Journal of Experimental Botany, Vol. 49, No. 318, pp. 1–11, January 1998
REVIEW ARTICLE
Light-dependent signal transduction and transient
changes in cytosolic Ca2+ in a unicellular green alga
G. Schönknecht1, C.S. Bauer and W. Simonis
Lehrstuhl Botanik I, Universität Würzburg, Mittlerer Dallenbergweg 64, D-97082 Würzburg, Germany
Received 1 May 1997; Accepted 15 August 1997
Abstract
Introduction
The physiological function and the molecular mechanisms of Ca2+-mediated signal transduction processes were studied in the unicellular green alga
Eremosphaera viridis by different electrophysiological
and microfluorimetric techniques. A sudden blockage
of photosynthetic electron transport by darkening or
inhibitors causes a transient hyperpolarization of the
plasma membrane. For the alga this transient hyperpolarization seems to be an important mechanism to
release monovalent ions and to drive the uptake of
divalent cations. The transient hyperpolarization is due
to the opening of K+ channels and is caused by a rapid
transient elevation of the cytosolic free Ca2+ concentration ([Ca2+] spike). Different agonists like caffeine
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or InsP which are known to release Ca2+ from internal
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stores in animal cells, also cause a transient hyperpolarization and a [Ca2+] spike, similar to darkening. In
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Eremosphaera the transient hyperpolarization can be
used as an indicator for [Ca2+] spikes. The InsP
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gated and the ryanodine/cADPR gated Ca2+ channels
which obviously both mediate Ca2+ release from
internal stores in Eremosphaera do not seem to be
involved in the dark-induced [Ca2+] spikes. Besides
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single [Ca2+] spikes, the addition of Sr2+ (or caffeine
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in the absence of divalent cations) causes repetitive
[Ca2+] spikes which may last hours and resemble
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[Ca2+] oscillations observed in excitable animal cells.
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These observations suggest that some principal
molecular mechanisms causing single or repetitive
[Ca2+] spikes are conserved from animal to plant
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cells.
Transient changes in the cytosolic free Ca2+ concentration
([Ca2+] ) play a key role for intracellular signal transduccy
tion processes (Bootman and Berridge, 1995; Bush, 1995;
Webb et al., 1996). In the last years it has become obvious
that these [Ca2+] changes frequently take the shape of
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a rapid transient increase, a so-called [Ca2+] spike. From
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animal cells it is known that [Ca2+] spikes are often
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organized in complex temporal and spatial patterns
( Thomas et al., 1996). In plant cells different stimuli were
shown to induce single ( Knight et al., 1991, 1992) or
even repetitive [Ca2+] spikes (McAinsh et al., 1995;
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Ehrhardt et al., 1996; Bauer et al., 1997). In this article,
current knowledge about the mechanisms and physiological significance of [Ca2+] spikes in the unicellular green
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alga Eremosphaera viridis is summarized. For a more
comprehensive view of calcium signalling in plant cells
the reader may consult recent reviews (Bush, 1995;
Trewavas et al., 1996; Webb et al., 1996).
The unicellular green alga Eremosphaera viridis has a
spherical shape and a diameter of 100–200 mm. This large
size makes it an ideal object for different electrophysiological techniques which were combined with the microinjection of effectors, inhibitors, and indicators. Especially
the microinjected Ca2+ indicator fura-2 dextran was used
to register changes in [Ca2+] (Plieth and Hansen, 1996;
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Bauer et al., 1997). With these techniques it was observed
that different stimuli and agents induce single or repetitive
[Ca2+] spikes in Eremosphaera which are always accomcy
panied by a transient hyperpolarization of the plasma
membrane. There is a physiological and a mechanistic
aspect of [Ca2+] spikes. The first part of this article,
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concerning the physiological aspect, describes how
[Ca2+] changes are involved in a signal transduction
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process. A light-off stimulus, which is received at the
Key words: Signal transduction, calcium, Eremosphaera
viridis, calcium spikes, membrane potential.
1 To whom correspondence should be addressed. Fax: +49 931 888 6158. E-mail: [email protected]
Abbreviations: [Ca2+] , cytosolic free calcium concentration.
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© Oxford University Press 1998
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Schönknecht et al.
thylakoid membrane, is coupled to a response at the
plasma membrane, namely the opening of ion channels,
by a [Ca2+] spike. The second part, concerning the
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mechanistic aspect, describes different processes which
give rise to a sudden increase in [Ca2+] . Different
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agents as caffeine, InsP , or Sr2+ which are known to
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release Ca2+ from intracellular Ca2+ stores in animal
cells, induce single or repetitive [Ca2+] spikes in
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Eremosphaera. Finally, the different mechanisms which
may contribute to cytosolic [Ca2+] spikes as a central
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element of intracellular signal transduction are
summarized.
running membrane potential is decreased to about
−180 mV. This transient potential change has a duration
of about 50 s and is observed after a light-off stimulus in
three out of four measurements. During the hyperpolarization the membrane resistance decreases and the membrane potential comes close to the Nernst potential for
K+. Different K+ channel inhibitors like TEA (1 mM )
( Köhler et al., 1983) or Ba2+ (>0.1 mM ) ( Thaler et al.,
1987) block the transient hyperpolarization. Obviously,
the transient hyperpolarization is caused by a transient
opening of K+ channels in the plasma membrane.
