1 Institute of Plant Biology & Zürich-Basel Plant Science Center,
University of Zürich, Zollikerstrasse107, 8008 Zürich,
Switzerland
2 Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724,
USA
3 Graduate Program in Genetics, State University of New York, Stony Brook, NY
11794, USA
* Author for correspondence (e-mail: grossnik{at}botinst.unizh.ch)
Accepted 26 February 2002
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SUMMARY |
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Key words: Arabidopsis thaliana, Double fertilization, Female gametophyte, feronia, Pollen tube invasion, Pollen tube reception, Sperm cell release, Supernumerary pollen tubes, Synergid degeneration
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INTRODUCTION |
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In many species the receptive synergid degenerates before the PT reaches
the FG (reviewed by Kapil and Bhatnagar,
1975; Russell,
1992
). Only one synergid is susceptible for signals induced by
pollination or derived from the approaching PT. It is not known how the
receptive synergid is selected. Synergid degeneration is considered to be
essential for fertilization (Drews and
Yadegari, 2002
; Jensen and
Fisher, 1968
; van Went and
Willemse, 1984
). Because PT reception and double fertilization are
rapid processes that involve inaccessible cells, studies about male-female
gametophytic interactions have been restricted to ultrastructural
investigations of pollen tube arrival, synergid degeneration and sperm cell
release (reviewed by Kapil and Bhatnagar,
1975
; Raghavan,
1997
; Russell,
1992
). The identification of mutants with defects in PT reception
and double fertilization is necessary to gain insights into the genetic
control and the molecular basis of these processes. In the last years
screening strategies have been developed in Arabidopsis thaliana to
identify mutants with female gametophytic defects
(Christensen et al., 1998
;
Howden et al., 1998
;
Moore et al., 1997
;
Shimizu and Okada, 2000
).
Although most mutations identified to date cause defects in embryo sac
development (Christensen et al.,
1998
; Moore et al.,
1997
), some appear to be implicated in PT guidance and reception
(Christensen et al., 2002
;
Shimizu and Okada, 2000
). For
instance, the Arabidopis mutant gfa2 fails in synergid
degeneration (Christensen et al.,
2002
). The PT is still attracted to the FG but the embryo sac
remains unfertilized. Synergid degeneration is, therefore, not required for PT
guidance but for PT reception. The GFA2 gene encodes a member of the
DnaJ protein family and is thought to function as a chaperone in mitochondria.
How the GFA2 protein controls cell death in the synergid is unknown.
We report the comprehensive phenotypic characterization of the Arabidopsis female gametophytic mutant feronia (fer/+). The mutant is named after the Etruscan goddess of fertility, because one half of the ovules remain unfertilized. PT reception is impaired in the feronia mutant, although embryo sac development is unaffected and synergid specification and differentiation appear to be normal. In feronia mutant FG, the PT continues to grow and invades the FG instead of arresting its growth and rupturing to release the sperm cells. Our analysis suggests that the feronia mutation disrupts the interaction between the male and the female gametophyte and, thus, defines a novel signalling process required for PT reception.
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MATERIALS AND METHODS |
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Molecular biology
Non-radioactive Southern blots using a digoxigenin-labelled probe (base
pairs 91 to 440 of the Ds element, accession number AF433043) were performed
according to the manufacturer's instructions. TAIL-PCR
(Liu and Whittier, 1995) to
isolate the sequences flanking Ds was performed as described previously
(Grossniklaus et al., 1998
).
Primers to amplify the molecular marker InDel D03, which shows a 6 bp size
difference between Ler and Columbia (Col) were NHP168
(5'-GGAGAGTAATCAGCAGCTGAG-3') and NHP169
(5'-GGAGAGTAATCAGCAGCTGAG-3').
Cleared whole-mount preparations and histology
For phenotypic characterization, seeds were cleared following the protocol
of (Yadegari et al., 1994).
GUS assays were performed as described previously
(Vielle-Calzada et al., 2000
).
