AndrewG.McCubbinandTeh-huiKaoarquivo.ufv.br/DBV/PGFVG/BVE684/htms/pdfs_revisao/sinais/... · The...

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September 6, 2000 15:21 Annual Reviews AR112-13

Annu. Rev. Cell Dev. Biol. 2000. 16:333–64Copyright c© 2000 by Annual Reviews. Al l rights reserved

MOLECULAR RECOGNITION AND RESPONSE

IN POLLEN AND PISTIL INTERACTIONS

Andrew G. McCubbin and Teh-hui KaoDepartment of Biochemistry and Molecular Biology, 403 AlthouseLaboratory,ThePennsylvania StateUniversity, University Park, Pennsylvania 16802-4500;e-mail: [email protected]; [email protected]

Key Words self/non-self discrimination, signal transduction, S-locus,receptor kinase, RNases

■ Abstract Many bisexual flowering plantspossess areproductivestrategy calledself-incompatibility (SI) that enables the female tissue (the pistil) to reject self butaccept non-self pollen for fertilization. Three different SI mechanisms are discussed,each controlled by two separate, highly polymorphic genes at the S-locus. For theSolanaceae and Papaveraceae types, the genes controlling female function in SI,the S-RNase gene and the S-gene, respectively, have been identified. For the Brassi-caceae type, the gene controlling male function, SCR/SP11, and the gene controllingfemale function, SRK, have been identified. The S-RNase based mechanism involvesdegradationof RNA of self-pollentubes; theS-proteinbasedmechanisminvolves asig-nal transduction cascadein pollen, including atransient risein [Ca2+]i and subsequentprotein phosphorylation/dephosphorylation; and the SRK (a receptor kinase) basedmechanism involves interaction of a pollen ligand, SCR/SP11, with SRK, followedby asignal transduction cascade in thestigmatic surfacecell.

CONTENTS

INTRODUCTION: Self-Incompatibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334SOLANACEAE TYPE SELF-INCOMPATIBILIT Y . . . . . . . . . . . . . . . . . . . . . . . . 336

TheS-RNaseGeneEncodes Pistil S-HaplotypeSpecificity . . . . . . . . . . . . . . . . . 336Structure-Function Relationships of S-RNases . . . . . . . . . . . . . . . . . . . . . . . . . . 337Approaches to Identifying theGene that Controls Pollen S-HaplotypeSpecificity . 338TheGeneration of New S-HaplotypeSpecificities . . . . . . . . . . . . . . . . . . . . . . . . 339Models for S-RNaseMediated Self-Incompatibility Response . . . . . . . . . . . . . . . 341Modifier Loci that Modulate theSI Response . . . . . . . . . . . . . . . . . . . . . . . . . . . 343

PAPAVERACEAE TYPE SELF-INCOMPATIBILIT Y . . . . . . . . . . . . . . . . . . . . . . 344TheS-GeneControls StigmaFunction in Self-Incompatibility . . . . . . . . . . . . . . . 344Structural Features of S-Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344Identification of Amino Acid Residues of S-Proteins Involved in Recognition . . . . 345Biochemical Responses in Pollen Following Self-Recognition . . . . . . . . . . . . . . . 345Protein KinaseActivity Implicated in theSI Response . . . . . . . . . . . . . . . . . . . . 346

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S-Protein-Binding Proteins in Pollen. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 347Model for Self-Incompatibility Interaction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348

BRASSICACEAE TYPE SELF-INCOMPATIBILITY . . . . . . . . . . . . . . . . . . . . . . 349TheSLGGene–a Polymorphic Stigma-Expressed Gene at theS-Locus. . . . . . . . . 349TheSRKGene Encodes StigmaS-Haplotype Specificity. . . . . . . . . . . . . . . . . . . 350TheSCR/SP11Gene Encodes PollenS-Haplotype Specificity. . . . . . . . . . . . . . . 352Structural Organization of theS-Locus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354Model for Self-Incompatibility Interaction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355

CONCLUSIONS AND FUTURE PERSPECTIVES. . . . . . . . . . . . . . . . . . . . . . . . 357

INTRODUCTION: Self-Incompatibility

Many flowering plant species that produce bisexual flowers have evolved mecha-nisms to circumvent the tendency toward self-fertilization, which is created by theclose proximity of the male (anther) and female (pistil) reproductive organs. Thesemechanisms are collectively termed self-incompatibility (SI). SI enables the pistilto distinguish between self (genetically related) and non-self (genetically unre-lated) pollen of the same species. Depending on the type of mechanism, rejectionof self pollen (tubes) by the pistil occurs either at the stigmatic surface or in thestyle. Non-self pollen is accepted by the pistil and its tube grows down throughthe style to reach the ovary where fertilization takes place. Thus SI prevents self-fertilization and consequent inbreeding and allows outcrosses to generate geneticdiversity within a species.

