The molecular and genetic basis of pollen–pistil interactions

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Review

Blackwell Science Ltd

Tansley review no. 128

The molecular and genetic basis of

pollen–pistil interactions

M. J. Wheeler, V. E. Franklin-Tong and F. C. H. Franklin

Wolfson Laboratory for Plant Molecular Biology, School of Biosciences, University of Birmingham,

Edgbaston, Birmingham B15 2TT, UK

Author for correspondence:

F. C. H. Franklin Fax:

+

44 121414 5925 Email: [email protected]

Received:

29 January 2001

Accepted:

1 June 2001

Summary

Over the past decade or so, there has been significant progress towards elucidatingthe molecular events occurring during pollination in flowering plants. This processinvolves a series of complex cellular interactions that culminates in the fusionbetween male and female gametes. The process also regulates crucial events suchas pollen adhesion, hydration, pollen tube growth and guidance to the ovules. Addi-tionally, in many instances, incompatibility mechanisms that control the acceptanceor rejection of pollen alighting on a recipient plant play a major role in the pollinationprocess. In this article we aim to review our current understanding of the componentsthat are implicated in enabling the pollen to deliver the male gametes to the ovaryand the molecular mechanisms by which they are thought to act.

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Contents

Summary 565

I. Introduction 565

II. Adhesion of pollen to the stigma 566

III. Pollen hydration 567

IV. Pollen germination and initial growth on the stigma surface 568

V. Pollen tube growth through the style and pollen tube guidance 569

VI. Control of pollen viability by incompatibility responses 572

1. Self incompatibility (SI) 573Gametophytic SI 573SI in the Solanaceae 573SI in Papaver 575Sporophytic SI 577SI in Brassica 577SI in Ipomoea 579

2. Interspecific incompatibility responses 579

VII. Conclusions and perspective 580

References 580

I. Introduction

Reproduction is a crucially important stage in the life cycle of allorganisms. As a consequence, there is an intense interest indetermining how this is controlled. The central importance ofreproduction in flowering plants has provided the justificationand driving force behind the extensive studies that have focusedon establishing the fundamental basis of this highly complex

process. Moreover, developments in molecular biology andgenetics will, no doubt, enable future applications based onthese fundamental studies, perhaps permitting the manipulationof reproductive processes, which could potentially have usesfor plant breeding.

In flowering plants one of the most important steps in thereproductive process is pollination. This has been the subjectof studies stretching back several centuries to pioneers such as

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Joseph Kolreuter who, in the 18th century, carried out extensivestudies of hybridizations between plant species. Later, CharlesDarwin was both fascinated and perplexed when noting that,in some instances, species with completely normal reproductiveorgans were unable to self-pollinate, yet were perfectlycapable of pollinating other individuals (Darwin, 1877). Theseproved to be the earliest observations of self-incompatibility.In this article we discuss the recent progress in elucidatingthe events that follow the arrival of a pollen grain on a recept-ive stigma. A cartoon of the route of the pollen from landingon the stigma, and its journey through the pistil to the ovaryis shown in Fig. 1, together with a summary of some of thecomponents identified as being involved in pollination, anddiscussed in this review. We also discuss the role of incompat-ibility systems, that act in a significant number of species tocontrol the acceptance or rejection of pollen alighting on arecipient plant, hence, playing a major role in the pollinationprocess.

II. Adhesion of pollen to the stigma

Initiation of pollination is dependent on the ability of thepollen grain to adhere effectively to the stigmatic surface. Thestigmatic surface of different plant species varies widely in bothmorphology and the presence or absence of exudates, andthese are likely determinants of the relative importance ofadhesion in any given species (Heslop-Harrison & Shivanna,1977). Control of pollen acceptance by adhesion is seen asparticularly important in species, such as those in the Brassicaceae,that have a dry stigma. By contrast, the surfaces of species, suchas those belonging to Solanaceae and Leguminosae that havewet stigmas appear to promote the adhesion of most pollenspecies (Zinkl & Preuss, 2000). Studies by Preuss

et al

. (1993)indicate that adhesion is under polygenic control. It is alsoapparent that, pollen adhesion in

Brassica

spp. increases overtime, requiring 30 min for maximum binding (Heizmann

et al.

, 2000). These two pieces of evidence suggest that thecontrol of pollen adhesion is likely to be complex (see Fig. 1).

In the Brassicaceae it is known that the pollen coat or tryphinecontains components involved in mediating cell–cell interactionsbetween pollen and stigma (Doughty

et al.

, 1993; Preuss

et al.

,1993; Dickinson, 1995). The presence of the pollen coat isessential for attachment of pollen to the stigmatic surface(Stead

et al.

, 1980; Elleman & Dickinson, 1986). It is also appar-ent that the application of isolated pollen coating invokesphysiological changes to the stigmatic papillae, notably a rapidexpansion of the outer layer of the stigmatic wall (Elleman &Dickinson, 1996). The interaction between stigma and pollenalso results in changes in the pollen coat enabling hydrauliccontinuity between the pollen grain and the stigmatic papillae(Elleman & Dickinson, 1986).

Most of the current research on pollen adhesion is focusedon factors affecting this process in

Arabidopsis thaliana

,

Brassicaoleracea

and

B. rapa

. Interactions between stigma and pollen

have been studied using either centrifugation (Luu

et al.

, 1997),detergent assays or spring displacement experiments (Zinkl

et al.

, 1999). It has been demonstrated that

A. thaliana

stigmasselectively bind

A. thaliana

pollen with much higher affinitythan pollen from related species and that this interaction occurswithin seconds of pollination (Zinkl

et al.

, 1999). In thesestudies, pollen from a range of monocotyledenous species wasfound to have little or no binding capacity to

A. thaliana

stigmas. Also, whilst pollen from several dicotyledonous speciesexhibited a range of binding capacities, in all instances it couldbe washed off by a detergent treatment that had little effect on

A. thaliana

pollen binding (Zinkl & Preuss, 2000). Interestingly,the study included

Brassica campestris

, which like

A. thaliana

is a member of the Brassicaceae. Despite this close relationship,the behaviour of

B. campestris

pollen binding was no differentto that of other dicotyledonous species, indicating a consider-able degree of specificity in this event.

A genetic screen using male sterile pistils, with the aim ofisolating mutants showing reduced pollen adhesion (Zinkl &Preuss, 2000), has resulted in the isolation of several

lap

(lessadherent pollen) mutants. The mutant

lap1

shows gross defectsin the exine, suggesting that it is the pollen coat that is importantin pollen adhesion. The fact that this mutant does not exhibitreduced fertility reveals that the adhesion process is independentof pollen hydration and growth. As yet, no genes have beenlinked to the

lap

mutants.Two stigmatic proteins have been implicated in playing

a role in adhesion in

Brassica

. A recent study of two membersof the

Brassica

self-incompatibility

S

gene family (see later),the S-locus glycoprotein (SLG) and the

S

-locus related pro-tein (SLR1) has suggested that they may both be involved inthis process. The force of adhesion between

Brassica

pollenand stigmas has been measured using a centrifugation assay(Luu

et al.

, 1997). In transgenic plants in which

SLR1

expres-sion was down-regulated by antisense suppression, a reduc-tion in adhesive force was found, whilst there was no effect onthe ability of the stigma to support pollen tube growth. A sim-ilar result was obtained when wild-type stigmas were treatedwith either anti-SLR1 or anti-SLG antibodies (Luu

et al.

,1999). The data from Luu

et al

. (1999), together with thelocalization of SLG and SLR1 within the cell wall rather thanthe pellicle (Kandasamy

et al.

, 1989; Umbach

et al.

, 1990),suggest that these proteins are involved in later steps of adhe-sion, rather than the initial binding event.

Both SLG and SLR1 have been shown to interact

in vitro

with pollen coat proteins (PCPs) (Doughty

et al.

, 1993; Hiscock

et al.

, 1995). The PCPs are an extensive family of gametophy-tically expressed, small, cysteine-rich proteins. Gel shift assayshave revealed that two family members PCP-A1 and PCP-A2,interact with SLG and SLR1, respectively (Doughty

et al.

,1993, 1998; Hiscock

et al.

, 1995). It is well established that, oncontact with the stigmatic surface, the pollen coat flows frombetween the baculae of the sporopollenin exine to form anadhesive foot at the papilla surface (Stead

et al.

, 1979; Elleman



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& Dickinson, 1990). Thus, it is reasonable to assume that thisevent would facilitate the PCP-SLR/SLG interaction

in vivo

(Doughty

et al.

, 2000). Initially, it had been suggested thatthe PCPs might be involved in some way in the

Brassica

incompat-ibility systems, which will be discussed later (Dickinson

et al.

,1997; Doughty

et al.

, 2000). Whilst their exact function hasnot yet been unequivocally established, at least some membersof the PCP family may play a role in pollen adhesion throughtheir interaction with SLR1/SLG.

III. Pollen hydration

Following adhesion of pollen to the stigmatic surface,successful pollen tube growth depends upon the hydration ofthe pollen grain (see Fig. 1). The processes involved in pollenhydration are, however, not as well characterized as thoseinvolved in adhesion. This, in part, is due to technical difficultiesassociated with the study of this process. The diverse nature of

the stigmatic surface in different angiosperm families sug-gests that hydration mechanisms are also likely to be divergent,although the results of hydration, notably a far-reaching reorgan-ization of the vegetative cell (Heslop-Harrison & Heslop-Harrison, 1992), are likely to be similar.