Specimens were observed using a Lecia DMR microscope (Leica Microsystems,
Bensheim, Germany) under bright-field Normarski optics. For preparation of
semi-thin sections, plant siliques were fixed overnight in 3.7%
paraformaldehyde in PHEM buffer (60 mM Pipes, 25 mM Hepes 10 mM EGTA, 2 mM
MgCl2, pH 6.9) on ice. Specimens were dehydrated in an ethanol
series (30%, 50%, 70%, 80%, 90%, 95%, 3x 100%) and transferred into
Technovit 7100 (Heraeus Kulzer, Wehrheim, Germany) according to the
manufacturer's instructions. The tissue was sectioned at 8 µm thickness on
a Leica RM 2145 microtome. After staining with 0.05% Toluidine Blue, sections
were observed under bright-field optics using a Leica DMR microscope.
For ultrastructural analysis silques were cut into 1 mm pieces and fixed on ice for 4 hours with 2.5% glutaraldehyde in 50 mM sodium phosphate buffer, pH 7.0. After three washes with sodium phosphate buffer the siliques were post-fixed in 2% buffered osmium tetroxide, washed and dehydrated in an acetone series (50%, 70%, 90%, 2x 100%) and infiltrated with Spurr's epoxy resin. Siliques were opened and ovules embedded in Spurr's epoxy resin between Teflon-coated microscope slides and polymerized at 60°C. Ultrathin sections were contrasted with 1% uranyl aceteate and Sato's lead solution. Specimens were examined in a Hitachi H 7000 TEM (Hitachi Ltd., Tokyo, Japan) at 60 kV.
Fluorescence staining of pollen tubes
For pollen tube staining, opened siliques were fixed overnight in
Lavdowsky's FAA (1.5% formaldehyde, 2% acetic acid and 30% ethanol) at 4°C
and washed in an alcohol series (70%, 50% 30% 10%), for 10 minutes each.
Tissue was softened with 10% chloralhydrate at 60°C for 10 minutes, rinsed
twice with sodium phosphate buffer (100 mM, pH 7) and then in 5 M NaOH at
60°C, for 5 minutes. Pollen tubes were stained with 0.1% Methyl Blue
(certified for use as Aniline Blue; Sigma, St. Louis, USA). Stained samples
were observed using a Zeiss Axioplan microscope (Carl Zeiss, Oberkochen,
Germany) equipped with an epifluorescence UV-filter set (excitation filter at
365 nm, dichroic mirror at 395 nm, barrier filter LP at 420 nm). Confocal
observations of stained samples were made using a Leica TCS-SP microscope. The
excitation wavelength was 430 nm and the spectral detection window was set as
450-580 and 700-800 nm. Images were acquired and processed using the Leica
Confocal Software, Version 2.0.
Image processing
All images were processed for publication using Adobe Photoshop 5.5 (Adobe
Systems Inc., San Jose, USA).
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RESULTS |
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The gene-trap line feronia had reduced seed set that varied from 45% to 60% in individual siliques. Three phenotypic classes of ovules/seeds were observed: on average (n>2000) 50% of the seeds were normal, 1% aborted early during seed development, and 49% of the ovules remained unfertilized and senesced (Fig. 2A,B). In a population of 446 plants the kanamycin resistance gene co-segregated with semisterility, suggesting that the Ds element is tightly linked to the feronia mutation. The segregation of kanamycin resistance and the result of a Southern blot hybridized with a Ds-specific probe (Fig. 2C) strongly suggest that a single Ds element is present in feronia. Thus, the kanamycin resistance gene could be used as a marker for the mutant feronia allele, greatly facilitating subsequent genetic analyses. However, reversion of the feronia phenotype through excision of the Ds element was unsuccessful, as was complementation with a genomic fragment containing the disrupted gene, indicating that the feronia mutant is not tagged.
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To confirm that the semisterile phenotype of feronia was indeed
caused by a gametophytic defect we analyzed the segregation ratio of the
kanamycin resistance gene in its progeny. For a fully penetrant gametophytic
mutation affecting specifically the female gametophyte, we expect a
segregation ratio of mutant to wild-type plants of 1:1, as half of the male
gametophytes carry the mutation in a heterozygote but only wild-type female
gametophytes are functional. Alternatively, reciprocal chromosome
translocations can lead to semisterility in both the male and female sex
(Ray et al., 1997). However,
while wild-type and semisterile plants are expected to segregate 1:1 in the
progeny of a plant carrying a reciprocal translocation, markers present on the
chromosomes involved in the translocation segregate in a Mendelian fashion.
