14
The difficult question of sex: the mating game
Vernonica E Franklin-Tong
There is currently an intense interest in understanding how
pollination and fertilization in flowering plants is controlled.
This is because of the central and crucial importance of sexual
reproduction in plant lifecycles. Plants have evolved many
complex mechanisms to prevent self-fertilization, and it is
thought that this may partially explain the great success of the
angiosperms. The journey of discovery in determining the
components and mechanisms involved in these processes has
been ongoing for some time. Recent data have provided fresh
insights into some aspects of what is involved in controlling
pollen germination and pollen-tube growth, both in normal
pollination and in self-incompatibility.
Addresses
Wolfson Laboratory for Plant Molecular Biology, School of Biosciences,
University of Birmingham, Edgbaston, Birmingham B15 2TT, UK;
e-mail: V.E.Franklin-Tong@bham.ac.uk
Current Opinion in Plant Biology 2002, 5: 14–18
1369-5266/02/$ — see front matter
© 2002 Elsevier Science Ltd. All rights reserved.
Abbreviations
calcium ion concentration
[Ca2+]
[Ca2+]i
intracellular [Ca2+]
cer
eceriferum
CRIB
Cdc42/Rac-interactive binding
ECM
extracellular matrix
GAP
GTPase-activating proteins
PCP
pollen-coat protein
rtg
raring-to-go
SAB
short actin bundle
SCA
stigma/stylar cysteine-rich adhesin
SCR
S-locus cysteine-rich protein
SI
self-incompatibility
SP11
S-locus protein 11
SRK
S-locus receptor kinase
S-RNase
self-incompatibility locus-RNase
Introduction
Pollination seems, on the face of it, to be a simple
process: pollen lands on a flower’s stigma, hydrates and
germinates, then grows through the pistil to the ovary,
where it achieves fertilization and produces seed. In
reality, however, it is extremely complex and involves a
series of events that are still relatively poorly understood
[1]. It is known (though the molecular basis for this is not
established) that there is tight regulation governing
whether pollen is accepted at an inter-specific level.
Often, there are also barriers to prevent self-fertilization.
Such barriers include self-incompatibility (SI), which is
genetically controlled by an S locus, whereby pollen
recognized as ‘self’ is inhibited. If the pollen tube gets
past this barrier, it must negotiate a long journey to the
ovule, where it needs to find an unpollinated ovule and
the route to the egg cell, where the sperm are delivered
and achieve fertilization. A cartoon outlining pollination
is shown in Figure 1.
It is apparent that the process of pollination involves many
interactions, recognition events, and signals, many of
which involve precise temporal and spatial regulation. We
are beginning to understand the complexity of some of
the mechanisms, components and genetic controls of
pollination and fertilization. Here, I review the significant
breakthroughs that have been made in improving our
understanding of the regulation of pollination over the
past year or so.
Beginning the journey: pollen hydration
and germination
Mature pollen grains are usually dehydrated by the time
they are shed from anthers so that they can remain viable
until they reach a suitable flower. When they alight on a
stigma, they draw water from the stigma; once hydrated,
they are activated and ready to go. Water flow is crucial, and
aquaporins, which are water-channel proteins [2], are not
only present in pollen but also regulate water flow to pollen
grains during hydration [3]. Pollen hydration is tightly
regulated, and several molecules are known to be involved
in stimulating pollen hydration. Long-chain lipids are
implicated as signal molecules: Arabidopsis mutants that are
defective in hydration, such as eceriferum (cer) and pop1,
have defects in lipid biosynthesis [4,5]. One of the cer genes
has recently been positionally cloned [6•], and it has
been demonstrated that a wild-type copy of this gene
complements the cer6-2 defect. In addition, a fertile
suppressor, cer6-2R, that partially restores pollen-coat lipids
has been identified. Analysis of this suppressor has provided
important evidence that small quantities of long-chain
lipids are sufficient for pollen hydration and germination.
Pollen-coat proteins (PCPs) also play an important role in
hydration. Severely delayed pollen hydration was detected
in an Arabidopsis mutant that had a defective GRP17
PCP [7]. It is therefore thought that there are genes that
promote efficient hydration. Whether GRP17 signals to
the stigma or modulates the activity of other molecules
remains to be established. More recently, Preuss’ group
analyzed several additional PCPs in Arabidopsis [8•].
Interestingly, the genes encoding these proteins are
clustered in the genome. One cluster encodes six lipases,
whereas the another comprised six lipid-binding oleosin
genes, which included the previously identified GRP17.
