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
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ABSTRACT |
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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.
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.
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
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
(
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).
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.
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
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.
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.
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.
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.
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+.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, 2.99 mM;
SO
, 0.1 mM;
BO
, 0.005 mM;
MnO
ex = 494 nm,
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.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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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; 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.
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.
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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).
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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.
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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.
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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
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ACKNOWLEDGEMENTS |
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We thank Dr. Darrell Schulze of the Purdue University Department of Agronomy for providing excellent technical assistance.
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FOOTNOTES |
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* 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.
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
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ABBREVIATIONS |
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The abbreviations used are: ER, endoplasmic reticulum; CaR, Ca2+-sensing Receptor; MS, Murashige and Skoog.
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