There is considerable evidence to suggest that long-chainlipids act as signals to stimulate pollen hydration. Mutantswith defects in pollen hydration, such as the

cer

mutants and

pop1

, have been shown to have defects in lipid biosynthesis (Preuss

et al.

, 1993; Hülskamp

et al.

, 1995a). Studies by Wolters-Arts

et al

. (1998) demonstrated that lipid-rich stigma exudatesfrom

Petunia

could restore normal pollen germination andgrowth in transgenic

Nicotiana tabacum

, in which the secretoryzone of stigmas was ablated using a cytotoxic

STIG1-barnase

gene (Goldman

et al.

, 1994). Application of one of the exudatelipids, the long chain lipid trilinolein, enabled

pop1

mutantsto germinate normally. It also enabled pollen to hydrate andgerminate on leaf tissue following removal of the cuticle, which

POLLEN COMPONENTS• pollen adhesion components eg. lap mutants• pollen hydration components eg. cer, pop mutants (lipid biosynthesis), grp17 mutant, aquaporins• pollen coat proteins (PCPs)• receptor-like kinases • components involved in SI eg. SCR, SP11, SBP, p26

Genes, gene products and other componentsidentified in various tissues as playing a role

in pollination processes

PISTIL COMPONENTS

• components that interact with pollen e.g. SCA• SI-related components involved in pollination • eg. SLG, SLR1, ARC1• components involved in SI eg. S protein, SRK

mponents• arabinogalactan proteins (AGPs) eg. TTS, NaTTS, GaRSGP• pistil specific extensin-like proteins (PELPs) eg. pt11, Pex1

• components involved in SI eg. S-RNases

pollenadhesion& hydration

Steps in the pollination processPartsof thepistil

stigma

style

ovary

transmitting tract

fertilization

pollen germination

pollen tube growth

pollen tube growth

ovules

OVULE COMPONENTS involved in POLLEN TUBE GUIDANCE

• mutations affecting embryo sac/pollen guidanceeg.sin1, bel1, 47H4, 54D12

• ECM co

Fig. 1 Structure of the pistil, pollination events, and components identified as playing a role in pollination. The left-hand side of this figure shows a generalized basic pistil structure (indicated in green, labelled in red). The basic steps in the pollination process are indicated in blue. The boxes indicate some of the components (genes, gene products and mutants) identified as being important in pollination.




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is normally a nonreceptive surface (Wolters-Arts

et al.

, 1998).Further support for the role of the exudate in hydration wasobtained in studies of

Nicotiana

, during which it was observedthat pollen grains alighting at the top of stigmatic papillae,that is above the level of the exudate, failed to hydrate (Lush

et al.

, 1998).Although the evidence indicates a key function for lipids as

signals for pollen hydration, it now seems likely that they arenot the only molecules involved in this process. Recently, an

A. thaliana

mutation that results in the loss of the oleosin-domain GRP17, the most abundant pollen coat protein, hasbeen described (Mayfield & Preuss, 2000). As a result of thismutation, pollen hydration was delayed almost three-foldcompared to wild-type pollen, which normally hydrates within4–5 min of alighting on the stigma. The delay was due to afailure to interact with the stigma, rather than failure to absorbwater

per se

, as once hydration had initiated, the mutant pollenexpanded at the same rate as wild-type pollen. Although the

grp17

pollen does eventually hydrate, the authors speculatethat the mutation substantially reduces its fitness comparedwith wild-type pollen. Whether GRP17 directly communicateswith the stigma or modulates the activity of other molecules,such as lipids, remains to be established (Mayfield & Preuss,2000).

One major breakthrough in our understanding of com-ponents that potentially regulate water flow to pollen grainsduring hydration, was the identification of an aquaporin thatis essential for pollen hydration (Ikeda

et al.

, 1997). Aquaporinsare water channel proteins (Chrispeels

et al.

, 1999). They areabundant components of the plasma membrane existing as atetramer of 27 kDa subunits, each comprised of six membrane-spanning domains. It is thought that each individual polypeptideforms a water channel, rather than the channel being formedby the tetrameric structure. Although the aquaporin describedby Ikeda

et al

. (1997) has been specifically implicated in thecontrol of self-incompatibility (see later), it seems apparentthat hydration in other species is likely to depend upon thepresence of similar proteins.

IV. Pollen germination and initial growth on the stigma surface

Once pollen is correctly hydrated it must then germinate. Itwill then grow through the stigma before it reaches thestylar tissues and eventually the ovules (see Fig. 1). The initialpenetration of the stigmatic surface has been studied in severalspecies with dry stigmas and appears to be quite variable. In

A. thaliana

and

B. oleracea

the pollen tube penetrates thestigmatic cuticle and enters a space between the outer layer ofthe cuticle and the main body of the fibrillar cell wall, whereit continues to grow until it reaches the base of the cell. It thenenters the stigma transmitting tissue where it grows inter-cellularly. In

Papaver rhoeas

the pollen tube grows underneaththe cuticle to the base of the papillar cell. In the Asteraceae

pollen tubes appear to grow extracellularly until they reach thebase of the papillae, although as they reach the base they thengrow through the middle lamella as is the case in the Brassicaceae(Elleman

et al.

, 1992).In the Solanaceae, it has been proposed that lipids direct

pollen tube growth by controlling water flow to pollen (Wolters-Arts

et al.

, 1998). The major components of the hydrophobicexudate in the Solanaceae are triglycerides (fatty acid chains– C18) (Cresti

et al.

, 1986). It is postulated that the hydro-phobicity of the exudate is a critical factor in the ability ofpollen to penetrate the stigma (Lush

et al.

, 2000). This estab-lishes a gradient of water in the exudate that is used as a guid-ance cue by the germinating pollen tube (Lush

et al.

, 1998).Lush and co-workers have attempted to reconstruct thestigmatic environment

in vitro

(Lush

et al.

, 2000). This workis based around the use of a two-phase system comprising ahydrophobic phase, representing the exudate, and a hydrophilicphase, the equivalent of the stigma cells and aqueous drops onthe stigma surface. The hydrophobic phase was created by theuse of exudate or specific oils, whilst the aqueous phase wasderived from the pollen growth media. These studies revealedthat the speed of pollen hydration and germination is relatedto the proximity between the aqueous phase and pollen grainssuspended in the exudate/oil phase. The pollen tube emergedfrom the aperture closest to the aqueous phase and was directedtowards this phase. This suggests that polarity of pollen tubegrowth may be determined by this simple physical cue. Sucha guidance mechanism that depends on the physical environ-ment in the stigma is independent of pistil components. Thissuggestion is supported by the observation that several oilswere an effective substitute for stigmatic exudate. This proposal,that the physical environment in the pistil guides pollen tubegrowth, was extremely contentious when first proposed becauseit appears to contradict the widely accepted view that thespecific chemical environment provides guidance cues.

Pollen tubes enter the stigma by growing through the inter-cellular spaces (Cresti

et al.

, 1986), and although they do notappear to be guided by high precision, their growth along thesurface probably provides a physical cue that increases the like-lihood of penetration occurring (Lush

et al.

, 2000). Mechanicalcues for signalling have been shown to be used by fungalhyphae, which, like pollen, also elongate by tip growth. Theyhave been shown to employ topographical guidance systemsto determine where their appressoria form, in a thigmotropicresponse. This has been established by using artificial, micro-fabricated surfaces to provide specific topographic patterns.Specific spacing and patterning are required for contact sensing,to which the hyphae respond by stimulating differentiation toform infection structures (Allen

et al.

, 1991; Perera

et al.

, 1997;Read

et al.

, 1997). Study of a wide range of fungi suggest thatthigmotropism is likely to be a general feature of fungal growth,rather than a specific pathogenic property (Perera

et al.

, 1997).More recently, evidence has been obtained, that suggests thisresponse involves calcium-dependent signalling (Watts

et al.

,



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1998). It is possible that this mechanism is involved in other tip-growing cells, such as pollen tubes, though to our knowledge,no one has investigated this possibility.

V. Pollen tube growth through the style and pollen tube guidance

Following penetration of the stigma, pollen tubes grow throughthe style towards the ovule (see Fig. 1). How an emergingpollen tube is accurately directed towards the ovule is a matterof considerable debate. That some form of guidance orstimulation mechanism is likely to be involved is largely basedon observations that

in vitro

-grown pollen does not generallyexhibit any inherent directionality, and the rate of growth isusually significantly slower than that of pollen growing

in vivo

( Jauh & Lord, 1995). In some species, such as maize and pearlmillet, for example (Heslop-Harrison

et al.

, 1985; Heslop-Harrison & Reger, 1988), it was proposed that the directionalityof tube growth is due to mechanical influences existing withinthe stylar tract. In this model of tube growth, the cellulararchitecture of the transmitting tissue provides the necessarytopographic guidance required by the elongating pollen tubeto successfully reach the ovary. This type of mechanism hasalso been suggested for the Solanaceae, where the tubes growthrough the intercellular spaces between the parallel files oftransmitting tract cells that comprise the rigid, solid stylesfound in these species (Lush

et al.

, 2000). However, in thecase of the Solanaceae, an alternative hypothesis has beenproposed based on chemical/biochemical guidance cues withinthe style that attract pollen tubes and promote growth towardsthe ovary.

