Journal of Experimental Botany, Page 1 of 13doi:10.1093/jxb/erq021
REVIEW PAPER
Pollen-pistil interactions regulating successful fertilization inthe Brassicaceae
Laura A. Chapman1 and Daphne R. Goring1,2,*
1 Department of Cell and Systems Biology, University of Toronto, Toronto, Canada M5S 3B22 Centre for the Analysis of Genome Evolution and Function, University of Toronto, Toronto, Canada M5S 3B2
* To whom correspondence should be addressed: E-mail: [email protected]
Received 30 November 2009; Revised 18 January 2010; Accepted 20 January 2010
Abstract
In the Brassicaceae, the acceptance of compatible pollen and the rejection of self-incompatible pollen by the pistil
involves complex molecular communication systems between the pollen grain and the female reproductive structures.
Preference towards species related-pollen combined with self-recognition systems, function to select the most
desirable pollen; and thus, increase the plant’s chances for the maximum number of successful fertilizations and
vigorous offspring. The Brassicaceae is an ideal group for studying pollen–pistil interactions as this family includes
a diverse group of agriculturally relevant crops as well as several excellent model organisms for studying both
compatible and self-incompatible pollinations. This review will describe the cellular systems in the pistil that guide thepost-pollination events, from pollen capture on the stigmatic papillae to pollen tube guidance to the ovule, with the
final release of the sperm cells to effect fertilization. The interplay of other recognition systems, such as the self-
incompatibility response and interspecific interactions, on regulating post-pollination events and selecting for
compatible pollen–pistil interactions will also be explored.
Key words: Interspecific crosses, pistil, pollen, pollen tube guidance, receptor kinases, self-incompatibility, signalling.
Introduction
The acceptance of compatible pollen and the subsequent
steps leading to successful fertilization is a complex and co-
operative process between the pollen and the receptive pistil.In the crucifer family (Brassicaceae), once a compatible
pollen grain lands on the stigma, pollen grain adhesion
occurs, followed by foot formation, pollen hydration, and
germination. The pollen tube emerges and penetrates the
stigmatic surface. It is then guided through the stigma, style,
and septum, and finally onto the funiculus to enter the
micropyle of the ovule where fertilization can occur (Fig. 1).
The sequential events from pollen adhesion to the path ofpollen tube growth through the pistil to the ovule for
fertilization have been carefully documented at the ultra-
structural level in Brassica spp. and Arabidopsis thaliana (Hill
and Lord, 1987; Elleman and Dickinson, 1990; Elleman
et al., 1992; Kandasamy et al., 1994; Hulskamp et al., 1995b;
Lennon et al., 1998). For all of these steps, there is the basic
requirement of proper reproductive tissue formation
(reviewed in Blackmore et al., 2007; Colombo et al., 2008;
Crawford and Yanofsky, 2008; Punwani and Drews, 2008;
Dickinson and Grant-Downton, 2009). In addition to de-velopmental mutants, plants defective in pollen tube growth
and guidance have been identified through genetic screens
leading to the identification of female and male components
regulating post-pollination events (Preuss et al., 1993;
Hulskamp et al., 1995a; Johnson et al., 2004; Boavida et al.,
2009). Finally, a number of species in the Brassicaceae are
known to have self-incompatibility systems which lead to the
recognition and rejection of ‘self’ pollen at a very early stageof pollen–pistil interactions (reviewed in de Nettancourt,
2001; Franklin-Tong, 2008). Thus, there is clearly a complex
level of communication taking place between the pollen and
pistil to ensure successful fertilization. This review discusses
the step-wise mechanisms involved in compatible pollen
responses for members of the Brassicaceae, and highlights
where species specificity to the pollen–pistil interactions is
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determined and how the self-incompatibility pathway inter-cepts the compatible pollen response.
Pollen capture and adhesion to the stigmaticpapillae
Members of the Brassicaceae have a dry stigma, which
refers to the absence of free-flowing surface secretions; one
of the interesting features of this trait is the early selectivity
of pollen capture following pollination (Heslop-Harrison
and Shivanna, 1977; Dickinson, 1995). Once pollen grains
come into contact with stigmatic papillae, only pollen grains
recognized as compatible are accepted, thus allowing plantsto ignore foreign pollen (Fig. 2). These compatible inter-
actions appear to be confined to species within the family,
but clearly can occur beyond the species level (Hulskamp
et al., 1995a). For example, successful pollinations, as
measured by pollen tube penetration into the stigma, have
been observed in interspecific and intergeneric crosses in the
Brassicaceae (Sampson, 1962; Hiscock and Dickinson,
1993; Lelivelt, 1993). However, these interactions are notstraightforward as there are other factors governing pollen
acceptance within the Brassicaceae. For instance, if present,
the self-incompatibility system is activated at the stage of pollen
adhesion, and self-pollinations, as well as reciprocal pollina-
tions between plants sharing the same self-incompatibility
alleles, are rejected (reviewed in Fujimoto and Nishio, 2007).