The segregation ratio of the kanamycin resistance gene in the progeny of
selfed feronia (fer/+) plants was 1.04:1.00 resistant to
sensitive seedlings in the F3 (n=501), and 1.18:1.00 in
the F4 generation (n>1000). The distorted segregation
ratio of the associated kanamycin resistance gene and the fact that no pollen
abortion was observed in fer/+ plants (data not shown) strongly
suggests that semisterility in feronia is caused by a gametophytic
defect and not by a reciprocal translocation or some other gross chromosomal
rearrangement.
feronia is a loss-of-function mutation predominantly
affecting the female gametophyte
While the segregation ratio distortion observed in fer/+ plants is
close to the expected distortion for a sex-specific gametophytic mutation, it
does not exclude the possibility that both sexes are partially affected. To
investigate the respective contribution of male and female gametophytic
defects, we determined the transmission efficiency of the feronia
mutant allele through either sex by reciprocal out-crosses to the wild type.
The analysis of the transmission efficiency of the kanamycin resistance gene
(Fig. 2D) demonstrated a slight
reduction through the male (TEmale=78.5%, n=930) but a
strong reduction through the female gametophyte (TEfemale=14.5%,
n=749). Despite a significant transmission of the kanamycin
resistance gene through both gametophytes, homozygous plants were never
recovered, indicating that the mutation causes zygotic lethality.
In order to make any conclusions about the wild-type function of a gene, it
is important to determine whether a mutation is recessive or dominant to the
wild type. Because dominance and recessiveness are defined as interactions
between two alleles in the same nucleus, it is not possible to investigate
this interaction in haploid gametophytes. Therefore, we generated tetraploid
plants, which produce diploid gametophytes, by repeatedly crossing
feronia to wild-type tetraploids. Siliques of tetraploid plants
potentially carrying between one and three feronia alleles were
analyzed microscopically for the feronia phenotype (see below). A
plant where approximately 50% of the relevant ovules showed the
feronia phenotype [50 wild type, 42 feronia, 36 arrested
ovules as typical of tetraploid Ler plants
(Grossniklaus et al., 1998)]
was analyzed further. As shown in Table
1, such a feronia segregation ratio is expected in either
a simplex tetraploid with feronia being dominant
(ferD/+/+/+) or a triplex tetraploid with feronia
being recessive (fer/fer/fer/+). While the
feronia phenotype is expected to segregate in a similar ratio among
the ovules in these two cases, the associated Ds element is expected
to segregate very differently among their progeny. As shown in
Table 1, the segregation
pattern of the Ds element is consistent with a triplex tetraploid
parent, strongly suggesting that feronia is a recessive,
loss-of-function mutation. Therefore, it should be possible to identify
insertional alleles in the area to facilitate the molecular isolation of the
gene. Taken together these segregation and transmission analyses strongly
suggest that the feronia mutation is recessive, affects predominantly
the female gametophyte, and causes zygotic lethality when homozygous,
consistent with the small percentage of early aborting seeds that we
observed.
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Taken together these results suggest that megagametogenesis and pollen tube
guidance are not affected in the feronia mutant but double
fertilization is. PT growth was not arrested in feronia mutant FGs
and the PT continued to grow, coil around and entangle itself within the
micropylar area (referred to hereafter as the `invading PT phenotype'). Since
the PT is a paradigm for polarized tip growth
(Hepler et al., 2001), it is
likely that the tip of the invading PT stays intact within feronia
mutant embryo sacs and that fertilization does not take place because the PT
fails to rupture and to release the sperm cells.
A defect in the embryo sac causes the aberrant behaviour of the
pollen tube
The analyses of the transmission efficiencies through both gametophytes of
the feronia (fer/+) mutant indicated a strong defect in the
FG, and a weaker defect in the male gametophyte
(Fig. 2B). Since the genetic
data supported a female gametophytic defect but the phenotype is manifest in
the male gametophyte, we asked to what extent the two gametophytes contribute
to the invading PT phenotype. To this aim, we performed reciprocal crosses
between wild-type and feronia (fer/+) plants and analyzed
the pistils 48 and 68 HAP.
