Fertilization in Torenia fournieri

Vol. 45 No. 2
SCIENCE IN CHINA (Series C)
April 2002
Fertilization in Torenia fournieri: actin organization and
nuclear behavior in the central cell and primary endosperm
YUAN Ming ( )1,2* , FU Ying ( )1,3*, WANG Feng ( )2,
HUANG Bingquan ()1, Sze-Yong Zee (
)1 & Peter K. Hepler4
1. Department of Botany, The University of Hong Kong, Pokfulam Road, Hong Kong, China;
2. Department of Plant Sciences, College of Biological Sciences, China Agricultural University, Beijing 100094,
China;
3. Research Center for Developmental Biology, College of Life Sciences, Wuhan University, Wuhan 430072, China;
4. Department of Biology, University of Massachusetts, Amberst, Massachusetts 01003, USA
Correspondence should be addressed to Sze-Yong Zee (email: [email protected])
Received December 6, 2001
Abstract Studies of the living embryo sacs of Torenia fournieri reveal that the actin cytoskeleton
undergoes dramatic changes that correlate with nuclear migration within the central cell and the
primary endosperm. Before pollination, actin filaments appear as short bundles randomly distributed in the cortex of the central cell. Two days after anthesis, they become organized into a distinct
actin network. At this stage the secondary nucleus, which is located in the central region of the
central cell, possesses an associated array of short actin filaments. Soon after pollination, the actin
filaments become fragmented in the micropylar end and the secondary nucleus is located next to
the egg apparatus. After fertilization, the primary endosperm nucleus moves away from the egg
cell and actin filaments reorganize into a prominent network in the cytoplasm of the primary endosperm. Disruption of the actin cytoskeleton with latrunculin A and cytochalasin B indicates that
actin is involved in the migration of the nucleus in the central cell. Our data also suggest that the
dynamics of actin cytoskeleton may be responsible for the reorganization of the central cell and
primary endosperm cytoplasm during fertilization.
Keywords: actin, embryo sac, central cell, Torenia.
In flowering plants, double fertilization involves the fusion of one sperm cell with an egg cell,
initiating the embryo, and the other sperm cell with the central cell leading to the formation of the
endosperm. In most plant species the process of syngamy between the male gamete and the central
cell involves a series of cytological and morphological events, including the fusion of polar nuclei
to form a secondary nucleus, migration of the polar nuclei or the secondary nucleus to the micropylar end of the central cell, and changes in cellular polarity. After fertilization, there is a migration of the endosperm nucleus back to the chalazal end of the endosperm and a reconstruction of
micropylar cell wall[1,2]. In spite of the importance of these early events for endosperm development, surprisingly little is known about their control mechanisms.
It is well known that nuclear migration occurs in the central cell during polar nuclei fusion,
* Yuan Ming and Fu Ying are considered joint first authors.
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and sperm and central cell syngamy. The fusion of the polar nuclei is brought about either by the
migration of the chalazal nucleus towards the micropylar nucleus[3] or the migration of micropylar
and chalazal nuclei towards the center of the central cell [4]. Pollination may accelerate the migration of the secondary nucleus towards the micropylar region of the central cell in preparation for
gametic fusion[5]. During fertilization, polarity in the central cell becomes established and this is
evidenced by the position of the nuclei and the distribution of the cytoplasm and organelles, which
invariably accumulate near the micropylar end of the embryo sac. The central cell can even retain
its polarity after isolation[6]. One of the two polar nuclei or the secondary nucleus is fertilized by a
single sperm nucleus, generating the primary endosperm nucleus. After fertilization the endosperm
nucleus moves away from egg apparatus[7,5]. This process may involve the reorganization of the
cytoplasm and the relocation of the cytoplasmic domains[8]. These events are induced not only by
the interaction of male and female gametes, but also by the activities of cytoskeleton[3,4].
It is well known that the cytoskeleton, especially the actomyosin system, is involved in the
ª
transport of organelles and male germ unit in pollen tubes[9 12]. By contrast, information on the
ª
behavior and function of the cytoskeleton during fertilization is relatively scarce [13 15]. Our preª
vious studies[14 17] have shown that reorganization of the actin cytoskeleton in the embryo sac
occurs simultaneously with a series of fertilization events including the reception of the pollen
tube and the migration of male gametes and their fusion with the target cells.
Although there are a few reports about localization of actin filaments in the embryo sac by
ª
conventional aldehyde fixation[13,14,18 20], it is technically difficult to preserve and visualize the
actin cytoskeleton in the relatively inaccessible embryo sacs. In addition, aldehyde fixation may
induce and trigger significant morphological changes in cell integrity during fertilization[21].
Microinjection of fluorescent phalloidin is an approach that may avoid those problems and has
ª
been successfully employed to label and visualize the actin cytoskeleton in living plant cells[22 25].
More recently, we reported that actin filaments organized into a distinct actin network in the cortex of the central cell after anthesis and became fragmented in the micropylar end of the central
cell after pollination by microinjection of Alexa 488-phalloidin into the central cell of Torenia
fournieri, but no details were given about the proceeding of the actin cytoskeleton reorganization
during fertilization[16]. In addition, we also showed that there was an actin cage around secondary
nucleus of the central cell. It is therefore of interest to investigate the role of the actin cytoskeleton
in nuclear migration.
