Measurement of Ca2+ Fluxes during Elicitation of the Oxidative Burst in Aequorin-transformed Tobacco Cells*

We have employed suspension cultured aequorin-transformed tobacco cells to examine the involvement of Ca2+ in signal transduction of the oxidative burst. Use of cultured cells for this purpose was validated by demonstrating that the cells responded to cold shock quantitatively and qualitatively similarly to the intact transgenic plants from which they were derived. Stimulation of the oxidative burst in the cell suspension was achieved by administration of oligogalacturonic acid, Mas-7 (a peptide known to activate G proteins and Ca2+ fluxes), hypo-osmotic stress, or harpin (a protein from the pathogenic bacterium Erwinia amylovora). The latter failed to promote any detectable increase in cytoplasmic Ca2+ concentration, whereas each of the former three triggered a rapid rise in cytosolic Ca2+followed by a return within seconds to basal Ca2+ levels. Peak Ca2+ concentrations induced by the former three elicitors were ∼0.7, 1.4, and 1.3 μm, respectively. Three lines of evidence suggest that the observed Ca2+pulses are essential to transduction of the oxidative burst signals by their respective elicitors: (i) inhibition of the Ca2+transients with Ca2+ chelators or Ca2+ channel blockers prevented expression of the oxidative burst, (ii) introduction of exogenous Ca2+ into the same cells initiated the burst even in the absence of other inducers of the response, and (iii) the observed Ca2+ transients often returned to near basal levels well before any H2O2 synthesis could be detected, suggesting that the Ca2+ influx is required to communicate the burst signal but not maintain the defense response. These data suggest that Ca2+ pulses serve frequently, but not invariably, to transduce an oxidative burst signal.

Because of its rapid expression, ease of assay, and similarity to the analogous defense response in human neutrophils (21), the oxidative burst has recently served as a model for exploring signal transduction pathways in plants. As the numbers of elicitors examined have risen, it has become increasingly clear that different elicitors may inaugurate independent signal transduction pathways that employ distinct sets of second messengers (1,22). Indeed, the pathway initiated by oligouronides appears to require participation of phospholipase C but not phospholipase A (12,23), whereas the pathway triggered by an elicitor from Verticillium dahliae requires exactly the converse (12). Although all of the independent signaling pathways are believed to converge with the assembly of an oxidase complex on the plasma membrane (24 -26), it remains uncertain as to whether the various pathways also share a need for specific kinases or Ca 2ϩ .
Ca 2ϩ is already believed to serve as a second messenger in such important plant processes as stomatal closure (27,28), geotropism (29), control of circadian rhythms (30), pollen tube elongation (31,32), regulation of plasmadesmata aperture (33), response to physical stress (34), and phytochrome phototransduction (35). Nevertheless, its accurate quantitation during cellular signaling events has until recently proven very difficult. Thus, 45 Ca 2ϩ binds avidly to cell walls, often masking the small changes in cytoplasmic 45 Ca 2ϩ that could indicate its function as a second messenger (36). Further, signal transduction pathways that might trigger release of Ca 2ϩ from intracellular organelles are largely invisible to 45 Ca 2ϩ -based methodologies, because no net change in cellular 45 Ca 2ϩ content occurs. Although Ca 2ϩ -sensitive fluorescent dyes have yielded useful information in a few plant systems (37), their general resistance to entry into plant cells has greatly limited their application (37). Indeed, most membrane-permeant esters of Ca 2ϩ -dependent fluorophores hydrolyze to their impermeant forms in the cell wall, preventing their penetration to their intracellular destinations. Fortunately in 1991, Knight et al. (38) transformed tobacco plants with a Ca 2ϩ -sensitive luminescent protein, termed aequorin, and demonstrated that resting levels of Ca 2ϩ were insufficient to induce photon emission but that stimulus-induced Ca 2ϩ increases generated readily measurable levels of bioluminescence. Since this initial discovery, aequorin-transformed plants have been exploited to quantitate intracellular Ca 2ϩ fluxes accompanying such diverse processes/ stimuli as direct mechanical perturbation (34,38), gusts of wind (39), cold shock (38,40), and circadian oscillations (30). Because the aequorin protein is expressed in the plant cell cytoplasm and because the intensity of light emitted by aequorin is directly proportional to Ca 2ϩ concentration, measurement of aequorin-derived bioluminescence enables direct quantitation of Ca 2ϩ transients in the transformed cytoplasm of the cell. In this report, we have employed suspension cultures of aequorin-transformed tobacco plants to monitor Ca 2ϩ transients during elicitation of the oxidative burst. We report here that several but not all elicitors of the oxidative burst induce a rapid and quantifiable influx of Ca 2ϩ and that this Ca 2ϩ is required for expression of the subsequent reactive oxygen species.