During growth towards the ovary, the pollen tubes are inintimate contact with the components of the extracellularmatrix (ECM) of the transmitting tract (Cheung, 1995). TheECM is comprised of a complex mixture of proteins, inparticular the arabinogalactan proteins, proline-rich glyco-proteins and extensin-like proteins. The arabinogalactanproteins (AGPs) are highly glycosylated, hydroxyproline-richglycoproteins that are implicated in various aspects of plantgrowth and development (Baldwin

et al.

, 1993; Lind

et al.

, 1994;Nothnagel, 1997). Studies of pollen tube growth through thetransmitting tract of

N.

tabacum

have resulted in the isolationof a class of AGPs, the transmitting-tissue specific (TTS) pro-teins (Cheung

et al.

, 1993; Wang

et al.

, 1993) that stimulatepollen tube growth

in vitro

. TTS proteins are encoded by twogenes,

TTS-1

and

TTS-2

, that exhibit a high degree of homology.They encode secreted polypeptides that, following cleavageof the signal peptide, have a molecular weight of

c

. 28 kDa(Cheung

et al.

, 2000). These polypeptide backbones aremodified by hydroxylation of the proline-rich domain locatedtowards their N-terminus and extensive glycosylation withN- and O-linked glycans. The most abundant mature TTSprotein in

N. tabacum

has an apparent molecular weight ofbetween 45 and 105 kDa. Interestingly, ectopic expression

studies have revealed that glycosylation of TTS is organ-specific, and is restricted to the transmitting tissues of the style(Cheung

et al.

, 1996). The TTS proteins are thought to be multi-functional, in that they are implicated in pollen tube adhesionin the style, pollen guidance and nutrition.

Evidence for the biological function of the TTS proteinswas obtained from both

in vitro

and

in vivo

studies (Cheung

et al.

, 1995; Wu

et al.

, 1995). Antibodies raised against TTSproteins were used in cyto-immunodetection studies with both

in vivo

and

in vitro

grown pollen. These revealed that the TTSproteins adhered to the cell wall and to the tip of the growingpollen tube. In addition, a proportion of the TTS proteins werefound to be incorporated into the pollen cell walls, therebyproviding a clear indication of the potential for ECM com-ponents to influence pollen tube growth. The observation thatthe addition of TTS proteins to pollen grown

in vitro

resultedin a threefold growth rate stimulation is also consistent witha direct role in pollen tube growth. Stimulation appeared to bedependent on the presence of glycan moieties, since chemicallydeglycosylated TTS did not stimulate growth. However,cautious interpretation is warranted since deglycosylation mayresult in denaturation of the protein. To investigate the role ofTTS

in vivo Cheung and co-workers constructed transgenicN. tabacum in which the level of TTS protein was reduced,using antisense suppression and sense cosuppression (Cheunget al., 1995). The reduction in TTS protein was found to leadto an overall reduction in seed set, the severity of which wasdependent on the degree of TTS down-regulation. This effectwas due to a reduced rate of pollen growth rate through thetransmitting tissues of the pistil, a finding entirely consistentwith the in vitro observations.

These studies additionally provided evidence that sug-gested that TTS proteins might provide directional cues toextending pollen tubes. Using a semi-in vivo system in whichpollen was grown through a cut style placed on growth medium,it was found that on emerging from the base of the cut-style,pollen tubes would grow in the direction of an agarose plugcontaining freshly prepared TTS proteins. This observationsuggested that TTS might be a signal for pollen tube guidancethrough the style to the ovary. Of course, the difficulty was toexplain how it might contribute to directional growth guid-ance in planta, as TTS is found throughout the extracellularmatrix of the stylar canal. One possible explanation, proposedby Cheung and co-workers, is based on their finding thatthere is a gradient in the level of glycosylation of TTS downthe style (Wu et al., 1995). The protein is most highly glyco-sylated at the ovarian end of the style and this is also reflectedin a corresponding increase in acidity. The TTS glycosylationgradient might also be modified through the interaction withthe elongating pollen tubes. This is based on two findings:TTS binds to the surface of growing pollen tubes, and possiblyacts as an adhesive substrate; and in vitro grown pollen tubesdeglycosylate TTS (Wu et al., 1995), suggesting that this mightalso occur in vivo. It is therefore proposed that pollen tubes



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grow through the transmitting tissue of the style along thegradient of increased TTS glycosylation. Furthermore, thedeglycosylation activity of the growing pollen would con-tribute by increasing the gradient in the vicinity of the extendingtubes and provide a potential nutrient source in the form ofthe released sugar molecules.

The proposed role of TTS is, however, controversial. Severallines of argument have been raised that question the exactfunction of TTS in pollen tube guidance. First, the theoreticalmaximum distance over which chemical cues can guide pollentubes is between 1.2 and 9.3 mm, compared with the 40–50 mm length of the N. tabacum style (Lush, 1999). Further-more, even over this shorter distance, gradients of 10 000-foldare required, which is in stark contrast to the fourfold gradientin TTS glycosylation from the top to the bottom of the style(Wu et al., 1995). Earlier studies have also demonstrated thatpollen tubes will grow through styles towards the stigma ifartificially introduced at the base or the middle of cut styles(Mulcahy & Mulcahy, 1987).

A further question concerning the exact role of TTS arosefrom the identification of a homologue of TTS, the galactose-rich stylar glycoprotein (GaRSGP) in N. alata (Sommer-Knudsenet al., 1996, 1998). GaRSGP has a polypeptide backbone that is97% identical to that of TTS. However, it is mostly locatedwithin the cell wall, not the intercellular space of the trans-mitting tract and crucially, unlike TTS, was found to be inactivein pollen tube growth assays (Sommer-Knudsen et al., 1998).This apparent discrepancy has recently been addressed. Stylartransmitting tissue glycoproteins were isolated from N. alatausing procedures applied in the isolation of TTS from N. tabacum(Wu et al., 2000), which resulted in the isolation of a set ofglycoproteins, referred to as NaTTS proteins that were virtuallyidentical to TTS from N. tabacum. Most importantly, NaTTSproteins were also found to enhance pollen tube growth andto attract tubes in the semi-in vivo assay used previously (Wuet al., 2000). It was suggested that the extraction proceduresused in the earlier study (Sommer-Knudsen et al., 1998) hadresulted in the isolation of a subset of less heavily glycosylatedglycoproteins typified by GaRSGP. Bearing in mind thatchemically deglycosylated TTS failed to promote pollen tubegrowth, it was proposed that the lower level of glycosylationof GaRSGP might explain why it too failed to promote growth(Wu et al., 2000).

Thus, whilst it appears that TTS proteins stimulate growththrough the style, whether or not they are providing guidancecues in vivo is less clear. The complexity of the ECM adds tothe difficulty in resolving this issue, particularly as other com-ponents may also have a function in pollen tube growth. It islikely that there are several different components that interact.In the case of N. tabacum, at least one other class, the class IIIpistil-specific extensin-like proteins (PELPs), are known toclosely interact with the pollen tube as it extends through thestylar canal (de Graaf et al., 1998). Extensins are a class ofhydroxyproline-rich glycoproteins found in plant cell walls

that are characterized by the presence of numerous repeats ofa Ser-(Hyp)4 motif. Studies indicate that the PELPs are actuallytranslocated from the matrix to the pollen tube wall. A similarsituation has been described in N. alata where a 120-kDa glyco-protein, which has both extensin-like and arabinogalactan-like properties, has been found to enter the pollen tube duringits growth through the style (Lind et al., 1996; Schultz et al.,1997). Similar types of protein have previously been reported inother species. For example, pt11 is a gene expressed specific-ally in the transmitting tissue of Antirrhinum pistils (Baldwinet al., 1992). Another extensin-like gene was reported in Zeamays, however, in this case, it was a pollen specific extensin-like protein, Pex1, that might potentially recognize and interactwith molecules within the style (Rubenstein et al., 1995).These studies suggest that extensin-like proteins may make animportant contribution to adhesion between the growingpollen tube wall and the ECM of the pistil transmitting tissueacross a wide range of plant families. This could directly influ-ence the efficiency by which the pollen tube negotiates thetransmitting tract and facilitate other signalling events betweenthe male and female tissues. These possibilities warrant furtherinvestigation.

A number of studies in other plant species have also pro-vided evidence that indicates a role for chemotrophic factorsin pollen tube guidance. In particular, they highlight the roleof the female gametophyte in a range of species (see Fig. 1). Inpeach, for instance, secretions from both the ovary and indi-vidual ovules appear to have a role in pollen tube guidance andpenetration of the ovule (Hererro, 2000). Studies have iden-tified mutants in which guidance of the pollen tube to theovule is impaired. Wilhelmi & Preuss (1996) screened 80 000A. thaliana plants before identifying two mutants ( pop2 andpop3 ) that were defective in genes involved in the targeting ofpollen tubes to ovules. Instead of growing towards the micropyleand avoiding already fertilized ovules, the pop mutants grewrandomly throughout the ovary locule. The pop mutantsappear to result from defects in both the pollen and pistil, andthe authors noted that there was a failure of pollen tubeadherence to the pistil. Also in A. thaliana, ovule mutantshave been described which appear to alter pollen tube growth(Hülskamp et al., 1995b). First of all, the growth path ofwild-type pollen was examined microscopically. This revealedthat in a situation where only limited numbers of pollen tubesgrew down the transmitting tract, there was a strong preferenceto emerge on the surface of the septum at the position of theovule closest to the stigma. Progressively fewer tubes emergedat ovules located at positions further down into the ovary. Thisgradient effect was only slightly reduced when the number ofpollen tubes growing through the transmitting tract increased.