Relation to photosynthesis
Dark-induced opening of K+ channels in the plasma
membrane
More than 10 years ago, it has been observed for the first
time that the unicellular green alga Eremosphaera viridis
responds to darkening with a transient hyperpolarization
of the plasma membrane (Fig. 1a) (Geisweid et al., 1982).
From a steady state value of about −90 mV the free
The light-off stimulus which triggers the transient hyperpolarization is received by the thylakoid membrane. This
was shown by the effect of different inhibitors of photosynthetic electron transport. The addition of DCMU
(3-(3,4-dichlorophenyl )-1,1-dimethylurea) ( Köhler et al.,
1983) or DBMIB (2,5-dibrom-6-isopropyl-3-methyl-pbenzochinone) (Förster, 1986) in the light, but not in the
dark, triggers a transient hyperpolarization. In the presence of the inhibitors a dark-induced transient hyperpolarization is blocked. This indicates that the transient
hyperpolarization of the plasma membrane is initialized
by the termination of photosynthetic electron transport.
There has to be a signal transduction chain from the
thylakoid membrane to the plasma membrane. This signal
transduction seems to take some seconds as a delay of
about 5 s is observed between the light-off stimulus and
the beginning of the membrane hyperpolarization ( Köhler
et al., 1985; Bauer et al., 1997).
Light-dependent changes in cytosolic ion activities
Fig. 1. Light-dependent changes of the membrane potential (a) and the
cytosolic ion activities (b, c, d ) in Eremosphaera. Recordings from
different measurements were combined. Each recording was started
under photosynthetically saturating white light, which was switched off
for 3 min (white and black bars at the top give the light protocol ). (a)
The electrical potential difference across the plasma membrane (E, mV )
was registered by an impaled microelectrode (Sauer et al., 1994). (b)
The cytosolic Ca2+ activity ([Ca2+] , nM ) was determined by
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fluorescence ratio imaging by means of the fluorescent Ca2+-sensitive
dye fura-2-dextran which was injected mechanically into the cytosol of
the alga (Bauer et al., 1997). This measurement was performed
discontinuous with a sampling interval of 1.5 s, as indicated by the
small open circles. (c) The cytosolic H+ (pH ) and (d) Cl− activity
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(—log[Cl−] ) were measured by ion-selective microelectrodes and are
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given on a logarithmic scale on a molar basis ( Thaler et al., 1992).
Possible candidates for the signal transduction between
thylakoid and plasma membrane are light-dependent
changes in cytosolic ion activities. By means of ion
selective microelectrodes, the intracellular ion activities in
Eremosphaera (which are summarized in Fig. 2) were
measured and the relationship between light-dependent
changes in cytosolic ion activities ( Fig. 1) and the lightdriven H+ transport across the thylakoid membrane were
examined.
Light-dependent cytosolic pH changes measured in
Eremosphaera (Fig. 1c) ( Köhler et al., 1986; Steigner
et al., 1988; Thaler et al., 1992) are comparable to those
measured in other green, photosynthetic active plant cells
( Felle and Bertl, 1986; Okazaki et al., 1994). Light-off
results in a transient acidification and a transient
alkalization is observed after light-on ( Fig. 1c). The transient cytosolic acidification after light-off precedes the
transient hyperpolarization by about 3 s (Steigner et al.,
1988). The light-dependent cytosolic pH changes are
directly caused by the light-driven H+ fluxes across the
thylakoid membrane ( Thaler et al., 1992; Hansen et al.,
Calcium in Eremosphaera
Fig. 2. External and intracellular ion activities for H+, K+, Ca2+, and
Cl− plus the electrical potentials (E ) of Eremosphaera at steady state
(Bethmann et al., 1995). The external medium contained 0.1 mM
KNO , 0.1 mM MgCl , 0.1 mM CaCl , and 2 mM MES adjusted to
3
2
2
pH 5.6 with NaOH. The ion activities and electrical potentials were
measured by ion-selective microelectrodes.
1993). After light-on, the protons pumped from the
chloroplast stroma into the thylakoid lumen are partly
compensated by a H+ flux from the cytosol into the
stroma, and after light-off some of the H+ released from
the thylakoid lumen are transported via the chloroplast
envelope into the cytosol. The transient nature of the
cytosolic pH changes is due to pH-stat mechanisms. The
addition of cyanide (0.5 mM ) which is known to reduce
the cytosolic ATP level results in permanent instead of
transient light-dependent pH changes ( Thaler et al.,
1992). Beside pH changes, light-dependent changes of the
cytosolic Cl− activity were measured in Eremosphaera
( Thaler et al., 1992; Bethmann et al., 1995). A cytosolic
Cl− activity of 2 mM in the light is increased to 3 mM
in the dark ( Fig. 1d ). The light-dependent Cl− fluxes are
likely to be caused by the parallel light-driven H+ fluxes
from the thylakoid to the cytoplasm, electrically counterbalancing each other (Schönknecht et al., 1988; Thaler
et al., 1992). In contrast to the light-dependent pH
changes, the light-dependent cytosolic Cl− activity
changes are permanent and not transient ( Thaler et al.,
1992). Compared to the very effective pH-stat mechanisms ( Fig. 1c) the homeostasis of the cytosolic Cl− activity
seems to be less perfect (Fig. 1d).
