Go Suzuki∗
Division of Natural Science, Osaka Kyoiku University, Kashiwara, 582-8582 Japan
Sexual reproduction is an important biological event not
only for evolution but also for breeding in plants. It is
a well known fact that Charles Darwin (1809–1882) was
interested in the reproduction system of plants as part
of his concept of ‘species’ and ‘evolution.’ His keen
observation and speculation is timeless even in the current
post-genome era. In the Darwin anniversary year of 2009,
I have summarized recent molecular genetic studies
of plant reproduction, focusing especially on male
gametophyte development, pollination and fertilization.
We are just beginning to understand the molecular
mechanisms of the elaborate reproduction system in
flowering plants, which have been a mystery for >100
years.
Keywords: Charles Darwin • Fertilization • Male gametophyte
development • Pollination • Reproductive barriers • Selfincompatibility.
Abbreviations: CMS, cytoplasmic male sterility; CRP,
cysteine-rich polypeptide; GSI, gametophytic selfincompatibility; LM, laser microdissection; PMC, pollen
mother cell; PPR, pentatricopeptide repeat; RLK, receptorlike kinase; SI, self-incompatibility; SSI, sporophytic selfincompatibility.
Introduction
The year 2009 is the bicentennial anniversary of Charles
Darwin’s birth, and Darwin tribute reviews have also been
published this year in the field of plant science (Fay and
Chase 2009, Holland et al. 2009, Hopper and Lambers 2009,
Moulia and Fournier 2009, McClure 2009). As is well known,
his contribution to plant science involves a wide range of
fields, such as plant speciation, orchid biology, phototropism, gravitropism and mating systems. In particular, his
great insight into sexual plant reproduction cannot be forgotten. He was fascinated by elaborate plant reproductive
∗Corresponding
Mini Review
Recent Progress in Plant Reproduction Research: The Story of
the Male Gametophyte through to Successful Fertilization
systems and the importance of outcrossing, and his pioneer
works on self-incompatibility (SI) are still well known among
present SI researchers (McClure 2009). Thus, the end of
Darwin’s anniversary year is a good time to summarize recent
progress in plant reproduction research. Therefore, in this
short review, I want to detail what has recently been discovered about plant reproduction, focusing especially on the
story of the male gamete up to fertilization in the angiosperm. It will allow us to recall Darwin’s innovative studies as
a plant scientist.
Pollen development and male sterility
The male gametophyte in flowering plants develops in the
anther of the stamen (Fig. 1). While the animal germline is
discriminated from somatic cells in the early developmental
stage, pollen mother cells (PMCs) are differentiated from
cells which are at the adequate location in the adequate late
developmental stage; these are called archesporial cells
(Scott et al. 2004). For successful reproduction, timing of
production of the male and female gametophytes is important, and male gametophyte development is synchronically
regulated in the same anther.
In the early stages of male gametophyte development,
PMCs undergo meiosis to form haploid tetrad cells. As in
other eukaryotes, a single DNA replication followed by
two characteristic divisions (meiosis I and II) results in a
reduction in ploidy in plant meiosis. During meiosis I, paired
homologous chromosomes undergo homologous recombination and segregate from each other, retaining sister
chromatid cohesion. As part of the research into a plantspecific phenomenon, several recent studies have attempted
to explain molecular mechanisms of wheat Ph1, which is
necessary for correct pairing of homologous chromosomes in
the polyploid genome (Griffiths et al. 2006, Sidhu et al. 2008).
In the subsequent meiosis II, sister chromatids are separated
and, finally, four haploid cells, the tetrad, are produced.
author: E-mail, [email protected]; Fax, +81-72-978-3660.
Plant Cell Physiol. 50(11): 1857–1864 (2009) doi:10.1093/pcp/pcp142, available online at www.pcp.oxfordjournals.org
© The Author 2009. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
All rights reserved. For permissions, please email: [email protected]
Plant Cell Physiol. 50(11): 1857–1864 (2009) doi:10.1093/pcp/pcp142 © The Author 2009.