Furthermore, interspecific or intergeneric pollen acceptance
can be regulated by a phenomenon called unilateral incom-patibility where pollen from one species is rejected by the
pistil of another species, yet the reverse cross is successful
(Sampson, 1962; Hiscock and Dickinson, 1993; Takada
et al., 2005). For example, no pollen tube penetration was
observed on self-incompatible B. oleracea pistils when
pollinated with different Brassicaceae species/genera pollen,
while self-fertile A. thaliana accepted a wide range of pollen
grains following the same survey (Hiscock and Dickinson,
1993). Typically, unilateral incompatibility occurs when the
female recipient is self-incompatible and the pollen donor is
from a different species that is self-fertile. Consistent with
unilateral incompatibility, pollinations between two differ-ent self-incompatible species are also typically unsuccessful
(Sampson, 1962; Hiscock and Dickinson, 1993). Thus, there
appears to be a basic ‘family-wide’ pollen recognition
system present in the Brassicaceae, but this recognition can
be attenuated in stigmas of self-incompatible plants for
interspecific or intergeneric pollen.
Fig. 1. Schematic of a pollinated A. thaliana pistil.
Fig. 2. Factors regulating pollen–stigma interactions in the Brassi-
caceae. Illustrations for the four post-pollination stages are shown,
and pollen and stigma factors that are implicated in each stage are
listed. Asterisks (*) indicate events that are inhibited or disrupted
during the self-incompatibility response. Please see the text for
further details and references.
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Two studies have specifically examined the early stages of
pollen capture and adhesion in interspecific pollinations, and
observed that pollen from plants outside the Brassicaceae
displayed much lower adhesion to B. oleracea (Luu et al.,
1998) and A. thaliana (Zinkl et al., 1999) stigmas as expected.
Despite the different approaches used to measure pollen
adhesive forces (reviewed in Heizmann et al., 2000), both
studies also found that crosses between Brassica spp. and A.thaliana tended to show poor pollen capture and adhesion
(Luu et al., 1998; Zinkl et al., 1999). Perhaps, some increased
specificity in pollen capture is modulated by different binding
affinities at this early stage. However, Luu et al. (1998)
surveyed several other Brassicaceae spp. and found high
levels of pollen adhesion following interspecific crosses.
Interestingly, the early stages of pollen capture occurred
irrespective of self-incompatibility or unilateral incompatibil-ity responses in play, but did not typically proceed beyond
the pollen adhesion stage (Luu et al., 1997a, 1998).
Pollen and stigma components required for pollencapture and adhesion
The early stages of pollen capture and adhesion involve the
exine (Gaude and Dumas, 1984; Zinkl et al., 1999), which is
the highly sculptured outer wall of the pollen grain,
composed primarily of sporopollenin (reviewed in Piffanelli
et al., 1998). Formation of the exine is essential for pollen
grain integrity as mutants with severe exine defects also
display reduced pollen viability (Paxson-Sowders et al., 2001;Guan et al., 2008). However, exine patterning is less critical
as a number of mutants with disrupted exine patterning have
been found to be viable (Nishikawa et al., 2005; Ariizumi
et al., 2008; Suzuki et al., 2008; Dobritsa et al., 2009a).
Interestingly, the exine was found to be the only component
required for the initial step of A. thaliana pollen capture onto
the A. thaliana stigma (Zinkl et al., 1999), and mutant pollen
grains with malformed exines have reduced adhesion to thestigmatic surface (Zinkl and Preuss, 2000; Nishikawa et al.,
2005; Dobritsa et al., 2009a). The chemical basis for this
exine specificity is not yet well understood; however, insight
into this specificity may emerge as the chemical nature of
sporopollenin becomes better known. Through the analysis
of A. thaliana mutant pollen grains that lack the exine layer,
recent progress has been made in identifying the candidate
biosynthetic enzymes required for exine/sporopollenin pro-duction (Morant et al., 2007; de Azevedo Souza et al., 2009;
Dobritsa et al., 2009b).
Following the initial adhesive interaction, the subsequent
stronger interactions between the pollen grain and stigmatic
papilla require proteins and lipids from both surfaces
(reviewed in Roberts et al., 1980; Dickinson et al., 2000). On
the pollen side is the requirement for the pollen coat that is
deposited in the interstices of the exine as shown in B.oleracea (Stead et al., 1980; Elleman and Dickinson, 1996).
Lipids are the main component of the pollen coat followed
by proteins, which include oleosin-like proteins, enzymes,
and small pollen coat proteins (PCPs) (Doughty et al., 1993;
Stephenson et al., 1997; Mayfield et al., 2001; Murphy, 2006).
On the stigmatic side, early studies on Brassica spp. showed
that the papillae have a waxy cuticle covered with the pellicle,
a thin proteinaceous layer, which is required for pollen
adhesion (Mattson et al., 1974; Stead et al., 1980; Zuberi
and Dickinson, 1985b; Elleman et al., 1992; Elleman and
Dickinson, 1996). The proteins and lipids in the pollen
coat and on the surface of the stigmatic papillae intermix
(termed foot formation) in a process that is essential for theacceptance of compatible pollen (Elleman and Dickinson,
1990; Kandasamy et al., 1994). Poor adhesion at this stage
was observed in A. thaliana mutants lacking the pollen coat
and in B. oleracea when the pollen coat was removed (Preuss
et al., 1993; Luu et al., 1997a; Zinkl et al., 1999).
