An Apoplastic Ca2+ Sensor Regulates Internal Ca2+ Release in Aequorin-transformed Tobacco Cells*

Stephen G. CessnaDagger and Philip S. Low§

From the Biochemistry and Molecular Biology Program and the Department of Chemistry, Purdue University, West Lafayette, Indiana 47904

Received for publication, August 2, 2000, and in revised form, December 5, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Removal of Ca2+ from tobacco suspension cell medium has two immediate effects on cytosolic Ca2+ fluxes: (i) externally derived Ca2+ influx (occurring in response to cold shock or hypo-osmotic shock) is inhibited, and (ii) organellar Ca2+ release (induced by a fungally derived defense elicitor, caffeine, or hypo-osmotic shock) is elevated. We show here that the enhanced release of internal Ca2+ is likely due to increased discharge from a caffeine-sensitive store in response to a signal transduced from an extracellular Ca2+ sensor. Thus, chelation of extracellular Ca2+ in the absence of any other stimulus directly activates release of intracellular Ca2+ into the cytosol. Evidence that this chelator-activated Ca2+ flux is dependent on a signaling pathway includes its abrogation by prior treatment with caffeine, and its inhibition by protein kinase inhibitors (K252a and staurosporine) and anion channel blockers (niflumate and anthracene-9-carboxylate). An unexpected characteristic of tobacco cell adaptation to low external Ca2+ was the emergence of a new Ca2+ compartment that was inaccessible to external EGTA, yet responsive to the usual stimulants of extracellular Ca2+ entry. Thus, cells that are exposed to EGTA for 20 min lose sensitivity to caffeine and defense elicitors, indicating that their intracellular Ca2+ pools have been depleted. Surprisingly, these same cells simultaneously regain their ability to respond to stimuli that usually activate extracellular Ca2+ influx even though all external Ca2+ is chelated. Because this gradual restoration of Ca2+ influx can be inhibited by the same kinase inhibitors that block EGTA-activated Ca2+ release, we propose that chelator-activated Ca2+ release from internal stores leads to deposition of this Ca2+ into a novel EGTA- and caffeine-insensitive compartment that can subsequently be activated by stimulants of extracellular Ca2+ entry.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A wide variety of stresses stimulate the expression of cytosolic Ca2+ transients in plant cells, presumably resulting in the acquisition of tolerance/resistance to the same stresses (1). Examples of such stresses include cold shock, which stimulates an influx primarily of extracellular Ca2+ (2, 3); pathogen infection and defense elicitor stimulation, which activate Ca2+ pulses largely from internal Ca2+ pools (4-6); and hypo-osmotic shock, which activates a biphasic Ca2+ transient deriving first from outside and then from inside the cell (7, 8). We have reported previously that, in the case of the hypo-osmotic shock-induced Ca2+ pulses, inhibition of either of the two Ca2+ pulses generally leads to greater Ca2+ entry during the uninhibited pulse (7). We have thus surmised that an internal Ca2+ store might receive information regarding the Ca2+ status of the external store and vice versa (7). In this way, a deficiency in the Ca2+ content of either the intracellular or extracellular Ca2+ pool could be compensated by additional release of Ca2+ from the undepleted pool.

It has already been established that animal cells can communicate the Ca2+ status of their internal compartments to the external Ca2+ pool. Thus, after stimulation of internal Ca2+ release, the resulting depletion of ER1 Ca2+ has been observed to induce a slow refilling of the ER by influx across the plasma membrane. This phenomenon has been referred to as store-operated calcium entry (9-11). The mechanism of activation of this process is still debated (12), but there is general agreement that some ER protein must monitor luminal Ca2+ levels and signal the plasma membrane to initiate Ca2+ influx when ER levels fall.

Conversely, vertebrate animal cells are also believed to communicate the availability of external Ca2+ across their cytoplasm to internal Ca2+ stores by signaling through a Ca2+-sensing Receptor (CaR) (13). CaR is a seven-transmembrane G-protein-coupled receptor expressed in the plasma membrane of several different cell types, most notably those involved in Ca2+ homeostasis (e.g. parathyroid) (14, 15). CaR is activated in the presence of high levels of extracellular Ca2+ and communicates with the cell's interior by a signal transduction cascade that often involves the release of Ca2+ from the ER (16). CaR-mediated signaling pathways modulate the secretion of various Ca2+-regulating hormones that maintain Ca2+ homeostasis in distal tissues (14, 16). Although it is clear that plants and vertebrates must have very different mechanisms of Ca2+ homeostasis, it is also likely that plant cells will likewise monitor both internal and external Ca2+ stores and develop some means of compensation when one of these stores becomes depleted.