1857
G. Suzuki
Fig. 1 Schematic representation of the reproductive organs in angiosperm. In the course of male gametophyte development (right), after meiosis,
the haploid microspore develops in the locule surrounded by the tapetum in the immature anther. After disintegration of the tapetum, the
pollen grain matures in the locule of the mature anther. At the time of flowering, anther dehiscence occurs to release the pollen, which is then
carried by wind or insects onto the stigma of the pistil. The compatible pollen germinates, and its pollen tube penetrates into the stigma,
elongates in the stylar transmitting tissue and is guided to the synergids of the embryo sac to achieve double fertilization (left). The two sperm
cells in the pollen tube finally fertilize the egg cell and the central cell to form the embryo and the endosperm, respectively.
Gene regulation of meiosis has been intensively studied in
yeast, and many plant orthologs of yeast meiosis genes share
similar functions in plants (summarized in Mercier and
Grelon 2008). This indicates that mechanisms of recombination and specialized chromosome segregation, which are
characteristics of meiosis, are fundamentally common in
eukaryotes. However, studies of plant orthologs sometimes
provide useful new information for research into meiosis of
other organisms (White 2008), and plant-specific meiosis
genes were also reported (Nonomura et al. 2004, Mercier
and Grelon 2008), suggesting the importance of plant
meiosis research.
In a locule of the anther, the tetrad haploid microspores
are released and mature to become pollen grains
(McCormick 1993, Fig. 1). At first, the uninucleate microspore
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develops into the bicellular pollen with a larger vegetative
cell and a smaller generative cell by an asymmetric mitosis.
Then, the generative cell undergoes a second mitosis to form
two sperm cells, which are finally used for double fertilization, as discussed below. In Arabidopsis, genetic studies using
various mutants were conducted to analyze such male
gametophyte development (summarized in Borg et al. 2009),
e.g. analysis of fbl17 mutants revealed the control of male
germ cell proliferation by the degradation of cell cycle
inhibitors (Kim et al. 2008).
The layer of the anther cells closest to the locule, termed
the tapetum, is metabolically active, and plays important
roles in pollen maturation (Fig. 1). The involvement of phytohormones, gibberellin and auxin in pollen development
was suggested by cell type-specific transcriptome analysis of
Plant Cell Physiol. 50(11): 1857–1864 (2009) doi:10.1093/pcp/pcp142 © The Author 2009.
Recent progress in plant reproduction research
rice microspore/pollen and tapetum (Hirano et al. 2008).
During pollen maturation, the sporophytic tapetum is acting
as a nutritive tissue by providing nutrition and materials for
pollen wall formation, and disintegrates in the later stage of
pollen development (Scott et al. 2004). In Brassica, tapetosomes, unique organelles in the tapetum, contain endoplasmic reticulum-derived vesicles including flavonoids, and
oleosin-coated lipid droplets including alkanes (Hsieh and
Huang 2007). After disintegration of the tapetum, these
flavonoids, alkanes and oleosins are discharged to the surface of the maturing pollen as pollen coats and might play
roles in pollen germination and pollen tube growth. Stable
intracellular oil bodies with unique oleosin and caleosin were
also found in lily mature pollen, which might serve as energy
reserves for germination (Jiang et al. 2007, Jiang et al. 2008).
Formation of the surface structure of pollen grains (pollen
walls) is an important process in pollen maturation (Scott
et al. 2004). The pollen walls consist of inner pectocellulosic
intine and outer sporopollenin-based exine. The exine provides the species-specific pollen surface structure and cavities storing the pollen coats, including lipids and proteins,
which are mainly supplied from the tapetum. Screening of
12 kaonashi mutants showing abnormal exine structure in
Arabidopsis makes it possible to understand more details of
the genetic regulation of exine formation (Suzuki et al. 2008),
in addition to other Arabidopsis exine mutants reported or
reviewed in recent papers (Ariizumi et al. 2008, Guan et al.
2008, Dobritsa et al. 2009, Wilson and Zhang 2009). Transcripts encoding potential proteins having some function in
exine synthesis were also observed from the transcriptome
analysis of rice anther (Huang et al. 2009).