When we pollinated wild-type plants with wild-type pollen, we did not
observe the invading PT phenotype. But when wild-type pistils were pollinated
with feronia (fer/+) pollen we noticed that PTs occasionally
invaded the FG (2 out of 110 at 48 HAP, 2 out of 229 at 68 HAP, see
Table 2). However, in these few
cases the PT coiling was not as pronounced as described above (data not
shown). In contrast, pollinated feronia (fer/+) pistils
always contained about 50% ovules invaded by the PT, regardless of whether the
pollen was derived from wild-type or feronia plants (see
Table 2). The frequency of PT
invasion corresponded to the observed semisterility. In a similar experiment,
we crossed feronia and wild-type pistils with pollen of a marker line
expressing the GUS reporter gene
(Jefferson et al., 1987) in
the PT (R. Baskar and U. G., unpublished). In the FG of all wild-type and half
of the feronia (fer/+) ovules the PT terminated in the
synergid (Fig. 3F). In the
unfertilized feronia FGs pollen tube growth failed to arrest
resulting in an intense GUS staining of the coiled PT in the micopylar area of
the mutant (Fig. 3G).
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Invading pollen tubes cause secondary embryo sac phenotypes
During the phenotypic characterization of the feronia mutant we
frequently observed invading PTs growing into the central cell
(Fig. 3H,I). In other cases, we
noticed the formation of endosperm or embryo-like structures, although the PT
was coiling at the micropylar end of the FG
(Fig. 3J). We determined the
frequency of these phenotypes in cleared whole-mount preparations. To
determine whether the ovules with formation of endosperm and embryo-like
structures were the homozygous feronia progeny that we expect to be
zygotic lethal, we analyzed pistils of reciprocal crosses between
feronia (fer/+) and wild-type plants 48 and 68
HAP.
The frequency of PTs entering the central cell in mutant feronia FG increased from 1-2% at 48 HAP to 3-5% at 68 HAP (see Table 2). The phenotype occurred regardless of whether mutant pistils were pollinated with pollen from wild-type or feronia plants. However, the number of PTs proceeding into the central cell was slightly higher when mutant pollen was used (see Table 2). In contrast to the tangled growth at the micropylar end, PTs that entered the central cell grew straight to the chalazal end of the FG (Fig. 3H,I). In some cases the PT proceeded to the chalazal part of the embryo sac and turned back to grow towards the micropylar tip (data not shown).
In about 10% of the invaded FGs the secondary endosperm nucleus of the
central cell started to divide. The frequency was independent of the pollen
source, wild-type or feronia, and did not change from 48 HAP to 68
HAP. In some cases we observed the formation of embryo-like structures
(Table 2), which always
arrested after a few cell divisions (data not shown). Embryo initiation was
observed more frequently at 68 HAP, which might be the result of the delay of
embryo development compared to endosperm formation
(Faure et al., 2002;
Mansfield and Briarty,
1991
).
In summary, the occurrence of PTs proceeding into the central cell and the formation of endosperm or embryo-like structures after PT invasion is independent of the pollen genotype and also occurs when wild-type pollen is used. Therefore, these seeds are not homozygous for the feronia mutation, and these phenotypes solely depend on the genotype of the female gametophyte. It is likely then that coiling pollen tubes occasionally burst and release sperm cells that effect a single fertilization event. The ability of the egg cell and the central cell to initiate cell and nuclear divisions, respectively, demonstrates that the female gametes in feronia are functional after PT invasion.
Mutant feronia embryo sacs attract supernumerary pollen
tubes
It is the common view that ovules in Arabidopsis receive only one
PT (Hülskamp et al.,
1995a; Hülskamp et al.,
1995b
; Shimizu and Okada,
2000
). Nevertheless, in hand-pollinated wild-type pistils we
observed five out of 526 ovules in which supernumerary PTs approached and
entered the FG (see Table 2).
These ovules were delayed in both embryo and endosperm development (data not
shown) suggesting that abnormal FGs permitted additional PTs to approach.
However, in the feronia mutant we consistently observed about 10% of
the invaded ovules attracting two or more PTs
(Fig. 4A,B,
Table 2). To determine whether
the PTs approached the micropyle simultaneously or sequentially we
investigated PT reception in hand pollinated feronia (fer/+)
plants at 12 and 24 HAP. At 12 HAP it was difficult to discriminate mutant and
wild-type FGs as only a few ovules already exhibited the invading PT
phenotype. Nevertheless, in three of 248 ovules analyzed an additional PT had
entered the micropyle, but in eleven ovules a second or third PT grew towards
the micropyle (Fig. 4D,E).