We report here that plant cells indeed respond to depletion of their extracellular Ca2+ reserves by activating the release of Ca2+ from internal stores. The route of communication between external and internal Ca2+ pools appears to require the function of protein kinases and anion channels. Further, after long term removal of external Ca2+ and the consequent depletion of the prominent internal store, plant cells appear to sequester their remaining Ca2+ in a caffeine-insensitive, EGTA-inaccessible compartment that can still be activated in response to stimuli that normally promote externally derived Ca2+ influx.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Plant Material-- Tobacco plants (Nicotiana tabacum L. var. Wisconsin-38) were transformed with the aequorin-encoding plasmid pMAQ2 (3) by the method of Liu et al. (17). Suspension cultures were then established and maintained as previously reported (5) by continuous shaking in Murashige and Skoog (MS) basal growth medium (Sigma; Ref. 18). MS medium contains the following ionic species in the given concentrations: NH<UP><SUB>4</SUB><SUP>+</SUP></UP>, 20.61 mM; K+, 20.045 mM; Ca2+; 2.99 mM; Mg2+, 1.05 mM; Na+, 0.101 mM; Mn2+, 0.1 mM; Fe2+, 0.1 mM; Zn2+, 0.029 mM; Cu2+, 0.0001 mM; Co2+, 0.0001 mM; NO<UP><SUB>3</SUB><SUP>−</SUP></UP>, 39.4 mM; Cl-, 2.99 mM; SO<UP><SUB>4</SUB><SUP>2−</SUP></UP>, 1.73 mM; PO<UP><SUB>4</SUB><SUP>3−</SUP></UP>, 1.25 mM; ethylenediaminetetraacetate4-, 0.1 mM; BO<UP><SUB>3</SUB><SUP>−</SUP></UP>, 0.1 mM; I-, 0.005 mM; MnO<UP><SUB>4</SUB><SUP>2−</SUP></UP>, 0.001 mM, pH 5.7 (18). Nonionic components include: sucrose (30 g/liter), enzymatic casein hydrolysate (1 mg/liter), and trace amounts of plant growth regulators, 2,4-dichlorophenoxyacetic acid (3 mg/liter), and kinetin (0.002 mg/liter). The osmolality of the medium was measured at 180-200 mosM with an osmometer.

Three different aequorin-expressing cell lines from three different transgenic parent plants were studied. Because these different cell lines expressed varying levels of aequorin protein, yet behaved similarly in each of the experiments performed, we conclude that incorporation and expression of the aequorin transgene did not alter plant cell responses.

Luminometry and Ca2+ Quantitation-- Luminometry of aequorin-transformed suspension cultures was performed as previously described, with some minor changes (4). Briefly, 24 h after subculture into fresh MS medium, cell cultures were incubated in 1 µM coelenterazine (Biosynth International, Naperville, IL) for 4-8 h. 1 ml of cell culture was then transferred to a luminometer cuvette that was placed in an LKB-Wallac Bio-Orbit luminometer chamber. Luminescence was recorded 10 times/s using Rainin Dynamax-LC software (Woburn, MA). EGTA and other treatments were added as indicated in the figure legends. A 90 mM stock solution of EGTA buffered to pH 5.7 (osmolality measured at 200 mosM) was used for all experiments. No measurable change in medium pH or osmolality occurred after EGTA additions to cell cultures up to [EGTA] = 45 mM. To quantitate the undischarged aequorin at the end of each experiment, cells were solubilized by injecting 200 µl of a solution containing 10% Nonidet P-40 and 500 mM CaCl2 into the luminometer chamber, and residual luminescence was recorded. Luminescence traces were then transformed by computer program (4) directly into [Ca2+]cyt using the equation described by Allen et al. (19).

