Download Fertilization in Flowering plants. New Approaches for an Old Story

Fertilization in Flowering plants. New Approaches for an
Old Story
Jean-Emmanuel Faure and Christian Dumas*
Ecole Normale Supérieure de Lyon, Laboratory of Plant Reproduction and Development, Unité Mixte de
Recherche 5667, Centre National de la Recherche Scientifique-Institut National de la Recherche
Agronomique-Ecole Normale Supérieure, Lyon-Université Claude-Bernard Lyon I, Lyon 69364 cedex 07, France
Our understanding of how plant fertilization operates, because of the complexity of the reproductive
structures and the highly diverse nature of the cellular
processes involved, has historically been very closely
linked to the developments in microscopy. In the 19th
century, the development of light microscopy equipment and protocols allowed S. Nawaschin and L.
Guignard (8) to discover independently that two fertilization events take place when the pollen tube meets
the embryo sac. One event involves the union of a
sperm with the central cell, from which the endosperm develops. The other event involves the union
of sperm and egg, giving rise to the embryo. The use
of the electron microscope led to a second series of
discoveries, in particular by the group of Jensen (10).
Sperm were shown to be real cells, despite their having been considered for decades to be no more than
naked nuclei. Observations also led to the idea that the
two fertilization events consist of plasmogamy and
karyogamy steps. However, further progress was
strongly limited by the inaccessibility of the gametes
due to their encasement in parental tissues.
PROGRESS FROM CELL AND MOLECULAR
BIOLOGY APPROACHES
New information came from the development of
techniques to isolate sperm cells from pollen grains
and the female gametes from the maternal tissues (5,
12). In vitro fusions of sperm cells with egg cells or
central cells were achieved by electrofusion (11) or
in Ca2⫹-containing media (6). Regeneration of the
sperm-egg fusion products into fertile plants in addition to in vitro divisions of the fertilized central
cells was obtained using feeder cells (12, 13). A major
benefit of in vitro fusion is that fertilization can be
directly visualized and manipulated. Isolated cells
can also be collected for molecular studies. The first
currently detectable cellular events that take place
after gamete fusion are an increase of the concentration of cytosolic Ca2⫹ (4) as in animal gamete fusion.
An influx of extracellular Ca2⫹ may contribute to this
cytosolic increase (1). However, an important ques* Corresponding author; e-mail [email protected];
fax 33– 4 –72–72– 86 – 00.
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tion that was extensively studied in animal fertilization still remains unanswered in plants: Is this Ca2⫹
elevation necessary and/or sufficient to trigger egg
activation and the initiation of development? Another similarity to animals is the establishment of a
block to polyspermy: Maize (Zea mays) sperm cannot
fuse with zygotes in vitro (6). This barrier is established as early as 45 s after the initial fusion in maize.
Cell wall deposition may mechanically contribute to
this block to polyspermy in analogy to the fertilization envelope that leads to slow block to polyspermy
in several animal species. However, this remains to
be demonstrated. The electrical properties of the gamete membranes also need to be characterized to
understand if an electrical block exists, i.e. a fast
block to polyspermy as occurs in several animal
species.
Unlike in animals (19), a molecule with a possible
role in sperm-egg interactions at the plasma membrane level has not yet been identified. Analogies
with other organisms will probably not be sufficient
to isolate these components because molecules involved in the reproduction process seem to have
diverged to a large extent between different taxonomic groups (19). The identification of such molecules is important for understanding how development is activated and for identifying any possible
difference in fertilization between the egg and central
cells. It should also help us to understand how far the
fertilization mechanisms have diverged between animal and plant species. New approaches hopefully
should allow the identification of such molecules in
the future. cDNA libraries have been prepared from
isolated maize gametes and in vitro zygotes. Subsequent differential screening has already provided a
few clones specifically expressed, or more highly
expressed, after fertilization (12). Libraries have also
been prepared from the mother cells of the two
sperms, i.e. the generative cells, in Lilium longiflorum
(22). Differential screening has led to the identification of LGC1, a gene expressed specifically in the
generative and sperm cells (22). It is interesting that
the product of LGC1 has been shown to localize at the
surface of the male gametic cells. Further studies will
be required to understand the role of all these newly
isolated genes. In addition, a major challenge in the
Plant Physiology, January 2001, Vol. 125, pp. 102–104, www.plantphysiol.org © 2001 American Society of Plant Physiologists
Fertilization in Flowering Plants
upcoming years will be to understand how far the in
vitro situation resembles the in vivo fertilization and
how far in vitro procedures can be used to understand the cellular events during gamete fusion.
NEW DEVELOPMENTS FROM
ARABIDOPSIS GENETICS
Screens to identify Arabidopsis mutants with fertilization defects have been done or are under
progress in several laboratories. The group of A.M.
Chaudhury has isolated three mutants, fis1, fis2, and
fis3 (for fertilization-independent seed), that show some
aspects of seed development without fertilization (3,
14). The group of R.M. Fischer similarly has isolated
fie, a fertilization-independent endosperm mutant (14,
15) allelic to fis3. They have also isolated f644, a
mutant allelic to fis1, to an embryo-defective mutant
emb173, and to the medea mutant from U. Grossniklaus’ group (9, 14). The three genes corresponding
to these described mutants are now often referred as
FIE (for fie or fis3), MEA (for medea, fis1, f644, or
emb173), and FIS2 (for fis2). MEA and FIE were
shown to encode proteins from the polycomb group,
whereas FIS2 encodes a zinc finger protein (9, 14).