These numbers correlate with the observed frequency of ovules receiving
supernumerary PTs at later time points. In about 10% (12 out of 113) of
invaded ovules additional PTs had entered the micropyle at 24 HAP, and similar
frequencies were observed at 48 HAP and 68 HAP
(Table 2).
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These experiments suggest that ovules containing mutant feronia FGs continue to attract additional PTs after they received the first PT. These supernumerary PTs are able to enter the micropyle, proceed towards the FG, and can potentially invade the embryo sac.
Synergids of feronia embryo sacs are normally specified and
differentiated
In feronia, PT growth failed to arrest and the PTs did not rupture
to release the sperm cells after entering the embryo sac. This would suggest
that a communication process between male and female gametophytes is
disrupted, which could either be a direct effect of disrupting a signalling
process between the synergid and the pollen tube, or a secondary effect due to
the abnormal development or differentiation of the synergids. To address the
question of whether PT reception failed because the synergids had developed
abnormally we crossed feronia into three independent enhancer
detector lines expressing the GUS reporter gene specifically in the synergids
(R. Baskar and U. G., unpublished).
In the wild type, the GUS signal was detectable after cellularization of the FG (Fig. 5A) and persisted in both synergids until fertilization. After fertilization the GUS signal decreased and became undetectable about 24 to 36 HAP (Fig. 5B). We tested whether the reduction in GUS expression was individually induced by the entry of the PT, or whether it was induced in all ovules by a general mechanism, e.g. a certain time after pollination. Therefore, we pollinated wild-type pistils with a limited number of pollen grains, so that only a few ovules were fertilized. GUS activity was readily detected in both synergids of unfertilized ovules, but was drastically reduced in ovules where a PT had entered the micropyle. This indicates that PT entry or fertilization induces the rapid decrease of the signal. In feronia mutants (fer/+) the GUS expression pattern of the three markers was indistinguishable from wild-type until fertilization (Fig. 5C). In fertilized ovules GUS activity disappeared within 24 HAP but it persisted in mutant feronia FGs (Fig. 5D).
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Degeneration of the receptive synergid appears normal at the
ultrastructural level
The PT normally enters the embryo sac through one synergid. In
Arabidopsis it was reported that this receptive synergid shows signs
of degeneration before or just around the time of PT entry
(Faure et al., 2002;
Murgia et al., 1993
). Synergid
degeneration is considered to be necessary for PT rupture and sperm cell
release in some species (Drews and
Yadegari, 2002
; Jensen and
Fisher, 1968
; van Went and
Willemse, 1984
). We investigated whether in the feronia
mutant the PT really entered the FG through one synergid and, if so, whether
the synergid showed the typical signs of degeneration.
We first analyzed semi-thin sections of feronia and wild-type
pistils stained with Toluidine Blue. We found that the coiling of the PT was
restricted to the micropylar area of the embryo sac
(Fig. 6A) and the PT did not
usually enter the central cell (Fig.
6B). To investigate cytological changes within the synergid, we
examined ultra-thin sections of wild-type and feronia siliques using
transmission electron microscopy. In material prepared 6 HAP we found no signs
of synergid degeneration, in either wild-type or feronia ovules (data
not shown). Wild-type ovules collected 24 HAP had enlarged, the zygote had
started to elongate and a few endosperm nuclei had formed (data not shown).
The unfertilized ovules in the feronia mutant remained smaller and
could easily be distinguished from fertilized ovules. In all analyzed mutant
FG the micopylar area was filled with multi-layered membrane invaginations
formed by the PT wall (Fig.
6C-F). The PT entered the embryo sac through or along the filiform
apparatus, a specialized cell wall structure of the synergids
(Fig. 6C,D). The receptive
synergid was electron dense, contained an increased number of spherosomes, its
nucleus was broken down and the organelles were disorganized. These structural
modifications resembled the cellular changes previously documented for
degenerated synergids in Arabidopsis
(Mansfield and Briarty, 1991;
Murgia et al., 1993
). The PT
often started to branch within the FG (Fig.