Cell–cell communication following pollen adhesion
One of the functions of the foot formation is likely to bring
the pollen and stigmatic signalling proteins together for the
putative ‘family-wide’ pollen recognition system (Fig. 2).
Candidates for this recognition process are two stigma-
specific proteins, the Brassica S-locus glycoprotein (SLG)and the Brassica S-locus related-1 (SLR1) proteins, which
have been implicated in pollen adhesion. Stigmas from B.
napus plants, with antisense suppressed SLR1, displayed
reduced pollen adhesion (Luu et al., 1997b, 1999). Further-
more, blocking either SLG or SLR1 by treating B. oleracea
stigmas with their respective antibodies also reduced pollen
adhesion (Luu et al., 1999). SLG was first identified as
a candidate S-locus gene for Brassica self-incompatibility,but is not essential for this trait (Nasrallah et al., 1985;
Takasaki et al., 2000; Silva et al., 2001). SLR1 was
identified by its sequence similarity to SLG (Isogai et al.,
1988; Lalonde et al., 1989; Trick and Flavell, 1989;
Watanabe et al., 1992). Interestingly, SLG and SLR1 are
secreted stigmatic glycoproteins with sequence similarity to
the extracellular domains of the large S-domain receptor
kinase family (Takayama et al., 1987; Isogai et al., 1988;Shiu and Bleecker, 2003). Thus, this raises the question of
whether they are functioning alongside a related receptor
kinase. In keeping with this, both SLG and SLR1 have been
found to bind to the small pollen coat proteins, PCP-A1 and
SLR1-BP, respectively (Doughty et al., 1998; Takayama
et al., 2000), and these interactions are proposed to mediate
pollen adhesion. With pollen adhesion and foot formation,
a hydrophilic environment is created for pollen hydration,and this would also allow the small PCPs to freely pass to
the stigmatic surface to interact with SLR1, SLG, and
perhaps other stigmatic receptors to mobilize the next steps.
Similar interactions occur at this stage following a self-
incompatible pollination (Fig. 2). There are two key
regulators of this response, the small pollen coat protein, S-
locus cysteine rich/S-locus protein 11 (SCR/SP11), and the
stigma-localized S Receptor Kinase (SRK). First isolated inBrassica spp., these proteins are encoded by two tightly-
linked and co-evolved polymorphic genes (reviewed in
Watanabe et al., 2008). The linked SCR/SP11 and SRK
alleles are referred to as S haplotypes, and pollen rejection
occurs between plants sharing the same S haplotype. SCR
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and SRK genes have also been identified in other self-
incompatible Brassicaceae spp. and characterized in more
detail for A. lyrata (reviewed in Sherman-Broyles and
Nasrallah, 2008). Following the attachment of ‘self’ pollen
to the Brassica stigma, SCR/SP11 binds to the membrane-
localized SRK, and this receptor–ligand interaction acti-
vates SRK (Kachroo et al., 2001; Takayama et al., 2001;
Shimosato et al., 2007). The SRK signalling pathway is thenactivated in the stigmatic papilla leading to rejection of the
‘self’ pollen (reviewed in Samuel et al., 2008; Haasen and
Goring, 2010). The intracellular signalling pathway includes
the Brassica M Locus Protein Kinase (MLPK), a plasma
membrane localized protein kinase, which interacts with
and is phosphorylated by SRK. MLPK is required for the
self-incompatibility response and is proposed to function in
a complex with SRK to activate downstream signallingproteins (Murase et al., 2004; Kakita et al., 2007a, b). The
next step in the pathway is proposed to be the ‘activation’
of the Brassica Arm-Repeat Containing-1 (ARC1) E3
ubiquitin ligase. ARC1 binds to the activated SRK kinase
domain and is required downstream of SRK for the self-
incompatibility response (Gu et al., 1998; Stone et al., 1999,
2003). ARC1, as a functional E3 ligase, is proposed to
inactivate factors in the stigmatic papilla that are normallyrequired to accept compatible pollen. Recently, one factor,
B. napus Exo70A1, has been identified as an ARC1 target
(Samuel et al., 2009). The result of the activated self-
incompatibility response is that pollen is inhibited as
described further in the following sections.
The unilateral incompatibility response, described above
with interspecific crosses, suggests that there are some
interactions occurring in the stigma between the proteinspromoting the self-incompatibility response and the ‘family-
wide’ compatible pollen recognition system. With related
signalling proteins involved, perhaps there are competitive
binding interactions taking place between SRK, which
promotes ‘self’ pollen rejection, and those receptors which
positively regulate pollen acceptance. However, Kandasamy
et al. (1994) reported that a Brassica line with an SRK
mutation was still able to reject Arabidopsis pollen, suggest-ing that SRK is not required for the unilateral incom-
patibility response. It is important to note that surveys
for unilateral incompatibility found several exceptions to
the rules of unilateral incompatibility suggesting that this
trait may be quite complex (Sampson, 1962; Hiscock and
Dickinson, 1993; Luu et al., 1998). A more complete picture
of the molecular interactions occurring as part of the
unilateral incompatibility response will clearly requirea broader understanding of the initial signalling in the
stigma for the ‘family-wide’ compatible pollen recognition,
at both the protein and genetic levels.