With Ca2+ selective microelectrodes no light-dependent
changes of the cytosolic Ca2+ activity ([Ca2+] ) could be
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registered in Eremosphaera (Bethmann et al., 1995). This
is in agreement with Trebazc et al. (1994), who also did
not observe light-dependent [Ca2+] changes in the livercy
wort Conocephalum conicum. In contrast to this, Miller
and Sanders (1987) reported relative large and permanent
light-dependent [Ca2+] changes in Nitellopsis. Recent
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measurements of [Ca2+] in Eremosphaera with furacy
2-dextran (Bauer et al., 1997) demonstrate that darkening
induces a rapid and transient increase in [Ca2+] ([Ca2+]
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spike) which lasts about 30 s ( Fig. 1b). Besides this
[Ca2+] spike no light-dependent changes in [Ca2+] were
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detected. Due its short duration and relative small amplitude the [Ca2+] spike after darkening was not detected
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in earlier measurements with Ca2+ selective microelectrodes. Whereas the light-dependent cytosolic pH and
Cl− activity changes are directly caused by the lightdriven ion fluxes across the thylakoid membrane, the
origin of the [Ca2+] spike after darkening is less obvious.
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Isolated chloroplasts have been shown to take up Ca2+
upon illumination and to release Ca2+ upon darkening
( Kreimer et al., 1985). The mechanism of these Ca2+
fluxes is unknown. Dark-induced Ca2+ release from
chloroplasts may be involved in the [Ca2+] spike
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observed in Eremosphaera upon darkening. However,
other internal Ca2+ stores as the vacuole or the endoplasmic reticulum may contribute as well.
An artificial cytosolic acidification (Steigner et al.,
1988) or an artificial increase in [Ca2+] ( Thaler et al.,
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1989) both induce a transient hyperpolarization of the
plasma membrane whereas an artificial increase of
the cytosolic Cl− activity ( Thaler et al., 1992) has no
effect. An artificial cytosolic acidification was brought
about by an external acidification from pH 5.5 to pH 3.1,
or by the addition of 1 mM Na acetate or 5 mM NH Cl
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( Köhler et al., 1985; Steigner et al., 1988). An artificial
increase in [Ca2+] was achieved by the addition of the
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Ca2+ ionophore A23187 ( Thaler et al., 1989) or by
microinjection of CaCl into the cytoplasm of
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Eremosphaera ( Förster, 1990). In the presence of external
Ca2+, A23187 caused an extremely prolonged transient
hyperpolarization.
This poses the question, does the [Ca2+] spike or the
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transient acidification after darkening or both cause the
transient hyperpolarization ( Fig. 1) of the plasma membrane? Whereas Köhler et al. (1985) and Steigner et al.
(1988) postulated that the transient cytosolic acidification
causes the transient hyperpolarization, recent measurements indicate that the [Ca2+] spike is both necessary
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and sufficient to induce a transient hyperpolarization. A
transient hyperpolarization was never observed in the
absence of a [Ca2+] spike and each [Ca2+] spike is
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accompanied by a transient hyperpolarization (Bauer
et al., 1997; see below). In contrast to this, a light-off
stimulus always induces a transient cytosolic acidification,
but this acidification is not always accompanied by a
transient hyperpolarization ( Thaler et al., 1992).
As Ca2+ and H+ at least in part bind to the same
substances inside the cytoplasm, a cytosolic acidification
results in some liberation of Ca2+ from these buffering
groups increasing [Ca2+] . However, such pH-dependent
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[Ca2+] changes are not likely to play a role under
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physiological conditions. Free H+ concentrations between
pH 7.7 and 7.2 corresponding to 20 to 60 nM are about
five times smaller compared to free Ca2+ concentrations
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Schönknecht et al.
which are in the range between 100 and 300 nM.
Therefore, light-dependent pH changes as described here
( Fig. 1c) are not sufficient for a considerable change in
[Ca2+] . Under physiological conditions changes of the
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cytosolic free H+ and Ca2+ concentration are not coupled
and both ions can independently be used as second
messengers inside the cell. This theoretical consideration
is supported by parallel measurements of cytosolic pH
and [Ca2+] . In Eremosphaera (Plieth et al., 1997) as well
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as in Vicia guard cells (Grabov and Blatt, 1997) a
moderate acidification of the cytosol to pH values >7.0
did not increase [Ca2+] . Only an artificial cytosolic
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acidification below pH 7.0 results in an increase of
[Ca2+] (Grabov and Blatt, 1997; Plieth et al., 1997).
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The artificial cytosolic acidification reported to induce a
transient hyperpolarization in Eremosphaera ( Köhler
et al., 1985; Steigner et al., 1988) results in pH values
below 7.0 (Steigner et al., 1988), and it is likely that the
artificial [Ca2+] increase due to this acidification, and
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not the acidification itself, caused the observed transient
hyperpolarization.
Besides the transient hyperpolarization of the plasma
membrane there exists another Ca2+-dependent lightinduced reaction in Eremosphaera. Blue-light induces a
systrophe (a chloroplast translocation to the centre of the
cell ) in the presence of external Ca2+ ( Weidinger and
Ruppel, 1985). Thaler et al. (1989) observed that an
artificial increase in [Ca2+] is accompanied by a systrocy
phe. An increase in [Ca2+] hyperpolarizes the plasma
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membrane and induces a chloroplast translocation to the
centre of the cell.
Ion fluxes during a transient hyperpolarization
The two electrode voltage-clamp technique was used to
investigate the ion fluxes involved in the transient hyperpolarization ( Köhler et al., 1989; Sauer et al., 1994). The
question was whether other ion currents besides those
carried by the K+ channel take part in this hyperpolarization. At maximum hyperpolarization the membrane
resistance is dramatically decreased and voltage-clamp
measurements result in ‘N-shaped’ I/V curves (Fig. 3a).
