2867
Development 124, 2867-2874 (1997)
Printed in Great Britain © The Company of Biologists Limited 1997
DEV0114
First evidence of a calcium transient in flowering plants at fertilization
C. Digonnet*, D. Aldon*, N. Leduc, C. Dumas and M. Rougier†
Ecole Normale Supérieure de Lyon, Reproduction et Développement des Plantes, UMR 9938 CNRS/ INRA/ ENS, Ecole Normale
Supérieure, 46 allée d’Italie, 69364 Lyon Cedex 07, France
*These authors contributed equally to this study
†Author for correspondence (e-mail: [email protected])
SUMMARY
We report here the first evidence of a transient elevation of
free cytosolic Ca2+ following fusion of sperm and egg cell in
a flowering plant by the use of an in vitro fertilization
system recently developed in maize. Imaging changes in
cytosolic Ca2+ at fertilization was undertaken by egg cell
loading with the fluorescent Ca2+ indicator dye fluo-3
under controlled physiological conditions. The gamete
adhesion step did not induce any cytosolic Ca2+ variation
in the egg cell, whereas the fusion step triggered a transient
cytosolic Ca2+ rise in the fertilized egg cell, lasting several
minutes. This rise occurred after the establishment of
gamete cytoplasm continuity. Through these observations,
we open the way to the identification of the early signals
induced by fertilization in flowering plants that give rise to
the calcium transient and to investigations of the role of
Ca2+ during egg activation and early zygote development
in plants, as has been reported for other better characterized animal and algae systems.
INTRODUCTION
Dresselhaus, 1996). Alternative methods were therefore
developed which allow sperm and egg to fuse selectively under
conditions that mimic natural fertilization. In vitro fusion of
isolated maize gametes has been achieved in the presence of
Ca2+ (Faure et al., 1994a) or in combination with high pH
(Kranz and Lörz, 1994). These improvements of in vitro fertilization in angiosperms provide the opportunity to study the
changes that occur in the fertilized egg cell (Kranz et al., 1995;
Tirlapur et al., 1995) as well as the signaling requirements
associated with sperm-egg fusion and egg activation (Rougier
et al., 1996).
A considerable body of informations is now available on
the cascade of signaling events induced by sperm-egg interaction in animals and on the mechanisms of egg activation
(for recent reviews see Nucitelli, 1991; Whitaker and Swann,
1993; Jaffe, 1996). Previous studies (Ridgway et al., 1977;
Steinhardt et al., 1977; Gilkey et al., 1978) and more recent
ones (reviewed by Jaffe, 1991; Whitaker and Swann, 1993;
Jaffe, 1996) performed on different models have shown that
the sperm triggers a transient rise of intracellular free Ca2+
levels in the fertilized egg. The role of Ca2+ in plant fertilization has been recently investigated in a brown algae,
Fucus (Roberts et al., 1994; Roberts and Brownlee, 1995). In
this lower plant model, a localized elevation of the cytosolic
Ca2+ concentration ([Ca2+]cyt) to micromolar levels seems to
be required for early fertilization events: the generation of the
fertilization potential and cell wall secretion. Brownlee
(1994) has suggested that, since higher plants reflect a high
degree of complexity and specialization in their reproductive
strategies, they may have strikingly different mechanisms for
signaling gamete fusion and egg activation than animals and
lower plants. This is the question that we have addressed.
The double fertilization characteristic of angiosperms
(reviewed by Russell, 1992) occurs internally, deep within
maternal tissues and involves two separate fusion events. A
sperm-egg cell fusion initiates the development of the embryo.
A sperm-central cell fusion leads to the development of the
nutritive tissue of the embryo or endosperm. In angiosperms,
sperm cells are not released free in the external medium but
are delivered close to the female gametes by the pollen tube
which enters the embryo sac through a degenerated synergid.
An understanding of how non-motile sperm cells recognize and
fuse with either the egg or the central cell is thus very difficult
to achieve in vivo, due to the inaccessibility of the gametes, the
simultaneity and rapidity of the fusion processes and the variability in the time course of double fertilization in maize (Mòl
et al., 1994).