Extracellular Ca2+ Measurements and Calculations-- External Ca2+ was measured with the fluorescent Ca2+-indicating dye Oregon Green BAPTA 5N (lambda ex = 494 nm, lambda em = 523 nm; Molecular Probes, Eugene, OR). Because of its relatively low affinity for Ca2+ (Kd ~ 20 µM Ca2+, at pH = 7), Oregon Green BAPTA 5N accurately reports Ca2+ concentrations ranging from <1 µM to >400 µM. To quantitate extracellular free Ca2+, 10 µg of dye/ml was added to the fluorimeter cuvette together with cell filtrates, and fluorescence was recorded.

Estimates of the free Ca2+ content of the medium after addition of EGTA (pKd EGTA-Ca = 7.39) were made using the MINTEQA2 environmental modeling software (United States Environmental Protection Agency, Center for Exposure Assessment Modeling, Athens, GA), assuming all ion concentrations listed in the Murashige and Skoog medium above, a suspension pH of 5.7, and no Ca2+ dissociation from the cell wall (20).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Compensation between Internally and Externally derived Ca2+ Pulses in Tobacco Cells-- We have previously shown that inhibition of external Ca2+ influx can lead to elevation of internally derived Ca2+ release (7). Thus, as shown in Fig. 1A, chelation of extracellular Ca2+ with EGTA not only inhibits the first phase of hypotonically stimulated Ca2+ entry (externally derived influx), but also elevates the second pulse (internally derived) of Ca2+ entry into the cytosol. Because elevation of the internally derived component was apparent whether we applied Ca2+ chelators (EGTA, EDTA, or BAPTA) or exchanged the cell medium for Ca2+-free buffers (data not shown), these reciprocal effects on the Ca2+ pulses are concluded to derive from removal of external Ca2+ rather than any Ca2+-independent effect of EGTA.



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Fig. 1.   Effect of external Ca2+ chelation on the cytosolic Ca2+ fluxes stimulated by hypo-osmotic shock, caffeine, or cold shock. Cytosolic Ca2+ concentrations were monitored in 1 ml of aequorin-transformed tobacco cells as described under "Materials and Methods." At the times indicated by the arrows, 3 mM EGTA was injected into the sample followed immediately by 1:1 dilution with double distilled H2O (A), 50 mM caffeine (B), or 1:1 dilution with ice-cold plant cell culture medium (C; Delta T = 12 °C). 1:1 dilution with isotonic medium at room temperature did not cause any measurable increase in Ca2+ concentrations (data not shown). All data are representative of at least three independent experiments in which similar results were obtained.

The compensatory increase in discharge of intracellular Ca2+ following chelation of extracellular Ca2+ implies some type of communication between the two Ca2+ pools. Conceivably, a cytosolic Ca2+ sensor (i.e. a Ca2+ and/or calmodulin-regulated protein kinase; Ref. 21) could detect the absence of the first (externally derived) Ca2+ transient and transmit that information to the intracellular store. Alternatively, the absence of extracellular Ca2+ could be directly detected by an extracellular sensor that signals to the intracellular store independent of any perturbation of the stimulated Ca2+ entry. To distinguish these two mechanisms, we examined the impact of external Ca2+ chelators on the discharge of intracellular Ca2+ following treatment with stimuli that have no effect on external Ca2+ influx. As shown in Fig. 1B, the cytosolic Ca2+ pulse stimulated by caffeine, a drug that activates the release of Ca2+ only from internal stores (7), is elevated when initiated immediately after EGTA addition. In contrast, cold shock activates a Ca2+ pulse that derives primarily from external stores (2, 3), and EGTA treatment potently inhibits its expression (Fig. 1C). It would thus appear that removal of external Ca2+ has two immediate effects on Ca2+ fluxes in tobacco cells: (i) it leads to inhibition of any externally derived Ca2+ influx, and (ii) it triggers elevation of internally derived Ca2+ release. Because the elevation of intracellular Ca2+ release proceeds even in the absence of any change in entry of extracellular Ca2+, we suggest that the Ca2+ detection system involves an extracellular sensor.