Ovule selection following emergence of the pollen tube onthe septum was also investigated. This revealed that a substantialnumber of tubes, some 39–46%, grew directly toward the mostproximally located ovule. Nevertheless, a significant propor-tion of the tubes grew toward more distant ovules. When



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comparable studies were carried out in four ovule mutants(bel1, sin1, 47H4 and 54D12) it was found that the normalbehaviour of the pollen tubes was perturbed (Hülskamp et al.,1995b). Emergence of the pollen tube on the surface of theseptum no longer favoured the ovule closest to the stigma. Inthe case of the sin1 and 47H4 mutants in which ovule develop-ment was most disrupted, the tubes emerged along the entirelength of septum with equal probability. After emerging onthe septum surface the ability of the pollen tube to target anovule was also adversely affected. Instead of directly growingtowards a funiculus and the micropyle of a selected ovule, thetubes grew randomly over accessible surfaces. These studiesdid not identify the exact tissue in the developing ovule thatprovides the directional signals for pollen tube growth. How-ever, they do provide a link between embryo sac developmentand pollen tube guidance. In the case of the 54D12 mutantsthat arrested at various stages in embryo sac development, itwas found that in the absence of any development, all pollentubes failed to find an ovule. In the ovules that arrested at anintermediate stage of development 28% were associated witha pollen tube, compared to the wild-type figure of 92%. It wasconcluded that the embryo sac, or associated tissues, generatesa long-range directional signal that influences the emergenceof the tube on to the septum surface, and subsequent guid-ance to an ovule. This signal did not appear to influence theearly stages in pollen tube growth as this was identical in bothwild type and mutant plants (Hülskamp et al., 1995b).

Other studies have provided additional evidence in supportof an important role for the embryo sac in pollen tube guidance.Ray et al. (1997) used a semisterile A. thaliana line, TL-1, thatwas heterozygous for a reciprocal chromosomal translocation.This resulted in plants with normal diploid somatic cells.However in this translocation line half of the meiotic productsreceived an abnormal chromosome complement, and con-sequently did not go on to develop normal embryo sacs. Whenthese plants were pollinated, the pollen tubes were guided tothe ovules containing normal embryo sacs but not to those withdegenerated embryo sacs. This again leads to the suggestionthat viable embryo sacs provide signals to guide the pollen tube.Whether this signal acts directly on the pollen tube or via aninfluence on the cells in the vicinity of the ovule remains to beresolved (Ray et al., 1997).

The role of the embryo sac in pollen tube guidance has alsobeen investigated in vitro (Higashiyama et al., 1998). In thesestudies pollen tubes and naked embryo sacs of Torenia fournieriwere cocultivated on solid growth media. The pollen tubeswere found to grow with a high degree of accuracy toward themicropylar end of the naked embryo sac. This behaviour wascompletely abolished by heat-treatment of the ovules, providingfurther evidence in support of a role for proteins produced by theembryo sac in pollen tube guidance. More recently, Shimizu& Okada (2000) have isolated an A. thaliana mutant with analtered phenotype in the female gametophyte. The mutant,magatama (maa), shows delayed female gametophyte develop-

ment. Pollen tube guidance also seems to be affected by themutation. Pollen tubes appeared to lose their way just beforeentering the micropyle, and the mutants also showed a tendencyto attract two pollen tubes rather than one. The authors proposea ‘monogamy’ model to account for this, where the femalegametophyte emits two attractants (a funiculus guidance cueand a micropyle guidance cue) whilst repulsive forces on thepollen tube prevent polyspermy. Thus, in the case of the maamutants both the micropyle guidance cue and the repulsivepollen signal are lacking.

The majority of the studies discussed above have focusedon the Solanaceae, which have solid stylar transmitting tracts.However, in the case of Lilium longiflorum, the organizationof the style is different. The lily style is hollow, with a liningof secretory cells that form the transmitting tract. Based ontheir studies over a number of years, Lord and co-workers havehypothesized that pollen tube guidance and growth rate throughthe style is dependent on a haptotactic (matrix adhesion-driven)mechanism (Lord & Sanders, 1992; Lord, 2000). They proposethat the pollen tubes adhere to the secretory cells via com-ponents in the style ECM and are thereby guided towards theovary. The influence of the style was suspected from circum-stantial evidence based on comparisons of in vivo and in vitrogrown pollen. Observations of pollinated styles after cryo-fixation revealed the apparent adhesion of the pollen tubes tothe secretory cell lining of the transmitting tract ( Jauh &Lord, 1995). By contrast to pollen grown in vitro, pollen tubesgrowing in vivo adhere to each other, forming star-shapedclusters of F-actin, reminiscent of focal adhesions in movinganimal cells. They also grow significantly faster than pollengrown in vitro ( Jauh & Lord, 1995, 1996). Furthermore, pre-vious studies had revealed that the transmitting tissue was capableof translocating inert latex beads in a manner reminiscent ofpollen tube growth (Sanders & Lord, 1989). These data havegiven rise to the proposal that pollen tubes are not just tip-growing cells, but act as a moving cell system, similar toneuronal cells, at least if they are in their natural environment,interacting properly with other components in the pistil. Thisanalogy has been explored in a recent review (Palanivelu &Preuss, 2000). These observations prompt the need for morestudies of pollen tubes in vivo, rather than in vitro, to obtaina better appreciation of what is really occurring with respectto control of pollen tube growth.

In order to take this model forward, it was necessary toidentify components within the style that were responsible forgenerating adhesion to the growing pollen. Recently, significantprogress towards this goal has been attained. An adhesion bio-assay, based on the binding of pollen tubes to a nitrocellulosemembrane in the presence of stylar extracts, has been developed( Jauh et al., 1997; Park et al., 2000). This has identified twofractions: one containing a high molecular weight componentand the other a small 9 kDa stigma/stylar protein, which wasidentified as a cysteine-rich adhesin (SCA), that in conjunc-tion with a pectic fraction, enabled pollen adhesion to occur.




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Cloning and subsequent nucleotide sequence analysis of SCA(Park et al. 2000) revealed that it has homology to plant lipidtransfer proteins (Kader, 1997). Immunolocalization con-firmed the localization of SCA within the stylar ECM and inpollen tubes growing in vivo, but not grown in vitro (Parket al., 2000). Further studies have resulted in the identificationof the other major component (Mollet et al., 2000). Sizefractionation indicated that this was a large molecule ofapprox. 1.5 MDa. Chemical analysis, together with the observa-tion that its pollen adhesion activity was sensitive to treatmentwith endopolygalacturonase, yet resistant to proteinase K,confirmed it as a member of the pectin family. Since it wasrecognized by the JIM5 antibody (Knox et al., 1990) it wasconcluded that it was in part composed of regions of low-esterified homogalacturonan.

Thus, together, the stylar pectin and SCA induce pollentube adhesion, both to other pollen tubes and to the epidermalcells of the stylar transmitting tract. The bioassay also revealedthat the two molecules bind to each other to promote pollenadhesion and stimulate the rate of pollen tube growth. Bindingwas found to be pH-dependent, with a significant reductionoccurring at pH 10, which coincided with a resultant loss inpollen adhesion capacity. These results suggest a role for chargeinteractions in this phenomenon. However, it appears thatthis in itself does not fully explain the adhesion activity, asSCA will also bind another negatively charged polymer, alginicacid, but this combination is inactive (Mollet et al., 2000).

These studies lend considerable support to the proposalthat a contact-stimulated guidance system facilitates pollengrowth through the style (Park et al., 2000). It is suggestedthat this defines a fixed track down the style, rather thaninvolving any diffusible guidance cue. Lord and co-workerssuggest that this mechanism may be considered analogous tothe laminin/netrin two-component guidance system thatdirects the path of neurone outgrowth (Hedgecock & Norris,1997; Park et al., 2000). In this case, the small, secreted netrinmolecule provides an interface between laminin and a receptorin the neurone. If the laminin/neurone analogy were correctthen it would be anticipated that there may be specific sites onthe pollen with which SCA-pectin complex interacts, possiblyinvolving receptor molecules.

Although there is no direct evidence to support the abovehypothesis at present, several receptor molecules have beenidentified in pollen. A pollen-specific receptor kinase, PRK1,has been identified in Petunia inflata (Mu et al., 1994). How-ever, this has since been implicated in the postmeiotic develop-ment of pollen, rather than the interaction of pollen with theextracellular matrix during pollen-tube growth (Lee et al., 1996).Perhaps of more relevance is the identification of a receptor-like kinase LePRK2, isolated from tomato (Muschietti et al.,1998). LePRK2, together with LePRK1, is a serine/threoninekinase found in the pollen plasma membrane/cell wall. Bothproteins possess an extracellular domain containing a leucine-rich repeat, a motif that is thought to mediate protein–protein

interactions (Kobe & Deisenhofer, 1994). LePRK1 is similarto PRK1 from Petunia (Mu et al., 1994), in that it is expressedduring pollen development. LePRK2 is also expressed late inpollen development. However, unlike LePRK1, the proteinincreases in abundance following pollination, and most inter-estingly is partially dephosphorylated in a specific response tostylar extracts (Muschietti et al., 1998). The SCA-pectincomplex may or may not interact with this class of receptor,nevertheless, these studies indicate that pollen receptors existthat respond to signals emanating from the female tissues. Itis anticipated that further pollen receptors will be identified inthe future. Finally, the analogy between pollen tube growthand neuronal growth mentioned above has gained additionalsupport recently. Guyon et al. (2000) have produced a studyof flavanol-induced pollen germination in Petunia that iden-tified a pollen cDNA encoding a protein whose sequenceexhibits similarity to neuromodulin, a neuropeptide implicatedin the regulation of axon tip guidance and growth (Zuberet al., 1989). This emerging concept has been reviewed recently(Palanivelu & Preuss, 2000).