Separate studies of sperm-egg and sperm-central cell fusions
are required to undertake a molecular dissection of the early
events and signaling steps associated with the double fertilization in flowering plants. The development of in vitro methods
in maize provides this opportunity (Faure et al., 1994b; Dumas
and Faure, 1995). Two kinds of in vitro fertilization procedures
have been developed (recently reviewed by Kranz and Dresselhaus,1996). Electrofusion of single isolated sperm and egg
cells leads to the production of zygotes (Kranz et al., 1991a)
which are able to undergo karyogamy (Faure et al., 1993) and
regenerate into plants (Kranz and Lörz, 1993). Since this
procedure involves forced fusions between sperm cells and
non-gametic cells (Kranz et al., 1991b), it is nonspecific and
therefore cannot be used to study the mechanisms of the physiological fusion processes between sperm and egg (Kranz and
Key words: maize, in vitro fertilization, calcium signaling, fluo-3
2868 C. Digonnet and others
In this study we were interested in investigating the Ca2+
signals induced by sperm-egg fusion in flowering plants using
the in vitro fertilization system developed in maize by Faure et
al. (1994a) and a Ca2+ imaging technique well adapted to the
delicate and easily burst egg protoplasts. We report here the
use of fluo-3 ester loading for the continuous monitoring of
cytosolic free Ca2+ in isolated maize egg cells during fertilization. We show that following sperm-egg fusion, a single Ca2+
transient is observed in all eggs investigated, indicating the
likely involvement of Ca2+ signaling in early zygote development in flowering plants.
MATERIALS AND METHODS
Plant material
Maize plants (Zea mays L.) of inbred lines A 188 used for gamete
isolation and HD 99 used for microspore culture, were grown in a
regulated growth chamber at the Ecole Normale Supérieure, Lyon,
France. The temperature ranged from 24°C (day) to 20°C (night) with
a 16-hour photoperiod at 70% relative humidity and a light intensity
of 450-500 µE/m2/second provided by high-pressure sodium lamps
(400 W, Philips, Belgium). In order to prevent pollination when plants
reached maturity, tassels were removed and the ears were bagged
before silk emergence. Ears were collected at a receptive stage when
emerged silks reached 12-13 cm in length (Dupuis and Dumas, 1989).
Female and male gamete isolation
Egg cells were isolated from ovules dissected from mature ears using
a method modified from Kranz et al. (1991a) and Faure et al. (1994a).
Half-ovules were incubated for 6 minutes at room temperature in an
enzymatic solution containing: 0.75% pectinase (Serva, Heidelberg,
Germany); 0.25% pectolyase (Sigma chimie, St Quentin Fallavier,
France), 0.5% Macerozyme Onozuka R10 (Yakult Honsha, Tokyo,
Japan); 0.5% Cellulase Onozuka R10 (Yakult Honsha), pH 5, adjusted
to 560 mOsm with mannitol. Ovule pieces were then washed with
0.57 M mannitol and egg cells were gently removed from the embryo
sac by manual microdissection under an inverted microscope. Egg
cells were only obtained in limited quantities (10-30 egg cells per ear
and per half day) and remained very fragile when transferred and
manipulated.
Sperm cells were released from freshly collected tricellular pollen
grains (Dupuis et al., 1987) after a pH/osmotic shock in 0.5 M
mannitol (Faure et al., 1994a).
Egg cell loading with fluo-3 AM
We chose to load the unfertilized egg cell with the acetoxymethyl ester
form (AM) of fluo-3 which is highly lipophilic and thus easily crosses
the plasma membrane by a non- disruptive route (Tsien, 1981; Kao et
al., 1989). The acetoxymethyl ester form of fluo-3 (fluo-3 AM) was
obtained from Molecular Probes (Eugene, OR, USA) and made up to
a 1 mM stock in anhydrous dimethylsulfoxide (DMSO) (SigmaAldrich chimie, St Quentin Fallavier, France).