EXPERIMENTAL PROCEDURES
Materials-BAPTA-AM and ionomycin were obtained from Calbiochem (La Jolla, CA), whereas ruthenium red was purchased from Sigma. Coelenterazine was obtained from Molecular Probes (Eugene, OR). All other chemicals were reagent grade or of higher purity and were obtained from major chemical suppliers.
Elicitors-An OGA 1 fraction that elicits the oxidative burst in all plant species tested to date was prepared as described previously (13). The OGA preparation used in these studies contained 0.5 mg/ml galacturonic equivalents as determined by the method of Blumenkrantz and Asboe-Hansen (41). Harpin, a potent inducer of both the oxidative burst (11,12) and the hypersensitive response (42), was a kind gift of Dr. Steven Beer (Cornell University). Mas-7, an active analog of mastoparan, and Mas-17, its inactive counterpart, were purchased from Calbiochem.
Aequorin-transformed Tobacco Suspension Cultures-Generation of aequorin-transformed tobacco (Nicotiana plumbaginofolia) plants has been previously described (38,43). Transgenic seeds from an F3 generation were a generous gift from Dr. Anthony J. Trewavas (University of Edinburgh). The seeds were sprouted on wet filter paper, and the seedlings were potted in soil. Leaves from these plants were surface sterilized using a solution of 8% calcium hypochlorite and 0.1% Tween 20 and transferred to W-38 agar to promote callus formation, as described previously (18). The calli were fragmented, transferred to liquid W-38 medium, and maintained in suspension by continuous shaking. The resulting suspension cultures were expanded by transferring 3 ml of culture to 25 ml of fresh media every 10 days. Except where noted in Fig. 4, all cell suspensions were studied between 16 and 30 h after transfer to fresh media, when the cells responded maximally to elicitor stimulation but displayed no activated characteristics in the absence of elicitation.
Reconstitution of Aequorin-In vivo reconstitution of aequorin was carried out by adding 25 l of coelenterazine (5 M final concentration) in ethanol to 3 ml of suspension cultured cells (Ϸ24 h old) and incubating overnight at 22°C in the dark. Control cells were treated similarly with an equivalent volume of ethanol alone.
Aequorin Luminescence Measurements-Luminescence measurements were made using a digital luminometer (LKB BioOrbit model 1250, Gaithersburg, MD). The luminometer was calibrated by setting the background counts to zero and a 0.26 Ci of 14 C internal standard to 10 mV. Coelenterazine-treated cultures (0.5 ml) were transferred to a luminometer cuvette and maintained in suspension by mild stirring. Luminescence was measured every 0.1 s for the duration of the experiment. All inhibitors and chelators were added at the start of stirring, whereas elicitors were added at the times indicated in the figures. At the end of the experiment, all of the unconsumed aequorin was discharged by injecting 250 l of a solution of 10% Nonidet P-40 and 100 mM CaCl 2 into the luminometer cuvette to determine the total light output.
Quantitation of Intracellular Ca 2ϩ Concentration-The kinetic profile of intracellular Ca 2ϩ concentration was obtained by transforming data of each luminescence curve using the equation described by 1 ⁄3 ]}. To determine the calcium concentration at each time point, a computer program was written to calculate L, the light intensity at time intervals of 0.1 s. L is obtained by integrating the luminometer output (mV) over these 0.1-s intervals. The sum of all L values is defined as L total (the total area under the curve). The sum of all L values from t ϭ 0 to any given time point is L cumulative and represents the aequorin that has already been discharged. L max is defined as L total Ϫ L cumulative at each time point, and L max represents the residual aequorin yet to be discharged. L max is not a constant but decreases steadily as aequorin is consumed during the experiment. Using the parameters L and L max , we calculated the Ca 2ϩ concentration corresponding to each time point in the luminescence curves. The figures present the raw computer transformations of the digitized luminescence data without any smoothing or noise reduction.