In this study, we have investigated in detail the process of the actin cytoskeleton reorganization in the central cell during fertilization, in particular, the participation of the actin cytoskeleton
in the control of nuclear migration. We address the following questions: (1) How do changes of
actin cytoskeleton proceed in the central cell? (2) What is the role of cytoskeleton in the central
cell during fertilization? (3) Is the migration of the secondary nucleus actin or microtubule dependent?
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To answer these questions we have specifically examined actin organization and behavior of
the secondary nucleus in the central cell and the primary endosperm of Torenia fournieri embryo
sacs during and after fertilization. Furthermore, we have also applied anti-actin and anti-microtubule drugs to determine the role of the microfilament and microtubule cytoskeleton in nuclear
migration in the central cell and primary endosperm. Our results show that the actin cytoskeleton
in the central cell undergoes dramatic changes during fertilization and is responsible for the reorganization of the cytoplasm and nuclear migration. These findings, along with previous studies
with Plumbago, Nicotiana and maize, strongly suggest that the actin cytoskeleton of the central
cell is actively involved in the fertilization process by inducing secondary nucleus migration and
the development of cytoplasmic polarity.
1
1.1
Materials and methods
Plant material
Plants of Torenia fournieri were grown in a growth chamber (Conviron, CMP 3244 Con-
trolled Environmats LTD) with 16-h photoperiods and 50% humidity at 25. Flowers were collected at different developmental stages, e.g., before anthesis, anthesis, 1, 2 d after flower opening,
and several hours after pollination. For pollination, flowers 2 d after opening were hand-pollinated
with fresh pollen and then harvested between 5 and 17 h after pollination.
1.2 Microinjection of the embryo sacs
Ovules were dissected and immediately immobilized onto a microscope slide in a thin layer
of 1% low temperature-gelling agarose, type VII (Sigma), which was prepared with the ovule culture medium containing 5 mmol/L HEPES, 1 mmol/L KCl, 1 mmol/L MgCl2, 0.1 mmol/L CaCl2,
3% sucrose, pH 7.0. The materials were then covered with a drop of inert oil (Voltalef PCTFE Oil,
Type 10S, Altochem, UK) to prevent the samples from drying. Glass microinjection needles were
pulled from 1.0 mm borosilicate glass capillaries (Warner Instrument Co.) with a Narishige PB-7
needle puller.
The experiments include a series of microinjections with embryo sacs isolated from flowers
at different stages as described above. Single or co-injection of Alexa 488-phalloidin (Molecular
Probes, Eugene, OR, USA) and propidium iodide (PI) (Molecular Probes) was conducted for labeling F-actin and nucleolus in the central cells. The red fluorescence of PI staining may only
represent nucleoli in cells as demonstrated by Kranz et al.[6]. It is adequate for our study here since
we only take it as an indication of the location of the nucleus. Microinjection of rhodamine tubulin
(Cytoskeleton Co., Denver, CO) was also performed to label the microtubules in the central cells.
The injection solution for staining of the actin filaments was prepared by drying down an
aliquot of 5 µL of Alexa 488-phalloidin stock solution in methanol (6.6 µmol/L) and then resuspending it in the injection buffer (100 mmol/L KCl, 2 mmol/L HEPES at pH 7.0) to make a final
concentration of 4 µmol/L Alexa 488-phalloidin. For the co-injection solution, 1 mg/mL stock
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solution of propidium iodide (PI) was added to make a final concentration of 4 µmol/L Alexa
488-phalloidin and 20 µg/mL PI. The solution was clarified by centrifugation at 13000 r/min for
15 min at room temperature, and then loaded in the capillaries of the injection needles according
to the procedure described by Staiger et al.[23].
The injection solution for visualizing microtubules was made to a final concentration of 1.5
mg/mL rhodamine tubulin diluted with the injection buffer (20 mmol/L glutamic acid, 2 mmol/L
MgCl2, pH 6.9 with KOH) from the original concentration of 10 mg/mL of the product. The loading of the injection solution in the capillaries was the same as described above.
Embryo sacs were injected following the procedure described in detail by Staiger et al.[23].
Microinjection experiments were carried out on a Leica DM IRB inverted microscope. The injection system (using oil-filled, hydraulic pressure injection) is controlled by a Narishige MN-188
three-dimensional manipulator. An estimated 1% of total cell volume was injected into the central
cells from the lateral region of the embryo sacs and 1020 central cells were injected at each developmental stage. The injected embryo sacs were immediately observed and the transmission and
fluorescent images were recorded using confocal laser scanning microscopy.
1.3 Anti-actin filament and anti-microtubule drug experiments
Flowers were hand-pollinated 2 d after opening and then collected 3 h after pollination.
Ovaries were dissected from the flowers and their walls removed allowing the ovules to be exposed. The materials were immediately immersed into culture medium (5 mmol/L HEPES,
1 mmol/L KCl, 1 mmol/L MgCl2, 0.1 mmol/L CaCl2, 3% sucrose) containing 10 nmol/L latrunculin A (Molecular Probes, Eugene, OR, USA) or 50 µmol/L cytochalasin B (Molecular Probes,
Eugene, OR, USA). After being cultured for 4 h, the ovules were dissected from the placenta.