Köhler et al. (1989) speculated that these N-shaped I/V
curves result from a complex voltage-dependent gating of
the plasma membrane K+ channel. However, in other
plant cells ranging from green algae (Shiina and Tazawa,
1987; Okihara et al., 1993) to higher plants like the guard
cells of Vicia faba (Schroeder, 1988; Keller et al., 1989;
Hedrich and Marten, 1993), similar N-shaped I/V curves
are described to result from a combination of different
currents, which are carried by different types of ion
channels. Based on investigations with varying external
ion concentrations and ion channel inhibitors, Sauer et al.
(1994) demonstrated that the N-shaped I/V curves measured at maximum hyperpolarization in Eremosphaera are
Fig. 3. A model for the different current components giving rise to the
N-shaped I/V curves measured in the peak of a transient hyperpolarization (Sauer et al., 1994). (a) I/V relationships measured with the two
electrode voltage-clamp technique at resting membrane potential (2)
and at maximum hyperpolarization ($) of the plasma membrane of
Eremosphaera. (b) The different current components at maximum
hyperpolarization were experimentally separated by the use of ion
channel blockers (Sauer et al., 1994). The K+ current (&) was
measured during a transient hyperpolarization with the inward cation
currents blocked by AlCl and shortly before the anion current
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appeared. The voltage-dependent anion current (+) results from the
difference of two I/V curves measured at maximum hyperpolarization
in the absence and presence of an anion channel blocker (A9C, NPPB
or Zn2+), respectively. The inward rectifying cation current (,) results
from the difference of two I/V curves measured in the absence and
presence of AlCl . (c) The summing up of the three current components
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displayed in (b) results in an N-shaped I/V curve which does not
significantly differ from an N-shaped I/V curve recorded in the peak of
a transient hyperpolarization (a).
due to the activation of different conductances: (1) an
outward cation current carried by the K+ channel
( Fig. 3b, &), (2) a voltage-dependent inward current
carried by anion efflux with a maximum at about
−220 mV ( Fig. 3b, +), and (3) an inwardly rectifying
cation current at hyperpolarizing voltages ( Fig. 3b, ,).
The transient opening of the K+ channels gives rise to
the increased conductance of the plasma membrane and
dominates the transient hyperpolarization. The opening
of voltage-dependent anion channels which are blocked
by A9C, NPPB, and Zn2+ carries an anion efflux which
Calcium in Eremosphaera
is only observed at maximum hyperpolarization thus
limiting its amplitude especially at low external K+. The
inward rectifying cation channels which open at hyperpolarizing voltages mediate the uptake of divalent cations,
and in contrast to the K+ channel they are blocked by
Al3+ but not by Ba2+ or TEA (Sauer et al., 1993, 1994).
Preliminary single channel recordings with the plasma
membrane of Eremosphaera (De Boer et al., 1994;
Schönknecht et al., 1995) showed the existence of an
outward rectifying K+ channel, an anion-selective channel, and an inward rectifying cation channel, in good
agreement with the two electrode voltage clamp
measurements.
When the electrophysiological data depicted in Fig. 3
are combined with the electrochemical potential gradients
of H+, K+, Ca2+, and Cl− shown in Fig. 2, it turns out
that during a transient hyperpolarization of the plasma
membrane K+ and anions leave the cytoplasm of
Eremosphaera while divalent cations may be taken up.
The net release of salt is likely to be involved in osmotic
adjustment. Due to the hyperpolarization to about
−180 mV, divalent cations may be taken up against a
concentration gradient of up to six orders of magnitude.
It was observed that in the absence of external divalent
cations appreciably fewer dark-induced transient hyperpolarizations are released ( Köhler et al., 1985). In the
natural environment of Eremosphaera (acidic Sphagnum
bogs) the activity of divalent cations is rather low (Gerdol,
1995), and a potent uptake system for divalent cations is
very important.
In summary, after darkening besides other changes of
cytosolic ion activities there is a [Ca2+] spike which is
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probably caused by Ca2+ release from the chloroplast.
This [Ca2+] spike results in a transient hyperpolarization
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of the plasma membrane which drives a net salt release
and a parallel uptake of divalent cations. The whole
process can be described as a Ca2+ release-induced Ca2+
uptake triggered by darkening.
[Ca2+] spikes in Eremosphaera
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Transient changes in [Ca2+] obviously play a key role
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in the light-dependent signal transduction from the thylakoid to the plasma membrane in Eremosphaera. To get
some insight into the molecular mechanisms generating
[Ca2+] spikes different agonists and inhibitors known to
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interfere with the cytosolic Ca2+ homeostasis in animal
cells were applied. To get a quantitative basis for these
investigations, first the Ca2+ activities and concentrations
in Eremosphaera measured by different techniques are
compared.
Intracellular calcium concentrations
The total Ca2+ concentration of Eremosphaera as determined by ICP-AES (induction coupled plasma-atomic
5
emission spectroscopy) is close to 2 mM growing in a
medium containing 110 mM Ca2+. When the cell wall was
separated from the broken cells by centrifugation a total
Ca2+ concentration of the protoplast of less than 0.5 mM
was measured (Bethmann et al., 1995). So, about threequarters of the total Ca2+ are bound to the cell wall, in
agreement with other plant cells (Demarty et al., 1984).
