Download gamete interaction in flowering plants

Cell–Cell Communication in Plant Reproduction
Let’s get physical: gamete interaction in flowering
plants
Stefanie Sprunck1
Cell Biology and Plant Biochemistry, University of Regensburg, Universitätsstrasse 31, 93053 Regensburg, Germany
Abstract
Fertilization comprises a series of precisely orchestrated steps that culminate in the fusion of male and female
gametes. The most intimate steps during fertilization encompass gamete recognition, adhesion and fusion.
In animals, some binding-effector proteins and enzymes have been identified that act on the cell surfaces of
the gametes to regulate gamete compatibility and fertilization success. In contrast, exploring plant gamete
interaction during double fertilization, a characteristic trait of flowering plants, has been hampered for a
long time because of the protected location of the female gametes and technical limitations. Over the last
couple of years, however, the use of advanced methodologies, new imaging tools and new mutants has
provided deeper insights into double fertilization, at both the cellular and the molecular level, especially for
the model plant Arabidopsis thaliana. Most likely, one consequence of inventing double fertilization may
be the co-evolution of special molecular mechanisms to govern each successful sperm delivery and efficient
gamete recognition and fusion. In vivo imaging of double fertilization and the recent discovery of numerous
female-gametophyte-specific expressed genes encoding small secreted proteins, some of whom were found
to be essential for the fertilization process, support this hypothesis. Nevertheless, recent findings indicate
that at least the membrane-merger step in plant gamete interaction may rely on an ancient and widely
used gamete fusion system.
Introduction
Sexual reproduction is a fundamental biological process
that depends on fertilization, the union of two gametes
produced by meiosis, leading to the formation of an individual
genetically distinct from its parents. In the majority of higher
eukaryotes, including plants, these two gametes differ in
size and/or form and are contributed by different parents.
By definition, the smaller gamete is considered as male
(sperm cell), whereas the larger gamete is regarded as female
(egg cell). For successful fertilization, the gametes need to
accomplish fundamental tasks: (i) the sperm cell has to be in
the immediate vicinity of the egg cell, (ii) the gametes have
to assure species-restricted gamete recognition and fusion,
and (iii) the egg cell has to prevent the fusion of more
than one sperm cell (block of polyspermy). The progressive
stages of gamete recognition and fusion are often collectively
termed as gamete interaction. However, depending on the
living environment of a sexually reproducing organism (i.e.
water or land) and the requirements for either internal or
external fertilization, distinct fertilization mechanisms have
evolved to control gamete attraction, recognition, adhesion
and fusion [1–3]. In animals and lower plants, including
mosses and ferns, most male gametes (sperm or spermatozoa)
are motile, being propelled by a long flagellum along a
Key words: Arabidopsis thaliana, double fertilization, egg cell, gamete recognition and fusion,
plasmogamy, sperm cell.
Abbreviations used: CLSM, confocal laser-scanning microscopy; CRP, cysteine-rich protein;
FG, female gametophyte; GCS1, GENERATIVE CELL-SPECIFIC 1; HAP2, HAPLESS 2; MG, male
gametophyte; mt, mating type; ZmES, Zea mays embryo sac.
1
email [email protected]
Biochem. Soc. Trans. (2010) 38, 635–640; doi:10.1042/BST0380635
gradient of chemoattractants, synthesized and secreted by
the egg or by somatic cells, such as follicle cells, associated
with the egg [3]. In contrast, the sperm cells of flowering
plants (angiosperms) differ from sperm of non-seed plants
and of most animals in their lack of flagella. A key innovation
that allowed angiosperms to carry out sexual reproduction
on land, without the need for water, is a form of internal
fertilization in which the non-motile sperms are delivered to
the female gametes via a pollen tube. Moreover, fertilization
in angiosperms is unique among all known organisms in that
not one, but two, female reproductive cells are fertilized in
a process called double fertilization. A complex mechanism
involving two male gametes (sperm cells) and two female
gametes (egg cell and central cell) results in two distinct
fertilization products, embryo and endosperm, that are both
required to achieve successful seed development [4,5].
Events preceding gamete interaction:
pollination and sperm cell delivery into
the FG (female gametophyte)
Plant gametes are, in contrast with animal gametes, not direct
products of meiosis, but differentiate within multicellular
haploid generations, the MG (male gametophyte) and the FG
respectively. In angiosperms, the gametophytic generations
are reduced to a few cells that are located within the diploid
sporophytic tissues of the flower. The FG (or embryo sac)
is enclosed by the ovule that is located within the ovary.