Control ovules were cultured in medium without LAT-A or CB. After the treatment, the ovules
were dissected and then stained with 2 µg/mL DAPI for 1 h. The number of ovules with a visible
secondary nucleus was counted under a Leica DM IRB upright fluorescent microscope. The actin
cytoskeleton was labeled by microinjection with 4 µmol/L Alexa 488-phalloidin and then observed using a Leica TCS NT confocal microscope (see below).
For the rescue experiments, latrunculin A was removed 1 h after LAT-A treatment and solution was exchanged four times with fresh culture medium with 1 h intervals. The procedure for the
observation of the secondary nuclei was as described as above.
The handling of ovaries after pollination followed the procedure described above. After removing the ovary wall, the exposed ovules were placed in the culture medium containing respectively 5, 25 and 50 µmol/L of oryzalin for 4 h. The control was cultured in the above medium
without oryzalin. The nuclear behavior of the central cell was observed as above. The microtubules were visualized by microinjection with 1.5 mg/mL of rhodamine tubulin and then observed using a Leica TCS NT confocal microscope (see below).
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1.4
Confocal microscopy
The microinjected embryo sacs were observed and recorded using a Leica TCS NT confocal
microscope with three channels and equipped with an argon-krypton laser. The 488-nm wavelength excitation line of the laser was used for Alexa 488-phalloidin fluorescence and 568-nm
wavelength excitation lines of the laser were used for PI and rhodamine-tubulin fluorescence.
About 1220 optical sections in 12 µm steps of transmission, and fluorescent images were
simultaneously collected and projected using a Leica TCS NT imaging software package. Images
were further processed using Adobe Photoshop 4.0 (Adobe, San Jose, CA).
2
Results
In this study, we describe actin organization and dynamics, and nuclear migration in the central cell and primary endosperm at different stages of embryo sac development from 1 d before
anthesis to 17 h after pollination. To facilitate description, the patterns of actin organization and
nuclear behavior are summarized in fig. 1 according to the time course of the main events during
fertilization. Five major patterns have been recognized, and are designated as Types A, B, C, D
and E. In each type, both actin organization and the disposition of the nucleus in the central cell
and primary endosperm (before and after fertilization) are shown. The details are described below.
Fig. 1. A diagram that shows the main events of actin organization and nuclear migration in the central cell and primary
endosperm of Torenia fournieri before and after fertilization. Five different types of actin distributional pattern have been
recognized. They are designated Types A, B, C, D and E. Gray filaments represent actin filaments and dark black refers to
nuclei. Dotted line indicates the outline of vacuoles. CC, Central cell; EA, egg apparatus; EN, primary endosperm nucleus;
ENDO, endosperm; HAP, hours after pollination; SN, secondary nucleus. Type A (see fig. 2(b) and 2(c)) is the dominant
pattern seen 1 d before anthesis. Actin filaments are organized into numerous short bundles largely distributed in the cortex of the central cell. The secondary nucleus derived from polar nuclei fusion is not visible because it locates at the chalazal end or the middle bent region of the embryo sac and is masked by the ovular tissue. Type B (see fig. 2(e), 2(f), 2(h)
and 2(i)) is most frequently seen 2 d after anthesis. At this stage, an actin network is distinctly present in the cytoplasm.
The secondary nucleus appears in the mid-region of the central cell. An array of actin filaments is associated with the
secondary nucleus (arrow). Type C (see plate I(6), (8)) is present in maximal frequency 5ü7 h after pollination. At this
stage actin filaments in the micropylar end and the perinuclear region of the central cell become fragmented into short
filaments and randomly distributed. The secondary nucleus is now located proximal to the egg apparatus. Type D (see
plate I(10) and (12)) is the pattern often seen (at 9ü10 h after pollination) during nuclear fusion between a sperm nucleus
and the secondary nucleus resulting in the formation of a primary endosperm nucleus. The actin filaments completely degrade into punctate structures throughout the endosperm. Type E (see plate II(2), (4)) is most frequently seen about 12ü
17 h after fertilization. Actin filaments reorganize into a distinct network in the cytoplasm of the endosperm. This network
appears quite similar to that seen in Type B. The endosperm nucleus has moved back to the chalazal end, accompanied by
the reappearance of a distinct array of actin filaments in the perinuclear region (arrow).