If we refer to pollen development, an explanation of cytoplasmic male sterility (CMS) is unavoidable, because it is an
important agricultural trait used in hybrid breeding. Plant
CMS shows a maternally inherited pollen sterility phenotype
and is regulated by interaction between mitochondria and
nuclei (reviewed in Chase 2007, Fujii and Toriyama 2008b).
Defective pollen development caused by mitochondrial
genomic organization can be restored by nuclear-encoded
Rf genes. In petunia, radish and rice, recently cloned Rf genes
encode mitochondria-targeted pentatricopeptide repeat
(PPR) proteins, and it is considered that the PPR proteins
play a role in post-transcriptional RNA modification of
CMS-determining genes in mitochondria. Interestingly,
more recently, another rice Rf gene against CW-type CMS
cytoplasm (Rf17) was cloned and shown to encode a novel
protein, RETROGRADE-REGULATED MALE STERILITY
(RMS), whose down-regulation caused fertility restoration
(Fujii and Toriyama 2009). Because there are many essential
mitochondrial genes in the nuclear genome, retrograde
signals from mitochondria to nuclei are undoubtedly important in plants (Fujii and Toriyama 2008b). Similar to the
known examples of retrograde regulation in yeast RTG
signaling, Drosophila cell cycle-related signaling and Arabidopsis plastid signaling, the plant CMS might be elaborately
regulated by typical retrograde signaling (Fujii and Toriyama
2008a, Fujii and Toriyama 2008b).
Pollination and SI
Mature pollen grains are then carried by wind or insects
onto the stigma of the pistil, also known as ‘pollination’
(Fig. 1). Pollination is an important gate before fertilization,
and involves cell–cell communication between haploid
(pollen) and diploid (stigma) cells. It must be noted that
haploid pollen is coated by the pollen coat from diploid
tapetum cells, as described above, so that the pollination
process includes communication between molecules
produced from parent diploid cells. These two different
kinds of behavior are well reflected in the SI system; some
species show gametophytic SI (GSI), while others show
sporophytic SI (SSI). In this section, I want to summarize
‘pollination and SI’ based on recent important findings.
As I mentioned in the Introduction, Darwin had a great
interest in plant reproduction, especially in pollination, SI
and the benefit of outbreeding. He might wonder why so
many plant species show self-sterility, even though only
advantageous variations are supposed to survive through
‘natural selection’ and the ‘struggle for existence’ based on
his theory of evolution. In his book ‘The Effects of Cross and
Self-Fertilisation in the Vegetable Kingdom’, published in
1876, he performed a large number of crossing experiments
and compared phenotypes of individuals obtained from
cross-pollination and self-pollination in various flowering
plants (Darwin 1876). Although he somehow misunderstood the evolution of SI as an incidentally acquired character dependent on environmental circumstances, it is
noteworthy that he realized the existence of heterosis and
the importance of SI from his exhaustive observations.
Now we know that SI is genetically regulated, although
Darwin did not have knowledge of Mendelian genetics at
that time. Darwin’s precise description about heterostyly in
Primula is famous, but, on the other hand, recent molecular
studies of SI have focused on homomorphic incompatibility.
Here I summarize the known molecular mechanisms of
three types of homomorphic SI systems: S-receptor kinase
(SRK)-based SSI systems in Brassicaceae; S-RNase-based
GSI systems in Solanaceae, Rosaceae and Plantaginaceae;
and S-glycoprotein-based GSI systems in Papaveraceae. For
readers who want to understand SI in more detail, a recently
published book edited by Vernonica E. Franklin-Tong
(Franklin-Tong 2008a) is an excellent resource.