Classic genetic studies carried out in the early 20th century revealed two majortypes of homomorphic SI systems, gametophytic and sporophytic (de Nettancourt1977). The term homomorphic indicates that all individuals of a self-incompatiblespecies produce flowers of the same morphological character. The self/non-selfdiscrimination between pollen and pistil is determined by one or more highly poly-morphic loci. This article focuses on two of the families that possess gametophyticself-incompatibility (GSI), Solanaceae and Papaveraceae, and one of the familiesthat possess sporophytic self-incompatibility (SSI), Brassicaceae. Although SI ineach of these families is controlled by a single genetic locus, theS-locus, the mech-anisms employed are completely different, at least at the level of recognition of selfand non-self pollen. It should be noted, however, that two other families that pos-sess GSI, the Rosaceae and Scrophulariaceae, most likely employ the same mecha-nism as the Solanaceae, based on the sequence similarity of the proteins they use tocontrol pistil function in SI (Sassa et al 1996, Xue et al 1996, Ishimizu et al 1998).

The S-locus of both the Brassicaceae and Solanaceae is now known to be amultigene complex, and theS-locus of the Papaveraceae is likely to be such acomplex. Thus the term haplotype is used to denote variants of the locus, and theterm allele is used to denote variants of a given polymorphic gene at theS-locus.

For self-incompatible species in the Solanaceae and Papaveraceae, the pistil dis-tinguishes between self and non-self pollen based on whether theS-haplotype ofthe haploid pollen matches either of the twoS-haplotypes of the diploid pistil.

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SELF-INCOMPATABILITY 335

That is, the SI phenotype of pollen (gametophyte) is determined by its ownS-genotype. When haplotypes match, pollen is recognized by the pistil as self andrejected, whereas if haplotypes differ, pollen is accepted for fertilization. Thuscrosses between two plants are compatible as long as theirS-genotypes differ inone of the twoS-haplotypes (Figure 1). In self-incompatible species of the Bras-sicaceae, the SI phenotype of pollen is determined by theS-genotype of its diploidparent (sporophyte). In the simplest case, the pollen is recognized by the pistilas self if either of the twoS-haplotypes carried by its parent matches one of thetwo S-haplotypes carried by the pistil. Thus crosses between two plants are pos-sible only if theirS-genotypes do not share any haplotype in common (Figure 1).However, often complex relationships exist between the twoS-haplotypes carriedby the pollen and pistil, such that one could be dominant over or recessive to theother, or they could interact to result in mutual weakening (Thompson & Taylor1966).

The following discussion centers on recent efforts to understand the molecularbasis of the self/non-self recognition between pollen and pistil, and the ensuing

Figure 1 Illustration of the genetic basis of gametophytic and sporophytic self-incompatibility. For the gametophytic type, the SI phenotype of pollen is determinedby theS-haplotype of its haploid genome, thus each pollen grain carries the product ofoneS-haplotype. For the sporophytic type, the SI phenotype of pollen is determined bythe S-genotype of its diploid parent, thus each pollen grain carries the products of twoS-haplotypes. In both types, matching of oneS-haplotype between pollen and pistil resultsin rejection of pollen. The scenario depicted for sporophytic SI assumes thatS1-haplotypeis co-dominant with, or dominant over,S3-haplotype.

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biochemical events that result in the inhibition of germination or tube growth ofself pollen.

SOLANACEAE TYPE SELF-INCOMPATIBILITY

The genera in the Solanaceae (nightshade) family includeNicotiana, Petunia,Solanum, andLycopersicon. The great majority of the wild species in this familyare self-incompatible. For example, of the 19 known taxa of thePetuniagenus,15 are self-incompatible and 4 are self-compatible (Tsukamoto et al 1998). How-ever, most commercial cultivars are self-compatible because the SI trait wasselected out at early stages of breeding in order to produce inbred lines ho-mozygous for desirable traits. For example, tomato (Lycopersicon esculentum),tobacco (Nicotiana tabacum), potato (Solanum tuberosum), and garden petunia(Petunia hybrida) are all self-compatible. Much of the molecular informationabout this type of SI has been obtained fromL. peruvianum, N. alata, P. inflata, andS. chacoense.

The S-RNase Gene Encodes Pistil S-Haplotype Specificity

The approach used to identify the gene that controlsS-haplotype specificity of thepistil was based on the prediction that proteins involved in self/non-self recognitionmust exhibit a high degree of allelic sequence diversity. This prediction was borneout when pistil proteins that co-segregate withS-haplotypes were identified basedon their differences in molecular mass and/or isoelectric point. For example, pistilsof S1S2 genotype ofP. inflataproduce a 24-kDa and a 25-kDa protein, the formerco-segregating with theS1-haplotype and the latter with theS2-haplotype (Ai et al1990). These proteins were initially called S-proteins but have been renamedS-RNases since the discovery that they have RNase activity (see below).

The S-RNase gene, in addition to being linked to theS-locus, also exhibits anumber of characteristics expected of the gene that determinesS-haplotype speci-ficity of the pistil. First, it is exclusively expressed in the pistil, with the proteinlocalized mostly in the upper segment of the style where inhibition of self pollentubes occurs. Second, it is expressed at a very low level in the pistils of immaturebuds, which are unable to reject self pollen, and the increase in expression duringsubsequent flower development coincides with the acquisition of SI by the pis-til. (This expression pattern makes it possible in some self-incompatible speciesto obtain plants homozygous for a particularS-haplotype by selfing at immaturebud stages.) Third, the sequences of its allelic products, S-RNases, are highlydivergent, with amino acid sequence identity ranging from 38 to 98%.

