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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.

2 of 13 | Chapman and Goring



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

Pollen–pistil interactions in the Brassicaceae | 3 of 13



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




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.




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.




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




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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