Spectrofluorimeteric Determination of H 2 O 2 Production-H 2 O 2 production in aequorin-transformed cells was monitored spectrofluorimeterically, as described earlier (13,45). Briefly, 1 ml of cultured tobacco cells were transferred to a gently stirred fluorimeter cuvette and treated with 7 l of pyranine (8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt; 0.2 mg/ml). Desired elicitors and inhibitors were added at the start of stirring. Loss of fluorescence due to peroxidase-dependent quenching of the dye pyranine ( ex at 405 nm, em at 512 nm) was continuously monitored. Incubation of cells with coelenterazine had no effect on either the oxidative burst or peroxidase activity.

Validation of the Use of Aequorin-transformed Tobacco Cells
to Measure Changes in Intracellular Ca 2ϩ -To study the role of Ca 2ϩ in signaling the oxidative burst, we generated suspension cultures from tobacco plants expressing the aequorin transgene, as described under "Experimental Procedures." However, before undertaking to measure elicitor-stimulated Ca 2ϩ transients, it was necessary to document that the suspension cultures behaved similarly to the transgenic plants from which they were derived. Three comparisons were undertaken to establish this similarity. First, earlier studies using seedlings from the transgenic tobacco plants established that cold shock is accompanied by a flux of cytoplasmic Ca 2ϩ that has a characteristic signature, i.e. a sharp rise that returns to basal levels within a minute (38). As can be seen in Fig. 1A, rapidly lowering the temperature of the suspension cultures (from 23 to 16°C) also induces a large increase in aequorin luminescence with an overall profile that is remarkably similar to that described for whole plants. Second, because the Ca 2ϩ transient in the seedlings could be partially inhibited by soaking the seedlings in millimolar concentrations of either La ϩ3 or EGTA (46), we next examined the sensitivity of our suspension cultures to the same reagents. As also shown in Fig. 1A, both La ϩ3 and EGTA blocked most of the cold shock-induced increase in cytoplasmic Ca 2ϩ concentration. Third, a crude proportionality was previously observed in the transgenic seedlings between the magnitude of the temperature decrease and the amplitude of the Ca 2ϩ pulse. Using the transgenic cell cultures, we observed an analogous proportionality, wherein the intensity of the luminescence spike increased with the magnitude of the temperature drop (data not shown). Together these data argue that the transgenic tobacco cell suspension cultures constitute a reasonable model of the transgenic plant.
Determination of True Intracellular Ca 2ϩ Concentration-Because the relationship between luminescence and Ca 2ϩ concentration is double logarithmic, presenting data simply as a change in luminescence can greatly exaggerate the extent of any change in intracellular Ca 2ϩ concentration. Therefore, we wrote a computer program to convert the observed luminescence signal directly into intracellular Ca 2ϩ concentration using the equation described under "Experimental Procedures." Fig. 1B presents the transformation of the data presented in Fig. 1A. This transformation reveals that in response to cold shock, the cytoplasmic Ca 2ϩ concentration peaks at around 2.7 M, a value in the range observed for whole plants. Furthermore, addition of La 3ϩ and EGTA reduced the peak Ca 2ϩ level to less than 1 M, once again comparable with the results obtained by Knight et al. (46).