In summary, the nature of pollen tube guidance within thestyle, which was, until recently, very poorly understood, remainsa contentious issue. A combination of genetic, molecular geneticand biochemical approaches has led to considerable progressin this area of research. There is certainly very good evidencefor complex glycoproteins playing an important role in pollentube guidance. Furthermore, we have not dealt with the issueof chemical guidance, though there is evidence for this in theliterature. Ideas that mechanical, chemical and biochemicalgradients may be involved are not mutually exclusive, and itmay be that all three play a role. However, it is clear that thereare unresolved questions. Part of the problem in this area is thecomplex nature of the style and the ECM, and the consequentlikelihood of further components involved in pollen tubeguidance that have yet to be identified. A summary of someof the components identified as involved in growth of pollenthrough the style is included in Fig. 1.

VI. Control of pollen viability by incompatibility responses

The interactions described above, between pollen and thefemale sporophytic tissue, have mostly been restricted to theevents occurring in compatible pollinations, the aim of whichis to ensure fertilization and seed set. The angiosperms have,however, developed means by which to discriminate betweendesirable pollen and undesirable pollen, which, if allowed toachieve fertilization, would have deleterious effects on thefitness of progeny. These mechanisms have been alluded toin the section describing control of pollen adhesion in theBrassicaceae, where there appears to be discrimination infavour of pollen derived from plants of the same species. In thissection we describe progress in our understanding of themechanisms whereby pollen that is ‘incompatible’ with the




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recipient plant may be recognized, and subsequently rejected.The first of these mechanisms is interspecific incompatibility,where pollen is rejected from donors of species different tothat of the recipient because it is too dissimilar. The secondmechanism is intraspecific incompatibility, generally referredto as self-incompatibility (SI) where pollen, although itoriginates from a donor plant of the same species, is rejectedbecause it is too similar, due to it originating from the same,or a genetically closely related plant. We will deal with thedifferent types of SI first, as it has been much more intensivelystudied, and in recent years considerable progress has beenmade in our understanding of this area of control of pollination.However, we will not deal with SI systems that operate at aheteromorphic level, where incompatibility is related to thepresence of flowers with differing morphology, such as distylyin Primula spp., since relatively little is known about themolecular basis of incompatibility in these systems. We willthen review some aspects of interspecific incompatibility, butit will be apparent that rather less is known about the mechanismsinvolved in this process in comparison to intraspecific SI.

1. Self incompatibility (SI)

Self incompatibility (SI) is by far the best understood mech-anism of pollen recognition and rejection. It is estimated tooccur in between 30 and 50% of flowering plant speciesenabling them to discriminate self-(incompatible) fromnonself-(compatible) pollen. Having been distinguished fromcompatible pollen, self-incompatible pollen is rejected at somepoint in the pollination process specific to that species. Thismay happen at hydration, germination, during growth throughthe style, in the ovule, or even postfertilization in some species.This suggests that there are a variety of mechanisms involvedin different SI systems and that the different mechanisms haveevolved in isolation. In most cases SI is controlled by a singlemultiallelic S-locus, which contains genes encoding at least astylar component and a pollen component. The interaction ofthese so-called S-gene products determines the fate of pollengrowing through the stigmatic and stylar tissues. SI is generallycategorized as being under sporophytic or gametophyticcontrol. In sporophytic SI, the pollen S-phenotype is deter-mined by the diploid S-genotype of the parent plant. Ingametophytic SI, however, the pollen S-phenotype is specifiedby its own haploid S-genotype. In some species, there is amore complex genetic control of SI, with the involvement ofother loci. For example, SI in the grasses is under control oftwo loci, S- and Z-. However, these systems will not be dealtwith here. In some species, detailed investigations of some ofthe processes controlling SI have been undertaken. Here wedescribe the better known SI systems, and what is currentlyknown about their mechanisms.

Gametophytic SI In the case of single-locus gametophyticself-incompatibility, two mechanistically different systems

have been studied in detail at the molecular level. The first ofthese is the S-RNase system, originally found and extensivelycharacterized in members of the Solanaceae (Anderson et al.,1986; McClure et al., 1989), and more recently reported in theRosaceae (Sassa et al., 1993), Scrophulariaceae (Xu et al., 1996),and Campanulaceae (Stephenson et al., 2000). A second, differ-ent gametophytic SI system is found in the Papaveraceae.

SI in the Solanaceae In the Solanaceae, in both compatibleand incompatible pollinations, the pollen germinates and apollen tube grows through the transmitting tract of the style.The growth of the incompatible pollen tube is, however,arrested by the time it has reached about one-third of the waythrough the style. Early studies investigated the stylar proteinsof different S genotypes to find proteins associated with SI.Analysis of stylar proteins from Nicotiana alata resulted in theidentification of a glycoprotein of c. 30 kDa that exhibitedgenetic linkage to the S locus (Anderson et al., 1986). Sub-sequent cloning and analysis of a large number of alleles of thisgene from different members of the Solanaceae revealed themto be highly polymorphic, exhibiting between 39 and 98%sequence identity (Anderson et al., 1989; Ai et al., 1990; Clarket al., 1990; Ioerger et al., 1990; Kheyr-Pour et al., 1990; Xuet al., 1990). Some S alleles from different species were foundto be more closely related than they were to S alleles from theirown species. This finding indicated that SI in the Solanaceaepredates the divergence of genera, which in the case of Nicotianaand Petunia is thought to have occurred 27 million years ago.Thus, the S-locus compares with other systems known to beunder frequency-dependent selection, such as the MHC lociin vertebrates (Hughes & Nei, 1988), in exhibiting trans-specificpolymorphism.

The S glycoproteins of the Solanaceae were found to exhibithomology to the catalytic domain of two fungal RNases, Rhfrom Rhizopus niveus and T2 from Aspergillus oryzae (McClureet al., 1989). Further studies confirmed the S glycoproteinswere indeed ribonucleases, and they have since been termedS-RNases (McClure et al., 1989; Clark et al., 1990; Xu et al., 1990;Gray et al., 1991; Singh et al., 1991). Transgenic studies, demons-trating gain and loss of function, established that catalyticallyactive S-RNases are crucial for rejection of incompatible pollen(Lee et al., 1994; Murfett et al., 1994). Thus, it was proposedthat S-RNases are S-allele-specific cytotoxins.

The identity of the pollen S protein in the Solanaceae remainsto be determined, but two suggestions as to its nature havebeen proposed (see Fig. 2). The first model suggests that it isa receptor which internalizes S-RNase molecules in an allele-specific manner (Fig. 2, model I). The S-RNase then degradespollen rRNA, leading to the cessation of pollen tube growth.In the alternative model (known as the inhibitor model), it isproposed that indiscriminate uptake of S-RNases occurs, andon entering the pollen tube, they interact with the pollen Sprotein that is proposed to be an RNase inhibitor. This recognizesand inhibits all S-RNases via an interaction with a low-affinity




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binding site, except when the S-RNase is of the same allelicspecificity. In this case, binding occurs via a high affinity,allele-specific site, which somehow prevents the interactionwith the low affinity site (Fig. 2, model II). This leaves theS-RNase fully active and able to inhibit pollen tube growth.Although there is no direct evidence to support either hypo-thesis, accumulating evidence favours the latter, since it canaccount for the observation that SI breaks down in tetraploidplants (Golz et al., 1999). It is also supported by recent immuno-cytochemical studies (Luu et al., 2000), which used a mono-specific polyclonal antibody directed against S11-RNase to labelpollen tubes growing in incompatible styles. Pollen from aplant homozygous for S12 was used to pollinate styles from aplant of genotype S11S12. Labelling with the anti-S11 antibodydetected markedly higher amounts of S11-RNase within thepollen tubes, compared to the extracellular matrix. This suggeststhat uptake of the S-RNase is independent of S genotype, afinding that adds weight to the inhibitor model.

Another important issue that remains to be addressed isthe basis of allelic specificity. Sequence comparisons revealthat S-RNases are organized into five conserved regions and twohypervariable regions that form a continuous surface on oneside of the protein. Whether S-specificity resides entirelywithin the hypervariable regions is debatable. Although theS11 allele of Solanum chacoense was successfully changed intoa closely related S13 allele (Matton et al., 1997), hypervariable

domain-swap experiments involving more diverged alleleshave not proved successful (Verica et al., 1998), but this maybe due to the failure of hybrid proteins to fold correctly. Thisproblem is unlikely to be resolved until the tertiary structureof S-RNases is characterized. Work is currently being under-taken towards this goal ( Ida et al., 2001; Parry et al., 1998).