Internal Ca2+ Release Is Activated after Removal of External Ca2+-- To further explore whether extracellular Ca2+ levels might be communicated to intracellular Ca2+ stores, EGTA was added directly to cell suspensions and cytosolic [Ca2+] was again monitored in the luminometer. As shown in Fig. 2A, chelation of external Ca2+ by addition of 9 mM EGTA results in an irregular Ca2+ transient that continues for more than 10 min. A measurable increase in cytosolic Ca2+ could even be detected after adding only 2 mM EGTA, which still leaves the extracellular concentration of free Ca2+ at roughly 1 mM (Fig. 2B). To more quantitatively evaluate the impact of external Ca2+ removal on the magnitude of the stimulated internal Ca2+ flux, a computer-generated estimate of the external free Ca2+ at various concentrations of added EGTA is also provided (Fig. 2B) (MINTEQA2, Ref. 20). This estimate takes into account the 2.99 mM Ca2+ and all other ionic species in the medium, but does not consider the Ca2+ stored in the cell wall, which, if substantial, could shift the dotted line to the right. In view of these considerations, the close reciprocal relationship between the two traces is remarkable and suggests some type of functional linkage between log[Ca2+ex]free and the amplitude of internal Ca2+ release, with half-maximal Ca2+ flux occurring when external Ca2+ concentrations are reduced to ~10-5.5 M.



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Fig. 2.   Observation of Ca2+ chelator-activated cytosolic Ca2+ fluxes. A, at the time indicated by the arrow, 9 mM EGTA was injected into a tobacco cell suspension culture and cytosolic Ca2+ was monitored as described under "Materials and Methods." B, a plot of the magnitude of intracellular Ca2+ release as a function of EGTA concentration. The experiments were conducted as in A. Because of the irregularity of the chelator-induced Ca2+ pulses, Ca2+ levels were monitored for 10 min, and the average cytosolic Ca2+ level over that period was calculated for each experiment. Basal Ca2+ levels varied from day to day from ~50 nM to 200 nM. Thus, the average increase in Ca2+ concentration above the basal level recorded on the same day is reported as an average of three independent experiments, ± S.D. The free Ca2+ content in MS medium after the addition of increasing concentrations of EGTA (dotted line) was calculated using the MINTEQA2 computer program (20) and is included in the figure for comparison. C, coelenterazine-treated, aequorin-transformed suspension cells were placed in the luminometer and [Ca2+]cyt was recorded as described under "Materials and Methods." Cells were treated with 9 mM EGTA at the time indicated by the first arrow. At the second arrow, 10 mM either CaCl2 or MgCl2 was injected into the cuvette. Data are representative of three independent experiments in which similar results were obtained.

To further demonstrate that the chelator-activated Ca2+ flux arises because of removal of external Ca2+, and not because of any other effect of EGTA, we conducted analogous studies using BAPTA, an unrelated Ca2+ chelator. When BAPTA was added to suspension cultures at a concentration of 5 mM, the cells responded with a cytosolic Ca2+ flux that reached an average of 0.083 ± 0.055 µM Ca2+ (n = 3). Although the magnitude of the BAPTA-induced Ca2+ flux was admittedly more variable than that measured after EGTA addition, the data still support the conclusion that removal of external Ca2+ results in a substantial cytosolic Ca2+ flux regardless of the method of Ca2+ chelation.

Although both EGTA and BAPTA prefer Ca2+ over many other bivalent cations, it was still conceivable that the Ca2+ fluxes might have been induced by removal of other cations. To examine this possibility, tobacco cells were first activated with EGTA (until the usual chelator-induced increase in cytosolic Ca2+ was observed), and then external Ca2+ was restored by injection of enough CaCl2 to compensate for the presence of EGTA. As shown in Fig. 2C, restoration of external Ca2+ to EGTA-treated cells returns the cytosolic Ca2+ activity to its basal level. Because addition of MgCl2 does not alter the EGTA-induced Ca2+ flux (Fig. 2C), we conclude that the chelator-activated Ca2+ flux is mediated by a Ca2+ sensor that cannot respond to Mg2+.

Chelator-induced Ca2+ Fluxes Derive from a Kinase- and Anion Channel-dependent, Internal Caffeine-sensitive Pool-- To characterize the Ca2+ pools involved in the chelator-induced Ca2+ transients, we first depleted the internal Ca2+ stores by incubating cells in caffeine for an extended period (7) and then tested whether EGTA could still induce an increase in Ca2+ levels. As shown in Fig. 3A, caffeine-treated cells no longer respond to EGTA with a transient rise in cytosolic Ca2+, indicating that the Ca2+ pulses induced by removal of external Ca2+ derive from caffeine-sensitive internal stores.