In Brassica SSI, a small cysteine-rich protein, S-locus
protein-11/S-locus cysteine-rich (SP11/SCR), in the pollen
coat acts as a ligand of SRK, which is localized on the cytoplasmic membrane of the stigma papilla cell. SP11/SCR and
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G. Suzuki
an extracellular receptor domain of SRK are highly polymorphic and can interact only when their S haplotypes are the
same (i.e. self-pollination), and they transduce ‘self-signals’
in the papilla cells with phosphorylation cascades (reviewed
in Takayama and Isogai 2005). Thus, as a result of the SRKmediated signal transduction, self-pollen is rejected on the
stigma papilla cells. On the other hand, non-self-pollen can
germinate, and its pollen tube is elongated and penetrates
into the pistils for successful fertilization. Even though almost
10 years have passed since the long-anticipated pollen S gene
was reported, signaling cascades between self-recognition
and rejection of self-pollen are still largely unclear. Gene
cloning from a self-compatible Brassica mutant revealed
that M-locus protein kinase (MLPK) has a role as a positive
regulator of SI and directly interacts with SRK in the plasma
membrane (Kakita et al. 2007). The receptor complex activated by phosphorylation on the membrane of papilla cells
might transduce the SI signal into the cells, a process which
is possibly followed by several steps of phosphorylation cascades and results in the inhibition of self-pollen germination.
ARM-repeat-containing protein (ARC1) is known as another
positive regulator of SI, and signaling pathways with similar
U-box/ARM-repeat-containing E3 ligases are conserved with
other kinase proteins in Arabidopsis (Samuel et al. 2008),
suggesting that common ubiquitin-mediated protein degradation pathways might be involved downstream of SRK and
other receptor-like kinase (RLK) signaling. In the latest interesting findings, Ivanov and Gaude (2009) reported that SRK
localizes predominantly to intracellular sorting endosomes
with a negative regulator THL1, and Tantikanjana et al. (2009)
showed the dual role of SRK in SI and pistil development by
using an Arabidopsis rdr6 mutant. Thus, there are now many
questions about the complicated SRK-mediated signal transduction leading to inhibition of self-pollination, though the
SI recognition itself is simple and easy to understand.
In contrast, the mechanism of arresting self-pollen tube
growth is simple to understand in the S-RNase-based GSI.
The arrest of the self-pollen tube growth in the style transmitting tissue can be explained by the effect of RNA degradation in the pollen tube catalyzed by RNase activity of the
self-S-RNase (reviewed in McClure 2009). However, recent
identification of the pollen S determinant, S-locus F-box
(SLF/SFB, Sijacic et al. 2004), makes it difficult to understand
the co-evolution of pollen and style S determinants and the
mechanisms of SI recognition (Newbigin et al. 2008, McClure
2009). In Antirrhinum, sequence divergence of SLFs is too
low between S haplotypes, indicating that polymorphisms of
SLF alleles have a much shorter evolutionary history than
that of the S-RNase alleles, which is in contradiction to
the long-held view of co-evolution of the SI genes (Newbigin
et al. 2008). From the viewpoint of molecular mechanisms,
both of two possible models, the ‘compartmentalization
model’ and the ‘S-RNase degradation model’, have some
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deficiencies for a perfect explanation of the S-RNase-based SI
(McClure 2009). Furthermore, two subfamilies of Rosaceae
(Prunoideae and Maloideae) possess different characteristics for the existence of competitive interaction, and the
copy number of SFB on the S-locus, suggesting the possibility
of mechanistic divergence of this GSI (Sassa et al. 2009).
Thus, some conflicts arise in understanding the puzzling
nature of S-RNase-based SI. If further experiments in the
future resolve these conflicts, whole mechanisms will be
easily clarified, because there is nothing downstream of
S-RNase.
The most exciting topic in recent SI studies is the identification of a pollen S product in the Papaver GSI system,
reported by Wheeler et al. (2009). It means that this third
set of the pollen/pistil SI factors was successfully identified
in Papaver. They are designated as Papaver rhoeas pollen S
(PrpS) and Papaver rhoeas stigma S determinant (PrsS). PrpS
is a transmembrane protein localized to the pollen tube
plasma membrane, and its putative extracellular loop segment interacts with PrsS, which is the stigma-expressed
S-glycoprotein. Although the precise protein function of
PrpS for activation of the SI response has not been determined, the S haplotype-specific interaction of PrpS and PrsS
might trigger the influx of Ca2+ into the shank of the pollen
tube, finally inhibiting pollen tube elongation (Franklin-Tong
2008b).