Comparable analyses with the trivalent cations La3+ and
Gd3+ demonstrate that the separation of the cell wall by
centrifugation is complete. Whereas concentrations of up
to 1.5 mM Gd3+ or La3+ were measured in the cell wall
the concentration in the cell sap after centrifugation was
below detection limit (<1 mM ) (Bauer et al., unpublished
results). The cytosolic free calcium concentration
([Ca2+] ) was determined to 163 nM by means of Ca2+cy
selective microelectrodes (Bethmann et al., 1995) and to
164 nM by fluorescence ratio measurements using the
Ca2+-dependent fluorescent dye fura-2-dextran (Bauer
et al., 1997) (Figs 1, 2). This [Ca2+] value is typical for
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the cytoplasm of eucaryotic cells (Bush, 1995). The
vacuolar free Ca2+ concentration as measured with Ca2+selective microelectrodes was 200 mM (Fig. 2). This is the
lowest value recorded in plant vacuoles up to now, and
probably reflects an adaptation to the low availability of
this ion in the natural environment of Eremosphaera.
Fluorescence ratio measurements of [Ca2+] , compared
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to Ca2+-selective microelectrodes, had a much higher
time resolution and a higher sensitivity (see above).
However, the absolute calibration is more reliable with
Ca2+-selective microelectrodes. Both techniques ideally
complement each other.
Agonist induced Ca2+ release
To understand which mechanisms in Eremosphaera are
involved in cytosolic Ca2+ homeostasis and the generation
of [Ca2+] spikes, agonists were applied which are known
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to release Ca2+ from internal stores.
Caffeine applied in the millimolar range (up to 40 mM )
releases Ca2+ from internal Ca2+ stores in animal cells
(Liu and Meissner, 1997). This Ca2+ release is mediated
by the ryanodine/cADPR receptor Ca2+ channel (Pozzan
et al., 1994). Little is known about the effect of caffeine
in plant cells ( Förster et al., 1989). Only recently Allen
et al. (1995) and Muir and Sanders (1996) presented
evidence for the presence of ryanodine receptor homologues in higher plants. Addition of 20 mM caffeine to
the external medium (0.1 mM Ca2+) induces a single
transient hyperpolarization in Eremosphaera (Förster
et al., 1989). Recent fluorescence ratio measurements of
[Ca2+] (Bauer et al., 1997) demonstrate that addition
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of caffeine induces a [Ca2+] spike parallel to the transient
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hyperpolarization (Fig. 4). La3+ and Gd3+ which block
Ca2+ uptake via the plasma membrane (see below) had
no effect on the caffeine-induced transient hyperpolariz-
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Schönknecht et al.
Fig. 4. Time-course of the membrane potential (E, mV, top) and the
cytosolic free Ca2+ concentration ([Ca2+] , nM, bottom) during the
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addition of caffeine (20 mM; bar on top gives perfusion protocol ) to
the external medium (Bauer et al., 1997). The membrane potential and
[Ca2+] were recorded in parallel with the same algal cell with an
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impaled microelectrode and by means of microinjected fura-2-dextran,
respectively.
ation indicating that the [Ca2+] spike was due to Ca2+
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release from internal stores. When ruthenium red, an
antagonist of the ryanodine/cADPR Ca2+ release channel
(Ma, 1993), was microinjected into the cytoplasm of
Eremosphaera 20 mM caffeine no longer induced a transient hyperpolarization (Bauer et al., unpublished results).
TMB (3,4,5-trimethoxybenzoic acid 8-diethylamino8
octyl ester) an antagonist of the InsP -induced Ca2+
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release (see below) had no effect (Bauer, 1994).
InsP (inositol 1,4,5-trisphosphate) is known to release
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Ca2+ from internal stores in animal (Berridge, 1993) and
plant cells (Bush, 1995; Webb et al., 1996). Direct patch
clamp measurements have shown that InsP opens Ca2+
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channels in the tonoplast (Alexandre et al., 1990; Allen
and Sanders, 1994; Allen et al., 1995). Microinjection of
InsP into the cytosol of Eremosphaera induces a transient
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hyperpolarization which is prolonged compared to a
dark-induced transient hyperpolarization (Förster, 1990).
Microinjection of InsP (inositol 1,4-bisphosphate), InsP
2
1
(inositol 2-monophosphate), Ins (inositol ) or F 2,6-P
2
(fructose 2,6-bisphosphate) had no effect. When BAPTA
was injected together with InsP no transient hyperpolar3
ization is observed. The removal of external Ca2+ had
no effect. TMB an antagonist of InsP -induced Ca2+
8
3
release in animal (Zhang and Melvin, 1993) and plant
cells (Schumaker and Sze, 1987) blocked the hyperpolarization. These data indicate that InsP in Eremosphaera,
3
like in other cells, causes Ca2+ release from internal stores
( Förster, 1990).
In the unicellular green alga Eremosphaera there exists
both an InsP - and a caffeine-induced Ca2+ release path3
way from internal stores. At the moment nothing is
known about the nature of the internal Ca2+ stores. Allen
et al. (1995) have shown that InsP gated and ryanodine
3
receptor homologue Ca2+ channels co-reside in the vacuolar membrane of beet storage roots. But other organelles
like endoplasmic reticulum ( Klüsner et al., 1995), chloroplast (see above), mitochondria or the nucleus (Santella,
1996) may play a role as internal Ca2+ stores in plant
cells as well.