A phenotype that opposite to that of grp17 mutants is seen
in an Arabidopsis gametophytic mutant raring-to-go (rtg),
which has defects in pollen hydration and germination. This
mutant was identified by its pollen phenotype: the pollen of
rtg can prematurely hydrate, germinate, and form pollen
tubes within the anther before dehiscence. These pollen
grains apparently either acquire or retain water within the
anther, bypassing the usual requirement for contact with
The difficult question of sex: the mating game Franklin-Tong
the stigma to hydrate and germinate. The pollen of rtg also
seem to have lost a guidance cue as they do not achieve
fertilization [9]. It is speculated that rtg may define a key step
during early pollination, but further analysis is required.
Figure 1
Pollen
transferred
to stigma
Pollen
hydration
Pollen
germination
and tube growth
Carrying on: Rop GTPases regulate
pollen-tube growth
Hydration initiates metabolic activity in the pollen grain.
This is accompanied by reorganization of cytoplasmic
components, and the establishment of an apical intracellular
Ca2+ concentration [Ca2+]i gradient. The pollen grain can
then germinate and grow. It has already established
polarity during germination and polar (i.e. tip) growth is
continued. Several elements, including the tip-localized
calcium ion concentration [Ca2+] gradient and an intact actin
cytoskeleton, are known to be crucial for this [1,10].
Significant understanding of the regulation of pollen-tube
tip growth has come from the identification and analysis of
the plant-specific Rop subfamily of Rho GTPases [11–13].
The Rho GTPases are key molecular switches in eukaryotic
signaling cascades, and several studies implicate a crucial
role for Rop in signaling to many important developmental
processes in plants, including tip growth in pollen tubes
[1,13]. It has been demonstrated that Rops play an
important role in the regulation of Ca2+-dependent pollentube growth [12,14]. In the past year, further insights have
been achieved. Using a two-hybrid approach to identify
potential Rop interactors, a family of Rho-GTPase-activating
proteins (GAPs) from Arabidopsis, termed RopGAPs, have
been identified and characterized [15••]. In addition to
having a GAP domain, RopGAPs contain a Cdc42/Racinteractive binding (CRIB) domain, a combination that is
apparently unique to RopGTPases. Biochemical analysis
of point mutations has revealed a novel CRIB-dependent
mechanism for the regulation of RopGAPs [15••]. Thus,
these findings provide strong evidence that Rop signaling
involves a unique GTPase regulatory mechanism.
A role for Rop in regulating the actin cytoskeleton in
pollen tubes has also been established recently by the
Yang group [16••]. Two important findings were reported.
First, this group demonstrated the existence of tip-localized
F-actin, which has, until now, been extremely controversial.
Second, they provided convincing evidence for the presence
of a population of F-actin bundles (named short actin
bundles [SABs]) at the extreme tip of living pollen tubes.
These SABs were found in addition to the sub-apical
actin ‘ring’ or ‘collar’ observed previously [10,17,18].
Furthermore, actin in the apical and sub-apical region of
growing pollen tubes was shown to be highly dynamic,
with the actin ‘ring’ and SABs oscillating over time. These
findings suggest not only that dynamic apical actin is
required for polarized tip growth but also that the
dynamics of the SABs are regulated by Rop signaling
[16••], thereby providing the first direct evidence linking
Rho GTPase to actin organization in controlling cell
polarity and polar growth in plants.
15
Stigma
Anther
Transmitting
tract
Pollen
shed
from
anther
Pollentube
growth
Style
Ovary
Ovule
Female
gametophyte
Synergids
Fertilization
Locule
Egg cell
Micropyle
Integuments
surrounding
female
gametophyte
Current Opinion in Plant Biology
Flower structure and the main events involved in pollination. Mature
pollen (yellow) is shed from the anthers. When it lands on a suitable
stigma, it hydrates, germinates and begins to grow. Pollen tubes extend
by tip growth, and in this way pass through the stylar tissue transmitting
tract (blue) to reach the ovary (dark green). Once there, the pollen tube
has to negotiate a route to find the micropyle. On approaching the
micropyle, the tube passes between the integuments, which ‘guard’ the
female gametophyte (bright green), and can then enter the gametophyte
and achieve fertilization upon release of its sperm cell nuclei. There are
many control points and signals that regulate crucial steps in pollination.
Only some of these have been identified to date. Some of the most
obvious ones have been indicated by the red double-headed arrows,
which suggest interactions between components contained or
secreted in/by the cells/tissues and the pollen grain/tube.