The seven-celled FG of the model plant Arabidopsis thaliana
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Figure 1 Structures of the A. thaliana FG and MG
(A) The seven-celled FG is enclosed by the outer integuments (oi) and inner integuments (ii) of the ovule, providing a small
opening (micropyle; mp) as entry for the pollen tube. The ovule is attached by the funiculus (fun) to the inner surface of
the ovary. The synergid nuclei (sn) are positioned towards the filiform apparatus (f) that forms at the micropylar pole of the
synergids. The egg cell nucleus (ecn) is positioned at the chalazal side of the egg, close to the central cell nucleus (ccn).
Three antipodals (ap) are located at the chalazal pole (chz) of the FG. Broken lines surrounding shaded areas indicate the
cell vacuoles. The big central cell vacuole (ccv) fills a large part of the FG. (B) Tricellular MG. The larger vegetative cell (vc)
encloses two small sperm cells (sc1, sc2) that are physically connected to each other and conjointly surrounded by the
plasma membrane of the vegetative cell [pm(v)]. The vegetative cell nucleus (vn) is less compact and irregularly shaped.
The sperm cell nuclei are indicated by black circles, the sperm cell plasma membranes [pm(s)] are coloured grey.
comprises two female gametic cells, the egg cell and the central
cell, which are flanked by accessory cells. Two synergid cells
neighbour the egg cell at the micropylar end of the FG, while
three antipodals are adjacent to the central cell at the chalazal
end of the FG (Figure 1A) [6]. The pollen that develops within
the anther represents the MG. Arabidopsis pollen is tricellular,
consisting of one larger vegetative cell encompassing the two
sperm cells (Figure 1B).
For double fertilization to succeed, the two non-motile
sperm cells must be transported through the pistil into
the FG, which strictly depends on the directional growth
of the pollen tube formed by the vegetative cell of the
MG. The initiation of pollen tube growth requires adhesion
of the pollen grain to a receptive female stigma and
hydration, providing first checkpoints for a species-restricted
interaction between the MG and the female reproductive
tract [7–9]. Once germinated, the tip-growing pollen tube
invades the stigma tissue and navigates across the female
reproductive tract towards the ovule, assisted by complex
communication between the tube cell and surrounding
sporophytic tissues [8–10]. Synergid cells secrete small and
species-specific proteins such as LURE1/2 and ZmEA1 (Zea
mays EGG APPARATUS 1), which are involved in the
last phase of pollen tube attraction, guiding tube growth
through the micropyle into the FG [11–13]. The first
molecular players FERONIA, LORELEI and ZmES (Z.
mays EMBRYO SAC) 1–4 found to be necessary for pollen
tube growth arrest and burst indicate that species-specific cell
recognition and signalling mechanisms also exist between the
receptive synergid and a compatible pollen tube, which may
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inside this synergid cell [14–17]. The released pollen tube
content mingles with the cytoplasm of the degenerating
synergid and spreads, as the synergid membrane disintegrates,
into a narrow space between the egg and central cell plasma
membranes [18,19]. Typically, only one pollen tube penetrates
each ovule, limiting FG entry to one sperm cell pair. Rapid
termination of pollen tube attractant(s) synthesis and/or
secretion or degradation after successful fertilization may
explain why the FG loses its ability to attract further pollen
tubes [20].