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Fig. 3.   The chelator-activated Ca2+ pulses derive from a caffeine-sensitive internal pool. A, tobacco cell suspensions were either treated with 50 mM caffeine or left unmodified (control) 10 min prior to stimulation with 9 mM EGTA (at the time marked by the arrow). Cytosolic Ca2+ levels were then measured as described above. Alternatively, tobacco cells were exposed to 9 mM EGTA for the indicated period and then stimulated with 50 mM caffeine (B) or 0.2% (v/v) V. dahlia elicitor (C). Both caffeine and the V. dahlia elicitor stimulate Ca2+ influx from intracellular pools only (5).

To further confirm that a caffeine-sensitive intracellular Ca2+ pool is specifically responsive to external Ca2+ removal, the ability of caffeine itself to trigger release of Ca2+ was evaluated as a function of time after EGTA addition. As seen in Fig. 3A, caffeine-induced Ca2+ release is prominent in tobacco cells immediately prior to Ca2+ chelation, but diminishes with time following EGTA addition until its disappearance by 20 min after chelation. V. dahlia elicitor-triggered Ca2+ pulses, which also derive from a caffeine-sensitive intracellular compartment (5), display the same kinetics of depletion (Fig. 3C). We, therefore, conclude that the EGTA-induced Ca2+ pulses derive from the same intracellular compartment that releases Ca2+ in response to both caffeine and Verticilium dahlia elicitor stimulation.

To begin to assess the mechanism by which a change in extracellular Ca2+ might be communicated to caffeine-sensitive intracellular Ca2+ stores, we next examined the impact of pharmacological agents that were previously found to regulate intracellular Ca2+ fluxes in tobacco cells (5). As shown in Fig. 4, cells that are treated with either the protein kinase inhibitor, K252a (Fig. 4A), or the anion channel blocker, niflumate (Fig. 4B), display little or no response upon subsequent stimulation with EGTA. Similar data are also obtained when another kinase inhibitor, staurosporine (4 µM), or a different class of anion channel blocker (anthracene-9-carboxylate, 200 µM) is employed (data not shown). We, therefore, suggest that the extracellular Ca2+ sensor requires participation of kinases and anion fluxes to promote release of Ca2+ from an internal Ca2+ pool.



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Fig. 4.   Effect of protein kinase inhibitors and anion channel blockers on the EGTA-induced Ca2+ flux. Aequorin-transformed tobacco cells were exposed to K252a (A) or niflumate (B) at the indicated concentrations for 10 min prior to EGTA addition (9 mM), and cytosolic Ca2+ was monitored as described under "Materials and Methods." Control cells were treated with 0.05% Me2SO to control for the solvent used to dissolve the inhibitors.

Restoration of Externally Derived Ca2+ Pulses after Long Term Deprivation of External Ca2+-- Although long term exposure to EGTA leads to the gradual depletion of internal Ca2+ stores, as evidenced by our inability to elicit release of Ca2+ from these compartments (Fig. 3), the same long term depletion of extracellular Ca2+ leads surprisingly to a gradual restoration of sensitivity to stimulants of external Ca2+ entry. Thus, cytosolic Ca2+ pulses activated by cold shock or hypo-osmotic shock, which are well documented to arise predominantly from external Ca2+ pools (2, 3, 7), gradually reappear following extended (~20 min) depletion of extracellular Ca2+. As seen in Fig. 5, the first phase of Ca2+ influx following hypo-osmotic shock converts from almost complete inhibition immediately following EGTA addition, to nearly normal Ca2+ entry after 20 min of EGTA treatment (Fig. 5, A and B). As the concentration of chelator was increased to as much as 36 mM, the first peak continues its return to control levels, whereas the second internally derived pulse becomes dramatically diminished (Fig. 5C). Importantly, similar results are also obtained when cold shock is assayed in place of hypo-osmotic shock (Fig. 5D), suggesting that the recovery of responsiveness after extended exposure to an extracellular chelator may be a general characteristic of any stimulated external Ca2+ influx.



