Fertilization: A Sticky Sperm Protein in Plants


Current Biology Vol 24 No 4
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1UMR144, CNRS- Institut Curie, 12 rue
Lhomond, 75005, Paris, France.
*E-mail: renata.basto@curie.fr
http://dx.doi.org/10.1016/j.cub.2013.12.054
Fertilization: A Sticky Sperm Protein
in Plants
During fertilization in eukaryotes, gametes of the opposite sex undergo a
complex series of interactions that culminate in cell fusion. A new study on
gamete interaction in plants has identified the first protein in multicellular
organisms shown by gene disruption to be essential for gamete membrane
adhesion.
Thomas Dresselhaus1,*
and William J. Snell2

Fusion of the plasma membranes of
gametes is among the most
species-specific events in evolution
and is likely an important component
of speciation. Fusion of lipid bilayers is
not a simple task and the evidence to
date indicates that nature has invented
membrane fusion only a limited number
of times [1]. So many species, so
few fusion proteins! Discerning the
evolutionary solution to this
conundrum has been difficult because
of the dearth of proteins shown by gene
disruption to be essential for gamete
fusion. Only two metazoan proteins
have been shown by in vitro assays
to be involved directly in sperm2egg
plasma membrane interactions and
by gene disruption to be essential
for fertilization: the hydrophobic
tetraspanin family member CD9 that
is expressed on mouse eggs (and
several other cell types) [2–4] and the
plasma membrane protein IZUMO1
expressed on mouse sperm [5]. CD9
likely plays a relatively non-specific,
facilitative role in fusion, and the
step in the membrane fusion
reaction that requires IZUMO1 (whose
presence is limited to vertebrates) is
uncertain [6].

The identification a few years ago
of the broadly conserved GENERATIVE
CELL SPECIFIC 1/HAPLESS 2 (GCS1/
HAP2) family of gamete-specific
proteins finally offered a potential
solution to the above puzzle. HAP2/
GCS1, first reported as a male sterility
gene, encodes a sperm protein
essential for fertilization in Arabidopsis
[7,8]. Studies of fertilization in two
protists that also express HAP2/GCS1,
Chlamydomonas and Plasmodium
demonstrated that gamete membrane
adhesion and membrane fusion are
distinct molecular events in the
membrane fusion reaction. Moreover,
it was shown that species specificity
during fertilization in the two
unicellular organisms derived from
species-specific membrane adhesion
[9,10]. HAP2/GCS1 is essential for
gamete fusion at a step after
membrane adhesion in both
organisms. Exciting new work
published recently in Current Biology
has now shown that gametes in
hap2/gcs1 mutants of Arabidopsis
are fully capable of membrane
adhesion [11]. Moreover, studies on
mutants defective in the sperm
protein GEX2 showed that it is
involved in membrane adhesion, thus
becoming the first protein in
multicellular organisms shown by gene
disruption to function in gamete
interactions before membrane merger,
and showed that it indeed undergoes
rapid evolution.
In contrast to most metazoans and

protists, fertilization in flowering plants
(angiosperms) seems almost unduly
complicated. First, the sperm are
immotile, and must be delivered to
the female ovule by the pollen tube.
And, plants actually do it in duplicate.
Pollen tubes deliver a pair of sperm
cells inside each ovule, and each
ovule contains two female gametes.
The outcome is a double fertilization
event. Thus, tightly coordinated
gamete interaction mechanisms are
required to ensure fusion of one
sperm cell with the egg cell and fusion
of the second sperm cell with the
central cell [12,13]. In spite of this
complexity, and in spite of the major
challenges to investigating plant
gamete interactions in vitro, our
understanding of cellular and
molecular mechanisms of plant
fertilization has begun to mature in
the past few years, and now surpasses
that of metazoans. For example,
although HAP2/GCS1 was known to be
essential for sperm2egg fusion in
Arabidopsis, it was not clear how it
could function in the membrane fusion
reaction, as HAP2/GCS1 in sperm in the
pollen tube is sequestered in the
endomembrane system and

mailto:renata.basto@curie.fr
http://dx.doi.org/10.1016/j.cub.2013.12.054
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GEX2-dependent adhesion gamete activation

Gamete activation GEX2-dependent adhesion

Egg cell Sperm cell

Egg cell Sperm cell

Egg cell Sperm cell

3

2
1

4

5

GEX2

HAP2/GCS1

2

1

5

3

4

6

Membrane fusion reaction

Current Biology

2

1

4

5

3

4

EC1

Figure 1. Possible schemes and order of events during gamete interactions in the model plant
Arabidopsis.

(Top) GEX2 could participate in the initial adhesion (1) that triggers female gamete activation
(2) and EC1 release (3) inducing sperm activation (4) leading to exposure of GCS1/HAP1 on