Late pre-zygotic events within the FG
In contrast with the molecular events involved in sperm
cell delivery into the FG, almost nothing is known about
later processes, covering the phase from sperm cell delivery
until gamete fusion (plasmogamy). Attempts to elucidate
these issues at the cellular and molecular levels are hampered
by the fact that angiosperm gamete interactions are shortlived events, taking place deeply embedded in the maternal
tissues of the ovule and ovary. Previous knowledge was
mainly based on cytological observations of fixed tissues
or after in vitro fertilization of isolated male and female
gametes [20,21]. From these studies, it is known, for
example, that the degenerating synergid exhibits a dramatic
F-actin (filamentous actin) reorganization and a significant
accumulation of calcium, which was suggested to contribute
to sperm cell transport to the fusion site and to the process
of gamete fusion respectively [19,20]. However, details about
Cell–Cell Communication in Plant Reproduction
Figure 2 CLSM analysis of growing pollen tubes and sperm cell delivery, plasmogamy and karyogamy during double fertilization in
A. thaliana
(A) In vitro-germinated pollen tube expressing the sperm cell marker HTR10-mRFP1, showing strong red fluorescence in
sperm cell nuclei (scn). The sperm cells are transported conjointly within the growing pollen tube. (B) In vitro-germinated
pollen tubes expressing the marker ARO1-GFP in the cytoplasm of the vegetative cell reveal the physical connection of the
two sperm cells (asterisk). (C–H) Fluorescence images (D, F and H) and respective merged bright-field images (C, E and
G) of FGs expressing the egg cell marker ARO1-GFP, analysed 5–9 h after hand-pollination using the sperm cell marker line
HTR10-mRFP1 as pollen donor. The micropyle is oriented towards the upper left corner in each picture. (C and D) Sperm
cell delivery into the degenerating synergid cell (dsy). (E and F) A sperm cell pair stays temporarily at the chalazal pole of
the degenerating synergid, in the narrow gap between the egg cell and the central cell which depicts the area of gamete
interaction. (G and H) Plasmogamy between the first sperm cell and the egg cell has occurred. The sperm cell nucleus 1
(sn1) is visible inside the egg cell, near the egg cell nucleus (en), whereas the second sperm cell (sn2) has not yet fused
with the central cell. (J and K) Visualization of karyogamy, after pollinating the egg cell marker line EC1pro :NLS-(3×)GFP with
the sperm cell marker line HTR10-mRFP1. Karyogamy between the second sperm cell nucleus and the central cell nucleus
(sn2/cn) is almost complete, indicated by the decondensation of the sperm cell chromatin (broken circles). In contrast, the
nucleus of the first sperm cell (sn1) is attached to the egg cell nucleus (en), but has not yet fused. Scale bars, 10 μm (A,
C–J); 5 μm (B and K).
the following plasmogamy and karyogamy have been fairly
unclear. Recently, advanced molecular and genetic approaches
and new imaging tools using living material provided deeper
insights into the last phase of double fertilization, at least
for the model plant Arabidopsis [4]. Male and female gamete
cell isolation and transcriptome-level analyses offered the
possibility of identifying both male and female gametespecific expressed genes [19,22–28]. Respective promoters
now serve as valuable tools to drive expression of fluorescent
proteins in Arabidopsis gametes, enabling live imaging of the
fertilization process. Figure 2 shows the events following
sperm cell delivery into the FG, analysed by CLSM (confocal
laser-scanning microscopy). Two egg cell marker lines, one
labelling the egg cytoplasm [19] and one line labelling the egg
cell nucleus [29], were pollinated with pollen of a sperm cell
marker line [23]. Sperm cells are produced after pollen mitosis
of the generative cell, and former electron microscopic work
on pollen of several flowering plant species revealed that a
traverse cell-wall-like structure connects the sperm cell pair
after cytokinesis (Figure 1B) [5]. Thus the two sperm cells
are transported conjointly within the growing pollen tube
(Figures 2A and 2B). Strikingly, the discharged sperm cells
appear to stay associated within the degenerating synergid
(Figures 2C–2F), despite the fulminating release of the pollen
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tube contents when the tube tip bursts. Back-to-back, both
sperm cells arrive at the chalazal pole of the degenerating
synergid and stays temporarily in the region between the
chalazal end of the egg cell and the micropylar end of
the central cell (Figures 2E and 2F). The first plasmogamy
appears to invariably occur between the egg cell and one
of the sperm cells, while the fusion of the second sperm
with the central cell is somewhat delayed (Figures 2G
and 2H). Nevertheless, karyogamy between this sperm cell
nucleus and the central cell nucleus takes place immediately
after plasmogamy, indicated by a rapid decondensation of the
male chromatin within the central cell nucleus. In contrast,
the nucleus of the first sperm cell stays attached to the egg cell
nucleus, but does not fuse immediately (Figures 2J and 2K).
It is known that male and female gametes must synchronize
their cell cycles for successful fertilization, and Arabidopsis
sperm cells are likely to be positioned in G2 -phase when they
meet their female counterparts [30]. Although it remains to be
determined conclusively, initial data suggest that the egg and
central cell reside in G1 /S and G2 /M transition respectively
[4]. Thus the observed delay of karyogamy between the egg
and sperm nucleus may reflect a phase in which the cell cycle
states of egg and sperm cell need to become synchronized.