Pollen hydration, following adhesion to thestigmatic papillae
The pollen hydration phase is an important step in the
compatible pollen response as it allows the quiescent,
desiccated pollen grain to regain its metabolic activity prior
to pollen tube emergence (Fig. 2). Compared to wet stigmas
on which pollen grains hydrate by default, the process of
pollen grain hydration on dry stigmas is highly regulated
(Zuberi and Dickinson, 1985a; Sarker et al., 1988). Pollen
hydration is one of the earliest steps blocked in a self-
incompatible pollination (Dickinson and Elleman, 1985;
Dickinson, 1995). The lipid and proteinaceous componentsof the pollen coat are essential to pollen hydration, and
during pollen foot formation, there are changes to the
pollen coat, termed coat conversion, that were initially
observed in B. oleracea (Elleman and Dickinson, 1986). In
this process, the lipids are thought to reorganize, perhaps
through the actions of the lipid-binding oleosin-like pro-
teins, to create a capillary system through which water can
flow from the stigma to the pollen grain (Murphy, 2006).
The role of lipids in pollen hydration
Clues to the importance of pollen coat lipids came from A.
thaliana mutants with defects in long-chain lipid metabolismsuch as the male sterile eceriferum (cer) mutants (Preuss
et al., 1993; Hulskamp et al., 1995a). In a strong cer6
mutant, the absence of long-chain lipids resulted in an
absence of pollen coat on the pollen grain surface and the
pollen failed to hydrate on the stigma. Interestingly, this
defect could be rescued by high environmental humidity or
the application of lipids to the stigma, both of which
allowed the pollen grain to hydrate and germinate leadingto successful fertilization (Preuss et al., 1993; Wolters-Arts
et al., 1998). A weaker cer6 mutant and the cer1 mutant
showed a reduction in the lipid droplets of the pollen coat
and also failed to hydrate on the stigma, but this defect
could be rescued by co-pollination with wild-type pollen
(Preuss et al., 1993; Hulskamp et al., 1995a). Further
characterization of these cer mutants and their respective
genes showed that CER1 was an enzyme needed for theconversion of long chain aldehydes to alkanes during the
process of wax biosynthesis (Aarts et al., 1995), that CER6
is required for the production of long chain fatty acids
(Fiebig et al., 2000).
In contrast to the aforementioned lipid mutants is the A.
thaliana fiddlehead (fdh) mutant that showed altered cuticle
composition over the entire shoot and allowed for pollen
germination and pollen tube growth on the shoot epidermis,but not on cotyledons or roots (Lolle and Cheung, 1993).
Intriguingly, Lolle and Cheung (1993) tested the species
specificity of this and found that pollen from other
Brassicaceae spp. had a similar response as A. thaliana
pollen, while pollen grains from plants outside of this family
adhered poorly to the fdh mutant shoot epidermis. Thus,
the ‘family-wide’ recognition system seems to be intact in
the shoot epidermis of the fdh mutant. When pollen grainsfrom the cer mutants were tested for hydration on the fdh
mutant shoot epidermis, no hydration was detected, in-
dicating that the pollen coat was required for this response
(Lolle et al., 1997). Lipid profiles from the fdh shoots
showed an increase in long chain fatty acids relative to the
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wild type and, consistent with this, the FDH gene is
predicted to encode a long-chain fatty acid elongase (Lolle
et al., 1997; Pruitt et al., 2000). Interestingly, the application
of lipids on wild-type leaves also promoted pollen germina-
tion and pollen tube growth (Wolters-Arts et al., 1998).
The pollen oleosin-like proteins have been implicated in
pollen hydration through the analysis of the A. thaliana
glycine-rich protein (grp)17 mutant (Mayfield and Preuss,2000). The grp17 mutant pollen had a pollen coat that was
normal in appearance, but was missing the GRP17 oleosin-
domain protein. The effect of this was a significant delay in
the initiation of hydration, in comparison to wild-type
pollen, although once initiated, a normal rate of hydration
was observed. A pollen coat enzyme has also been
implicated in pollen hydration through the analysis of a T-
DNA insertion mutant for the A. thaliana extracellular
lipase 4 (EXL4) gene (Updegraff et al., 2009). The exl4
mutant pollen is morphologically the same as wild-type
pollen grains and initiated hydration at roughly the same
time as the wild-type, but at a slower rate, resulting in
a significantly longer time for hydration. The exl4 mutant
pollen was found to have reduced esterase activity, relative
to the wild type, suggesting that EXL4 is an esterase with
a role in lipid modification (Updegraff et al., 2009).