The products of the three genes are thought to form
a complex that represses the genes involved in seed
development (3). The phenotype of the mutants is
complex. It is interesting that if the female gametophyte has a mutant medea allele, for example, embryo
development does not proceed normally (9). Therefore, the corresponding genes are thought to have a
gametophytic maternal effect on the development of
the embryo. Although the precise role of these master
genes needs to be identified, as does their integration
into the signaling that leads to seed development,
these data have important consequences for our understanding of fertilization. They outline the importance of the female gametophyte in embryo development, in contrast with the long-held belief that genes
newly expressed in zygotes were more important in
early development. Besides this important new perspective, it should be noted that this could constitute,
in particular, a limitation to the strategies of differential screening described above to identify genes
involved in early zygote development.
It has been suggested that the observed maternal
effect for the MEA gene is actually due to the silencing of the paternal alleles by genomic imprinting (21).
It is possible that FIS2 and FIE are also regulated in
that manner. Methylation may be the basis for such a
differential expression because crosses with pollen
from the ddm1 mutant or MET1 antisense plants rescue seeds with a maternal copy of medea (7, 21). The
group of U. Grossniklaus recently suggested that the
whole paternal genome is actually transiently silenced after fertilization and that embryogenesis and
endosperm development is mainly under maternal
control (20). These authors focused on 20 genes exPlant Physiol. Vol. 125, 2001
pressed early in the endosperm and/or embryo.
They studied the expression of the maternal or paternal copies of these genes using ␤-glucuronidase
and allele-specific reverse transcriptase-PCR and
proposed that the paternal copies are silenced during
the first few days following fertilization. From these
data they argued that an unequal contribution of the
parental genomes is not restricted to MEA, FIS2, or
FIE, but is true for the whole genome. These data are
exciting but will need further examination. It is difficult to extend these observations made with a few
genes to the whole genome. There may exist very
subtle and critical differences from one gene to another, and also between the embryo and the endosperm. In addition, one of the 20 genes studied by
the group of U. Grossniklaus was PROLIFERA, but
data on the expression of this gene just published by
Springer et al. (18) have been interpreted in a conflicting way.
AT RANDOM OR PREFERENTIAL?
The two male gametes have two different target
cells, the egg and the central cells, with two very
different developmental fates, the embryo and the
endosperm. In addition, observations suggest sperm
cell dimorphism in several species (17). It is therefore
reasonable to ask if the sperm cells fuse at random or
selectively, i.e. if there is a preferential fertilization,
and if this could be important for further development. The possibility of a preferential fertilization of
one sperm with the egg cell has been suggested from
a very small number of studies performed in maize
and Plumbago zeylanica (16). In vitro fertilization may
now be used to test if the two male gametes from the
same pollen grain can fuse with egg cells. However,
the data supporting a general paternal silencing
would argue against any differential role of the
sperm in early development, except the possible inheritance of specific organelles or molecules.
THE ORIGIN OF DOUBLE FERTILIZATION?
The evolutionary significance of double fertilization in flowering plants has been questioned during
the last ten years. Original works from the early
1900s, suggesting that two karyogamy events occur
in parallel in the female gametophyte of some gnetales, were reinvestigated and confirmed by Friedman (8). A rudimentary process of double fertilization was observed in Ephedra nevadensis, Ephedra
trifurca, and Gnetum gnemon (8). It consists of the
fusion of one sperm nucleus with the egg cell nucleus
and of a second sperm nucleus brought by the same
pollen tube with the mitotic sister nucleus of the egg.
The author proposed that this process developed in a
common ancestor of the gnetales and the flowering
plants and that one of the fusion products later
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Faure and Dumas
evolved into the endosperm of angiosperms. However, recent data separate angiosperms from all gymnosperms and put the gnetales as the closest relatives
to conifers (2). This implies that double fertilization
arose independently in gnetales and angiosperms.
Molecular evidence is therefore now required to determine whether the model proposed for the origin of
double fertilization and the evolutionary equivalence
of the two fertilization events is valid, or if double
fertilization emerged differently in the angiosperms.
CONCLUSIONS
The last 15 years have been characterized by a burst
of new information from cell biology and genetics.
Important new ideas have emerged: (a) Some fertilization steps and cellular processes, such as the Ca2⫹
increase, seem to be very similar to the equivalent
events in animals, at least in the egg. Future investigations will be required to identify the molecules
involved, and to understand if these steps are similar
in the central cell; (b) Genes with maternal effects
seem to be important for early seed development;
and (c) There may be a broad imprinting of genes and
an early silencing of the paternal genome. Therefore,
the precise contribution of the male gametes to the
very early development will have to be identified. In
the future, a greater understanding of fertilization
should be gained from further technological advances, such as confocal microscopy techniques and
the use of green fluorescent proteins to visualize
structures in living materials.
ACKNOWLEDGMENTS
We are grateful to Dr. Charlie Scutt (Centre National de
la Recherche Scientifique, Lyon, France) and Professor
Sheila McCormick (University of California, Berkeley) for
their critical reading of the manuscript.
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