Isolated egg cells were loaded for 6 minutes at room temperature,
in the dark, with 1 µM final concentration of fluo-3 AM prepared in
0.55 M mannitol. Loaded egg cells were subsequently rinsed 3 times
in 0.55 M mannitol and 3 times again in the fusion medium composed
of 0.55 M mannitol containing 5 mM CaCl2. These loading and
washing steps were particularly delicate because each isolated egg cell
had to be transferred from the incubation medium to the different
washing media using a microcapillary. All the incubating media were
sterile and distributed into microdroplets under mineral oil (Merck,
Darmstadt, Germany).
In vitro fertilization (IVF)
In vitro fertilization was performed according to Faure et al. (1994a),
in the presence of 5 mM CaCl2, conditions in which almost 80% of
sperm-egg pairs fused. Fusion of fluo-3 loaded egg cells with sperm
was particularly sensitive to experimental manipulation. To preserve
the high percentage of fusion, it was necessary to wash the fluo-3
loaded egg cells six times in order to remove extracellular dye completely.
Spatial localisation of fluo-3 in egg cells
In order to control the intracellular distribution of fluo-3 and check
the retention of the probe in the cytoplasm without sequestration into
organelles (see Read et al., 1992; Callaham and Hepler, 1991),
optical sections of fluo-3 AM loaded egg cells were obtained and
processed using a confocal laser scanning microscope (Axioplan,
Carl Zeiss, Oberkochen, Germany) with a 488 nm excitation from
an argon ion laser. A water immersion objective (×16, Plan-Neofluar,
Zeiss) was used for optical sectioning with zoom setting at 100. Zseries images were acquired at spacing steps of 1 µm in the dual
channel splitscreen mode which allows a simultaneous display (side
by side) of light and fluorescence confocal images as illustrated in
Fig. 1A,B. Observations were made at the Centre Commun de Quantimétrie at Lyon I University.
Imaging of cytosolic free Ca2+
For real time dynamics of the [Ca2+]cyt changes, fluo-3 AM loaded
cells were observed with an Olympus IMT2 inverted microscope
(Olympus France, Rungis, France), using a Nikon Fluor 40× oil
immersion objective (1.3 numerical aperture) (Nikon France,
Champigny sur Marne, France) and a non-fluorescent immersion oil
(Cargille Laboratories, Cedar Grove, USA). The excitation wavelength was determined using an interference filter (500 nm ±14;
Ealing, Les Ulis, France) mounted in a motor-driven excitation filter
wheel lambda-10 (Sutter Instruments, Novato, CA) in front of the
Biologic 150 W xenon light source (Biologic, Claix, France). The
excitation light was delivered to the microscope through an optic fiber.
Fluorescence from the dye was monitored during fertilization using
an Olympus fluorescent cassette containing a 510 nm dichroic
reflector and 520 nm long-pass glass emission filter. The fluorescence
emission was imaged using a C2400 low light level imaging camera
from Hamamatsu and was acquired at the rate of one image every 4
seconds using an ARGUS-50 image analysis system (Hamamatsu
Photonics, Massy, France). Images were captured at a resolution of
256×256 pixels and digitized to 256 gray levels. Each image recorded
was the mean of eight individual frames. The camera dark signal was
recorded prior to each experiment and substracted on-line. Data were
then corrected for photobleaching attenuation using a commercially
available program (BioPatch analysis 3.2, Biologic). No autofluorescence from the unfertilized or fertilized egg cell was observed at the
excitation wavelength of 500 nm.
Since fluo-3 emits fluorescence in response to Ca2+ ions, the
increase in the fluorescence intensity is directly related to an increase
in free Ca2+ concentration (Minta et al., 1989; Kao et al., 1989).
However, accurate calibration of absolute free Ca2+ level is difficult
to perform when using single wavelength probes (Stricker et al., 1992;
Read et al., 1992) and was unsuccessful when using fluo-3 loaded egg
cells. For these reasons, we have chosen to describe the Ca2+
dynamics during fertilization using absolute fluorescence intensity
measurements expressed in arbitrary units.
Imaging of fluorescence variation during fertilization was
performed with an Olympus IMT2 inverted microscope (Japan) as
described for imaging of cytosolic free Ca2+ with fluo-3. After each
recording (fluo-3) we checked the occurrence of fertilization by performing DAPI staining of the nuclei. Fusion products were placed
in a solution of 14 mM DAPI (4′,6-diamidino-2-9 phenyl-indole,
Sigma Chimie) prepared in 0.5 M mannitol-Triton X-100 (0.001%
v/v) and observed under an inverted IM 35 Zeiss microscope at 395
nm. Photography was performed using a colour Kodak 400 ASA
film.