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Actin organization and nuclear behavior in the central cell before fertilization
Fig. 2(a) shows a living embryo sac of Torenia fournieri that protrudes from ovular tissue,
exposing most of its micropylar part. The
chalazal end of the embryo sac is masked by
the opaque ovular tissue and hence is not
visible under the light microscope. From 1 d
before anthesis to 2 d after flower opening,
the female germ unit in the exposed part of
Fig. 2. Organization of actin cytoskeleton in the central cell of
Torenia fournieri before and after anthesis. Fig. 2(b), 2(c), 2(e),
2(f), 2(h) and 2(i) are fluorescent images. Fig. 2(a), (d) and (g) are
transmission images collected simultaneously with their corresponding fluorescent images. CC, Central cell; DSy, degenerated
synergid; E, egg; SN, secondary nucleus; Sy, synergid. Bar = 10
µm. An embryo sac (one day before anthesis) protrudes from the
ovular tissue showing an egg apparatus (one egg and two synergids) and a central cell. (b) An optical section of the same embryo
sac shown in (a). Note the presence of actin filaments in the cortex (arrowhead) of the central cell. (c) A projected confocal image
of the same embryo sac shown in (a). Numerous short and randomly distributed actin bundles (arrowhead) are present in the
cytoplasm of the central cell. Note that the distribution is particularly dense in the region near the egg apparatus (arrow). (d) An
embryo sac 2 d after anthesis. Two degenerated synergids are
obvious in the micropylar end of the embryo sac. (e) An optical
section of the same central cell shown in (d). Note that thick actin
bundles (arrow) are present in the micropylar cortex. (f) A projected image of the same embryo sac shown in (d). An actin network (arrowhead) is distributed throughout the cytoplasm. Actin
filaments are particularly dense in the micropylar region and they
organize into numerous thick bundles (arrow) near the egg apparatus. (g) Another embryo sac 2 d after anthesis. Note the presence of a number of vacuoles in the central cell. (h) An optical
section of the same embryo sac shown in (g) at the center of the
central cell, showing actin filaments aligned in a cytoplasmic
strand (arrowhead). (i) A projected image of the central cell of the
same embryo sac shown in (g). Note the presence of a distinct
actin network (arrowhead) in the cortex of the central cell.
the embryo sac can be easily observed. Like
other typical embryo sacs, it consists of two
synergids and an egg cell at the micropylar
end and a large central cell in the center of
the embryo sac. In central cells, which were
injected with Alexa 488-phalloidin and examined by confocal microscopy, numerous
short actin bundles are observed distributed
primarily in the cell cortex. They are particularly dense in the region adjacent to the
egg apparatus (fig. 2(b) and 2(c)). This pattern of actin organization (designated as
Type A in fig. 1) in the central cell is commonly present in embryo sacs from 1 day
before anthesis to 2 d after anthesis. An
ovary of T. fournieri contains more than 200
ovules, and the developmental stages of
these ovules can vary. However, the Type A
pattern of actin filament distribution (fig. 1)
is dominant 1 d before anthesis, reaching a
frequency of 93.3% (table 1), which declines
greatly after pollination (table 1). Before
anthesis, the secondary nucleus in the mature
embryo sac has already formed by the fusion
of the two polar nuclei in the central cell[5].
The secondary nucleus is rarely visible before anthesis because it usually locates in the
chalazal end or the middle bent region of the
embryo sac and is therefore masked by the
opaque ovular tissue (table 2, also see fig.
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2(a) and 2(c)).
Table 1 Various types of actin organization observed in the central cells and the primary endosperms
of the embryo sac of T. fournieri
Type Ac(%)
Type Bc(%)
Type Cc(%)
Type Dc(%)
Type E c(%)
Total number of
injected cells
−1 DAAa)
14 (93.3%)
1 (6.7%)
0 (0%)
0 (0%)
0 (0%)
15
0 DAA
19 (67.9%)
8 (28.6%)
1 (3.6%)
0 (0%)
0 (0%)
28
1 DAA
13 (65.0%)
5 (25.0%)
2 (10.0%)
0 (0%)
0 (0%)
20
2 DAA
5 (20.8%)
18 (75%)
1 (4.2%)
0 (0%)
0 (0%)
24
After pollination
5-7HAPb)
1 (5.3%)
1 (5.3%)
14 (73.7%)
3 (15.8%)
0 (0%)
19
During fertilization
9-10HAP
1 (5.9%)
1 (5.9%)
4 (23.5%)
11 (64.7%)
0 (0%)
17
0 (0%)
0 (0%)
6 (25.0%)
6 (25.0%)
12 (50%)
24
Before
pollination
After fertilization
12-17HAP
The numbers and frequencies in bold represent the highest percentage of each type observed in the embryo sacs at various
stages before and after fertilization. a) DAA = Days after anthesis (-1DAA = One day before anthesis). b) HAP = Hours after
pollination. c) Various types (A, B, C, D, E) of actin distribution pattern seen in the central cell or primary endosperm are shown
in fig. 1.
After anthesis, the synergid begins to degenerate. In most instances only one of the two synergids degenerates, although the degeneration of both has been observed (fig. 2(d)). At this stage,
the actin cytoskeleton in the cytoplasm of the central cell displays a distinct network, in which
individual filaments are longer than those observed in the embryo sacs 1 d before anthesis (fig.
2(e) and 2(f)). The filaments aggregate adjacent to the egg apparatus (fig. 2(e) and 2(f)). Figs. 2(g),
2(h) and 2(i) show the actin network in the central cell viewed from the micropyle 2 d after anthesis. Actin filaments are also found longitudinally aligned in the cytoplasmic strands (fig. 2(h)).
This pattern of the actin cytoskeleton, seen in the central cell of the embryo sac 2 d after anthesis,
is designated as Type B (fig. 1), and is observed most frequently (about 75%, see table 1) in embryo sacs 2 d after anthesis.