Some Elicitors of the Oxidative Burst Induce a Ca 2ϩ Flux-With the ability to characterize the kinetics and magnitude of Ca 2ϩ fluxes in cultured tobacco cells established, it was of interest to examine whether an intracellular Ca 2ϩ pulse might participate in an elicitor-induced oxidative burst. We focused our investigation on the elicitors OGA, Mas-7, hypo-osmotic shock, and harpin primarily because of our previous experience with these elicitors. Specifically OGA, a component of the plant cell wall thought to be released during pathogen attack or wounding, was selected because it rapidly induces activation of phospholipase C (23) and, presumably, release of intracellular Ca 2ϩ through the action of inositol trisphosphate. Mas-7, an activator of heterotrimeric G proteins, was also chosen because it similarly stimulates the oxidative burst via activation of phospholipase C (23). Hypo-osmotic shock, a novel inducer of the burst (17), was selected primarily because it is known to trigger oxidant production via some type of mechanosensor (17), i.e. a pathway commonly inaugurated by Ca 2ϩ channels (47). Finally, harpin, a heat stable protein from the bacteria E. amylovora was chosen, because it induces the oxidative burst (11,12) and may promote the hypersensitive response (42,48) in tobacco. Importantly, in addition to serving as potent inducers of the oxidative burst, all of the above elicitors are available in relatively pure form, confirming that any Ca 2ϩ transients observed will have been induced by the same component in the preparation that is responsible for stimulating the oxidative burst.
The impact of elicitors on intracellular Ca 2ϩ levels was evaluated by mixing the elicitors with reconstituted aequorin cells and monitoring the increase in aequorin luminescence. As seen in Fig. 2A, OGA induces an influx of Ca 2ϩ into the cytoplasm that begins immediately following OGA addition and ends within 20 s of its initiation. The peak Ca 2ϩ concentration achieved in the experiment displayed here was 0.67 M; however, this value varied from 0.28 to 0.9 M, depending on the responsiveness of the particular flask of cells employed. Mas-7 also induces a rapid increase in cytoplasmic Ca 2ϩ , although the magnitude (1.4 M) and duration of the pulse are much larger than observed with OGA (Fig. 2). Mas-17, an inactive analog of Mas-7, has little effect on aequorin luminescence, confirming that the Mas-7 effect is indeed specific. Osmotic shock, unlike the other elicitors, triggers two contiguous phases of Ca 2ϩ release that last for a total of ϳ4 -5 min (Fig. 2). The peak Ca 2ϩ concentration is 1.34 Ϯ 0.39 M for the first pulse and 1.44 Ϯ 0.12 M for the second (n ϭ 5). Interestingly, harpin, even at concentrations sufficient to induce both the burst and the hypersensitive response does not cause an increase in aequorin luminescence, even when the luminescence measurements are extended to 40 min following stimulation. Because the signal to noise ratio in these studies is greater than 50:1 (actual data are shown without any noise reduction), it can be concluded that Ca 2ϩ is not necessary for transduction of the harpin-stimulated oxidative burst.
Using untreated cells from the same flasks, we simultaneously monitored the oxidative burst initiated by the above elicitors using a fluorimeteric assay. Although the oxidative burst patterns for these elicitors have been previously published (12), they are presented here to allow a direct comparison of the kinetics of the Ca 2ϩ pulse with the kinetics of oxidant production by the same cells. Fig. 2B shows that the different elicitors, including harpin, induce bursts characterized by different lag periods following elicitor addition and by different rates of H 2 O 2 production. Nevertheless, in all cases where a Ca 2ϩ transient is observed (Fig. 2A), the Ca 2ϩ influx precedes the biosynthesis of H 2 O 2 (Fig. 2B). In fact, for each of the three Ca 2ϩ -mediated pathways, the concentration of cytoplasmic Ca 2ϩ is seen to return to its unstimulated level before production of the oxidative species terminates. Importantly, this rapid expulsion of Ca 2ϩ from the cytoplasm cannot be an artifact of aequorin depletion, because cell permeabilization at the end of each experiment demonstrated that only a small fraction of the intracellular aequorin had been consumed (see "Experimental Procedures"). Further, the Ca 2ϩ influx cannot be dependent on oxidant production, because addition of sufficient catalase to eliminate any detectable H 2 O 2 had no effect on the Ca 2ϩ influx FIG. 1. Effect of cold shock on aequorin-transformed tobacco suspension cultures. A, coelenterazine-treated aequorin-transformed tobacco cells (0.1 ml) were transferred to a luminometer cuvette, and the basal luminescence was monitored following protocols described under "Experimental Procedures." At the time indicated (arrow), the cells were subjected to cold shock by submersing the cuvette into a 1°C ice bath for 5 s, allowing the temperature of the suspension to decrease from 23 to 16°C. The cuvette was then transferred back to the luminometer, and the measurements were continued. The effect of EGTA (5 mM) and La 3ϩ (10 mM) were evaluated by adding these compounds to the suspensions directly before subjecting the cells to cold shock as described above. Traces shown here are representative of data obtained in three independent experiments. Note: luminescence data were not collected during the 10-s interval of cold shock administration (doubleheaded arrow), and no smoothing or noise reduction of the data were performed. B, traces shown here represent the computer transformation of the luminescence data displayed in A to yield actual cytoplasmic Ca 2ϩ concentrations using the equations described under "Experimental Procedures." (data not shown). Taken together, these data suggest that the Ca 2ϩ pulses associated with elicitor stimulation exhibit the properties of a true second messenger in that they precede initiation of the biological response but are not required to maintain it, nor do they depend on it.