6E). In one case we observed the PT to bifurcate while entering
the FG (Fig. 6C,D). The central
cell and the egg cell appeared to be intact although the egg cell was often
firmly clasped by the PT invaginations
(Fig. 6F).
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DISCUSSION |
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After entering the synergid, the PT continued to grow, coiled and sometimes
bifurcated (Fig. 3B,D;
Fig. 6). Thus, the PT tip must
be intact, because PTs elongate by polarized tip growth
(Franklin-Tong, 1999;
Hepler et al., 2001
).
Consequently, the PT must fail to rupture and discharge the sperm cells in
feronia FGs, imparing double fertilization. The feronia
mutant phenotype clearly shows that in Arabidopsis the FG
participates actively in PT reception. The FG is known to control long-range
guidance of the PT to the ovule
(Higashiyama et al., 2001
;
Hülskamp et al., 1995b
;
Ray et al., 1997
) and into the
micropyle (Shimizu and Okada,
2000
). These interactions are likely to involve the secretion of
chemo-attractive compounds by the FG
(Lush, 1999
). In contrast, PT
reception is achieved within the synergid by an immediate interaction of the
PT with the FG. The defect in PT reception observed in the feronia
mutant must, therefore, be the result of a deficient synergid function, i.e. a
failure in the communication between the synergid and the PT.
Synergid specification and degeneration in feronia appear
normal
The autolytic degeneration of one of the synergids is thought to be a
prerequisite for PT rupture (Jensen and
Fisher, 1968). An abnormal degeneration of the synergid could
explain the defect in PT reception observed in the feronia mutant.
However, based on our studies with synergid-specific markers, the synergids in
feronia appear to be specified and differentiated normally
(Fig. 5A,B). In wild-type
plants, the GUS signal decreases in both synergids within 24 HAP, while the
staining persists in feronia FGs. Taken together these data suggest
that either synergid degeneration or normal pollen tube reception is required
for the down-regulation of GUS marker gene activity. However, the
investigation of the ultrastructure of mutant feronia FGs indicated
that the receptive synergid shows the typical characteristics of degeneration
(Fig. 6C-F). Both normal
expression of synergid markers before PT entry and ultra-structural changes in
the synergid after PT entry indicate that there is no basic defect in synergid
development and differentiation in mutant feronia FGs. It is more
likely that the persisting GUS activity after PT invasion is a consequence of
unsuccessful PT reception in feronia FGs, as discussed below.
Synergid degeneration is not necessary for PT attraction as demonstrated by
the gfa2 mutant in Arabidopsis in which the synergids fail
to degenerate (Christensen et al.,
2002). Despite normal PT guidance to the ovule, the PT does not
enter the FG and mutant gfa2 embryo sacs remain unfertilized. In
feronia the PT enters the FG by its normal route through the
receptive synergid and the remains of the degenerated synergid cover the PT.
Thus, the PT is exposed to the enzymatic environment that is responsible for
the autolytic degeneration of the synergid. Therefore, in
Arabidopsis, synergid degeneration might facilitate the entry of the
PT into the embryo sac and possibly the targeting of the sperm cells to the
female gametes, as suggested previously
(Drews and Yadegari, 2002
;
Russell, 1992
), but is not
sufficient to accomplish PT rupture and sperm cell release.
The feronia mutation disrupts signalling between male and
female gametophytes
The failure in PT reception in feronia is likely to be caused by
the disruption of a signalling process between the male and female
gametophyte. This view is consistent with the observed failure of PT reception
in crosses between different rhododendron species. In these interspecific
crosses, PT growth does not arrest and results in PT overgrowth in the embryo
sac similar to the feronia phenotype
(Kaul et al., 1986;
Williams et al., 1986
). This
defect can be explained by the evolutionary divergence in specific, co-evolved
recognition systems (Hogenboom,
1984
) required to accomplish PT reception. The failure in PT
reception observed in feronia mutants can be interpreted as the
absence or change of a female gametophytic component involved in the direct
communication between the female and the male gametophyte.