Stigmatic responses regulating pollen hydration
Recently, Exo70A1 was identified as a protein required in the
stigma for pollen hydration in B. napus and A. thaliana. Eitherthe absence or a reduction of Exo70A1 in the stigma resulted
in poor pollen hydration for both A. thaliana and B. napus. In
A. thaliana, an RFP-tagged B. napus Exo70A1 construct was
able to rescue this defect, and, interestingly, RFP:Exo70A1
was localized to the plasma membrane in the stigmatic
papillae of mature flowers (Samuel et al., 2009). Initially,
Exo70A1 was isolated as a substrate for the Brassica ARC1
E3 ubiquitin ligase, and the reduced pollen hydrationassociated with the loss of Exo70A1 fits with ARC1’s role in
the inhibition of compatibility factors, such as Exo70A1, to
promote the self-incompatibility response. Consistent with
this, the overexpression of RFP:Exo70A1 in B. napus stigmas
was able to partially overcome the self-incompatibility re-
sponse, favouring self-compatibility (Samuel et al., 2009).
Exo70A1 is a putative subunit of the plant exocyst complex
proposed to be involved in the tethering of post-Golgisecretory vesicles to the plasma membrane (reviewed in He
and Guo, 2009; Zarsky et al., 2009). Thus, Exo70A1, in its
role with the exocyst complex, may be involved in tethering
secretory vesicles to the stigmatic papilla plasma membrane at
the pollen contact site to deliver other stigmatic factors
required for pollen hydration. Vesicle-like structures have
been observed in B. oleracea stigmatic papillae following
treatment with pollen coat (Elleman and Dickinson, 1996).The signalling events that regulate the stigmatic responses
to compatible pollen at this stage involve calcium. Follow-
ing pollination, real-time imaging of calcium levels in the A.
thaliana stigmatic papillae led to the discovery of three
cytosolic calcium increases in the apical region of the papilla:
the first following pollen hydration, the second increase prior
to pollen germination, and the third increase when pollen
tube penetration of the stigmatic cell wall occurred (Iwano
et al., 2004). In B. rapa, changes in the actin cytoskeleton
were also documented following pollination (Iwano et al.,
2007). The application of either compatible pollen grains or
pollen coat, induced actin polymerization in the apical region
of the stigmatic papilla. This resulted in the increasedformation of actin bundles at the apical tip at a time when
the pollen grains were undergoing hydration (Iwano et al.,
2007), an observation consistent with the idea of secretory
vesicles being delivered along the actin cytoskeleton to the
apical tip/pollen contact site for exocyst tethering and
membrane fusion (He and Guo, 2009). In addition, changes
in the tubular vacuolar network in the apical region of the
stigmatic papilla were observed with the large central vacuoleorienting towards the pollen contact site with compatible
pollen, perhaps to promote pollen hydration (Iwano et al.,
2007). By contrast, the application of self-incompatible
pollen grains or pollen coat was associated with a decrease
in actin filaments in the apical region and a more disorga-
nized appearance of the tubular vacuolar network in the
stigmatic papillae (Iwano et al., 2007).
Pollen germination and pollen tube growththrough the stigmatic surface
Once the pollen grain has successfully hydrated, the pollen
germinates and a pollen tube emerges (Fig. 2). The pollentube typically grows through the foot to penetrate the
stigmatic cuticle and then enters the outer layer of the
stigmatic cell wall (Elleman et al., 1992; Kandasamy et al.,
1994). For B. oleracea, the pollen tube grows through the
inner and outer layers of the cell wall (Elleman et al., 1992).
A. thaliana pollen tubes also grow through the two layers of
the stigmatic cell wall, but were observed to grow between
the cell wall and the plasma membrane as well (Ellemanet al., 1992; Kandasamy et al., 1994). The invasion of the
pollen tube through the stigmatic cuticle and cell wall would
be predicted to require enzyme modification of these layers,
to allow further pollen tube growth. Searches for cutinases
or esterases that could break down the stigmatic cuticle
have uncovered esterase activities in both Brassica pollen
and the stigma extracts, and treatment of stigmas with
a serine esterase inhibitor blocked pollen tube penetration(Hiscock et al., 1994, 2002; Lavithis and Bhalla, 1995). The
stigmatic cell wall at the pollen contact point has been
observed to be expanded, presumably due to the action of
cell wall-modifying enzymes (Elleman and Dickinson, 1990;
Kandasamy et al., 1994). Similarly, cell wall-modifying
enzymes such as polygalacturonases and pectin esterases
have also been identified in Brassica pollen (Kim et al.,
1996; Dearnaley and Daggard, 2001). The initial expansionof the stigmatic cell wall, however, is more likely to be due
to enzymes secreted by the stigmatic papilla and is
consistent with observation by Elleman and Dickinson
(1996) that ER, Golgi, and vesicle-like structures are
associated with the sites of stigmatic cell wall expansion.
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Exo70A1 is also required in the stigma for the penetration
of compatible pollen tubes. When B. napus stigmas with
reduced levels of Exo70A1 mRNA were pollinated with
compatible pollen, very little seed set was observed and most
pollen tubes failed to penetrate the stigmatic barrier,
although pollen tubes could be seen growing over the surface
of papillae cells unable to penetrate the cuticle or cell wall
(Samuel et al., 2009). Similar to the role of the exocyst inpollen hydration (described in the previous section), the
tethering of secretory vesicles by the exocyst complex to the
stigmatic papilla plasma membrane at the pollen contact site
may be required for the delivery of enzymes required for
pollen tube penetration through the stigmatic cuticle and cell
wall. Once this process is initiated, the pollen tube probably
produces its own cell-wall modifying enzymes and is able to
partially support its continued growth through the cell walllayers of the stigmatic papillae.