Ca2+ transients in plant fertilization 2869
Zygote division after in vitro fertilization
15 minutes after in vitro fertilization, zygotes were transferred to a
drop of liquid culture medium, which consisted of 50% v/v NBM
solution (Mòl et al.,1993) supplemented with zeatin (1 mg/l), 2,4-D
(1 mg/l) and 50% mannitol 0.5 M in 0.75% w/v low melting point
agarose (type IX, Sigma Chimie, St Quentin Fallavier, France). This
medium was laid over NBM medium containing the same growth
regulator combination as above and solidified with 0.8% w/v Sea
Plaque agarose (FMC BioProducts, RocKland, ME). Fusion products
were kept in the dark at 25°C overnight, then were transferred to a 12
mm diameter Transwell insert (Costar Corp., Cambridge, MA) placed
inside a well of a 12-well cluster dish containing 1 ml of a suspension of maize microspores (genotype HD 99, density 2.103
microspores per ml) undergoing androgenesis as described by Leduc
et al. (1996). Fusion products and microspores were cocultured for at
least 48 hours in NBM medium containing zeatin (1 mg/l) and 2,4-D
(1 mg/l) and kept in the dark at 25°C.
In order to check whether nuclear or cell division had occurred
during culture, fusion products were stained with DAPI, as previously
described (Leduc et al., 1996).
RESULTS
Loading of egg cells with fluo-3 AM and subcellular
distribution of the Ca2+ probe
Our first objective was to be able to image cytosolic Ca2+ in
maize eggs. Microscopic observations indicated that fluo-3
AM uptake in egg cells started immediately after incubation.
This loading time was very short in comparison to those
reported for loading other plant protoplasts, generally 1 to 2
hours (Gehring et al., 1990; Irving et al., 1992; Fallon et al.,
1993).
Using confocal analysis, we showed that dye-loaded egg
cells were morphologically indistinguishable from untreated
cells. After isolation, the egg cell appeared round in shape,
highly vacuolated and devoid of cell wall, as shown in the
bright-field image in Fig. 1A. This protoplast contained a dense
cytoplasm surrounding the nucleus from which cytoplasmic
strands were directed towards the plasmalemma.
No dye sequestration into the vacuole and the nucleus was
A
P
V
NC
B
found to have occurred when egg cells were loaded with 1 µM
fluo-3 AM for 6 minutes. In the fluorescent image (Fig. 1B) of
the fluo-3 loaded egg cell shown in Fig. 1A, only the nucleusassociated cytosol and the cytoplasmic strands were fluorescent. This dye distribution remained unchanged for at least
2 hours after transferring the cells into a dye-free medium
allowing kinetic studies of Ca2+ changes during gametic
fusion.
Influence of the loading procedure on the ability of
fluo-3-loaded egg cells to fuse in vitro and divide
We checked the ability of isolated egg cells to fuse with sperm
after loading with fluo-3. This control revealed the importance
of the washing step after the loading procedure in preserving
a high percentage of fusion (80%). An additional control was
performed to verify that intracellular Ca2+ homeostasis (Gilroy
et al., 1993) required to maintain normal growth and development was not disturbed by fluo-3 buffering of free Ca2+ in the
egg. We developed a culture system allowing early steps of
zygote development in vitro. In these culture conditions, egg
cells loaded in the presence of 1 µM or 5 µM fluo-3 AM
proceeded to cleave, following fertilization, in a manner similar
to that observed in non-loaded egg cells used as controls. Fig.
2 illustrates one of the multicellular structures obtained from
loaded egg cells fertilized in vitro. These observations repeated
three times, support the view that the Ca2+ dynamics reported
here occurred in cells that were capable of subsequent division.
Loaded egg cells that failed to fuse with a male gamete (20%)
did not cleave when subjected to the same culture procedure.