Single [Ca2+]
spikes as they are observed in
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Eremosphaera ( Figs 1, 4) are observed in a variety of
plant cells. [Ca2+] spikes can be induced by mechanical
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signals, cold-shock, or elicitors in plant cells and a correlation between mechanical signal strength and amplitude
of the resulting [Ca2+] spike was shown ( Knight et al.,
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1991, 1992; Trewavas et al., 1996). The role of single
[Ca2+] spikes in signal transduction in plant cells
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becomes more and more obvious (Trewavas et al., 1996;
Webb et al., 1996). In Eremosphaera the [Ca2+] spike
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after darkening is part of a signal transduction chain
from the thylakoid to the plasma membrane, i.e. from
the cell interior to the surrounding membrane. Most
signal transduction chains are the other way round, they
transduce signals which are received at the plasma membrane to the cell interior. This demonstrates that [Ca2+]
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spikes are involved in many different signal transduction
processes in plant cells (Trewavas et al., 1996; Webb
et al., 1996) and poses the question of how a stimulus
specificity is achieved. In a single [Ca2+] spike informacy
tion may be encoded in the amplitude and kinetic, and
in animal cells it has been shown that micromolar and
submicromolar [Ca2+] spikes regulate distinct cellular
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functions ( Ito et al., 1997). In more and more eukaryotic
cells repetitive [Ca2+] spikes or [Ca2+] oscillations are
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observed ( Fewtrell, 1993; Petersen et al., 1994) raising
the additional possibility to encode information in the
frequency of [Ca2+] oscillation.
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Baseline spiking [Ca2+] oscillations
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In Eremosphaera regular repetitive [Ca2+] spikes are
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observed under certain conditions. When divalent cations
were omitted from the external medium (0.1 mM EGTA,
no divalent cations added) Förster et al. (1989) observed
that the addition of 20 mM caffeine induce repetitive
transient hyperpolarizations. Recent [Ca2+] measurecy
ments demonstrated that 20 mM caffeine at low external
Ca2+ (0.3 mM ) induce repetitive [Ca2+] spikes in
cy
Eremosphaera which cause repetitive transient hyperpolarizations ( Fig. 5) (Bauer et al., 1997). The caffeine-induced
[Ca2+] spikes and hyperpolarizations had a frequency
cy
of about 1 min−1 and a duration of up to 60 min. The
application of the known Ca2+ channel blockers Gd3+
(see Fig. 5), La3+ (both 100 mM ) or verapamil (10 mM )
reversibly inhibited repetitive [Ca2+] spikes and transient
cy
hyperpolarizations at low external Ca2+ (Bauer et al.,
1997). In contrast, single [Ca2+] spikes induced by
cy
Calcium in Eremosphaera
7
Fig. 5. Time-course of the membrane potential (E, mV, top) and the cytosolic free Ca2+ concentration ([Ca2+] , nM, bottom) during the addition
cy
of caffeine (20 mM; bars on top gives perfusion protocol ) at a low external concentration of divalent cations (0.1 mM EGTA; 0.3 mM Ca2+)
(Bauer et al., 1997). The additional perfusion of 100 mM GdCl for 5 and 7 min, respectively, reversibly inhibited the membrane potential as well
3
as the [Ca2+] oscillations. The membrane potential and [Ca2+] were recorded in parallel with the same algal cell with an impaled microelectrode
cy
cy
and by means of microinjected fura-2-dextran, respectively.
caffeine at higher external Ca2+ concentrations, were not
affected by these Ca2+ channel blockers (see above). This
indicates, that for sustained caffeine-induced [Ca2+]
cy
oscillations a certain Ca2+ permeability of the plasma
membrane is necessary. Especially during the addition
and wash out of Ca2+ channel blockers the perfect
synchronism of [Ca2+] spikes and repetitive transient
cy
hyperpolarizations was obvious (Fig. 5) (Bauer et al.,
1997). After microinjection of InsP into the cytosol of
3
Eremosphaera, Förster (1990) sometimes observed repetitive transient hyperpolarizations which, however, decayed
after 5 to 6 repetitions and did not show a dependency
on the external Ca2+ concentration or the amount of
injected InsP .
3
The divalent cation Sr2+ due to its similarity to Ca2+
is transported by most Ca2+ ATPases and Ca2+ channels.
Sr2+ is taken up into intracellular Ca2+ stores ( Kwan
and Putney, 1990), and Sr2+ entry may induce Ca2+
release from internal stores in animal cells mimicking the
so-called Ca2+-induced Ca2+ release (Grégoire et al.,
1993). In Eremosphaera addition of Sr2+ (≥100 mM ) to
the external medium induces repetitive transient hyperpolarizations (Thaler et al., 1989). Ba2+ at micromolar
concentrations has a similar effect, whereas, at millimolar
concentrations Ba2+ inhibits transient hyperpolarizations
by blocking the K+ channel ( Thaler et al., 1987). Recent
[Ca2+] measurements based on microinjected furacy
2-dextran (Bauer et al., 1997) demonstrated that addition
of 1.0 mM Sr2+ induces repetitive [Ca2+] spikes and
cy
parallel repetitive transient hyperpolarizations in
Eremosphaera. In contrast to caffeine-induced [Ca2+]
cy
oscillations (see above), neither the probability to induce
oscillations by Sr2+ (95%) nor their duration or frequency
were influenced by the external concentration of Ca2+ or
Mg2+ (Bauer et al., unpublished results). The Sr2+induced [Ca2+] spikes and hyperpolarizations appeared
cy
with a frequency of about one every 2 min. The duration
of a single [Ca2+] spike during Sr2+-induced repetitive
cy
changes was comparable to caffeine-induced repetitive
changes, 25 to 30 s. The 2-fold lower frequency under
Sr2+ compared to caffeine is mainly caused by longer
intervals between the single Sr2+-induced [Ca2+] spikes
cy
compared to caffeine-induced [Ca2+] spikes. A duration
cy
of more than 2 h was frequently observed for Sr2+induced oscillations (Bauer et al., 1997).