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Fig. 5.   Restoration of externally derived Ca2+ pulses after long term external Ca2+ removal. [Ca2+]cyt levels were monitored as described under "Materials and Methods." A, hypo-osmotically activated Ca2+ pulses were recorded after treatment with 9 mM EGTA for the indicated periods. The arrow marks the time at which cells were hypo-osmotically shocked by 1:1 dilution with double distilled H2O. B, as in A, cells were subjected to varying periods of EGTA treatment (9 mM), and then hypo-osmotically shocked. The % restoration of the first peak of osmotically triggered Ca2+ influx is then plotted as a function of the duration of exposure to excess external EGTA (average ± S.D., n = 3). 100% restoration is taken as the magnitude of the Ca2+ peak exhibited by Ca2+-bathed control cells. C, cell suspensions were treated with the indicated concentrations of EGTA for 20 min prior to hypo-osmotic shock at the time indicated by the arrow. D, cells were treated with 9 mM EGTA for the indicated periods prior to a cold shock generated by injection of 1 ml of ice-cold cell culture medium into the suspension, at the time indicated by the arrow. E, cells were treated with 9 mM EGTA for 20 min followed by 50 mM caffeine (or buffer as a control) and then subjected to cold shock (as in Fig. 5D) at the indicated time point. Data are representative of three independent experiments in which similar results were obtained.

Because the mobilizable internal Ca2+ stores in plants are probably vast (1), it was conceivable that restoration of the externally derived Ca2+ pulses following long term removal of external Ca2+ was due to the release of enough Ca2+ from internal pools to replenish the extracellular medium. To explore this possibility, we monitored Ca2+ levels in the extracellular medium after addition of 9 mM EGTA. However, no significant replenishment of external Ca2+ could be detected, even following 2 h of EGTA application (data not shown). Furthermore, addition of an extra 9 mM EGTA to cells that had already been incubated in EGTA for 20 min did not prevent activation of the restored cold shock-stimulated Ca2+ peak (data not shown). These data demonstrate that the return of responsiveness to stimulants of external Ca2+ entry is not due to restoration of external Ca2+. Additionally, the revived Ca2+ influx also does not appear to derive from the previously characterized caffeine-sensitive internal Ca2+ pool. As shown in Fig. 5E, caffeine injected into the cuvette 20 min after EGTA treatment does not prevent a cold shock-stimulated Ca2+ flux. Thus, it appears that long term removal of external Ca2+ results in the sequestration of Ca2+ into a third compartment that is EGTA-inaccessible and caffeine-insensitive, and which releases Ca2+ into the cytosol in response to stimuli that normally activate externally derived influx in Ca2+-bathed cells.

Finally, to demonstrate that the Ca2+ released from internal stores after the depletion of external Ca2+ might be responsible for filling this new compartment, signaling modulators that were previously found to block the EGTA-activated release of intracellular Ca2+ were again tested for their abilities to prevent the filling of this EGTA-inaccessible Ca2+ store. As shown in Fig. 6, K252a (and staurosporine; data not shown) also prevents the restoration of the cold shock-activated Ca2+ pulse (Fig. 6). Importantly, these modulators do not affect the cold shock-induced Ca2+ pulse either when added to Ca2+-bathed (control) cells, or when administered to Ca2+-depleted cells that have been exposed to EGTA for sufficient time to permit filling of the EGTA-inaccessible compartment (data not shown). These observations suggest that gating of Ca2+ from neither the apoplastic space nor the unidentified compartment is altered by the kinase inhibitors. Rather, it would appear that a kinase-dependent pathway must mediate some step in communicating the depletion of external Ca2+ stores to the machinery involved in filling an EGTA-inaccessible Ca2+ pool at the expense of the more prominent intracellular Ca2+ store.



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Fig. 6.   Restoration of the cold shock-stimulated Ca2+ peak after external Ca2+ removal is sensitive to inhibitors of EGTA-induced Ca2+ release. Suspension cultured tobacco cells were treated with either K252a or 0.05% Me2SO (control) for 10 min prior to recording [Ca2+]cyt in the luminometer. At the time indicated by the first arrow, 9 mM EGTA was added into the cuvette. At the second arrow, cells were diluted 1:1 with ice-cold medium. Inset, the percentage of restoration of EGTA-treated cells to the cold shock-induced Ca2+ amplitudes measured in Ca2+-bathed control cells on the same day. Data shown are representative of three independent experiments in which similar results were obtained.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have presented data to suggest that a change in the Ca2+ content of an extracellular Ca2+ store can be rapidly communicated to an intracellular compartment that responds by releasing Ca2+ into the cytosol (Fig. 2). If a stress-related signal to discharge Ca2+ is received by the internal organelle soon after removal of the external Ca2+, the magnitude of Ca2+ release is heightened (Fig. 1A). If, in contrast, the signal to discharge Ca2+ arrives after the store of organellar Ca2+ has already been depleted, no further release of the cation is noted (Fig. 3C). Thus, some type of mechanism for sensing extracellular Ca2+ appears to modulate the behavior of a major intracellular Ca2+ storage compartment. In view of previous data demonstrating communication from the intracellular Ca2+ pool to the extracellular Ca2+ store (7), we conclude that these two Ca2+ compartments are well aware of the Ca2+ status of the other.