We concluded from these data that fluo-3 had no cytotoxic
effect on the egg cells in our experimental conditions. After
loading, these eggs preserved their cellular characteristics, their
ability to fuse in vitro with a male gamete and their capacity
to initiate development.
Ca2+ measurement during adhesion and fusion
steps
Since the in vitro fertilization of fluo-3-loaded egg cells with
a sperm cell was clearly possible, we were in a position to
follow cytosolic Ca2+ dynamics during fertilization. Under our
experimental conditions, the period between the gamete
contact and the beginning of the plasma membrane fusion
varied between 30 seconds and 2 minutes. During the first 3040 seconds of gamete adhesion, we optimized the imaging
system to the level of dye fluorescence emission in the egg cell.
N
CS
Fig. 1. Microscopic characteristics of a single maize egg cell loaded
with fluo-3 AM. (A) Bright-field image. The isolated egg cell is a
protoplast characterised by an outlying developed vacuoles (V). The
most part of the cytoplasm is associated with the nucleus (CS,
cytoplasmic strands; NC, nucleus-associated cytoplasm; P,
plasmalemma). (B) Confocal image of Ca2+cyt localization in the
same egg cell. The dye is mainly restricted to the nucleus-associated
cytoplasm and to peripheral cytoplasmic strands (CS). Bar, 10 µm.
Fig. 2. In vitro division of a zygote derived from IVF of a fluo-3
AM-loaded egg cell with a male gamete. (A) Phase contrast
micrograph of a 48-hour old divided structure. (B) Fluorescence
micrograph of the same structure showing 4 nuclei (arrowheads)
after DAPI staining. Bar, 10 µm.
2870 C. Digonnet and others
0
A
250
0s
4s
8s
12 s
24 s
41 s
Fig. 3. Evidence for an intracellular Ca2+ elevation triggered by in
vitro fertilization of Zea mays egg cells loaded with fluo-3 AM.
(A) Time sequence of pseudo-color images illustrating temporal and
spatial distribution of the Ca2+ transient triggered by sperm-egg
fusion. (B) Magnification of time course of the early events occurring
during sperm-egg fusion described in A. The graphs, on the right,
report changes in fluorescence intensity measured along an axis (line
ab) passing through the egg cell at the site of adhesion of the
unstained male gamete (first image, 0 s). Arrows and arrowheads
shows the position of male gamete incorporation. The egg cell
diameter is 60 µm.
100
65 s
89 s
0
29 min 36 s
250
B
Intensity
b
40
20
b
0
0
0
a
4s
60
100
0s
a
Fluorescence Emission
80
b
100
10
20
30
40
a
50
60
Time (min)
Fig. 4. Temporal changes in fluorescence emission monitored during
IVF using egg cells loaded with fluo-3 AM. Transient Ca2+ rise in a
fluo-3 AM-loaded egg cell. (a) Typical positive response resulting
from successful gamete fusion. (b) Recording obtained from egg cell
that failed to fuse. Fluorescence emission is the relative changes in
fluorescence in arbitrary unit.
0
8s
100
0
12 s
100
0
Thus, the first image (0 seconds, s) shown in Fig. 3 was
recorded 40 seconds after the establishment of the close contact
between the two gametes.
The time course of IVF illustrated in Fig. 3 shows that the
adhesion step (0 seconds; s) did not induce any variation of
[Ca2+]cyt in the maize egg cell. Moreover we observe in the
temporal analysis illustrated in Fig. 4A, that adhesion between
the two gametes could continue for 30 minutes without further
membrane fusion during which time no variation in fluorescence emission signal occurred.
The first sign that fusion had occurred was a sudden influx
of fluo-3 into the unloaded male gamete (Fig. 3A,B, 4 s). This
is evident as a shoulder on the graph corresponding to the fluorescence emitted along the axis a-b passing through the fusion
site (Fig. 3B, 4 s). As soon as cytoplasmic continuity had been
established between the two adhered gametes, fluo-3 diffused
from the egg into the unstained sperm making it visible as illustrated in Fig. 3B (4 s, arrow). Diffusion of the dye into the
fusing sperm cell increased in the subsequent image (8 s). At
the same time, an increase in the fluorescence emission level
represented by red dots occurred in the nucleus-associated
cytosol of the fusing egg cells. The origin of the increase could
not be established due to limitations of the method used to
interpret spatial data.