Attempts have recently been made to identify othercomponents involved in the SI response in the Solanaceae.McClure et al. (1999) used a differential screening approachto address this problem. A cDNA library was constructed fromN. alata stylar tissue. This library was then hybridized with stylarRNA from N. alata and the closely related, but self-compatiblespecies, N. plumbaginifolia. A clone was found that hybridizedto N. alata RNA only. The clone designated HT was found toencode a 101 residue asparagine-rich protein. RNA blot analysissuggests that HT transcript accumulates in the style beforeanthesis with a slight lag behind S-RNase expression. Trans-genic experiments demonstrated that HT is essential in the SIresponse. N. plumbaginifolia × N. alata Sc10Sc10 hybrids trans-formed with an antisense construct of HT lost the ability toreject Sc10-pollen, whereas untransformed control plants wereable to reject this pollen. Unfortunately, database searcheshave failed to detect homology to any known proteins, and,as yet, the function of HT is unknown. The detection ofnonS-linked factors involved in the Solanaceae SI responsesuggests that a true picture of the mechanics of this response

Secreted pistil S-RNases

S1 receptor

S1 pollen

rRNA degradation

S1

S2

Model II Model I

S2

lowS1 receptor

high

S1

S2

S1

rRNA degradation

Fig. 2 Model for the mechanism of pollen inhibition for the Solanaceae. The products of the female S gene, the pistil S-RNases (dark green and dark blue) are secreted into the transmitting tissue of the style. Pollen tubes growing through the style encounter S-RNases. In the case of pollen that carries an S allele corresponding to either of the alleles present in the style, inhibition occurs. Two models have been proposed for the inhibition mechanism. In Model I (indicated on the right-hand side) the S-RNase (S1 here) enters the S1 pollen tube (shown in yellow) via an allele-specific receptor. The S-RNase then degrades the rRNA within the incompatible pollen tube, and arrest of pollen growth occurs by the time it has grown about a third of the distance through the style. In Model II (indicated on the left-hand side) all S-RNases enter the pollen via a nonspecific transporter. On entry, they encounter the pollen S-receptor, which has two ligand-binding sites. These are a low-affinity site that binds and inhibits the S-RNases in an allele-independent manner (shown here as an interaction with S2 RNase), and a high-affinity site, which is allele specific. Binding to this site prevents binding of the S-RNase (S1 here) to the low-affinity site, hence the protein remains active and degrades the pollen rRNA. As discussed in the text, recent evidence favours the inhibitor model (Model II). Adapted, with permission from Macmillan Reference Ltd.




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is, as yet, incomplete and current searches are underway toidentify other elements of the response (McClure et al., 1999).

In conclusion, despite extensive efforts by a number ofgroups the identity of the pollen S-gene in the Solanaceae andother species with an S-RNase SI system remains to be resolved.Although current evidence now favours the inhibitor model(Fig. 2, model II), a complete understanding of the mechanismof SI in the Solanaceae is still some way off. Whilst isolationof the pollen S-gene remains a major goal, studies by McClureand co-workers have identified other aspects of this systemthat require further investigation. As mentioned above, theirstudies have revealed that one or more factors other than theproducts of the S locus are required for a functional SI system.In addition, they have obtained evidence that reveals a role forS-RNases in interspecific SI (see below).

SI in Papaver A gametophytic system of SI control is alsofound in the field poppy, Papaver rhoeas. However, studies atthe molecular and biochemical level have revealed that thestigmatic S gene, and the mechanisms involved in pollen inhibi-tion, are completely different from that of the Solanaceae (seeFig. 3). Unlike in the Solanaceae, where the SI response involvesthe action of cytotoxic proteins, the SI response in P. rhoeas ismediated by a complex signalling cascade. The response doesnot involve the degradation of rRNA (Franklin-Tong et al.,1991). Instead, several biochemical and cytological changes aretriggered in the pollen that are likely to contribute to the inhibi-tion of its growth. The response also has some notable physi-ological differences to that found in the Solanaceae. P. rhoeaslacks a style and rejection of pollen takes place on the stigmaticsurface. As well as this, P. rhoeas has a dry stigmatic surfacecompared to the wet, lipid-rich exudate found on the surfaceof the Solanaceae (Elleman et al., 1992). Inhibition of incompat-ible pollen in P. rhoeas is rapid compared with the relativelyslow inhibition in the Solanaceae, acting on a time scale ofminutes rather than hours. These fundamental differenceshave led to suggestions that gametophytic SI systems haveevolved independently several times. Research into SI in Papaverhas been helped considerably by the development of an invitro bioassay (Franklin-Tong et al., 1988). The bioassay haspermitted the SI response to be reproduced in vitro, and hasenabled many downstream events involved in SI-inducedpollen inhibition to be analysed in detail. A summary of theP. rhoeas SI response is shown in Fig. 3.

The stigmatic S proteins of P. rhoeas are small (c. 15 kDa)extracellular signalling molecules. In the order of 66 S allelesare estimated to exist in this species (Lane & Lawrence, 1993).Several alleles (S1, S3, S8 and Sn1 from P. nudicaule) of thestigmatic S gene have now been cloned (Foote et al., 1994;Walker et al., 1996; Kurup et al., 1998). Although the exactbasis of allelic specificity remains to be elucidated, site-directedmutagenesis of the S1 protein has established that certainresidues located in hydrophilic surface loops are crucial for therecognition of S1 pollen (Kakeda et al., 1998; Jordan et al.,

1999). These stigmatic S proteins interact with the pollen S geneproduct, which is believed to be a plasma membrane receptor.The nature of the pollen receptor is unclear at present. Onecandidate, an S protein binding protein (SBP) has been iden-tified (Hearn et al., 1996). SBP is a pollen plasma membraneglycoprotein of 70–120 kDa. Binding to the S protein is atleast partly dependent on the glycan moiety of SBP. WhilstSBP specifically binds S proteins in vitro studies suggest thatthis binding is not S-allele specific. Hence, SBP may be anaccessory receptor rather than the pollen S receptor itself. How-ever, as analysis of S protein mutants has revealed that allmutants that exhibit reduced ability to inhibit incompatiblepollen also have reduced SBP binding activity ( Jordan et al.,1999), it is conceivable that SBP is the S receptor. Resolutionof this issue awaits the cloning of SBP and/or the S receptor.

It is well established that inhibition of incompatible pollenin P. rhoeas is mediated by the activation of a calcium-basedsignal transduction pathway in the pollen. It is thought thatan increase in cytosolic free Ca2+ ( [Ca2+]i) is the initial step inthe signalling cascade, and that the SI reaction is a receptor-mediated response, with the S protein acting as a signalmolecule that triggers increases in [Ca2+]i. Evidence for thiscomes from calcium imaging studies that demonstrated thatincreases in [Ca2+]i were stimulated within a few seconds of anincompatible interaction and preceded the inhibition of incom-patible pollen tube growth (Franklin-Tong et al., 1993, 1995,1997). Confirmation that S proteins alone were sufficient to elicitthis response came from use of recombinant S proteins, whichshowed that they acted as signal molecules (Franklin-Tonget al., 1995). As Ca2+ is important for the regulation of pollentube growth and is known to act as a second messenger in plantcells (Franklin-Tong, 1999a, 1999b; Rudd & Franklin-Tong,1999; Rudd & Franklin-Tong, 2001), these findings areconsistent with expectations.

Downstream of the initial Ca2+ signals, SI-induction resultsin the phosphorylation of several proteins in a S-specificmanner. This strongly suggests that they are involved in SI inPapaver pollen. Two of these proteins have been named p26and p68 (Rudd et al., 1996, 1997). Phosphorylation of p26,which occurs within 90 s of challenge, is Ca2+-dependent andmost likely involves a calcium-dependent protein kinase,CDPK (Rudd et al., 1996). The recent cloning and character-ization of p26 indicates that it is a soluble inorganic pyrophos-phatase. The pyrophosphatase activity of the p26 recombinantprotein is strongly inhibited by phosphorylation. It is pro-posed that this reduces the rate of pollen tube growth due toan adverse effect on the biosynthetic capacity of the pollen( J. J. Rudd, V. E. Franklin-Tong, F. C. H. Franklin, unpublished).Phosphorylation of p68, in contrast, is Ca2+-independent (Ruddet al., 1997), and provided the first hint that the SI signallingcascade mediating the SI response is complex, having both aCa2+-dependent and a Ca2+-independent phase. Additionalsupport for this idea comes from recent data that providesevidence that a mitogen activated protein kinase (MAPK) is