Pollen tube guidance to the femalegametophyte
When pollen tubes emerge at the base of the stigmatic
papillae, they grow intercellularly down to the style and
then through the transmitting tissue of the style and septum
(a central tissue that runs to the base of the ovary) (Hill and
Lord, 1987; Lennon et al., 1998). Once the pollen tube
emerges from the septum, it grows over the surface of the
septum to a funiculus (which attaches the ovule to theseptum). The pollen tube then grows along the funicular
surface to the micropylar opening of the ovule where it
enters to release the sperm cells (Fig. 1) (reviewed in
Yadegari and Drews, 2004). The transmitting tissue is
a specialized column of cells producing extracellular matrix
(ECM) material through which the pollen tubes navigate en
route to the ovules. It provides chemical gradients and
nutrients for pollen tube guidance and growth, and propertransmitting tract formation is essential for efficient pollen
tube guidance (reviewed in Crawford and Yanofsky, 2008).
For example, the No Transmitting Tract (NTT) gene in A.
thaliana is required for normal transmitting tract develop-
ment, and in ntt mutants, pollen tubes are no longer
restricted to growth through the septum. In addition, pollen
tube elongation was slower and more jagged in this mutant,
in comparison to the wild type (Crawford et al., 2007).Despite the abnormal transmitting tract in the ntt mutants,
pollen tubes were able to grow through the style and
fertilize the ovules at the apex of the ovary. This shows that
some long-range pollen tube guidance systems are still
functional, since the pollen tube can be directed to the
funiculus from what remains of the transmitting tract
(Crawford et al., 2007).
Pistil factors regulating pollen tube guidance
In recent years, it has become more clear that chemo-
attractant gradients in the pistil play an important role in
guiding pollen tubes to the ovules (Fig. 3) (reviewed in
Johnson and Lord, 2006; Crawford and Yanofsky, 2008;
Higashiyama and Hamamura, 2008). One of the first
molecules proposed to guide pollen tubes was c-aminobutyric acid (GABA) that was identified through the
analysis of the A. thaliana pollen-pistil (pop)2 mutant
(Wilhelmi and Preuss, 1996; Palanivelu et al., 2003). The
pop2 mutant displayed abnormal pollen tube guidance. The
pop2 pollen tubes were less able to find the micropyle and
frequently displayed an atypical behaviour with more than
one pollen tube present on a single funiculus. This defective
pollen tube guidance required both the pollen and pistil tocarry the recessive pop2 mutation, as wild-type pollen tubes
behaved normally on pop2 pistils (Wilhelmi and Preuss,
1996). The pop2 mutant was found to lack a functional
GABA transaminase, which led to excess levels of GABA in
the pistil. In wild-type pistils, GABA was found to be
present as a gradient, starting from the stigma and
increasing in concentration to the inner integument of the
ovule, and this gradient was proposed to guide the growingpollen tube (Palanivelu et al., 2003). Further advances in the
understanding of chemo-attractants were provided by the
discovery of chemocyanin in the lily system (Kim et al.,
2003), followed by the identification of the structurally-
related plantacyanin in A. thaliana (Dong et al., 2005). Both
peptides exist in increasing gradients along the style, and
Fig. 3. Model of pollen tube guidance to the female gametophyte
in A. thaliana. An illustration of a pollen tube growing to an ovule is
shown, with the guidance cues and genes that are proposed to
regulate pollen tube guidance and perception overlaid on this
diagram. If expression patterns are known, gene names are
coloured to match the cells where they are expressed. Coloured
boxes indicate steps that are disrupted in mutants. Please see the
text for further details and references.
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transmission tract leading to the ovary. When plantacyanin
was overproduced in A. thaliana pistils, wild-type pollen
tubes had difficulties at the earliest stages of pollen tube
guidance, circling the stigmatic papillae and even growing
away from the style (Dong et al., 2005).
During the course of the last decade, the understanding
of the role of the female gametophyte in producing pollen
tube guidance cues has increased immensely with thediscovery of funicular and micropylar guidance systems
(Fig. 3). Initially, genetic screens for pollen guidance defects
uncovered mutants in which the absence of the female
gametophyte results in impaired guidance of pollen tubes to
the ovule (Hulskamp et al., 1995a; Ray et al., 1997). The
novel Gamete-Expressed (GEX)3 gene, which is expressed
in the egg cell, is one factor that acts at this stage. Reduced
GEX3 expression in the pistil resulted in wild-type pollentubes being unable to locate the micropyle following growth
along the funiculus (Alandete-Saez et al., 2008). A compa-
rable phenotype was also observed in the A. thaliana
magatama1 (maa1) and magatama3 (maa3) mutants, in
which female gametophyte development was delayed, and
pollen tubes were unable to find the micropyle (Shimizu and
Okada, 2000). Interestingly, Shimizu and Okada (2000)
found similar phenotypes when examining the path ofpollen tubes following interspecific crosses with wild-type
pistils. When either B. napus or Orychophragmus violaceus
pollen was applied to A. thaliana pistils, the pollen tubes
grew to the septum, but very few were guided to the ovules,
with some pollen tubes mimicking the maa phenotype.
