Maternal Control of Male-Gamete Delivery in Arabidopsis
Involves a Putative GPI-Anchored Protein Encoded by the
LORELEI Gene
W
Arnaud Capron,
a,1
Mathieu Gourgues,
b,2
Lissiene S. Neiva,
b,3
Jean-Emmanuel Faure,
b,4
Frederic Berger,
b,5
Gabriela Pagnussat,
a
Anjali Krishnan,
a
Cesar Alvarez-Mejia,
c
Jean-Philippe Vielle-Calzada,
c
Yuh-Ru Lee,
a
Bo Liu,
a
and Venkatesan Sundaresan
a,6
a
Department of Plant Biology, University of California, Davis, California 95616
b
Ecole Normale Supe´rieure de Lyon, Unite´ Mixte de Recherche 5667, Reproduction et De´veloppement des Plantes, Lyon cedex
07, France
c
National Laboratory of Genomics for Biodiversity, Cinvestav, Guanajuato, Mexico
In Angiosperms, themale gametes are delivered to the female gametes through thematernal reproductive tissue by the pollen
tube. Upon arrival, the pollen tube releases the two sperm cells, permitting double fertilization to take place. Although the
critical role of the female gametophyte in pollen tube reception has been demonstrated, the underlying mechanisms remain
poorly understood. Here,wedescribe lorelei,anArabidopsis thalianamutant impaired in spermcell release, reminiscent of the
feronia/sire`nemutant. Pollen tubes reaching lorelei embryo sacs frequently do not rupture but continue to grow in the embryo
sac. Furthermore, lorelei embryo sacs continue to attract additional pollen tubes after arrival of the initial pollen tube. The
LORELEI gene is expressed in the synergid cells prior to fertilization and encodes a small plant-specific putative
glucosylphosphatidylinositol-anchored protein (GAP). These results provide support for the concept of signalingmechanisms
at the synergid cell membrane by which the female gametophyte recognizes the arrival of a compatible pollen tube and
promotes sperm release. Although GAPs have previously been shown to play critical roles in initiation of fertilization in
mammals, flowering plants appear to have independently evolved reproductive mechanisms that use the unique features of
these proteins within a similar biological context.
INTRODUCTION
In plants, the gametes are not directly generated as products of
meiosis, but result from division and differentiation of the multi-
cellular haploid gametophytes. In angiosperms, the gameto-
phytes are the pollen and the embryo sac, producing the male
and female gametes, respectively. Furthermore, the male gam-
etes are nonmobile and are instead carried, after germination of
the pollen grain on the female stigma, by the pollen tube. The
pollen tube emanates from the pollen grain and finds its way in
the female tissues, ultimately delivering two sperm cells to the
embryo sac, at the mycropylar end. The canonical mature em-
bryo sac, as in Arabidopsis thaliana, is composed of seven cells:
the egg cell and the central cell, which will receive the two sperm
cells, two synergids at the micropylar end, and three antipodal
cells at the chalazal end. The proper delivery of themale gametes
relies on a series of signals from the embryo sac. Through laser
ablation experiments in Torenia fournieri, Higashiyama et al.
(2001) showed that the synergid is the direct source of attractant
for the pollen tube, while recent work in maize (Zea mays) has
shown that the egg cell may also play a role in this guidance
(Dresselhaus, 2006). Furthermore, work by Chen et al. (2007) has
shown an involvement of the central cell in the guidance of the
pollen tube in Arabidopsis. Upon arrival at the embryo sac, the
pollen tube releases the sperm cells for the double fertilization to
proceed. Furthermore, the embryo sac loses its ability to attract
pollen tubes after fertilization is initiated.
Recently, the identification in Arabidopsis of the receptor-like
kinase FERONIA has shed some light on potential signaling
mechanisms responsible for these processes. In feronia and
sire`ne mutants, the pollen tube can reach the embryo sac, but
instead of arresting and delivering the two sperm cells, the pollen
tube does not arrest and continues to grow and invades the
1
Current address: Department of Cell Systems Biology, University of
Toronto, 25 Willcocks St., Toronto, ON M5S 3B2, Canada.
2
Current address: Unite´ Mixte de Recherche, Interactions Plante Micro-
organisme et Sante´ Ve´ge´tale,CentreNationaldelaRecherche
Scientifique/Institut National de la Recherche Agronomique/Unice, 400
Route des Chappes, Sophia Antipolis, F-06903 France.
3
Current address: Department of Molecular Neurogenetics, McGill
University/Montre´al Neurological Institute, 3801 Rue University, Mon-
treal, Quebec H3A 2B4, Canada.
4
Current address: European Commission, Research Directorate Gen-
eral, Research Infrastructures (Unit B3) Office SDME 1/133, B-1049
Brussels, Belgium.
5
Current address: Temasek Life Sciences Laboratory, 1 Research link,
National University of Singapore, Singapore 117604.
6
Address correspondence to sundar@ucdavis.edu.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described
in the Instructions for Authors (www.plantcell.org) is: Venkatesan
Sundaresan (sundar@ucdavis.edu).