Ca2+ explosion in the fertilized egg cell
At 12 seconds (Fig. 3A, frame 4), the male gamete was no
Ca2+ transients in plant fertilization 2871
We report here the first description of a fertilization-associated
Ca2+ transient in a flowering plant by showing that a Ca2+
transient is triggered by sperm-egg fusion in maize. We have
been able to investigate these Ca2+ dynamics by applying a
sensitive Ca2+-imaging procedure to a recently developed in
vitro fertilization system gaining experimental access to the
analysis of sperm-egg fusion at the cellular and molecular
levels.
[Ca2+]cyt in isolated maize egg cells during fertilization
appeared to be ester loading using the Ca2+ indicator fluo-3.
As recently outlined by Malho and Trewavas (1996), although
the use of such a single-wavelenght dye precludes accurate calibration, it does not prevent assessment of differences that
result from signaling. In our case, this kind of information was
the most crucial.
By demonstrating the ability of fluo-3 to permeate intact egg
cells under the form of acetoxymethylester (fluo-3 AM) we
confirm and extend earlier data collected from intact plant cells
(Gehring et al., 1990) or plant cell protoplasts (Shacklock et
al., 1992; Ranjeva et al., 1992; Graziana et al., 1993). However,
artefacts may be induced by loading plant cells with AM fluorescent compounds. As previously reported by Gilroy et al.
(1991), an unequivocal interpretation of imaging data can only
be clearly established if the dye is localized in the cytosol, the
cell remains unperturbed by the dye loading and analysis procedures and the cellular response is not altered by the measurement procedure. Our experimental conditions almost
fulfilled all these criteria. Within the window of time necessary
for imaging Ca2+ kinetics, we observed no sequestration of the
dye into vacuoles which are the main calcium sequestrating
organelles in plants (Blumwald and Gelli, 1993; Johannes et
al., 1992). Since egg cells are highly vacuolized protoplasts,
we consider, as previously outlined by Oparka and Hawes
(1992), that the major risk of dye sequestration was in this
organelle. We also showed that morphology of egg cells and
organelle distribution were not disrupted by dye-loading and
that loaded egg cells preserved their ability to fuse and to
divide.
Successful egg cell loading with fluo-3 as a prelude
to Ca2+ imaging
In this study, we show that, contrary to previous reports
(Tirlapur et al., 1995), imaging cytosolic Ca2+ in egg cells is
possible. However, owing to the fact that isolated egg cells are
fragile protoplasts available only in low numbers and highly
sensitive to osmotic pressure, touch and chemicals, the choice
of experimental procedure was critical. Although the use of
ratiometric indicators such as fura-2 is recommended to allow
accurate calcium measurements in plant cells (Read et al.,
1992; Graziana et al., 1993), we were unable to apply such a
methodology to egg cells. Several assays using acetoxymethylesters of fura-2 (fura-2 AM) to load unfertilized egg cells led
to their bursting during the loading procedure. Direct loading
of plant cells with fura-2 AM has also been previously reported
to be unsuccessful due to either extracellular or incomplete
internal hydrolysis (Graziana et al., 1993). Another possible
method of dye introduction was to directly inject fura-2 into
the cytosol. However, microinjection, with its attendant
problems, represents a more intrusive protocol, especially
when applied to plant protoplasts (Read et al., 1992). Successful microinjections of dyes and reporter genes have been
performed in our laboratory (Leduc et al., 1996) using isolated
maize zygotes fertilized in planta. However appropriate conditions including immobilization of zygotes in agarose and
regeneration of a cell wall were required. Such conditions
cannot be applied to unfertilized isolated egg cells which are
devoid of cell wall and must be maintained free in the fusion
medium to allow in vitro fertilization. Therefore the best
adapted technology for detecting qualitative changes in
Dynamics of Ca2+ rise at fertilization
Since the Ca2+ signal reported here in fertilized maize eggs is
single and transient, lasting several minutes, it differs markedly
in its form and duration from the transients induced by various