Angiosperm gamete interactions may
require unique molecular mechanisms
Unlike any other taxa, flowering plants have to face particular
problems caused by the unique feature of double fertilization:
successful seed development is dependent on fertilization of
both the egg and central cell. As typically only one
pair of sperm cells is delivered into the FG, the gamete
recognition and fusion system needs to be as efficient
as possible. It seems plausible that specific molecular
mechanisms co-evolved to ensure that one sperm fuses
with each female gamete in a timely manner. However,
the molecular players of angiosperm gamete interaction are
largely uncharacterized, and many questions remain to be
answered. It is, for example, not yet clear whether the
two male gametes randomly choose their female interaction
partner, or whether there is preferential fusion with a
particular female gamete. Also unclear is how polyspermy
is prevented, and whether the delivered sperm cells become
activated before they fuse. Available Arabidopsis mutants
were utilized to address some of these questions. Recent data
on the retinoblastoma-related 1 (rbr1) mutant, producing
supernumerary but functional eggs, revealed that the
isomorphic sperm cells of Arabidopsis are both functionally
equivalent for fertilizing the egg cell [29], arguing against
preferential fusion in this species. Fertilization experiments
with different mutants producing single sperm-like cells,
however, yielded contradictory results [31,32], but may need
closer examination. Evidence for an in vivo polyspermy
block on the egg cell, but not on the central cell, was
given by fertilization experiments using the polyspermic
tetraspore (tes) mutant [33], indicating that the exclusive polyspermy block on the egg may be not only to avoid
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each female gamete receives one of the two sperm cells [34].
Moreover, the physical association between the two sperm
cells arising during mitosis of the generative cell may be
an important mechanistic element of double fertilization,
as it may be important for both a concerted transport of
the two sperm cells within the growing pollen tube and
synchronous delivery to the site of gamete interaction. The
spatial partitioning of the two sperm cells may also help
to avoid spontaneous sperm–sperm fusion when the cells
are released into the calcium-rich micro-environment of the
degenerating synergid [20]. In consequence, the precisely
timed separation of the sperm cells may be one prerequisite
for successful double fertilization. In Arabidopsis, this appears
not to take place until the sperm cells have reached their fusion
site (Figures 2E and 2F).
Gamete adhesion and fusion
Membrane fusion events generally require molecules that
tether and dock membranes and bring them into close
proximity. Furthermore, molecules are required that locally
disturb the lipid bilayers in order to reduce the energy
barriers for fusion [35]. Still, gamete fusion is a specialized
form of membrane fusion that is, like other cell–cell fusion
events, substantially different from intracellular membrane
fusion [36]. Studies on fertilization in vertebrate and
invertebrate species, as well as on mating in the unicellular
eukaryotic model organisms Chlamydomonas reinhardtii and
Saccharomyces cerevisiae provided important insights into the
process of gamete fusion [2,37–40]. Although the details of
fertilization are highly variable for the different taxa, gamete
fusion exhibits some common principles. After gametes of
opposite sex or mating types come into close proximity,
the subsequent events generally comprise (i) pre-fusion
attachment, where adhesive interactions are formed between
the plasma membranes of the two gametes, and (ii) the
coalescence of the two juxtaposed lipid bilayers (membrane
merger). Molecules that act on the surface of the gametes, e.g. as cell adhesion molecules mediating physical gamete
interactions, as ligand–receptor pairs involved in signalling,
or as fusogenic proteins mediating membrane fusion, are
regarded as key components of these well-co-ordinated
processes.