After anthesis, the secondary nucleus of the central cell begins to move to the central region
of the embryo sac. However, the number of embryo sacs with a visible secondary nucleus is low
with a frequency of about 2.5% at the day of anthesis, about 6.6% 1 d after anthesis and 14.2% 2 d
after anthesis (table 2). 7 h after pollination, the frequency of the embryo sacs with a visible secondary nucleus in the central region or at the site adjacent to the egg apparatus increases from
14% to 58% (table 2). This indicates that pollination may accelerate the movement of the secondary nucleus from the chalazal region to the micropylar region of the central cell. As the
secondary nucleus moves towards the micropylar region of the central cell it is surrounded by an
array of actin filaments (plate I(1), (2)) forming a perinuclear ring or cage. A well-organized
network of actin filaments is still present in the cytoplasm of the central cell (plate I(2)). Moreover,
actin filaments (plate I(4)) have also been found in the cytoplasmic strand that extends from the
secondary nucleus to the cortical cytoplasm near the egg apparatus (plate I(3)). Active organelle
movement also has been observed in the cytoplasmic strand (unpublished data).
When the pollen tubes approach the embryo sac (plate I(5)), the actin filaments at the micropylar end of the central cell become fragmented (plate I(6)). As the secondary nucleus migrates
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near the egg apparatus (plate I(7)), the perinuclear actin array also fragments into short filaments
(plate I(12)). This type of actin filament distribution is seen in the central cell of the embryo sac
about 5ü7 h after pollination and is designated as Type C (fig. 1). This type reaches a frequency
of about 73.7% (table 1), 5ü7 h after pollination.
Table 2 Embryo sacs showing visible secondary nuclei in the central cell before and after pollination
Stages
Total No. of embryo sacs observed
No. of embryo sacs showing
visible secondary nuclei
Frequency (%)c)
1 DAAa)
132
0 DAA
159
1 DAA
140
2 DAA
147
7 HAPb)
143
0
4
10
21
86
0%
2.5%B3.3
6.6%B9.4
14.2%B3.5
58.8%B8.3
a) DAA, Days after anthesis. b) HAP, Hours after pollination. Pollination was performed 2 d after anthesis. c) Values are
meansBSD.
2.2 Actin organization and nuclear behavior in the central cell and primary endosperm during
and after fertilization
About 9 to 12 h after pollination, syngamy between one sperm cell and the central cell normally takes place. The prominent features at this stage are the penetration of the pollen tube into
one of the synergids and migration of the secondary nucleus towards the site adjacent to the egg
cell and the degenerated synergid (plate I(9)). Soon after the pollen tube has discharged the male
gametes into the degenerated synergid, syngamy followed by karyogamy between sperm and central cell takes place (plate I(10)). Co-microinjection of Alexa 488-phalloidin and propidium iodide
(PI) in the central cell at this stage revealed that a sperm nucleus is about to fuse with the secondary nucleus (plate I(10)). The actin network in the central cell becomes completely fragmented.
In the cortex and the perinuclear region, abundant short actin fragments are present (plate I(10)).
When nuclear fusion occurs, the actin cytoskeleton in the central cell further degrades into puncta
(plate I(11), (12)). This type of actin distribution is designated as Type D (fig. 1), and shows a frequency of about 64.7% in the embryo sacs 9ü10 h after pollination (table 1).
About 12ü17 h after pollination, the primary endosperm nucleus is about to move from the
egg apparatus back to the chalazal end (plate II(1), (3)). At this stage, the actin filaments rapidly
reorganize into a distinct network in the cytoplasm of the primary endosperm (plate II(2), (4)). An
array of short actin filaments surrounding the primary endosperm nucleus reemerges (plate II(2),
(4)). In the rest of the cytoplasm of the primary endosperm, numerous actin filaments are densely
distributed (plate II(5), (6)). This type of actin distribution is designated as Type E (fig. 1), and is
observed at a frequency of about 50% in the primary endosperm about 12ü17 h after pollination.
The pattern of organization of actin filaments seen in Type E is very similar to the actin network
(viz. Type B) 2 d after anthesis, indicating that the actin cytoskeleton has been rapidly restored
after fertilization.
2.3 The effects of cytoskeletal inhibitors on nuclear migration of the central cell
To determine if the secondary nucleus migration in the central cell is microfilament or
microtubule dependent, we treated the embryo sacs with anti-actin (latrunculin A, LAT-A; cyto-
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chalasin B, CB) and anti-microtubule (oryzalin)
drugs. Latrunculin A and cytochalasin B are
known to disrupt actin filaments and oryzalin
ª
to disrupt microtubules[26 28]. Actin filaments
are fragmented after the embryo sac has been
treated with 10 nmol/L latrunculin A (fig. 3(c)).
Treatment with LAT- A or CB blocks cytoplasmic streaming in the central cell and disrupts the transvacuolar strands (fig. 3(b)). The
effect of LAT-A or CB on the movement of the
secondary nucleus was examined by counting
the ovules with a visible secondary nucleus in
the central cell after treatment. The frequency
of ovules with a visible secondary nucleus
dropped from about 49.6% to 12.7% after
LAT-A treatment and from about 51.8% to
15.4% after CB treatment (table 3). Microinjection of Alexa 488-phalloidin into treated
cells shows that the actin cytoskeleton is depolymerized with the treatment of LAT-A (fig.
3(c)) and CB (see table 3), when compared
with control embryo sacs (fig. 3(a)), indicating
that there is a structural and functional relationship between actin microfilaments and the
secondary nucleus migration.