Generally, in compatible pollination, normal growth of
the pollen tube requires lipids on the stigma (Wolters-Arts
et al. 1998) and a precise cytoplasmic Ca2+ concentration in
pollen tube tips (Iwano et al. 2009). In the growing pollen
tube, mitochondria and Golgi vesicles are distributed differently, and their movement is regulated differently by microtubule-dependent and actin filament-dependent motors
(Romagnoli et al. 2007). Successful pollen germination and
pollen tube elongation in the stylar transmitting tissues lead
to fertilization, the final step of the story of the male gametophyte (Fig. 1). How are the pollen tubes containing the
sperm cells accurately guided to the embryo sac with the egg
cell? Here, I now want to describe pollen tube guidance and
fertilization.
Pollen tube guidance and fertilization
One of the most impressive findings in plant science reported
this year is undoubtedly the pollen tube attractant molecules reported by Okuda et al. (2009). There are many
mysterious phenomena in the fertilization process (reviewed
in Berger et al. 2008). Since double fertilization was first
identified by Sergius Nawaschin and Leon Guignard at the
end of the 19th century (Nawaschin 1898, Guignard 1899),
the mechanism of how to the pollen tube is attracted to the
embryo sac has long been a mystery, because of the difficulty
of observing the embryo sac embedded in the ovule (Fig. 1).
Plant Cell Physiol. 50(11): 1857–1864 (2009) doi:10.1093/pcp/pcp142 © The Author 2009.
Recent progress in plant reproduction research
By using the in vitro fertilization system of Torenia,
Higashiyama and colleagues clearly demonstrated that the
synergid cell is necessary for the short-range pollen tube
guidance before fertilization (Higashiyama et al. 2001) and,
after extensive experimentation, the authors finally identified molecules which can attract the pollen tube and are
secreted from synergid cells (Okuda et al. 2009). The pollen
tube attractants, designated as LURE1 and LURE2, are small
cysteine-rich polypeptides (CRPs). It is interesting that both
LUREs and SP11/SCR are categorized as CRPs as the signaling
molecules involved in plant reproduction. This similarity
inspired us to image the molecular recognition mechanism
by which LUREs might act as ligands of unidentified RLKs
localized on a tip of the pollen tube. Many CRPs were found
in the Torenia synergid cells (Okuda et al. 2009), and several
RLKs expressed in synergid cells and pollen tubes were
recently identified as female and male factors, respectively,
controlling pollen tube behavior in Arabidopsis (EscobarRestrepo et al. 2007, Boisson-Dernier et al. 2009, Miyazaki
et al. 2009), suggesting that complex direct communication
between female and male cells before fertilization might be
carried out via RLK-mediated signaling pathways.
After pollen tube arrival, growth arrest and pollen tube
discharge, which are caused by interaction between synergids and pollen tubes as described above, the two released
sperm cells migrate and fuse to the egg cell and central cell
to accomplish double fertilization. In this step, the GENERATIVE CELL SPECIFIC1 (GCS1) protein localized on the membrane of generative cells is known to have an essential role in
gamete attachment and fusion (Mori et al. 2006). GCS1 was
first identified in the lily, and homologs were observed in
Arabidopsis, other angiosperms, algae and parasites, suggesting its fundamental role in membrane fusion during
fertilization. In most plant species, the two sperm cells are
morphologically indistinguishable, and there is some debate
as to whether or not the decision for each sperm cell to fertilize either the egg cell or the central cell is already accomplished in the pollen tube (Berger et al. 2008). A recent report
by Ingouff et al. (2009) proposed that each of the two sperm
cells shares an equal ability to fertilize the egg cell, using an
Arabidopsis RETINOBLASTOMA RELATED 1 (rbr1) mutant
having an abnormal embryo sac with two egg cells.
To date, various molecular players, whose disruption
affects pollen tube guidance, have been reported. In Arabidopsis, the MYB98 transcription factor is necessary to form
the filiform apparatus of synergids, which might be essential
for secretion of pollen tube attractants (Kasahara et al. 2005).