Pollen-tube guidance in the pistil
For many years, there have been contentious discussions
about the guidance of pollen tubes in the pistil. Although
these issues are by no means resolved, several recent studies
have hinted at the identity of some of the components
involved in guidance. It has been suggested that the
embryo sac has a role in pollen-tube guidance, and recent
data from an A. thaliana mutant have revealed that several
proteins produced by the embryo sac are likely to be
16
Growth and development
involved (see also Update). The mutant magatama (maa),
which has delayed female gametophyte development,
also has a defective pollen-tube-guidance system [19•].
Although pollen tubes are directed towards the female
gametophytes for much of their journey in maa mutants,
they lose their way before entering the micropyle.
Furthermore, whereas ovules usually attract a single pollen
tube, the ovules of maa mutants show a tendency to attract
two. A model has been suggested in which the female
gametophyte provides guidance signals to direct the pollen
tube to the funiculus and the micropyle, as well as a signal
to prevent polyspermy.
Other important clues about how pollen-tube growth and
guidance in the pistil are regulated have been provided
by Lord and co-workers (see [20] for a recent review).
For many years, these researchers have hypothesized
that pollen-tube guidance and growth through the stylar
extracellular matrix (ECM) is dependent on a matrixadhesion-driven mechanism. They suspected the importance
of the style because of differences between in-vivo- and
in-vitro-grown pollen. Lord and colleagues suggested that
pollen tubes are not just tip-growing cells, but act as a
moving cell system (similar to neuronal cells) if they are
in their natural environment (i.e. the style) where they
can interact properly with components in the pistil.
Components in the style extracellular matrix were therefore
thought to be important for pollen-tube adherence and
adhesion to cells in the style, and responsible for guidance
towards the ovary.
Significant progress has recently been made towards the
identification of stylar components involved in adhesion to
the growing pollen tube. An adhesion bioassay to measure
the binding of pollen tubes in the presence of stylar
extracts [21] has identified two pistil components that are
implicated in adhesion. One was a small 9-kDa pistil
protein that is a stigma/stylar cysteine-rich adhesin (SCA).
Immunolocalization located SCA in the stylar ECM as
expected. Importantly, SCA was also localized in pollen
tubes growing in vivo but not in vitro [22••]. More recently,
the other stylar component has been identified as a
pectin [23••]. The bioassay established that both of these
components induce pollen-tube adhesion, both to other
pollen tubes and to the epidermal cells of the stylar
transmitting tract. The two molecules also bind to each
other to promote pollen adhesion and stimulate the rate of
pollen-tube growth [23••].
These findings support the existence of a contact-stimulated
guidance system that facilitates pollen-tube growth
through the pistil. Lord proposes that this guidance system
defines a fixed track down the style, and suggests that this
mechanism may be considered to be analogous with the
laminin–netrin two-component guidance system that
directs the path of neuron outgrowth [22••]. Parallels
between mechanisms involved in pollen-tube and axon
guidance have recently been reviewed by Palanivelu and
Preuss [24•], and are of interest, especially, as there seem
to be no adhesion molecules in animals and plants that are
related by their primary sequence. If this analogy is correct,
future work will perhaps identify specific sites on the
pollen that interact with the SCA–pectin complex.
Rejection of self: how does it work?
There have been several recent advances in our understanding of the components involved in the rejection of
self pollen in Brassica. A stigmatic S-locus receptor kinase
(SRK) is known to be involved [25–27]. The male component,
an S-locus cysteine-rich protein (SCR), is a PCP and has
been shown to confer pollen S specificity in Brassica
oleracea [28]. Recent analysis of two alleles of a corresponding
pollen gene from Brassica rapa, S-locus protein 11 (SP11)/SCR,
have established that this gene is the sole male determinant
of S-specificity in Brassica [29•]. It is proposed that the
interaction of SCR/SP11 and SRK triggers a signal transduction cascade in the stigma that results in rapid
inhibition of pollen growth (see also Update). Recently, it
has been demonstrated that SRK is phosphorylated in vivo
within 60 minutes of self-pollination [30••]. This seems to
be remarkably slow for a signaling response, suggesting
that something else happens upstream of this phosphorylation. Nevertheless, this is the first demonstration of a
plant receptor-like kinase being phosphorylated in response
to a specific stimulus in vivo. Related data also indicate
that autophosphorylation of SRK is inhibited by a stigma
thioredoxin THL1 and activated by PCPs [30••]. By
analogy to animal receptor kinases, whose ligands activate
their receptor by inducing their oligomerization and
transphosphorylation, it is suggested that the activation
of SRK by PCPs may involve receptor oligomerization.
This is consistent with a previous study showing that
transphosphorylation occurs between SRK molecules [31].