The Observed Ca 2ϩ Pulses Participate in Transducing an Oxidative Burst Signal-The observation that harpin can induce a burst without participation of Ca 2ϩ as a second messenger raises the question of whether the Ca 2ϩ influx stimulated by the other three elicitors is an epiphenomenon or an essential step in signaling their oxidative bursts. To investigate this issue, we decided to block the influx of Ca 2ϩ induced by OGA, Mas-7, and osmotic shock, and then monitor the effect on H 2 O 2 biosynthesis. As shown in Fig. 3, A-F, incubation of cells with ruthenium red, a Ca 2ϩ channel inhibitor known to be active in plant cells (49), eliminates most of the elicitor-evoked Ca 2ϩ flux as well as most of the oxidative burst for all three elicitors. Furthermore, treatment of the cell suspensions with BAPTA-AM, a membrane-permeable intracellular Ca 2ϩ chelator, eliminated all oxidant production (data not shown). As expected, neither treatment with ruthenium red nor Ca 2ϩ chelator affected the harpin-induced burst, i.e. consistent with its inability to mobilize Ca 2ϩ . These data suggest that Ca 2ϩ is indeed necessary for the Mas-7, OGA, and hypo-osmotic stress-stimulated oxidative bursts and that prevention of its influx is sufficient to block oxidant biosynthesis.
Effect of Ca 2ϩ Influx on Burst Activity-To learn whether Ca 2ϩ influx is itself sufficient for induction of the oxidative burst, we examined the effect of ionomycin, a Ca 2ϩ ionophore known to be active in both plants and animals, on generation of H 2 O 2 by the coelenterazine-treated tobacco cells. As seen in Fig. 4, A and B, addition of 20 M ionomycin to the aequorintransformed cells induces both a rise in intracellular Ca 2ϩ levels (Ͼ4 M) as well as a burst in H 2 O 2 biosynthesis. In contrast, when EGTA is present in the culture medium, ionomycin-mediated induction of the oxidative burst is prevented (data not shown). These data suggest that Ca 2ϩ influx alone is sufficient to initiate a burst in responsive tobacco cells. However, in the course of these studies, we also observed that as the suspension cultures age (Ͼ36 h after transfer to fresh medium) and enter their elicitor-insensitive stage (13,45), they also lose their ability to respond to ionomycin with oxidant biosynthesis (data not shown). That is, although ionomycin is still capable of inducing the anticipated Ca 2ϩ influx in these insensitive cells (as is also hypo-osmotic stress), the cells fail to respond with an oxidative burst. These data suggest that competency factors downstream of the Ca 2ϩ influx remain important in regulating the response of a plant to elicitation.