W
Online version contains Web-only data.
www.plantcell.org/cgi/doi/10.1105/tpc.108.061713
The Plant Cell, Vol. 20: 3038–3049, November 2008, www.plantcell.orgã 2008 American Society of Plant Biologists

embryo sac (Huck et al., 2003; Rotman et al., 2003). In addition,
feronia/sire`ne embryo sacs can attract supernumerary pollen
tubes (Escobar-Restrepo et al., 2007). FERONIA encodes a
receptor-like kinase and is expressed in the filliform apparatus,
on the surface of the synergid cells (Escobar-Restrepo et al.,
2007).FERONIAmay thusbe part of a signaling cascade from the
embryo sac to the pollen tube causing growth arrests and
allowing spermcells release. In thismodel, the ligandof FERONIA
can be produced by the pollen. Alternatively, the ligand could be
produced the embryo sac itself and FERONIA would be involved
in the maturation of functional synergids able to interact with the
pollen tube. The abstinence bymutual consent (amc)Arabidopsis
mutant (Boisson-Dernier et al., 2008) displays an absence of
release of the spermcells from thepollen tube similar towhatwas
observed in feronia/sire`ne. However, this mutant differs from
feronia in that the defect appears only when a pollen tube from an
amc pollen grain encounters an amc embryo sac, and mutant
embryo sacs are fully competent to receive wild-type pollen. The
AMC gene encodes a peroxin expressed in a wide range of
vegetative tissues and strongly expressed throughout both male
and female gametophytes. AMC appears to be necessary for
PTS-1–dependent peroxisome import andmay be involved in the
processing of a ligand or some other signaling steps in the
recognition pathway for both the pollen and the embryo sac. In
this study, we describe an Arabidopsis mutant, lorelei, whose
phenotype is reminiscent of the feronia/sire`ne mutation. lorelei
embryo sacs show an impairment of fertilization caused by an
inability of the pollen tube to release the sperm cells upon arrival
at the lorelei embryo sac. The pollen tube subsequently experi-
ences a continuous growth, resulting in an invasion of the embryo
sac. The LORELEI gene encodes a putative plant-conserved
glucosylphosphatidylinositol (GPI)-anchored protein (GAP) and
is expressed in the synergid cells of the embryo sac. We thus
identify a second femalegametophyte-specific component of the
signaling pathway or pathways required for fertilization in higher
plants.
RESULTS
The loreleiMutation Causes an Invasive Pollen Tube
Phenotype and Failure of Fertilization
To obtain more data on the role of the embryo sac in pollen tube
guidance, we performed a genetic screen to isolate mutants
deficient in pollen tube reception and pollen tube arrest typical of
the sire`ne class represented by the two alleles srn and fer. Plants
from apopulation of Columbia (Col) wild-type seeds irradiated by
gamma rays were used as pollen donors to fertilize wild-type
ovules. We did not obtain male gametophytic mutants defective
for fertilization among 2000 lines. However, we isolated mutant
lines that showed >25% of seeds arrested at early stages after
self-fertilization. The line GM474 showed a phenotype similar to
srn and fer. Ovules from self-fertilized lre-1/+ plants showed
Figure 1. Phenotype of lorelei Mutants.
(A) to (F) Aniline blue staining of ovules from different lorelei mutants
pollinated with wild-type pollen.
(A) Wild-type arrival of the pollen tube (arrow) to the micropyle (my).
There is no fluorescence inside the ovule.
(B) Pollen tube curling at the micropyle of an lre-1 ovule.
(C) Pollen tube invading the central cell (cc) and turning back toward the
micropyle of an lre-2 ovule.
(D) Pollen tube curling inside the micropylar end of an lre-3 mutant.
(E) Pollen tube entering the central cell of an lre-4 ovule and heading
back toward the micropyle.
(F) An example of two pollen tubes reaching the same lre-1 ovule. The
two pollen tubes crawl along the funiculus and enter the embryo sac
before forming coils at the micropylar end.
(G) to (I) DIC pictures of ovules the same lre-2/+ silique pollinated with
wild-type pollen.
(G) An example of pollen tube entering deep inside the central cell and
turning back toward the micropyle.
(H) Pollen tube coiling at the micropylar end of the embryo sac.
(I) Wild-type-looking ovule with a developed endosperm (en) and an
embryo at the dermatogen stage (asterisk).
Arrow, pollen tube; cc, central cell; my, micropylar end; ch, chalazial end;
en, endosperm; *, embryo. Bars = 50 mm.
Table 1. Transmission of lre for the lre-1 and lre-2 Deletions
Ovule 3 Pollen lre/+ Wild Type Ratio lre-1/Wild Type
lre-1/+ 3 Col 19 224 0.08:1
Col 3 lre-1/+ 142 181 0.78:1
Kan
r
Kan
s
Ratio Kan
r
/Kan
s
lre-2/+ self 983 902 1.09:1
lre-2/+ 3 Ler 43 361 0.12:1
Ler 3 lre-2/+ 224 266 0.84:1
The genetic transmission of the lre mutation (ovule donor listed first) of
the lre-1 and lre-2 deletions was established by observation of lorelei
phenotype (invasive pollen tube) occurrence for lre-1 and by assaying
the transmission of the linked nptII gene in lre-2.
A GPI-Anchored Protein in Angiosperm Sperm Cell Release 3039