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Fig. 3 SI in Papaver. (a) A model for the mechanism of self-incompatibility in Papaver. Stigmatic S1 proteins (indicated in dark blue) and S2 proteins (green) are secreted by a S1S2 pistil, and encounter an S1 pollen tube (pale yellow). The pollen is assumed to have a S1 receptor (light blue), which interacts with S1 proteins, but not S2 proteins. Here the S receptor is depicted as a single entity, but it may be a receptor complex, with S protein binding protein (SBP). The interaction with incompatible S proteins results in an immediate increase of cytosolic free calcium [Ca2+]i (indicated in red) within the pollen. Subsequent to this, the tip-focused [Ca2+]i gradient is lost. It is postulated that many of the signalling events triggered within the pollen are Ca2+-dependent. Several protein kinases are activated, resulting in protein phosphorylation, notably of p26 and p68, and a calcium dependent protein kinase (CDPK) is thought to be involved in the case of p26. SI induction also results in extensive rearrangement of the actin cytoskeleton, which is now thought to be a major target for the SI signalling cascades. There is evidence that a programmed cell death(PCD)-signalling cascade is triggered by self incompatibility (SI), eventually resulting in the fragmentation of nuclear DNA. It is thus thought that the initial ligand–receptor interaction sets off a cascade of events that may be interrelated, giving rise to several different mechanisms, all of which may contribute to inhibition of pollen tube growth. Adapted, with permission from Macmillan Reference Ltd. (b) Timescale of events in the Papaver SI pollen response. Following addition of incompatible S proteins there is a virtually immediate increase in [Ca2+]i in the pollen which lasts for c. 10 min. Within 1 min, the tip-focused calcium gradient dissipates and tip growth is concomitantly arrested. p26, the inorganic pyrophosphatase, is phosphorylated by 90 s and the level of phosphorylation has increased by 400 s. Phosphorylation of p68 occurs before 240 s and is still increasing at 400 s. Phosphorylation of p52-MAPK is detected by 5 min, peaking at 10 min. Alterations to the actin cytoskeleton occur almost instantaneously, with F-actin detected at the tip, marginalization and fragmentation of F-actin within a few minutes; punctate foci of actin form rather later, and continue to ‘grow’ over c. 1–2 h. Nuclear DNA fragmentation, which is a hallmark of PCD, is first detected around 4 h after SI induction, and increases over the subsequent 10 h. It is thought that there may be three ‘phases’ to the SI response: ‘early’, ‘commitment’ and ‘late’.

Loss of Ca 2+

tip gradient [Ca2+]iCDPK

Ca2+

PCD cytoskeleton

Secreted stigmatic S proteins S2 S1

S1-receptor S1 pollen

p68PK

p26

(a)

h

(b)

F-actin alterations

Tip growtharrested

Increases in [Ca2+]i

Incompatible S proteins added

DNA fragmentation

p26 phosphorylation

p68 phosphorylation

p52 MAPK activation

Early events

Commitment phase?

Late events

Time

p52




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activated during the SI response in incompatible pollen. Theactivation of this MAPK appears to be SI-specific and occursdownstream of increases in [Ca2+]i ( J. J. Rudd, F. C. H. Franklin,and V. E. Franklin-Tong, unpublished).

A recently identified target for the SI response in P. rhoeas isthe actin cytoskeleton. During the response rapid and dramaticchanges occur in the actin cytoskeleton of incompatiblepollen tubes (Geitmann et al., 2000; Snowman et al., 2000a,b).Within 1–2 min, and possibly within 30 s, there is a reorgan-ization of filamentous (F)-actin to the pollen tube apical region,which is normally relatively free of F-actin bundles. Further-more, F-actin also accumulates in the cortical region adjacentto the plasma membrane. Concomitant with this is an apparentdecrease in the number of F-actin bundles in the lumen of thepollen tube, and by 10 min fine fragments of actin are seenthroughout the cytoplasm. Later alterations are seen fromc. 20 min up to several hours later in the SI response wherepollen F-actin is reorganized into punctate foci, which enlargeover this time period (Geitmann et al., 2000; Snowman et al.,2000a,b). These data strongly suggest that the actin cytoskeletonis a target for the SI signalling cascades. Further downstreamof these events, S-specific nuclear DNA fragmentation hasbeen detected 4–12 h after induction of the SI response ( Jordanet al., 2000). This is a hallmark feature of programmed celldeath (PCD). Nuclear DNA fragmentation is a key target forPCD signalling cascades, and these findings suggest that PCDis triggered by the SI response in Papaver pollen. Thus, theultimate death of incompatible pollen in this species takesseveral hours.

A model for how these components and events mightparticipate to elicit SI in Papaver is shown in Fig. 3(a). Anindication of the timescale of these events is shown in Fig. 3(b).It is proposed that there are perhaps three phases in thePapaver SI response. First, a very rapid inhibition of tip growthtakes place. Since this is known to be reversible, it is thoughtthat a ‘commitment’ phase follows, during which processesare triggered that lead to the irreversible degradative processesdetected in the ‘late’ phase.

Broadly speaking, two issues of particular importancerequire resolution. First, as mentioned above, the nature of theS receptor remains to be determined. Second, it is clear thatthe mechanism of inhibition of incompatible pollen is highlycomplex. Substantial progress has been made towards defin-ing aspects of the signal transduction cascade that mediatesthe SI response, and a number of associated cellular eventshave been identified. Nevertheless, it still remains to be estab-lished how these events, and the signals responsible for them,are interrelated and regulated to bring about the irreversibleinhibition of incompatible pollen.

Sporophytic SI As well as different gametophytic SI systems,it is apparent that there are at least two distinct sporophyticSI systems. One of these is utilized by members of the Con-volvulaceae. However, the most intensively studied of the

sporophytic systems is that of the Brassicaceae, particularlyB. oleracea. Physiologically, the Brassica SI response dependsupon the presence of a ‘dry’ stigmatic surface (Dickinson, 1995).Incompatible pollen alighting on the stigma usually fails tohydrate and germinate, the response occurring at the stigmaticsurface within minutes of pollination (Ferrari & Wallace, 1975).The growth of any incompatible pollen that succeeds inpenetrating the stigmatic surface halts following the depositionof callose produced in the stigmatic papillar cells (Dickinson &Lewis, 1973). These callose deposits associated with the SIresponse are not, however, required for the response to take placeper se (Sulaman et al., 1997). The molecular mechanisms under-lying this response are now beginning to be characterized.

SI in Brassica Molecular studies have revealed that the S locusin Brassica oleracea and B. campestris is complex, extending,in some instances, over several hundred kilobases. Studies ofa range of S haplotypes from amongst the 50 or so that arepredicted to exist, have identified two stigmatic glycoproteins,a secreted S-locus glycoprotein (SLG) of around 57 kDa(Nasrallah et al., 1985) and a 120-kDa S-receptor kinase (SRK),which contains a region of high homology to SLG (Steinet al., 1991). The exact role of SLG in Brassica SI is unknown,and remains to be ascertained (see below). Meanwhile, it has beendemonstrated that SRK is a key element in the Brassica SIsystem (Nasrallah et al., 1985; Nasrallah et al., 1988; Kandasamyet al., 1989; Stein et al., 1991). It is related to a large family ofplant receptor-like kinases (Walker & Zhang, 1990; Steinet al., 1991; Walker, 1993), and direct evidence that the SRKgene encodes a functional serine/threonine protein kinase hasbeen obtained (Goring & Rothstein, 1992).

Nucleotide sequencing reveals that SRK genes can beclassified into two groups that correlate with the phenotypicdominance/recessive relationships in plants carrying various Sallele combinations. Class I alleles exhibit a strong SI pheno-type and are generally dominant or codominant, whereasClass II alleles are usually recessive and exhibit a weaker SIphenotype. Alleles within each group exhibit c. 90% homology,and approx. 70% homology with members of the othergroup. Initially, sequencing indicated that for any given S-haplotype, the extracellular domain of SRK was highly similarto the SLG encoded by the same S -haplotype. However, morerecent studies indicate that in some S-haplotypes there issignificant sequence divergence. Initially, it was generallybelieved that SRK and SLG worked in conjunction in the SIreaction, possibly as a receptor complex. The analysis of self-compatible mutant lines has confirmed that a functional SRKis required for rejection of incompatible pollen (Goring et al.,1993; Nasrallah et al., 1994; Takasaki et al., 2000).

Most S-haplotypes appear to encode a functional SLGgene, and in most cases it appears to be expressed. Recentstudies have attempted to ascertain the function of SLG. Dixitet al. (2000) demonstrated that expression of SLG alongsideSRK was necessary for the accumulation of physiologically




Review578

significant quantities of SRK in transgenic tobacco plants.Without coexpression of SLG and SRK, SRK formed highmolecular weight aggregates. This implies a role for SLG inmaintaining the solubility and stability of SRK. Transgenicstudies by Takasaki et al. (2000) also showed a potential rolefor SLG in the enhancement of the SI response, although SLGalone was unable to confer S-specificity. However, in certainS-haplotypes SLG activity may be dispensable, as a number ofSI lines have been identified that lack detectable SLG protein(Roberts et al., 1994; Gaude et al., 1995).

A further member of the S gene family, SLR1 (S-locusrelated), has also been identified. However, the SLR1 genedoes not map to the S locus, and is therefore, not directlyinvolved in SI. Instead, as mentioned earlier, it appears to havea role in the adhesion of pollen to the stigma (Lalonde et al.,1989; Trick & Flavell, 1989; Boyes et al., 1991).

Since the identification of pistil S genes from Brassica spp.,all of the major groups investigating SI in Brassica have beensearching for the male counterpart, the pollen S gene. Untilrecently, very little was known regarding the male side of theinteraction. As functional SI requires that both male andfemale components exhibit complete genetic linkage, it wasconsidered likely that they were in close physical proximity toone another. Having isolated part of the S locus containingSRK, it was then theoretically possible to identify potentialpollen S candidates by sequencing regions around SRK.Nevertheless, the physical size and complex nature of the Slocus has meant that this route was employed only recently,following the delineation of the S locus by Casselman et al.(2000). Subsequently, a gene, SCR (S locus cysteine rich pro-tein), mapping to the S locus has been cloned and shown intransgenic plants to confer pollen S specificity (Schopfer et al.,1999). The identification of SCR was achieved duringsequencing of the 13 kb region between the S8 SRK and SLGgenes. The SCR is secreted as a mature hydrophilic protein of8.4–8.6 kDa, and is characterized by the presence of 8 cysteineresidues (Schopfer et al., 1999).