In addition to such synergid-mediated regulation, the
CENTRAL CELL GUIDANCE (CCG) nuclear protein in the
central cell (Chen et al. 2007), the membrane-bound HAP2
(GCS1) protein in the sperm cells (von Besser et al. 2006) and
MAGATAMA3 (MAA3) helicase, which regulates female
gametophyte development (Shimizu et al. 2008), also play
a role in pollen tube guidance in Arabidopsis. It is expected
that an integrative explanation of all the molecular mechanisms of pollen tube guidance will be found.
Species and reproductive barriers
This year is also known for the fact that it is the 150th anniversary of the publication of The Origin of Species (Darwin
1859). In this famous work of Darwin, he refuted the fixed
definition of species as distinct creations of God. Categories
of species, subspecies and variety are all part of a continuous
lineage, and are defined arbitrarily. As is known, the most
common biological definition of a species is an isolated
group, whose members can cross with each other and produce fertile offspring. However, fertility is also defined arbitrarily and is determined differently by different researchers.
In this regard, reproductive barriers might merely be spinoffs of evolution. Nevertheless, reproductive barrier, like
interspecific incompatibility is still an attractive theme for
researchers of plant and animal reproduction, because
breaking the reproductive barriers will give us new important products. A classical Dobzhansky–Muller model explains
a simple scenario to produce genetic incompatibility
between isolated populations, and Brideau et al. (2006)
showed a molecular-level explanation of the Dobzhansky–
Muller model in Drosophila. In plants, interestingly, Bikard
et al. (2009) reported intraspecific incompatibility in
Arabidopsis thaliana caused by the Dobzhansky–Muller-like
epistatic interaction. Reciprocal silencing of essential duplicated genes causes genetic incompatibility within species
and could contribute to reproductive isolation and speciation. In general, reproductive barriers also exist in the
pollination and fertilization steps described here. The
relationship between interspecific incompatibility and SI
(Hiscock and Dickinson 1993, Murfett et al. 1996) and species preference of the pollen tube attractant from synergid
cells (Higashiyama et al. 2006) are both known. In addition
to these reports, molecular mechanisms of reproductive
barriers, in connection with the molecular evolution of key
players of pollination and fertilization, will be uncovered in
the near future.
Future prospects
Molecular techniques and bioimaging technology are making
remarkable progress day by day in dissecting cell-level gene
regulation and in visualizing the dynamic state of organelles,
molecules and other materials. Laser microdissection (LM)
technologies enable us to perform cell type-specific gene
expression profiling in plants (Ohtsu et al. 2007). In fact, in
plant reproduction research, LM microarray analysis was
applied to the cell-type specific transcriptomes of the male
gametophyte and tapetum in rice (Hobo et al. 2008, Suwabe
et al. 2008, Watanabe 2008). Whole transcriptome analysis of
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G. Suzuki
a single cell using a powerful next-generation sequencing
technology (F. Tang et al. 2009) can also be applied to complex plant sexual tissues. In bioimaging analysis, visualizing
mitochondria and plastids in the living pollen provides us
with useful information (Matsushima et al. 2008, L. Y. Tang
et al. 2009), and precise monitoring of actin dynamics in
papilla cells and Ca2+ dynamics in pollen tubes has been
successful in pollination studies (Iwano et al. 2007, Iwano
et al. 2009). By endless scientific trials with these emerging
technologies, our molecular understanding of plant reproduction will advance step by step from one level to the next.
Although we tend to consider the phenomena of life at
a micro level, we should continue research with a broader
perspective, as Darwin did.
Funding
The Ministry of Education, Culture, Sports, Science, and
Technology of Japan (MEXT) [Grants-in-Aid for Special
Research on Priority Areas (No. 18075003)].
Acknowledgments
The author thanks Drs. Takeshi Ishimizu (Osaka University),
Keita Suwabe (Mie University) and Masumi Miyano (Tohoku
University) for critical reading of the manuscript.
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(Received October 4, 2009; Accepted October 9, 2009)