Interspecific pollen guidance to the micropyle was also
observed to be inefficient in a study with Torenia fournieri
(Higashiyama et al., 2006). Thus, interspecific barriers mayexist at the level of the micropylar guidance system to
prevent unproductive fertilization events. Another pheno-
type observed at high frequency in the A. thaliana maa
mutants was two pollen tubes approaching a single ovule.
This suggested that repulsive signals from the ovule to deter
the approach of more than one pollen tube at the funiculus
were disrupted (Shimizu and Okada, 2000). Recently, the
MAA3 gene was cloned and predicted to encode a helicasewith a role in RNA processing and metabolism (Shimizu
et al., 2008).
In searching for repulsive signals that would restrict more
than one pollen tube from growing on the funiculus, nitric
oxide has been suggested as a candidate for this signal
(Crawford and Yanofsky, 2008) on account of its ability to
cause a sharp turn in growing lily pollen tubes (Prado et al.,
2004). More recently, support for the role of nitric oxide inmicropylar guidance has emerged in a study with the A.
thaliana nitric oxide synthase (nos)1 mutant that is defective
in nitric oxide production and displays reduced fertility
(Guo et al., 2003; Prado et al., 2008). Wild-type pollen tubes
were able to grow in proximity to nos1 ovules, but became
deformed as they approached the micropyle (Prado et al.,
2008). In addition, staining for nitric oxide in wild-type
ovules showed an asymmetric distribution around themicropyle, perhaps indicating a function in funnelling
pollen tube at the micropyle (Prado et al., 2008).
The analysis of mutants defective in micropylar guidance
established the presence of this guidance system, but the
question then arose as to which female gametophyte cells
were the source of this signal. At maturity, the female
gametophyte is composed of two synergid cells, the egg cell,
a central cell and three antipodal cells (reviewed in Punwani
and Drews, 2008). Laser ablation studies in Torenia four-
nieri, in which different female gametophytic cells weresystematically destroyed, provided compelling evidence for
a micropylar guidance signal originating from the synergid
cells (Higashiyama et al., 2001). More recently, the attrac-
tant signal being secreted from the T. fournieri synergid cells
was identified as a group of proteins, termed the LURE
proteins, which belong to the defensin-like subgroup of
cysteine-rich polypeptides (Okuda et al., 2009). Interest-
ingly, the Brassica SCR/SP11 protein, which functions asa pollen ligand in the self-incompatibility response, also
belongs to this defensin-like family of proteins (Mishima
et al., 2003).
In A. thaliana, support for the role of synergid cells in
guiding pollen tubes to the micropyle came from the
analysis of the myb98 mutant (Kasahara et al., 2005). This
mutant displayed normal female gametophyte development,
with the exception of the synergid cells. As a result, pollentubes would grow to the funiculus, but were unable to find
the micropyle, suggesting that the affected synergid cells
were not producing the required signal for this guidance
step (Kasahara et al., 2005). MYB98 is a transcriptional
regulator expressed specifically in the synergid cells where it
functions to activate the expression of genes required for
both pollen tube guidance and the formation of the synergid
cell filiform apparatus (Punwani et al., 2007). The MYB98-regulated genes included predicted genes for small cysteine-
rich proteins, similar to the T. fournieri LURE proteins
(Jones-Rhoades et al., 2007; Punwani et al., 2007, 2008).
Another transcriptional regulator, Central Cell Guidance
(CCG), expressed in the central cell of the ovule, has also
been found to be necessary for pollen tube guidance to the
micropyle in A. thaliana (Chen et al., 2007).
Pollen tube perception in the ovule
Once the pollen tube enters the micropyle, the final stage is
pollen tube perception, where the pollen tube penetrates
a synergid cell and bursts to release the two sperm cells forfertilization (Fig. 3). In the A. thaliana feronia (fer)/sirene
(srn) mutant, the pollen tubes enter the micropyle but fail to
stop growing and are unable to rupture which results in the
formation of pollen tube coils within female gametophyte
(Huck et al., 2003; Rotman et al., 2003). The FER/SRN
gene encodes a receptor kinase belonging to the CrRLK1
subfamily of receptor kinases and is expressed in the
synergid cell in A. thaliana (Escobar-Restrepo et al., 2007;Hematy and Hofte, 2008). Interestingly, Escobar-Restrepo
et al. (2007) also tested interspecific crosses between A.
thaliana and two other Brassicaceae species, A. lyrata and
Cardamine flexuosa, and observed interspecific barriers at
the level of pollen tube perception. When the more closely
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related A. lyrata pollen was applied, roughly half of the
pollen tubes successfully fertilized A. thaliana ovules, while
most of the remaining A. lyrata pollen tubes displayed the
overgrown fer/srn-like phenotype. For the more distantly
related C. flexuosa pollen, many fewer A. thaliana ovules
attracted C. flexuosa pollen tubes, and the C. flexuosa
pollen tubes either coiled outside the micropyles, stopped
just after entering the micropyles, or entered the micropylesand continued to grow in the fer/srn-like phenotype in the
wild-type A. thaliana ovules (Escobar-Restrepo et al., 2007).