Dispatch
R165
cytoplasmic vesicles (Figure 1). A
report last year resolved this seeming
paradox and also uncovered a unifying
theme in fertilization — Sprunck
and colleagues showed that sperm
arrival in the vicinity of both female
gametes triggered the egg cell to
secrete a set of EC1 proteins, which in
turn activated the sperm to expose
their previously cryptic HAP2/GCS1 at
the cell surface [14]. Thus, as in
metazoans and protists, initial
interactions between gametes are
required to render the gametes
competent for cell2cell fusion. In
another demonstration of the likely
conservation of fertilization
mechanisms, members of the
tetraspanin family (TET), which are
characterized by four transmembrane
domains and two extracellular loops,
might also be implicated in gamete
interaction in plants. TET11 and TET12
localize to the sperm cell plasma
membrane and TET9 was detected
recently in the plasma membrane of
both the egg and central cell [14,15],
raising the possibility that they may
function as facilitators of the
membrane fusion reaction similar to the
proposed function of mammalian CD9.
The 17 members of the Arabidopsis
tetraspanin family, however, are
expressed throughout the organism
and gene disruption studies have not
been reported yet.
In this new report, Mori and

colleagues [11] present yet another
major advance for the field. In a
screen for plants defective for
double fertilization, they uncovered a
male sterile mutant gene whose
disruption interfered with fertilization.
Several hours after pollination of
wild-type ovules with wild-type pollen,
the egg and central cell become
fertilized by the sperm pairs, and no
free sperm pairs are detectable. In
the new mutant, however, sperm
cell pairs remained visible between the
egg and central cell well after
pollination. Genetic mapping and
mutant rescue experiments showed
that the mutation was in GAMETE
EXPRESSED 2 (GEX2; At5g49150),
the sperm surface. (Middle) GEX2 might
alternatively be accessible on the cell surface
only after gamete activation and function
(124) in post-activation adhesion (5) as part
of (Bottom) the membrane fusion reaction
(6). Numbers indicate order of events and
black boxes represent unknown receptors
and cell surface interactors.



Current Biology Vol 24 No 4
R166
previously identified as an Arabidopsis
sperm-specific homologue of a maize
sperm gene of unknown function [16].
The wild-type GEX2 gene encodes a
single-pass transmembrane protein of
1087 amino acid residues containing
two filamin repeat domains and a
carboxy-terminal transmembrane
domain. Thus, it is likely that almost
the entire protein is exposed on the
surface of (activated) sperm cells.
Importantly, GEX22GFP rescues
fertilization in the mutant. GFP
imaging studies showed membrane
localization, with some associated with
the plasma membrane and substantial
amounts internally surrounding the
nucleus.

Using a novel cell adhesion assay
in which cell wall material was digested
in situ to generate round gamete
protoplasts and in which markers were
used to visualize cell boundaries, Mori
and colleagues [11] then showed that
in some cases one, and in some cases
both, gex2 mutant sperm failed to
attach to the egg and central cell
protoplasts. This result was in striking
contrast to the fusion-defective hap2/
gcs1 mutant, in which both sperm
bound to female gamete protoplasts.
Thus, the authors concluded that
HAP2/GCS1 is required for a step after
adhesion and that GEX2 functions in
sperm2egg and sperm2central cell
adhesion. As the authors indicate, the
finding that disruption of gex2 does
not completely block gamete adhesion
and fusion indicates that the protein
likely acts redundantly with other gene
products.

Moreover, the authors’ bioinformatic
analyses uncovered intriguing
properties of GEX2. The ratio between
the number of non-synonymous
substitutions per non-synonymous
site and the number of synonymous
substitutions per synonymous site
(Ka/Ks), which is a measure of the
rate of evolution, showed that GEX2
was diverging more rapidly than
HAP2/GCS1. Thus, as in protists, this
higher plant gamete adhesion protein
likely confers species specificity to
sperm2egg/central cell interactions.
The most rapidly evolving region
of GEX2 proteins from different
Arabidopsis species turned out to
be in the ectodomain in the first
filamin repeat domain, which is
predicted to form immunoglobulin-like
folds found in many cell adhesion
proteins [17]. In another nod to
conserved features of fertilization,
mammalian IZUMO1 also possesses
an immunoglobulin-like domain in
its predicted ectodomain [5], and
the FUS1 protein in Chamydomonas
also contains an amino-terminal,
filamin-like domain in its predicted
ectodomain [9]. FUS1 is the
species-specific protein expressed
only on plus gametes of
Chlamydomonas that is essential for
the membrane adhesion step in the
Chlamydomonas gamete membrane
fusion reaction.

These exciting findings have
generated a number of new questions.
Is GEX2 a bona fide adhesion molecule
that directly binds to egg cell surface
proteins, or does it facilitate the
adhesion activity of other as yet
unidentified proteins? Which additional
proteins are responsible for the
residual gamete adhesion observed
in the gex2 mutants? At which step
in gamete interactions does GEX2
act — does it act in the initial
interactions that trigger egg activation,
or does GEX2 act later and become
exposed on the sperm surface after
gamete activation similar to GCS1/
HAP2 (Figure 1)? It can also not be
excluded that GEX2 plays a late role
in the membrane fusion reaction. It
should be straightforward to test
whether EC1 is released upon contact
with gex2 mutant sperm and if HAP2/
GCS1 is sequestered or located on
the surface of gex2 sperm. The ability
to isolate living sperm and female
gametes either by FACS or manually
[18,19] also now makes in vitro
experiments possible in Arabidopsis to
investigate the specificity and strength
of the interaction. Finally, the major
task for the future is the identification
of the GEX2 interaction partner(s) at
the egg and central cell surfaces.
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1Cell Biology and Plant Biochemistry,
Biochemie-Zentrum Regensburg, University
of Regensburg, Universitätsstraße 31, 93053
Regensburg, Germany. 2Department of Cell
Biology, University of Texas Southwestern
Medical School, Dallas, TX 75390, USA.
*E-mail: thomas.dresselhaus@ur.de
http://dx.doi.org/10.1016/j.cub.2013.12.044

mailto:thomas.dresselhaus@ur.de
http://dx.doi.org/10.1016/j.cub.2013.12.044

	Fertilization: A Sticky Sperm Protein in Plants
	References