Initial identification of SCR as a candidate for the pollen Sgene was undoubtedly aided by it possessing a similar structureto the PCPs. The PCPs, although not S-linked, had previouslybeen shown to interact with SLG (see Section II). Just beforethe publication of the SCR sequence, Suzuki et al. (1999)reported the analysis of a B. campestris S-haplotype, and com-mented that one of the ORFs, SP11 (here 11 refers to the sizeof the polypeptide and not the S-haplotype), also had similaritiesto the PCPs previously identified. Comparison of this genewith the SCR allele suggests that SP11 is, in fact, the pollen SIdeterminant in B. campestris S9. Indeed, this has now beenconfirmed by studies in which application of recombinantSP11 from the S9 haplotype of B. campestris has been shownto elicit the SI response on S9 stigmas in the presence of crosspollen (Takayama et al., 2000).

A model for SI in Brassica has been proposed (see Fig. 4),although it is clear that several components remain to be

identified. It is proposed that interaction of SCR and SRKtriggers a signal transduction cascade in the stigmatic papillarcell, which results in rapid inhibition of pollen growth (as shownin Fig. 4). Several stigmatic cellular targets have been iden-tified, using yeast two-hybrid screening to find proteins thatinteract with the kinase domain of SRK (Bower et al., 1996;Gu et al., 1998). One of these, ARC1, interacts with SRK ina phosphorylation-dependent manner (Gu et al., 1998). Anotheris THL1, which has been reported to regulate autophosphoryla-tion of SRK (Cabrillac et al., 2001). Since down-regulation of

pollen grain

PCP

SCR/SP11

aquaporin

stigmatic papilla

?

SLG

SRK

arc1

P

P

Fig. 4 Model for the mechanism of pollen inhibition for Brassica. In Brassica the self incompatibility (SI) response occurs within the stigmatic papillar cells. When a pollen grain (yellow) alights on the papilla surface (green), the pollen coat (dark yellow), containing pollen coat proteins (PSPs) that include PCPs (black trapezoids) and the pollen S ligand, SCR/SP11 (dark blue circles), flows to form a layer (shown in dark yellow) between the pollen and stigma. In an incompatible reaction, the SCR of the pollen coat and SRK on recipient stigma are encoded by the same S–haplotype and interaction between SCR and the extracellular domain of SRK takes place. This interaction results in activation of the intracellular ser-thr protein kinase domain of SRK (shown as a dark green star). The role of the S locus glycoprotein (SLG), which has the same structure as the extracellular domain of SRK is unclear. Following activation, SRK phosphorylates ARC1 (shown in mauve). This appears to be the first step in an intracellular signalling cascade within the papillar cell. Although a detailed analysis remains to be undertaken, there is evidence that suggests this signalling cascade may ultimately regulate the activity of aquaporins in the stigmatic papillae to limit the availability of water to the incompatible pollen. Adapted, with permission from American Society of Plant Physiologists.




Review 579

ARC1 in transgenic plants results in the partial breakdown ofSI, this suggests that it plays an important role (Stone et al.,1999). However, knocking-out ARC1 expression does not resultin full self-compatibility, as the level of seed set is not equivalentto that obtained from a cross-pollination. This suggests thatother unidentified components participate in the rejectionmechanism. The identification of a self-compatible line of Brassicathat is defective in an aquaporin–like stigmatic gene, mod,suggests the SI response may involve the regulation of watertransfer from the stigmatic papillae to the pollen (Ikeda et al.,1997). This component is not linked to the S-locus, so itprobably acts as a modifier gene. However, since it has beenshown to be required for SI to take place, it clearly plays animportant role in the response, most likely very early, at thepollen hydration stage.

SI in Ipomoea Investigations are also currently underway todetermine the molecular nature of sporophytic SI in theConvolvulaceae using Ipomoea trifida, a close relative of sweetpotato. As the SI system in the Convolvulaceae has a geneticalcontrol that is identical to the Brassicaceae (Kowyama et al.,1980) it might be expected to employ similar molecules in thiscell–cell recognition process. It appears, however, that Ipomoeadoes not employ the same genes and mechanisms as Brassicato control SI. Although SRK homologues have been identifiedin I. trifida, none of these has been demonstrated, thus far, tobe S-linked. It is thought, for this reason that the underlyingmechanisms of these two families are unrelated (Kowyama et al.,1996), and that SI in the Brassicaceae and the Convolvulaceaeprobably evolved independently.

Attempts to identify the molecular basis of SI in this familyhave concentrated on the use of two strategies. First, 2D-SDS-PAGE gel analysis of proteins derived from the stigmatictissues of plants of different S-genotypes identified polymorphicprotein spots of c. 70 kDa, designated as SSPs (S-locus-linkedstigma proteins). Full-length cDNA clones corresponding tothese polymorphic proteins were then isolated. Homologysearches with the sequence of the four SSP genes identifiedthem as having structural homology to nonmetallo short-chainalcohol dehydrogenases (SCADs). However, linkage analysislater showed recombination between SSP and the S-locus.Second, AFLP-fingerprinting has been attempted, using stigmacDNA derived from plants of several different S-genotypes.From these studies 11 putative S-linked fragments were iden-tified, one of which was found to be SSP. RFLP analysisis currently being carried out on the remaining AFLP clones,although, as yet, no candidate S-genes have been documentedand thus the molecular basis of SI in the Convolvulaceaeremains unknown (Kowyama et al., 2000).

2. Interspecific incompatibility responses

By contrast to intraspecific incompatibility (SI), inter-specific incompatibility has received far less attention. Hence,

comparatively little is known regarding the molecularmechanism that underlies this process. Two general modelshave emerged to describe how angiosperms distinguish con-specific pollen from that produced by donors of foreignspecies. The incongruity model, proposed by Hogenboom(1975), suggests that interspecific incompatibility is theresult of barriers determined by evolutionary divergence ofphysiology or morphology between species. This is especiallylikely in those species that are only distantly related. Incon-gruity then, can be seen as an essentially passive process.The alternative model contrasts with incongruity, since it isproposed that cross-hybridization is prevented by an activeprocess that inhibits what would otherwise be a compatiblepollination (de Nettancourt, 1977). These interspecificincompatibility responses have been observed within severalplant families.

A connection between the underlying mechanisms behindinterspecific incompatibility and SI has been suggested (Lewis& Crowe, 1958). Within many plant families, there are closelyrelated species exhibiting both SI and self-compatibility (SC).An important feature of interspecific crosses in a number ofgenera is that they allow for crosses in one direction only, withthe reciprocal cross being unsuccessful (Mutschler & Liedl,1994). For instance, it has frequently been observed thatpollen from self-compatible species is often rejected by pistilsof self-incompatible species, whilst the reciprocal crosses areviable. This is generally referred to as the SI × SC rule. How-ever, this apparent interrelationship between interspecificincompatibility and SI is not clear-cut. There are exceptionsto the SI × SC rule and the timing and nature of the responsein interspecific incompatibility is not necessarily equivalentto that in the SI response (Ascher & Peloquin, 1968). Never-theless, there is evidence suggesting that factors involved ininterspecific incompatibility are linked to the S locus. Forexample, Pandey (1981) demonstrated differences in abilityto reject foreign pollen between S alleles of the same Nicotianaspecies, whilst mapping studies have confirmed a role forinterspecific incompatibility for the S locus in Lycopersicon(Chetelat & de Verna, 1991). Recent evidence points to a morecomplex picture regarding interspecific incompatibility. Forexample, a cross between N. alata and the closely relatedspecies, N. tabacum shows an exception to the SI × SC rule inthat although this cross is rejected, the same cross using aself-compatible cultivar of N. alata is also nonviable. Trans-genic studies, examining the role of S-RNases in interspecificincompatibility, indicate a requirement for both S-RNasesand additional factors that remain to be identified (Murfettet al., 1996). Thus, even in systems where there is substantialevidence for a link between SI and interspecific incompatibil-ity, the S-locus describes only a part of the system (McClureet al., 2000). The challenge facing workers in this field is toestablish the nature of those other factors and to determinethe extent of the involvement of the S-locus in interspecificincompatibility.




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VII. Conclusions and perspective

We have attempted to review the considerable progress madeover the past decade in elucidating the components andmolecular processes involved in pollination events. What isapparent is that this involves highly complex cellular interactionsthat regulate a series of crucial events, such as pollen adhesion,hydration, pollen tube growth and guidance to the ovulesbefore fertilization may be achieved (as summarized in Fig. 1).Over and above this, there are incompatibility mechanisms(see Figs 2, 3 and 4) that provide barriers to fertilization inmany instances. This review shows that in many areas thereare just a few studies, which although very informative, do notgive a very coherent picture overall. Our knowledge in someaspects is therefore rather fragmented. Furthermore, thecomplexity of both the intercellular and intracellular inter-actions is only just beginning to be revealed. Although it isapparent that much progress has been made in recent years inour understanding of pollen–pistil interactions, there stillremains huge gaps in our knowledge.

Acknowledgements

Work in the authors’ labs is funded by the BBSRC and theGatsby Technical Education Project.

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The molecular and genetic basis of pollen–pistil interactions