Fig. 3. The effect of anti-actin and anti-microtubule treatments
on the actin and microtubule cytoskeleton in the central cell. (a),
(c), (d) and (f) show the fluorescent images. (b) and (e) are transmission images collected simultaneously with their corresponding
fluorescent images. CC, Central cell; Sy, synergid; E, egg cell; SN,
secondary nucleus. Bar = 10 µm. (a) The actin cytoskeleton in the
central cell of an embryo sac 2 d after anthesis without LAT-A
treatment. (b) The transmission image of an embryo sac treated
with LAT-A. Note that there are no transvacuolar strands in the
central cell. (c) The actin cytoskeleton in the central cell of the
embryo sac shown in (b). Note that after LAT-A treatment the
actin filaments have shortened or fragmented (arrow). (d) A cortical microtubule array in the central cell microinjected with rhodamine-tubulin. Note that all microtubules (arrow) align longitudinally with the axis of the central cell. (e) A transmission image
of an embryo sac after treatment with oryzalin. Note that there are
a few transvacuolar strands (arrow) present. Moving vesicles are
visible in the cytoplasmic strands of the central cell. (f) An embryo sac after oryzalin treatment. Note that the microtubules have
completely disrupted (arrowhead) in the central cell.
Table 3 Frequency of ovules harvested 3 h after pollination showing visible secondary nuclei in the central cells after latrunculin A and cytochalasin B (CB) treatments
Total No. of embryo sac
observed a) b)
No. of embryo sacs showing
visible secondary nuclei a) b)
Frequency (%) c)
After LAT
washed out
Control
50 µmol/L CB
Control
10 nmol/L LAT-A
507
1209
100
162
250
158
48
82
21
49.6% ± 5.2
12.7% ± 2.2
48%
51.8% ± 4.9
15.4% ± 4.7
133
a) Total of 3 experiments. b) The numbers represent the number of embryo sacs observed. c) The % frequencies are presented as means ± SD.
To confirm such effect of the F-actin disruption on nuclear movement in the central cells,
LAT-A was removed by washing out and exchange with fresh culture medium after the treatment.
The nuclear migration was indeed rescued after LAT-A had been removed. The frequency of
ovules with a visible secondary nucleus increased to 48% (table 3), 4 h after washing out LAT-A
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treated ovules with fresh culture medium.
Microtubules, which have been revealed through the incorporation of microinjected rhodamine-tubulin, are longitudinally aligned in the cortex of the central cell (fig. 3(d)). To test if
microtubules play a role in the movement of the secondary nucleus, the central cell is treated with
the ovules with different concentrations of oryzalin 2 d after anthesis. Microtubules are disrupted
into punctate structures throughout the cytoplasm of the central cell. The effect of oryzalin in the
central cell is thus confirmed by microinjection of rhodamine-tubulin (fig. 3(e) and (f)). The results indicate that, unlike the treatments with LAT-A or CB, oryzalin causes no apparent changes
of cell organization in the central cell and the transvacuolar strands remain normal. Moreover, the
frequency of the secondary nucleus migration observed in the central cells is 52.2% to 53.8% in
treated ovules, compared with 58.5% in the control (table 4). In addition, the effects of oryzalin on
the secondary nucleus migration at different oryzalin concentrations (from 5 µmol/L to 50
µmol/ L) show almost no difference (the variation is between 52.2% and 53.8%, table 4). Taken
together these data indicate that microtubules do not seem to participate in secondary nucleus migration in the central cell of Torenia fournieri.
Table 4 Frequency of ovules harvested 3 h after pollination showing visible secondary nuclei in the
central cells after oryzalin (Ory) treatment
Control
Total No. of embryo
sacs observed a) b)
No. of embryo sacs
showing visible
secondary nuclei a) b)
Frequency (%)c)
281
162
58.5% ± 4.0
50 µmol/L Ory
232
123
52.9% ± 5.1
25 µmol/L Ory
5 µmol/L Ory
251
251
136
131
53.8% ± 4.6
52.2% ± 5.7
a) Total of 5 experiments. b) Number of embryo sacs observed. c) Frequency (%) presents as means ± SD.
3
Discussion
3.1 The actin cytoskeleton in the central cell and endosperm displays a conspicuous reorganization before and after fertilization
With the successful application of microinjection into the living embryo sacs of T. fournieri,
we have been able to visualize dramatic changes in the organization of the actin cytoskeleton in
Torenia central cells during fertilization. Five major patterns of actin organization (fig. 1) have
been recognized. Before pollination, the maturation of the central cell is accompanied by a reorganization of actin cytoskeleton from short, randomly aligned bundles (see fig. 1. Type A) to a
distinct network distributed in the cytoplasm (see fig. 1, Type B). This network, containing numerous long filaments, is distributed mainly in the cell cortex, with some filaments aligned in the
cytoplasmic strands. A similar network has also been observed in the central cell of maize[16],
Plumbago[13], and Arabidopsis[19]. Although the role of these actin arrays in the central cell is not
fully understood, the actin network, accompanied by the microtubules, appears to provide a structural scaffold for the highly vacuolated central cell and, together with myosin, generates cyto-
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FERTILIZATION IN T. fournieri
221
plasmic streaming. These conclusions are supported by the application of LAT-A and CB, which
results in the collapse of the central cell (unpublished observations) and the inhibition of streaming.