stimuli described so far in other plant protoplasts or cells
(Bush, 1993, 1995). Among them, mechanical touch (Knight
et al., 1991; Trewavas and Knight, 1994) or osmotic shock
recently investigated in Fucus rhizoid (Taylor et al., 1996)
might have been expected to trigger Ca2+ changes within eggs
either during manual adhesion of gametes or when incubating
egg cells in the loading or fusion media. In addition to the
striking differences between the Ca2+ signal depicted in maize
egg cells and those induced by these two stimuli in plant cells,
no [Ca2+]cyt changes could be observed during the adhesion
step. They were only measured after completion of sperm-egg
fusion. This data also proves that the Ca2+ contained in the
fusion medium is not responsible of the induction of Ca2+
signaling in contrast to data from guard cells showing that
exposure to 1 mM CaCl2 can cause large cytoplasmic Ca2+
oscillations (McAinsh et al., 1995). Thus we can be certain that
modifications in [Ca2+]cyt result from physiological modifications of the egg cell brought about by fertilization and do not
originate from any artefactual stimulus. When compared to
other calcium signals induced by fertilization in various
animals, the single transient signal reported here in a flowering
plant is more similar in form, magnitude and duration to the
Ca2+ transient imaged using fluo-3 in the sea urchin egg
(Swann and Whitaker, 1986; Stricker et al., 1992; Gillot and
Whitaker, 1993) than to the repetitive transients or oscillations
reported in mammals (reviewed by Swann and Ozil, 1994) and
longer visible, plasmogamy was achieved and the male nucleus
was incorporated in the egg cell. The Ca2+ increase triggered
by fusion reached its peak amplitude 85 seconds after the
beginning of the gametic fusion process. The fluorescence
signal remained uniformly elevated for 2 minutes and then
decreased to reach the resting [Ca2+]cyt level after 29 minutes
of recording (Fig. 4). We have observed this transient increase
in the [Ca2+]cyt in all fertilized eggs investigated (n=11). In
loaded egg cells maintained in Ca2+ enriched fusion medium
which failed to fertilize during Ca2+ measurement, fluorescence level remained unchanged (Fig. 4).
Analysis showed that at fertilization, the increase in signal
amplitude representing the increase in the [Ca2+]cyt is relatively
variable from pair to pairs of male and female gametes
analyzed (n=11). There was also some variations in the
duration of the Ca2+ increase (10-30 minutes) (n=11). This may
reflect varying degrees of egg cell maturity as reported by Mòl
et al. (1994) from in vivo studies.
DISCUSSION
2872 C. Digonnet and others
ascidians (Speksnijder et al., 1989). By contrast, the Ca2+
elevation recorded here in all fertilized maize eggs so far
analyzed differs from the smaller, cortical Ca2+ elevation
reported in Fucus in 30% of the fertilized eggs investigated
(Roberts et al., 1994; Roberts and Brownlee, 1995). Thus,
although fertilization mechanisms differ markedly between
flowering plants and animals, they all involve a calcium signal
at the single egg cell level after addition of sperm, as illustrated
in the present study, although this signal may show different
characteristics.
Imaging such Ca2+ changes in fertilized maize eggs was
greatly facilitated by the use of the in vitro fertilization system
recently developed in our laboratory (Faure et al., 1994a)
allowing direct access to the site of sperm-egg fusion and the
precise determination of fusion time. The results illustrated in
Fig. 3 indicate that in maize as in sea urchin (Figs 1 and 2 in
Swann et al., 1994) and more recently in mouse (Lawrence et
al., 1997), diffusion of the dye into the sperm from the egg
cytoplasm precedes the Ca2+ increase in the egg. These
findings support the view that sperm-egg fusion is the prelude
to the egg’s early Ca2+ transient. However, further analyses
using confocal microscopy will have to be performed in order
to gain spatial data and elucidate the mode of propagation of
the Ca2+ transient in fertilized maize eggs. Such analyses
would help to indicate whether the rise in [Ca2+]cyt becomes
uniformly elevated throughout the entire fertilized egg or propagates through it in a wave-like fashion as illustrated in sea
urchin (Swann and Whitaker, 1986; Stricker et al., 1992; Gillot
and Whitaker, 1993).