DISCUSSION
The advent of transgenic aequorin plants has significantly improved the technology for monitoring Ca 2ϩ fluxes in suspension cultured cells. Thus, in contrast to the more conventional use of 45 Ca 2ϩ and/or Ca 2ϩ -sensitive fluorophores, the aequorin methodology (i) requires no harsh methods for loading cells or preparing them for observation, (ii) is neither toxic to the plant cells nor to the investigator, (iii) allows for the collection of quantitative data at short (0.1 s) time intervals, thereby permitting accurate evaluation of the kinetics of a Ca 2ϩ transient, (iv) permits localization of the Ca 2ϩ -sensitive protein in desired internal organelles by use of organelle targeting sequences, and (v) generates crude data with a signal to noise ratio generally exceeding 50:1, enabling characterization of processes involving even very small changes in cytoplasmic Ca 2ϩ . With such desirable attributes, the aequorin technology should serve as the method of choice for investigating the involvement of Ca 2ϩ in many cellular signaling events.

FIG. 2. Effect of plant defense elicitors on intracellular Ca 2؉ concentrations and the oxidative burst.
A, coelenterazine-treated cells (0.5 ml) were stimulated with the following elicitors 2 min after the start of stirring: OGA (25 g/ml), Mas-7 (5 g/ml), Mas-17 (5 g/ml), harpin (100 g/ml), and osmotic shock (1:1 dilution with water). Luminometry was conducted as described under "Experimental Procedures." The traces presented here were chosen from several trials (n Ն 5) to best represent the average response. Even though the harpin response is only displayed for 7 min, no increase in luminescence was observed for the full 40-min duration of the experiment. While 25 g/ml harpin readily activates the oxidative burst in our suspension cultures, 100 g/ml is required for induction of the hypersensitive response. B, a separate aliquot (1 ml) of the same cells employed in A was simultaneously treated with the same elicitors at the same concentrations at the start of stirring. The oxidative burst triggered by these elicitors was monitored using the fluorimeteric assay described under "Experimental Procedures." Since the use of suspension cultured plant cells to investigate molecular aspects of plant behavior, the question has frequently arisen regarding the relevance of single cell studies to whole plant physiology. Unfortunately, responses to this concern have been largely inadequate, primarily because valid comparisons between whole plants and single cells/cell clusters have been difficult to design. Although much still remains to be resolved on this issue, the similar responses to cold shock of the aequorin transgenic plants and their derived suspension cultures suggests that single cells can in some cases yield data that very accurately reflect the behavior of the whole plants. Thus, Knight et al. (46) observed a Ca 2ϩ transient in coldshocked tobacco seedlings that was characterized by a 10-s width at half-height that rose abruptly to ϳ2.3 M Ca 2ϩ and fell slowly to its basal (nanomolar) level over a period of ϳ30 s. We also observed a cold shock-induced Ca 2ϩ pulse in the trans-genic suspension cultures with an ϳ8-s width at half-height that increased precipitously to 2.6 M Ca 2ϩ and then declined more slowly to basal levels over a ϳ30 s total time period. Ca 2ϩ fluxes in both the cell suspension cultures and whole seedlings were similarly inhibited by EGTA and La 3ϩ , and in both systems the magnitudes of the cold shocks and Ca 2ϩ transients were roughly proportional. Although physical barriers/inequities prevent similar comparisons of Ca 2ϩ transients following mechanical perturbation, elicitor stimulation, or osmotic stress, the observation that these signals also trigger Ca 2ϩ fluxes in both single cell and whole plant systems (38,39,50,51) adds further weight to the contention that cultured cells can serve as useful models when properly validated. With this motivation in mind, it should be possible to explore in greater detail the role of Ca 2ϩ pulses in signaling a variety of defenserelated responses. Although the studies presented here provide the first quantitative data on the kinetics and magnitudes of the Ca 2ϩ transients induced by elicitors of a defense response, they represent by no means the first evidence for Ca 2ϩ involvement in defenserelated signaling. Indeed, several groups have reported that extracellular EGTA can inhibit the mobilization of selected disease resistance mechanisms (52,53). Other workers have shown that introduction of exogenous Ca 2ϩ into resting cells can initiate a defense response in the absence of the usually required elicitors (16,54). Still other laboratories have demonstrated that Ca 2ϩ channel blockers such as La 3ϩ can prevent expression of a normal pathogen resistance mechanism when conditions are otherwise designed to stimulate its induction (52,54). Nevertheless, it has never been determined wherein Ca 2ϩ actually exerts control over processes leading to disease resistance. In the case of the oxidative burst, data presented here demonstrate that Ca 2ϩ must enter the tobacco cell cytoplasm to initiate the burst, but the cation must not remain to sustain it. Thus, for the OGA-induced burst, cytoplasmic Ca 2ϩ rises to ϳ0.7 M and returns to basal levels within ϳ20 s (Fig. 2). H 2 O 2 biosynthesis, in contrast, is initially detected ϳ2 min following OGA addition, i.e. well after the Ca 2ϩ concentration has returned to its resting level. Even with ionomycintreated cells, where the primary stimulus for oxidant production is the ionophore-catalyzed entry of Ca 2ϩ into the cell, the Ca 2ϩ stimulus is seen to disappear long before the end product of the pathway (i.e. H 2 O 2 ) is observed (Fig. 4). Clearly, these data indicate that Ca 2ϩ serves only a transient role of communicating an upstream signal to a downstream effector. After performing this messenger function, Ca 2ϩ appears to be no longer needed. Moreover, it can be argued that the proximal Ca 2ϩ -dependent effector in the signaling pathway may be similarly transiently activated, returning to its resting state when the activating Ca 2ϩ is removed. Signaling components such as the Ca 2ϩ -dependent protein kinases (55,56), calmodulin (57) and its responsive kinases (58,59), Ca 2ϩ -dependent phosphatases (60), Ca 2ϩ -gated channels (61), and Ca 2ϩ -activated phospholipases (62) are obvious candidates for this downstream effector.
As mentioned previously, elevation of elicitor concentration stimulates a roughly proportional increase in the amplitude of the Ca 2ϩ transient and the magnitude of its downstream oxidative burst. In contrast, when the same comparison is conducted among unrelated elicitors of the oxidative burst, no such proportionality is observed. Indeed, Mas-7 was found to induce the largest Ca 2ϩ influx yet stimulate the weakest oxidative burst. Because Ca 2ϩ appears to be essential for transduction of the burst signals of at least three of the elicitors, it must be concluded that factors in addition to Ca 2ϩ can significantly modulate the strength of a burst signal and that these unidentified factors are differentially activated by the various elicitors. Such regulatory characteristics require the involvement of bifurcating and reconverging signal transduction pathways.
During preparation of this manuscript, another article appeared describing the use of aequorin-expressing suspension cultured cells to evaluate Ca 2ϩ transients during hypo-osmotic shock (51). Although their aequorin transgene was introduced directly into the cultured tobacco cells by infection with Agrobacterium tumefaciens, the response to low osmolarity was nevertheless very similar to that shown in Fig. 2, except the first Ca 2ϩ peak was very small and no quantitative data on Ca 2ϩ were presented. Because no evaluation of the consequent oxidative burst was conducted (51), further comparisons between the two studies are not possible.
Finally, it was admittedly surprising that harpin was found to trigger no Ca 2ϩ influx during its stimulation of the oxidative burst (Fig. 2) and presumed induction of hypersensitive cell death (4). Obviously, its signaling pathway must be very different from those initiated by OGA, Mas-7, and hypo-osmotic stress. Previous work has also shown that harpin signaling is also distinct from that of a V. dahliae elicitor (12). Nevertheless, Ca 2ϩ -independent transduction of an oxidative burst signal is not without precedence, because concanavalin A, in contrast to most other stimuli, induces human neutrophils to FIG. 4. Effect of ionomycin on intracellular Ca 2؉ levels and H 2 O 2 production. A, 20 M ionomycin was added to coelenterazinetreated aequorin cells at the time indicated (arrow), and the increase in intracellular Ca 2ϩ concentration was monitored by luminometry. B, coelenterazine-treated aequorin cells (1 ml) were transferred to a fluorimeter cuvette and treated with 20 M ionomycin, and its effect on H 2 O 2 biosynthesis was monitored by the oxidative quenching of pyranine. Similar results were obtained in three independent trials. synthesize reactive oxidants in the absence of any change in cytoplasmic Ca 2ϩ (63). Clearly much additional research will be required before the diversity of mechanisms that initiate and regulate the plant oxidative burst is fully understood.