After pollination and during fertilization, one conspicuous change is the fragmentation of the
actin network in the central cell. About 5ü7 h after pollination, but before the arrival of the pollen
tube, the actin network becomes fragmented into numerous short filaments in the micropylar end
around the secondary nucleus (see fig. 1, Type C). As nuclear fusion occurs between a sperm nucleus and the secondary nucleus, the actin cytoskeleton becomes completely degraded into randomly dispersed punctate structures (fig. 1. Type D). The degradation of the actin cytoskeleton
during karyogamy between a sperm nucleus and the secondary nucleus suggests that these
changes may be involved in reorganization of cytoplasm of the primary endosperm. A similar
process has been shown in the syncytial embryo of Drosophila, in which the actin network partially disassembles around the nuclei during axial expansion, coupled with cytoplasmic streaming
and nuclear migration along the anterior-posterior axis[29].
Soon after karyogamy, the actin network is rapidly re-established in the cytoplasm of the
primary endosperm (fig. 1, Type E) as the nucleus migrates towards the chalazal end. Since the
development of the endosperm normally precedes embryogenesis in most of the angiosperm species, the reorganization of the actin network in the cortex of the primary endosperm may be involved in relocation of the cytoplasmic domains[8].
3.2 Structural and functional studies indicate that actin participates in nuclear migration in the
central cell and primary endosperm
Fusion of the polar nuclei and migration of the secondary nucleus are the crucial events for
fertilization and later endosperm development. In some angiosperms, fusion of the polar nuclei
and migration of the secondary nucleus occur prior to fertilization[1]; Torenia fournieri belongs to
this category. Fusion of the polar nuclei occurs during maturation of the embryo sac and migration
of the secondary nucleus initiates after anthesis. The migration of the nucleus appears to be accelerated by pollination[5]. We found that the frequency of nuclei positioned near the egg apparatus
increases from 14.2% after anthesis to 58.8% after pollination (table 2). This finding is consistent
with previous observations by Higashiyama et al.[5], suggesting that the interaction between male
and female gametes may accelerate the migration of the secondary nucleus towards the egg apparatus. The migration of the secondary nucleus in close proximity to the egg cell provides the
sperm cells a short distance over which to effect simultaneous fertilization.
During fertilization, dynamic changes and reorganization of the actin cytoskeleton in the
central cell appear to coincide with the migration of the secondary nucleus for fusion with the
male gamete. After anthesis and before pollination, two distinct arrays of actin filaments have
been observed in the central cell, including an array of actin filaments in the cytoplasmic strands
and an actin network in the cortex (see fig. 1, Type B of actin organization). After pollination,
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Vol. 45
however, remarkable changes occur. Two distinct arrays of 4 actin filaments become associated
with the secondary nucleus when it descends towards the egg apparatus: (i) an actin cage enwraps
the secondary nucleus; and (ii) thick bundles emanate from the secondary nucleus to the micropylar cortex of the central cell adjacent to the egg cell. These arrays are fragmented into puncta when
the secondary nucleus moves to the site adjacent to the egg apparatus. However, when the primary
endosperm nucleus migrates back towards the chalazal end after fertilization, the actin cage surrounding the nucleus reemerges. Similarly a tightly configured cage of actin has been recognized
surrounding the nucleus in the desmid Micrasterias[30]. Given this close relationship with the migrating nucleus, the actin cytoskeleton has been postulated to play a role in maintaining the proper
location of the nucleus and in generating the motive force for nuclear movement[30]. Similarly, we
postulate that these arrays of actin are responsible for the nuclear migration in the central cell and
primary endosperm before and after fertilization.
In addition to the structural data above, the results gained with different inhibitors, LAT-A,
CB and oryzalin, all consistently indicate that actin microfilaments exert a primary role in nuclear
migration. LAT-A and CB greatly inhibit nuclear migration in the central cell, whereas oryzalin,
an anti-microtubule drug shows no significant effects. A similar role for the actin cytoskeleton in
nuclear migration has been reported in the pollen tube. Not only is an analogous structural configuration of actin observed but in addition the process is sensitive to CB[12]. Moreover, myosin,
the motor protein for actin filament motility[11] has also been detected on the surface of the vegetative nucleus and generative cell. Its interaction with actin microfilaments could provide the necessary motive force for organelle and nuclear migration in the pollen tube [10]. Thus, the current view
holds that the actin cytoskeleton may play an important role in organelle and nuclear transportation in the pollen tube.
By contrast, in many other tip-growing cells, microtubules, rather microfilaments, have been
reported to be involved in nuclear migration. For example, in fungal hyphae, nuclear translocation
ª
appears to be microtubule-dependent[31 34]. Involvement of microtubules in nuclear migration has
also been reported in the germination of the fern spores[35], and in growing root hair cells[36].
However, our data indicate that disruption of microtubules with oryzalin only has a slight effect on
nuclear migration in the central cell. Taken together, these observations suggest that the control of
nuclear migration by different cytoskeletal elements may be cell-type and species dependent. In
some other systems, both actin filaments and microtubules appear to participate in the movement
of the nucleus. In green algae, nuclear movement is only inhibited by the application of both CB
and colchicine[37]. Menzel et al.[38] postulated that actin is an essential cytoskeletal element for the
nuclear movement whereas microtubules likely function as a trailing anchor. According to our
results, although microtubules seem to have no significant effect on the nuclear migration in the
central cell, they may be utilized as a scaffold for an auxiliary support for more fragile actin filaments.