The A. thaliana LORELEI (LRE) gene, which encodes
a putative glucosylphosphatidylinositol-anchored protein, is
also expressed in the synergid cells, and the lre female
gametophyte mutant displays impaired sperm cell release,
similar to the fer/srn mutant (Capron et al., 2008).
Pollen factors required for pollen tube guidance andperception
While the discussion has largely focused on pistil factors
required for compatible pollinations, pollen tube guidance,
and fertilization, there is also a basic requirement of in-
tact cellular processes within the pollen tube to drivingrapid polar growth (reviewed in Cheung and Wu, 2008;
Moscatelli and Idilli, 2009). In addition, the pollen tubes
need to perceive the guidance cues within the pistil to direct
growth towards the female gametophyte. Therefore, genetic
screens have also focused on identifying male mutants
which display disrupted pollen tube guidance to the ovule
as an approach to finding these predicted pollen tube
receptors (Johnson et al., 2004; Boavida et al., 2009). Forexample, the Lycopersicon esculentum pollen-specific recep-
tor kinase, LePRK2, is required for pollen germination and
tube growth, and plays a role in responding to growth-
promoting signals from the pistil (Zhang et al., 2008). A
candidate for this growth-promoting signal is LeSTIG1,
a small cysteine-rich pistil protein, which binds to the
extracellular domain of LePRK2 and promotes pollen tube
growth in vitro (Tang et al., 2004). In A. thaliana, thecharacterization of the FER/SRN receptor kinase in the
synergid cells has recently led to the identification of two
closely related members functioning in the pollen tube to
regulate sperm cell release (Boisson-Dernier et al., 2009;
Miyazaki et al., 2009). The ANXUR1 (ANX1) and
ANXUR2 (ANX2) genes are expressed at highest levels in
the pollen, and double anx1/anx2 mutants produce pollen
tubes which rupture prematurely. Thus, ANX1 and ANX2are proposed to function in the pollen tube to co-ordinate
the timing of pollen tube rupture and release of the sperm
cells, in conjunction with the FER/SRN receptor kinase
signalling in the synergid cells (Boisson-Dernier et al., 2009;
Miyazaki et al., 2009). Given the late stage at which these
receptor kinases function, there are presumably additional
pollen tube receptors required for sensing guidance cues in
the transmitting tissue. The genetic screens for pollen tubeguidance defects have identified male mutants with disrup-
ted pollen tube guidance at earlier stages, and look
promising for uncovering new players in this process
(Johnson et al., 2004; Boavida et al., 2009).
Conclusions
At the stigmatic surface of Brassicaceae pistils, there are
complex pollen recognition signalling systems at play to
determine whether a pollen grain should be accepted. There
is evidence to support a ‘family-wide’ pollen recognition
system that allows for interspecific and intergeneric crosses;
however, some preference for species-specific pollen may
occur by different initial binding affinities of pollen grains
(Zinkl et al., 1999; Hiscock and Dickinson, 1993). As such,the identification of all protein and lipid factors contribut-
ing to pollen adhesion and hydration efficiency will be
essential to understanding these interactions more fully. In
addition, the self-incompatibility system is intrinsically
linked to the compatible pollen–pistil interactions and
functions to override the compatible pollen responses when
activated (Samuel et al., 2009). Thus, increasing our un-
derstanding of compatible pollen–pistil interactions willprovide an insight into how the self-incompatibility path-
way functions in the stigmatic papillae to block what would
otherwise be recognized as compatible pollen.
Once the pollen tube has penetrated the stigmatic surface
and continues to grow towards the ovule, there appear to be
other recognition systems in play for species selectivity.
Little support currently exists for a species-specific filter in
the septum; however, species specificity has been proposedin the form of variable concentrations of chemo-attractant
molecules and the differential abilities of pollen tubes from
different species to detect such compounds (Johnson and
Lord, 2006). A family-wide comparison of chemo-attractant
molecules would be needed to evaluate such an hypothesis.
Nevertheless, there is evidence for other layers of species
discrimination in regulating the final stages of pollen tube
guidance. This includes the micropylar guidance system thatfunctions most efficiently with species-specific pollen tubes
(Shimizu and Okada, 2000). In addition, barriers in the
female gametophyte act at the pollen tube perception stage
to prevent fertilization between more distantly related
species in the Brassicaceae (Escobar-Restrepo et al., 2007).
Finally, while not discussed in this review, there are
reproductive barriers taking place post-fertilization, such as
with interploidy crosses, which lead to non-viable embryos(for a review see Dumas and Rogowsky, 2008).
Acknowledgements
We thank members of the Goring laboratory for criticalreading of the manuscript. LAC is supported by a graduate
scholarship from the Natural Sciences and Engineering
Research Council of Canada (NSERC), and research in the
laboratory of DRG is supported by grants from NSERC
and a Canada Research Chair to DRG.
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