In plant cells little is known about [Ca2+] oscillations.
cy
Phytohormone-induced [Ca2+] fluctuations reported
cy
earlier were strongly damped and ceased after a few
repetitions ( Felle, 1988; Schroeder and Hagiwara, 1990).
Only recently stable [Ca2+] oscillations were reported
cy
for plant cells (Johnson et al., 1995; McAinsh et al., 1995;
Ehrhardt et al., 1996; Bauer et al., 1997). There is a
physiological and a mechanistic aspect of [Ca2+] oscillacy
tions. The physiological aspect is about the involvement
in signal transduction and the importance for biological
processes, whereas the mechanistic aspect focuses on
questions such as: Which internal Ca2+ stores are
involved? Which Ca2+ fluxes increase [Ca2+] ? How is
cy
Ca2+ rapidly removed from the cytosol again? In plant
cells first information is emerging about the physiological
function of [Ca2+] oscillations and how they are involved
cy
in signal transduction (Johnson et al., 1995; McAinsh
8
Schönknecht et al.
et al., 1995; Erhardt et al., 1996). However, nearly nothing
is known about the mechanism of Ca2+ oscillations in
plant cells. Our experiments with Eremosphaera using
caffeine and Sr2+ are aimed at elucidating the mechanism
of [Ca2+] oscillations in pant cells.
cy
In animal cells different patterns of [Ca2+] oscillations
cy
were classified which are likely to represent also mechanistically distinct types (Fewtrell, 1993; Thomas et al.,
1996). Besides [Ca2+] fluctuations which do not display
cy
a regular symmetric pattern, [Ca2+] oscillations display
cy
either a regular sinusoidal pattern or a clear baseline with
discrete repetitive [Ca2+] spikes. For baseline spiking
cy
[Ca2+] oscillation the frequency and latency period, but
cy
not the amplitude, are determined by the agonist dose,
whereas for sinusoidal [Ca2+] oscillations the agonist
cy
dose regulates the amplitude, but not the frequency
( Thomas et al., 1996). Furthermore, [Ca2+] oscillations
cy
with a baseline spiking pattern continue for a long period
of stimulation. In Eremosphaera the frequency increases
and the latency period decreases with increasing external
Sr2+ concentrations (agonist dose) whereas the amplitude
does not depend on the external Sr2+ concentration
(Bauer et al., unpublished results). Moreover, Sr2+induced repetitive [Ca2+] spikes in Eremosphaera last
cy
very long. This indicates that baseline spiking [Ca2+]
cy
oscillations in plant and animal cells may be based on
similar mechanisms.
Recent experiments corroborate this view. In animal
cells Sr2+ is known to induce Ca2+ release from internal
stores via the ryanodine/cADPR Ca2+ release channel
(Meissner, 1994; Pozzan et al., 1994). This is probably
also the case in Eremosphaera since Sr2+-induced oscillations are blocked by ruthenium red (Bauer et al., unpublished results) which is known to block ryanodine/cADPR
Ca2+ release channels in animal (Ma, 1993) and plant
cells (Allen et al., 1995). After an initial rise in [Ca2+]
cy
additional mechanisms come into play to establish sustained [Ca2+] oscillations. A central element is a periodic
cy
Ca2+ uptake and release by internal Ca2+ stores
( Fewtrell, 1993; Petersen et al., 1994; Thomas et al.,
1996). In Eremosphaera, the inhibitors DBHQ (2,5-ditert-butylhydroquinone) and CPA (cyclopiazonic acid )
blocked Sr2+-induced repetitive [Ca2+] spikes (Bauer
cy
et al., unpublished results). Both inhibitors were shown
to act on Ca2+ ATPases of the endoplasmic reticulum in
plant cells (Logan and Venis, 1995; Hwang et al., 1997).
This indicates, that comparable to animal cells, a repetitive Ca2+ release and Ca2+ re-uptake by the endoplasmic
reticulum generates baseline spiking [Ca2+] oscillations
cy
in Eremosphaera in the presence of Sr2+. Moreover, Ca2+
fluxes across the cell membrane are necessary for sustained
oscillations in most cases (Fewtrell, 1993; Petersen et al.,
1994). A Ca2+ influx across the plasma membrane is
also necessary for sustained [Ca2+] oscillations in
cy
Eremosphaera as shown by the effect of the plasma
membrane Ca2+ channel blockers La3+ and Gd3+
( Fig. 5). The picture emerging for [Ca2+] oscillations in
cy
Eremosphaera (Bauer et al., unpublished results) shows
remarkable parallels to models developed for [Ca2+]
cy
oscillations in animal cells. Thus, the baseline spiking
[Ca2+] oscillations in Eremosphaera do not only display
cy
the same rhythmic spiking pattern commonly found in
excitable animal cells, they are probably also caused by
similar mechanisms.