However, despite gamete fusion being such a fundamental
and ancient process, surprisingly few candidate proteins
have been identified that play essential roles in gamete
adhesion and fusion. Major players in mammalian sperm–egg
interaction are the ubiquitously expressed tetraspanin family
members CD9 and CD81 which possess four transmembrane
domains and act on the egg surface, and the sperm-specific
protein IZUMO, an Ig superfamily single-transmembranedomain protein. Putative additional players in mammalian
gamete interaction are members of the large family of
disintegrin-domain-containing ADAM (a disintegrin and
metalloproteinase) proteins on sperm, integrins and yet
unidentified GPI (glycosylphosphatidylinositol)-anchored
Cell–Cell Communication in Plant Reproduction
proteins on the egg plasma membrane, as well as family
members of the CRISPs (cysteine-rich secretory proteins)
associated with the sperm surface [40]. In the unicellular
biflagellated haploid green alga Chlamydomonas, mt (mating
type) recognition and initial adhesion of the flagella of
mt(+) and mt(−) gametes is achieved by mt-specific
agglutinins, fibrous proteins that belong to the family of
HRGPs (hydroxyproline-rich glycoproteins) residing on
the surface of the flagellar membrane. The membrane
fusion event of Chlamydomonas gametes is dependent on
at least two consecutively acting proteins [37,41]. FUS1,
a transmembrane protein containing five Ig-like repeats
in its extracellular domain, is located at the apex of the
fertilization tube of activated mt(+) gametes. Although
FUS1, which is not found in other species, is essential
for prefusion attachment between the mating structures
of activated mt(+) and mt(−) gametes, the membrane
merger step is dependent on the single-transmembrane
protein GCS1 (GENERATIVE CELL-SPECIFIC 1)/HAP2
(HAPLESS 2) [41], being mainly expressed in mt(−)
gametes [24]. Interestingly, GCS1/HAP2 of Chlamydomonas
represents a homologue of a sperm-cell-specific membrane
protein in flowering plants that was termed GCS1 [24] or
HAP2 [42] respectively, the first identified key player
in Arabidopsis gamete interaction. Although not yet shown
for Arabidopsis gametes, GCS1/HAP2 is probably involved
in the membrane merger of flowering plant gametes, similar to
its Chlamydomonas homologue [41]. As GCS1/HAP2 is not
specific for flowering plants, but can be found in a wide range
of organisms, including green and red algae, slime moulds,
ciliates, choanoflagellates, cnidarians and parasites such as
Plasmodium falciparum, it may represent an ancient and
highly conserved component of a gamete fusion machinery.
In contrast, the molecules involved in gamete recognition and
the pre-fusion adhesion processes appear to be subjected to
more rapid evolution, probably to establish species-specific
fertilization barriers [2].
Species-specific gamete interaction in
flowering plants?
It may be argued that angiosperm gametes do not need
species-specific recognition mechanisms at the level of
gamete interaction, as pre-zygotic barriers efficiently operate
during pollen germination, pollen tube growth and pollen
tube discharge [7–9,14,16]. However, elaborate cell–cell
interactions can be postulated based on the vast number of
FG-specific expressed genes encoding small and potentially
secreted polymorphic proteins such as various subgroups
of the CRP (cysteine-rich protein) superfamily [11,25,43–
45]. For instance, the two CRPs LURE1 and LURE2 are
specifically secreted by Torenia fournieri synergids and act
as chemoattractants on Torenia pollen tubes [11], whereas
ZmES4 is secreted from maize synergid cells to induce pollen
tube burst [16]. Accordingly, gamete-specific expressed CRPs
may fulfil as-yet-unknown but important functions during
double fertilization, e.g. as signalling molecules involved
in intercellular communication. One example is a small
subfamily of egg-cell-specific CRPs of Arabidopsis that is
specifically secreted upon sperm cell arrival. After knocking
down the whole gene family, the Arabidopsis gametes are
unable to fuse, indicating an essential role for these CRPs
during gamete interaction (S. Sprunck, S. Rademacher and
T. Dresselhaus, unpublished work). Although the functional
redundancy of the various polymorphic proteins generated
by the FG cells hamper their functional analysis, their
specific expression pattern and first examples of candidate
gene down-regulation suggest that they play key roles in
the complex process of double fertilization, including gamete
interaction(s).
Conclusions
In flowering plants, we still know almost nothing about the
late events of double fertilization, as most attention had been
directed until now towards the understanding of the mechanisms involved in sperm cell delivery towards and into the FG.
However, gamete recognition, adhesion and fusion are key
for reproductive success. Unravelling underlying molecular
mechanisms will help us to solve long-standing questions
about how flowering plant gametes achieve the two gamete
fusion events during double fertilization in such an efficient
and timely manner. It will be exciting to identify additional
players of flowering plant gamete interaction and to study
their evolutionary history to find out: (i) to what extent the
successive steps of gamete interaction are conserved between
eukaryotic organisms, (ii) whether these processes represent
additional hybridization barriers, and (iii) whether the
knowledge generated can be used to overcome such putative
levels of pre-zygotic fertilization barriers. Progress is likely
to be made in the near future, as plant gametes including those
of the model systems A. thaliana and rice are now accessible
for transcriptomic-based approaches ([46], L. Šoljić, T.
Dresselhaus and S. Sprunck, unpublished work, and M. Sen,
Y. Zhang, X. Gou and S.D. Russell, personal communication).
Acknowledgements
I thanks Thomas Dresselhaus for critical reading of the manuscript
and Fred Berger for providing the sperm cell marker line HTR10mRFP.
Funding
The work in my laboratory is supported by the German Research
Council (DFG) [grant number SP 686/1].
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Received 10 September 2009
doi:10.1042/BST0380635