A question obviously arises regarding the function of the above communication, and more specifically, how the active release of intracellular Ca2+ upon depletion of extracellular Ca2+ might benefit the cell. Although many plausible explanations might be offered, we suggest that different Ca2+ compartments in the cell might have different signaling functions (1, 11). As a consequence, uncompensated emptying of one compartment could conceivably eliminate an entire group of signaling pathways essential for plant survival. Sharing of Ca2+ reserves from a filled compartment to an empty compartment would avoid this potential dysfunction. In this scenario, the release of intracellular Ca2+ upon removal of the external Ca2+ could have evolved to allow replenishing of the depleted extracellular stores or their substitutes.

Because our Ca2+ depletion studies were conducted in the presence of sufficient EGTA to prevent resupply of extracellular stores, we were unable to evaluate whether the external Ca2+ pool might normally be replenished by the above mechanism. We did observe, however, that in the presence of excess apoplastic chelating capacity, a new Ca2+ compartment was formed that could substitute for extracellular Ca2+ in transducing cold shock and hypo-osmotic stress-induced signals. This new Ca2+ pool was not in the culture medium, since it could neither be detected by Ca2+ indicator dyes nor depleted by treatment with additional EGTA. It was also not likely associated with the primary intracellular store, since it was still functional after this caffeine- and elicitor-responsive store had been emptied. Although we currently have no information on the identity of this new compartment, we hypothesize that it may reside in an internal organelle located near the signaling machinery that triggers entry of extracellular Ca2+. In this way, it could readily substitute for the depleted extracellular Ca2+ in mediating cold shock and hypo-osmotic stress signals to the nucleus.

Because release of intracellular Ca2+ in response to chelation of extracellular Ca2+ is so immediate, we suspect that the putative Ca2+ sensor is extracellular. As such, it is conceivable that the sensor might be homologous to animal CaRs, which are G-protein-coupled receptors that signal in response to elevated serum [Ca2+] via phospholipase C and inositol trisphosphate (14). In fact, the Arabidopsis genome contains several open reading frames with substantial homology to the animal CaRs (e.g. emb CAB63012.1). The function of these proteins in plants has, unfortunately, not yet been elucidated. However, because the EGTA-induced Ca2+ fluxes in tobacco cells are insensitive to inhibitors of phospholipase C (i.e. neomycin sulfate and U73122; data not shown), but are inhibited by modulators of anion channels and kinases (Fig. 4), it appears that the Ca2+ sensor in our studies must differ at least in its downstream effector components from the animal CaRs (14). An alternative possibility is that the plant extracellular Ca2+ sensor involves the cell wall itself. He and colleagues (22) have recently identified a family of transmembrane cell wall-integrated receptor-like protein kinases, the extracellular domains of which interact with the pectic portion of the cell wall in tobacco. Because pectin is known to bind large amounts of Ca2+ and to dramatically change conformation upon Ca2+ removal (23, 24), it is possible that Ca2+-regulated changes in cell wall-kinase interactions could also initiate the signaling cascade leading to release of organellar Ca2+.


    ACKNOWLEDGEMENTS

We thank Dr. Darrell Schulze of the Purdue University Department of Agronomy for providing excellent technical assistance.


    FOOTNOTES

* This work was supported by National Science Foundation Grant MCB-9725934.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: Depts. of Biology and Chemistry, Eastern Mennonite University, Harrisonburg, VA 22802.

§ To whom correspondence should be addressed: Dept. of Chemistry, Purdue University, 1393 Brown Bldg., West Lafayette, IN 47904. Tel.: 765-494-5273; Fax: 765-494-0239; E-mail: plow@purdue.edu.

Published, JBC Papers in Press, January 3, 2001, DOI 10.1074/jbc.M006989200


    ABBREVIATIONS

The abbreviations used are: ER, endoplasmic reticulum; CaR, Ca2+-sensing Receptor; MS, Murashige and Skoog.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES


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