Origin and significance of Ca2+ rise?
Key questions remain to be addressed regarding the origin and
the mechanisms of fertilization-associated Ca2+ signaling in
maize. Whether extracellular Ca2+ is solely or partly responsible for the calcium increase or whether there is a role for intracellular stores remains to be determined. To date we know that
(1) decreasing extracellular Ca2+ in fusion medium strongly
reduces the percentage of fusion in our in vitro fertilization
model (Faure et al., 1994a); (2) Ca2+ appears to be important
for gametes themselves, either for maintaining egg cell
membrane stability (Holm et al., 1994) or for influencing
sperm cell integrity (Zhang et al., 1995) in vitro; (3) high levels
of total Ca2+ has been reported in vivo in synergids of various
species in which male gametes are delivered close to their
female targets (Chaubal, 1990, 1992). These data are not sufficient to prove that Ca2+ is required for fusion but favour the
possibility that extracellular Ca2+ might help to initiate the
intracellular Ca2+ response as reported in different fertilization
models such as Fucus (Roberts and Brownlee, 1995). However,
since the fertilization-induced Ca2+ rise in maize lasts several
minutes, we cannot exclude a contribution of vacuolar calcium
to its propagation. The vacuole is an important component of
egg cells and is known to be the principal intracellular Ca2+
store in plants (Blumwald and Gelli, 1993; Johannes et al.,
1992).
This [Ca2+]cyt elevation represents one of the earliest physiological responses that have been observed following in vitro
egg-sperm fusion in maize. Among these, the early responses
preceding karyogamy (Faure et al., 1993) include cell wall
synthesis that is completed within 10 minutes in electrofused
eggs (Kranz et al., 1995) and 20 minutes in Ca2+-fused eggs
(unpublished data). This is the period during which [Ca2+]cyt
elevation occurs. At much later stages of zygote development,
other responses include membrane-bound calcium and calmodulin changes (Tirlapur et al., 1995) or abundant expression of
a calreticulin gene (Dresselhaus et al., 1996). The questions
remaining to be addressed are: are the early calcium transient
reported here and these early and later events correlated and
how might they combine to contribute to egg activation and
zygote development. The results of numerous experiments
mostly performed in animals indicate that the transient rise in
intracellular Ca2+ following sperm-egg fusion is essential for
the subsequent events that constitute egg activation (Jaffe,
1996). In Fucus, fertilization is also followed by a sequence of
activation events including the occurrence of a fertilization
potential similar in many ways to that found in several animal
eggs, and the rapid onset of cell wall production correlated with
the localized Ca2+ increase (Roberts et al., 1994). After a few
hours, these events lead to the production of a differentiated
polarized zygote with typical properties. Investigations using
our experimental model are currently in progress to establish
whether similar links between the Ca2+ fertilization response
and other events presumed to trigger egg activation also occur
in flowering plants.
In conclusion, new strategies will have to be developed to
elucidate how the transient Ca2+ elevation reported here at fertilization in flowering plants is generated and what the precise
function and consequences of this Ca2+ signal on zygote development are. Although it is presently unclear which responses
are triggered by this newly observed Ca2+ signal, its discovery
contributes significantly to understanding of the cellular and
molecular responses triggered by egg-sperm fusion in
flowering plants. Moreover, the present study highlights the
similarities that do exist between plant and animal fertilization
responses and increases our fundamental knowledge of this
early and essential event at fertilization both in plants and
animals. Finally, this study provides a new contribution to the
importance and diversity of Ca2+ functions in plant development (reviewed by Hepler and Wayne, 1985; Kauss, 1987;
Bush, 1995).
We thank Colin Brownlee, Catherine Leclerc and Marc Moreau
for helpful discussions and we are especially greatful to Michael
Whitaker for his help and comments during preparation of the manuscript. We thank R. Blanc for growing the maize plants, F. Rozier
and M. Tomkowiak for technical assistance, and P. Audenis for photography. This work was supported by the Centre National de la
Recherche Scientifique (CNRS), the Institut National de la
Recherche Agronomique (INRA), and the Ecole Normale
Supérieure de Lyon.
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(Accepted 29 May 1997)