So far, it was unresolved whether the PT accomplishes growth arrest and PT
rupture on its own or whether the FG, in particular the receptive synergid,
actively participates in these processes. Investigations of PT reception in
cotton (Jensen and Fisher,
1968) and spinach (Wilms,
1981
) demonstrate that PT rupture is a controlled process. In
cotton, sperm cell discharge is accomplished by a sub-terminal, and in spinach
by a terminal, opening, which is sealed afterwards by a callose plug. If
growth arrest and PT rupture are PT-intrinsic processes, then the PT requires
a signal supplied by the FG to trigger these processes after entry into the
FG. This signal might be absent in mutant FGs of feronia. But if
arrest of growth and PT rupture need the active participation of the FG, then
there are two possible interpretations for the observed phenotype in
feronia: Either the mutant is defective in a novel recognition
pathway enabling the FG to respond to the entering PT, or mutant FGs lack an
essential component that either directly, e.g. by an enzymatic reaction, or
indirectly, e.g. by inducing a signal transduction cascade, accomplishes PT
growth arrest, rupture of the PT tip, and sperm cell release.
Other signalling events fail after pollen tube entry
It is known that PT reception immediately triggers several responses in the
embryo sac. These include the movement of the egg nucleus
(Faure et al., 2002), the
targeting of the sperm cells to the female gametes
(Huang et al., 1993
;
Huang and Sheridan, 1998
), the
degeneration of both synergids (Russell,
1996
), and the loss of PT attraction by the FG. Mutant
feronia FGs display a defect in at least two of these processes.
First, the two synergids do not senesce normally, because we observed a
persisting expression of synergid-specific gene expression in mutant FGs
(Fig. 4D), and second, a
distinct fraction of the mutant FGs attracts supernumerary PTs
(Fig. 4).
The PT reaches the FG about 6-8 HAP in plants of the Columbia ecotype
(Faure et al., 2002) but there
will be some differences depending on ecotype and growth conditions. When we
analyzed the expression of three independent synergid-specific molecular
markers in wild-type plants, the GUS signal disappeared within 24 HAP in both
synergids. As the half-life of GUS in the FG is rather short (12 hours after
blocking protein biosynthesis with cycloheximide GUS activity is barely
detectable; R. Baskar and U. G., unpublished data), the down-regulation in
both synergids must be controlled by a general mechanism that is activated
immediately after the PT enters the embryo sac. How this rapid response is
accomplished remains unclear. In mutant feronia FGs GUS activity
persists after PT invasion. Therefore, the normal signalling pathway leading
to the repression of the GUS signals must be interrupted. One possibility is
that mutant FGs do not respond to the entry of the PT, as discussed above.
Alternatively, the global repression of gene activity in the synergids might
be induced by components of the PT that are not discharged into mutant
feronia FGs.
Whereas only one PT generally approaches the micropyle
(Hülskamp et al., 1995b;
Shimizu and Okada, 2000
), we
observed that
10% of mutant feronia FGs attracted two or more
PTs (Fig. 5). The PTs were able
to enter the micropyle and invade the FG. How the FG prevents the arrival of
supernumerary PTs is not understood (Lush,
1999
; Smyth,
1997
). Repulsive interactions between two PTs have been discussed
as a possible mechanism (Lush,
1999
; Shimizu and Okada,
2000
). But the observation that two or more PTs can enter mutant
FGs in feronia mutants argues against such a repulsive interaction.
Another explanation is an immediate loss of PT attraction after functional PT
reception, provided that simultaneous growth of PTs to the same FG is
prevented either because the amount of the chemo-attractant is very
limited or is immediately sequestered by the approaching PT. Mutant
feronia FGs do not repress synergid-specific GUS markers, indicating
that a failure in PT reception does not induce a global repression of gene
activity in the synergids. Therefore, it is likely that the expression of a
synergid-borne attractant persists as well and results in the attraction of
supernumerary PTs.
The characterization of the female gametophytic mutant feronia demonstrates that the FG controls PT behaviour after entry into the synergid. Because there is no developmental defect in mutant feronia FGs and the synergids undergo normal differentiation and degeneration, the feronia mutation must affect an, as yet unidentified, active communication process between PT and the synergid. The isolation of the FERONIA gene will provide more information about the complex interactions between the male and female gametophyte necessary to accomplish the process of double fertilization in angiosperms.
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ACKNOWLEDGMENTS |
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Footnotes |
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While our paper was in press, the description of sirene, a mutant
with a similar phenotype, was published
(Rotman et al., 2003).
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