Apart from this report, little progress has been made in
the analysis of the signal transduction cascade that is
assumed to be triggered by SRK–SCR interaction.
Further analyses of two thioredoxins that interact with
SRK in a yeast two-hybrid assay have indicated that the
reducing activity of the thioredoxins is important [32],
which is consistent with other data [30••]. Finally, an
additional role for SRK is suggested by data that indicate
that SRK is responsible for determining the dominance
relationships of S alleles in the pistil [33•].
In the past year, not much has been published that
advances our understanding of other SI systems. Two other
major SI systems are well characterized. In the Papaver
system, a signaling cascade involving Ca2+ is triggered in
incompatible pollen [1]. Targets for this cascade include
the actin cytoskeleton [18] and it is thought, because DNA
fragmentation is detected in incompatible pollen, that a
programmed cell death cascade is triggered [34]. In the
SI system found in the Solanaceae, self-incompatibility
locus-RNases (S-Rnases) are known to encode the female
S-component, and two models for this SI system were
suggested some time ago. One model is that the S-RNases
The difficult question of sex: the mating game Franklin-Tong
enter incompatible pollen tubes via a specific receptor so
that their RNA is specifically degraded, resulting in
inhibition of pollen-tube growth. The other model proposes
that S-RNases enter pollen tubes regardless of their allele
specificity, and that the S-RNases are distinguished when
internalized in the pollen. A major advance in our understanding of this SI system is the demonstration that
S-RNases enter compatible pollen tubes [35•], suggesting
that uptake of the S-RNase is independent of S genotype.
At the end: (if you’re lucky!) fertilization
At the end of its journey — if it’s lucky — the pollen tube
reaches the egg cell and double-fertilization takes place.
Not much is known about this process, although a major
breakthrough came a few years ago in the form of evidence
of an increase in [Ca2+]i following fusion of sperm and egg
cell upon in vitro fertilization in maize [36], suggesting that
a specific signal transduction cascade was triggered by this
event. Strikingly, the [Ca2+]i signal appeared (at least
superficially) similar to the [Ca2+]i-wave motifs triggered
by fertilization in other systems [37,38]. More recently, a
long-lasting Ca2+ influx has been demonstrated in the
vicinity of the sperm entry site, which spreads to the plasma
membrane of the whole zygote [39•]. These findings constitute an important step forward in our understanding of
fertilization in higher plants. However, we still do not know
if the increases in [Ca2+]i are due to the Ca2+ influx, or what
their roles in triggering post-fertilization events are.
Conclusions
There has been considerable activity in the field of plant
reproductive biology over the past few years, and a number
of important advances have been made over the past year
or so. Although we still have no clear overview of all the
components involved in pollen-tube guidance or of the
signals encouraging or preventing pollen tubes from
growing through the pistil tissues, we are beginning to
get a much better idea of some of the major players in this
important mating ‘game’.
Update
It has recently been demonstrated that SCR/SP11 interacts
with SRK in vitro in an S-allele-specific manner [40,41].
When bound to SRK, SCR/SP11 induces SRK autophosphorylation [41]. Furthermore, evidence that SLG (a
stigmatic component whose role in the SI response has
been questioned) interacts with SRK to form a high-affinity
receptor complex for SCR/SP11, has established that
SLG does, indeed, directly participate in the SI response.
Further evidence of the guidance of pollen tubes by the
components of the embryo sac has been obtained.
Interestingly, this evidence suggests that the egg cell is
not responsible for providing guidance signals in Torenia
fourenieri. By studying the effects of laser-ablation of different
cells in the embryo sac on directional growth of the pollen
tube, it has been ascertained that the two synergids, located
either side of the egg cell, are largely responsible for the
guidance signals [42].
17
Acknowledgements
Work in the author’s laboratory is funded by the Biotechnological and
Biological Science Research Council (BBSRC).
References and recommended reading
Papers of particular interest, published within the annual period of review,
have been highlighted as:
• of special interest
•• of outstanding interest
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•
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The cloning of CER6 by positional mapping reveals that this gene is identical
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18
Growth and development
depolarization or inhibition of growth. RopGAP1 overexpression recovered
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•
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•
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The analysis of transgenic plants suggests that although S-locus dominance
relationships in the stigma appear to be determined by SRK, surprisingly,
this protein does not seem to determine the levels of stigma expression of
self-incompatibility.
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fragmentation triggered in the self-incompatibility response in
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•
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that some sort of discriminatory event takes place within the pollen so that
only incompatible pollen tubes are inhibited.
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The vibrating-probe technique was used to demonstrate that Ca2+ influx is
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