition of caffeine in the absence of any other stimulus induced substantial entry of Ca²⁺ into the cytosol of the tobacco cell (Fig. 4A). Since these caffeine-activated Ca²⁺ transients were found to be insensitive to membrane-inpermeant Ca²⁺ chelators and to competition with extracellular Mn²⁺ or Mg²⁺ (data not shown), we reason that this Ca²⁺ signal indeed derives from the caffeine-induced emptying of an intracellular Ca²⁺ pool.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   The effect of intracellular Ca2+ modulators on Ca2+ fluxes. Aequorin luminescence recordings and Ca2+ calculations were performed as outlined under "Experimental Procedures." A, 0.5 ml of suspension-cultured cells were treated with either 100 or 40 mM caffeine at the time indicated by the arrow. Aequorin luminescence recordings of control cells treated with isotonic sucrose did not exceed 0.15 µM cytosolic [Ca2+] (data not shown). B, 0.5 ml of suspension-cultured cells were treated with either 40 mM caffeine (from an isotonic 180 mM stock solution) or isotonic sucrose (control) for 10 min before a 1:1 dilution with distilled water at the time indicated by the arrow. C, cells were treated with either 100 or 500 µM niflumate or with a corresponding volume of Me2SO alone (control) 10 min before hypo-osmotic stimulation in the luminometer. Cells were diluted 1:1 with distilled water at the time indicated by the arrow.

The Two Channels Regulating Ca²⁺ Influx during Hypo-osmotic Shock Communicate-- It did not escape our attention that inhibition of the first Ca²⁺ transient generally resulted in enlargement of the second (Fig. 2A). Only in the case of Mg²⁺ addition was this compensating increase in the trailing Ca²⁺ pulse not consistently observed. Conversely, enhancement of the initial phase of Ca²⁺ influx by supplementation of the medium with extra Ca²⁺ invariably reduced the amplitude of the latter (Fig. 2B). Furthermore, inhibition of the second phase of Ca²⁺ uptake by niflumic acid consistently enhanced the first (Fig. 4B). Similar but somewhat reduced effects were also seen with moderate concentrations of caffeine (Fig. 4A). Taken together, these data argue that some type of communication must occur between the two phases of osmotically stimulated Ca²⁺-gating and that inhibition or enhancement of one phase of influx somehow leads to a compensating Ca²⁺ flux during the other. It will be interesting to explore the pathways along which such communication travels.

	DISCUSSION

Top Abstract Introduction Procedures Results Discussion References

We have provided evidence that the two phases of Ca²⁺ entry into the cytoplasm of osmotically shocked tobacco cells sequentially involve (i) the influx of extracellular Ca²⁺ and (ii) the release of intracellular (compartmentalized) Ca²⁺. Thus, prevention of external Ca²⁺ entry by the addition of EGTA or competing cations (i.e. Mg²⁺, Mn²⁺, Fe²⁺) diminished the first phase of Ca²⁺ uptake, whereas augmentation of the extracellular Ca²⁺ pool specifically enhanced this same wave of Ca²⁺ uptake. Taken together with data on the blockade of the second phase of Ca²⁺ influx by caffeine and niflumate, i.e. probable modulators of intracellular Ca²⁺ release, a strong case for the above Ca²⁺-gating assignments can now be made. This contention is also bolstered by observations that Mg²⁺, which blocks only the initial phase of Ca²⁺ influx, prevents externally added ⁴⁵Ca uptake, and niflumic acid, which eliminates only the second phase of Ca²⁺ influx, does not.

We have also observed that communication may occur between the two waves of Ca²⁺ entry into the cytoplasm of a stimulated tobacco cell. Unfortunately, there are currently few clues in the literature regarding the mechanism through which this communication might occur. Although hypo-osmotic stress has been frequently observed in eukaryotic cells to activate release of both intracellular and extracellular Ca²⁺ stores, little data regarding cross-talk between these two Ca²⁺ depots has yet been presented (53-58). Furthermore, the gating and signaling pathways activated by hypo-osmotic stimulus in nonplant systems may be very different from those in tobacco, since, opposite to the order of Ca²⁺-gating in tobacco cells, internal release of Ca²⁺ appears to precede influx of externally derived Ca²⁺ in mammalian cells (55-58). This order of Ca²⁺ channel activation in animal cells is reminiscent of the phenomenon of capacitative Ca²⁺ entry, in which intracellular release of Ca²⁺ leads to the activation of plasma membrane Ca²⁺ channels and the refilling of internal stores (27, 59-61). Although the mechanisms of communication between internal and external Ca²⁺ stores during capacitative Ca²⁺ entry are currently an area of intense research, no consensus mechanistic theory has been put forth (61). In osmotically stressed tobacco cells, a Ca²⁺-sensing mechanism that would adjust the Ca²⁺ efflux from the second (intracellular) Ca²⁺ store depending on the quantity of Ca²⁺ delivered during the first (extracellular) phase of Ca²⁺ entry can be easily envisioned. The intracellular Ca²⁺ gate would simply need to be negatively regulated by Ca²⁺ or a Ca²⁺-activated enzyme (e.g. a kinase) (62, 63). It will be important in the future to look for such a signaling mediator following treatment of stimulated cells with excess Ca²⁺ or EGTA (Figs. 2, A and B).

In contrast to the feed-forward signaling mechanism hypothesized above, identification of a communication pathway that responds to the readiness state of the second phase of Ca²⁺ entry and pre-emptively modulates the first is more difficult to imagine. Nevertheless, pretreatment of osmotically stimulated tobacco cells with moderate concentrations of caffeine or niflumic acid invariably augments the initial Ca²⁺ transient before eliminating the latter (Fig. 4, A and B). However, Takahashi et al. (39) have presented evidence that the general serine/threonine protein kinase inhibitors K252a and staurosporine selectively inhibit the second phase of Ca²⁺ influx without a compensating increase in the first phase. Thus, this modification of extracellular Ca²⁺ influx by internal Ca²⁺ transport capability may not be a general phenomenon and may depend on the type inhibitors used. There are clearly complexities in these Ca²⁺ signaling pathways that will require considerable research to resolve. It will be interesting to explore the area of Ca²⁺ channel communication in plant cells and to possibly identify at both the protein and DNA level the Ca²⁺ channels and other molecules involved.

It was encouraging to observe that several inhibitors could be identified that selectively block cytosolic influx of Ca²⁺ from either intracellular or extracellular Ca²⁺ stores. Although lanthanides and ruthenium red were found to inhibit both pathways of Ca²⁺ influx, the other modulators detailed above were highly selective for just one pathway. Based on these observations, it should now be possible to resolve which downstream consequences of hypo-osmotic shock rely on which phases of Ca²⁺ entry for signal propagation. It is conceivable, for example, that pathways designed to adjust cellular volume/turgor pressure during osmotic stress (14, 23, 64, 65) might depend on a different Ca²⁺ signal than pathways evolved to initiate the osmotically induced oxidative burst (8, 66). Indeed, preliminary studies indicate that only the second of the two phases of Ca²⁺ entry is required to stimulate reactive oxygen biosynthesis following hypo-osmotic stimulation.²