In every case where in Eremosphaera a ratiometric
[Ca2+] measurement was performed in parallel to a
cy
membrane potential measurement, the synchronism of
the two parameters was perfect. Under conditions that
induce a single transient hyperpolarization we always
observed a synchronous [Ca2+] spike (Figs 1, 4).
cy
Repetitive transient hyperpolarizations are always accompanied by parallel repetitive [Ca2+] spikes (Fig. 5), and
cy
a transient hyperpolarization in the absence of a [Ca2+]
cy
spike was never observed. This close correlation makes it
possible to use the transient hyperpolarization of the
plasma membrane as a qualitative indicator for a transient
increase of [Ca2+] in Eremosphaera.
cy
Although the close correlation between [Ca2+] and
cy
membrane potential is obvious, it is not known how a
[Ca2+] spike causes a transient hyperpolarization. Does
cy
Ca2+ act directly on the plasma membrane K+ channel
or are there are additional signal transduction steps in
between? The first plant outward rectifying K+ channel,
KCO1, which was recently cloned and sequenced was
shown to be activated by cytosolic Ca2+ in an expression
system at physiological activities showing a very steep
Ca2+ dependency (Czempinski et al., 1997). The voltagedependence ( Fig. 3) and the blockage by millimolar Ba2+
concentrations are comparable for KCO1 and the K+
channel in the plasma membrane of Eremosphaera.
Moreover, in Eremosphaera a relative small increase in
[Ca2+] (Figs 1, 4, 5) results in a dramatic increase of
cy
the K+ conductivity of the plasma membrane (Fig. 3a)
pointing to a very steep Ca2+ dependency as well. On the
other hand, there are preliminary evidences for additional
signal transduction steps. First, the hyperpolarization
lasts about 10 s longer than the [Ca2+] increase does.
cy
Second, all calmodulin inhibitors tested so far, namely
calmidazolium, chlorpromazine, trifluoperazine or
W7 (N-(6-aminohexyl )-5-chloro-1-naphthalenesulphonamide) blocked transient hyperpolarizations (Fig. 6)
regardless of the stimulus (darkening, caffeine, Sr2+)
(Bauer, unpublished results).
Summary
The single [Ca2+] spikes and accompanying transient
cy
hyperpolarizations observed in Eremosphaera during
caffeine-or Sr2+-induced oscillations have an amplitude
and kinetic comparable to the [Ca2+] spike and transient
cy
Calcium in Eremosphaera
9
Fig. 6. A schematic model for the different processes contributing to [Ca2+] spikes and accompanying transient hyperpolarizations of the plasma
cy
membrane in Eremosphaera. Solid arrows indicate ion transport processes and fluxes. Dashed lines indicate regulatory interactions, dashed arrows
mean activation and dashed T-shaped lines mean inhibition. CM stands for calmodulin and RR for ruthenium red, all other abbreviations see text.
The symbolic Ca2+-ATPases were included to indicate that some kind of active Ca2+ transport is necessary to build up the electrochemical Ca2+
gradients which drive the passive Ca2+ fluxes.
hyperpolarization observed after light-off. In every case
the transient hyperpolarization of the plasma membrane
comes close to the Nernst potential for K+ and could be
blocked by TEA or Ba2+. On the other hand, the darkinduced transient hyperpolarization in Eremosphaera is
neither influenced by TMB (Bauer, unpublished results)
8
which blocks InsP -induced Ca2+ release nor by ruth3
enium red (Bauer et al., unpublished results) which blocks
caffeine- or Sr2+-induced Ca2+ release. Therefore, neither
the InsP gated nor the ryanodine/cADPR gated Ca2+
3
channel, both of which can mediate Ca2+ release from
internal stores in Eremosphaera, seem to be involved in
the [Ca2+] spike after darkening. These numerous
cy
different Ca2+ release mechanisms which are established
in the same algal cell may contribute to stimulus specificity
in Ca2+-mediated intracellular signal transduction processes. Figure 6 summarizes the different processes which
are likely to be involved in a sudden increase in cytosolic
free Ca2+ and outlines how a cytosolic [Ca2+] spike
cy
could give rise to a transient hyperpolarization. Cessation
of photosynthetic electron transport by light-off or by
inhibitors (DCMU, DBMIB) may cause Ca2+ release
from the chloroplast. Both, caffeine (Figs 4, 5) and InsP
3
cause Ca2+ release from intracellular stores in
Eremosphaera. The Ca2+ release pathways activated by
InsP or by caffeine and Sr2+ are likely to be the InsP
3
3
gated or the ryanodine/cADPR gated Ca2+ channel,
respectively. Whether these Ca2+ release pathways reside
on the same or on different Ca2+ stores is not known.
There are indications that caffeine- and Sr2+-induced
[Ca2+] oscillations are due to repetitive Ca2+ release
cy
and re-uptake by the endoplasmic reticulum (Bauer et al.,
unpublished results). A better understanding, how
[Ca2+] spikes are generated, and how the increased
cy
[Ca2+] interacts with the ion channels of the plasma
cy
membrane will help to understand the role of Ca2+ in
intracellular signal transduction not only in
Eremosphaera.
Acknowledgements
We would like to thank Drs C. Plieth and U.-P. Hansen for
technical support and fruitful co-operation. This work was
financially supported by the Deutsche Forschungsgemeinschaft
(SFB 176, TP B11).
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