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FERTILIZATION IN T. fournieri
223
Acknowledgements This research was supported by a RGC grant from the Research Grants Council of Hong Kong and
a CRCG grant from University of Hong Kong to B. Q. H, a CRCG grant to S. Y. Z, NSF grant No. MCB-9601087 to P. K. H.,
and the National Key Basic Research Project of China (No. G19990117) and grants from the National Natural Science Foundation of China (Grant Nos. 39625003 and 19890380) to M.Y. We thank Dr. C.J. Staiger and Prof. E. C. Yeung for critical reading
of the manuscript.
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YUAN Ming et al.: Fertilization in Torenia fournieri
Plate I
Actin organization and the secondary nucleus migration in the central cell during fertilization. (2), (4), (6), (8), (10) and (12)
show the fluorescent images, where the green color represents the actin filaments labeled by Alexa 488-phalloidin and the red
color represents nucleoli of the secondary nuclei stained by PI. (1), (3), (5), (7), (9) and (11) are transmission images collected
simultaneously with their corresponding fluorescent images. CC, Central cell; DSy, degenerated synergid; E, egg cell; EN, endosperm nucleus; ENDO, endosperm; PT, pollen tube; SN, secondary nucleus. Bar = 10 ? m. (1) An embryo sac after pollination
showing an egg cell at the micropylar end and a large central cell in the center. Note that the secondary nucleus is present in the
chalazal end of the exposed part of the central cell. (2) A projected image of the same embryo sac shown in (1). Long actin filaments (arrowhead) are seen in the cytoplasm of the central cell. Some actin filaments (arrow) circle the secondary nucleus. (3)
An embryo sac after pollination but before the arrival of the pollen tube. An egg and a central cell are evident in the median
optical section. Note that there is a cytoplasmic strand (arrowhead) between the secondary nucleus and the micropyle. (4) A
projected image of the median optical sections of the central cell shown in (3). Note the presence of actin bundles in the cytoplasmic strand (arrowhead). Some actin filaments are also present at the micropylar cortex adjacent to the egg cell (large arrow).
The small arrow indicates the egg nucleus. (5) An embryo sac showing a degenerated synergid. The other degenerated synergid is
not in focus. (6) Actin filaments in the central cell of the same embryo sac shown in (5). Note that some actin filaments have
fragmented (arrowhead) in the micropylar end of the central cell while long actin filaments are still distinct in the chalazal end of
the cell (arrow). The secondary nucleus is visible in the central region of the cell. (7) An embryo sac before pollen tube arrival.
Note that the secondary nucleus has now migrated to the region of the central cell adjacent to the egg cell. (8) Actin filaments and
the secondary nucleus in the central cell of the same embryo sac shown in (7). Note that the secondary nucleus is located at the
position adjacent to the egg cell. Some actin filaments in the perinuclear region of the secondary nucleus have fragmented (arrow). (9) An embryo sac during fertilization. Note that a pollen tube (not in focus) has penetrated into the embryo sac. The secondary nucleus in the central cell has located in the micropylar region adjacent to the egg. (10) A projected image of the same
embryo sac shown in (9). A sperm nucleus (small arrow) has moved to the proximity of the secondary nucleus. Short actin filaments are found sparsely distributed in the central region (arrow) and densely align at the micropylar end (arrowhead) of the
central cell. (11) An embryo sac during karyogamy. Note that pollen tube has penetrated into a degenerated synergid. (l2) An
optical section of the same embryo sac shown in (11). Note that a sperm nucleus is fusing with the secondary nucleus (arrow) and
the actin network has degraded into punctate structures (arrowhead).
YUAN Ming et al.: Fertilization in Torenia fournieri
Plate II
Actin organization and nuclear behavior in the primary endosperm after fertilization. (2), (4) and (6) show the fluorescent images,
where the green color represents the actin filaments labeled by Alexa 488-phalloidin and the red color represents nucleoli of the
secondary nuclei stained by PI. (1), (3) and (5) are transmission images collected simultaneously with their corresponding fluorescent images. DSy, Degenerated synergid; EN, endosperm nucleus; ENDO, endosperm; PT, pollen tube; Z, zygote. Bar = 10
? m. (1) A transmission image of an embryo sac 16 h after pollination, showing a zygote and the primary endosperm with its
nucleus in the median region of the embryo sac. Note that the endosperm nucleus is near the zygote. (2) A projected image of the
same endosperm shown in (1). A distinct actin network (arrowhead) reappears in the cortex of the primary endosperm. (3) A
transmission image of another embryo sac after fertilization. Note the presence of a pollen tube in the degenerated synergid. (4) A
projected image of median optical sections of the same endosperm shown in (3). Note the presence of an actin array (arrow) in
the perinuclear region. Dense actin filaments are evident in the cortex (arrowhead) of the developing endosperm. (5) A transmission image of an embryo sac after fertilization. Note that the pollen tube (PT) is still present in the degenerated synergid after
gametic fusion. (6) The array of actin filaments in the same endosperm shown in (5). Long actin filaments (arrowhead) are
abundant in the intervacuolar cytoplasm of the endosperm. Note that the endosperm nucleus has now migrated back to the chalazal part of the primary endosperm.