The function of the S-RNase gene in SI has been directly confirmed by gain-of-function and loss-of-function experiments. These experiments showed that theS-haplotype specificity of the pistils of transgenic plants could be altered by theexpression of a sense or antisense S-RNase transgene (Lee et al 1994, Murfett

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et al 1994). For example, inP. inflata, when the S3-RNase gene was introducedinto plants ofS1S2 genotype, those transgenic plants that produced S3-RNase inthe pistil gained the ability to rejectS3 pollen. Conversely, when an antisenseS3-RNase gene was introduced into plants ofS2S3 genotype, transgenic plants inwhich the production of S3-RNase in the pistil was suppressed lost the ability torejectS3 pollen. These experiments thus demonstrated that theS-RNase gene issolely responsible for the S-haplotype specificity of the pistil.

Structure-Function Relationships of S-RNases

Despite the sequence diversity among S-RNases, five regions of sequence conser-vation, C1 to C5, have been identified (Ioerger et al 1991, Tsai et al 1992). C2and C3 share a high degree of sequence similarity with the corresponding regionsof two fungal RNases, RNase T2 and RNase Rh. It is this similarity that led tothe discovery that S-proteins are RNases (McClure et al 1989). The finding thatRNases are employed by the pistil to reject self pollen raised the possibility thatthe RNase activity is responsible for growth inhibition of self pollen tubes. Toexamine this possibility inP. inflata, a mutant S3-RNase gene, with the codonfor one of the two catalytic histidines replaced with an asparagine codon, wasintroduced intoS1S2 plants, and the transgenic plants were analyzed for theirability to rejectS3 pollen (Huang et al 1994). The results showed that produc-tion of this mutant S3-RNase did not confer the ability to rejectS3 pollen on thetransgenic plants. Thus the RNase activity is an integral part of the function ofS-RNases.

S-RNases are glycoproteins with one or more N-linked glycan chains. Thestructure of the glycan chains of some S-RNases has been determined (Woodwardet al 1992, Oxley et al 1998, Parry et al 1998), and the similarities in the structurefor different S-RNases suggest that the glycan chains are unlikely to encode theS-haplotype specificity. Indeed, when an engineered S3-RNase gene ofP. inflata,with the asparagine codon for the only N-glycosylation site of the protein replacedwith an aspartic codon, was introduced intoP. inflataplants ofS1S2 genotype, thisnon-glycosylated S3-RNase functioned as well as wild-type S3-RNase in rejectingS3 pollen (Karunanandaa et al 1994). Thus theS-haplotype specificity determinantof S-RNases resides in the protein backbone and not in the glycan side chains.

Sequence comparison of S-RNases has revealed two hypervariable regions,termed HVa and HVb, which are also the most hydrophilic regions of S-RNases(Ioerger et al 1991, Tsai et al 1992). These two characteristics led to the hypothesisthat HVa and HVb are the prime candidates for the determinant ofS-haplotypespecificity. Consistent with this hypothesis, HVa and HVb form a continuous sur-face on one side of S-RNases in a three-dimensional structure model ( Parry et al1998) based on the coordinates of RNase Rh (Kurihara et al 1992), which sharesimilar predicted secondary structures with S-RNases. To examine the role of HVa,HVb, and other regions of S-RNases inS-haplotype specificity, chimeric S-RNasegenes were constructed and introduced into transgenic plants for analysis of the

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S-haplotype specificity displayed by each of these hybrid S-RNases. In construct-ing these chimeric genes, one allele of the S-RNase gene was used as the backbone,and the sequence for a region of another allele was swapped into the correspond-ing region of the backbone allele. When pairs of S-RNases with a high degreeof sequence diversity were used for domain swapping, every region swapped(including HVa and HVb) led to the loss of theS-haplotype specificity of theallele used as the backbone. Moreover, no gain of the newS-haplotype specificityof the allele source for the newly introduced region was found, despite the factthat all these hybrid S-RNases exhibited normal levels of RNase activity (Kao &McCubbin 1996, Zurek et al 1997). These results also suggest that the RNaseactivity of S-RNases is necessary but not sufficient for their function in SI.

Two S-RNases ofS. chacoense, S11-RNase and S13-RNase, which are among themost similar pairs of S-RNases characterized so far (Saba-El-Leil et al 1994), werealso used for construction of chimeric S-RNase genes (Matton et al 1997). Thesetwo S-RNases differ only in 10 amino acids, 3 of which are located in HVa and 1in HVb. When the amino acids of HVa and HVb of S11-RNase were changed tothose of S13-RNase, transgenic plants that produced this hybrid S-RNase rejectedS13 pollen, but notS11 pollen. These results appear to suggest that HVa and HVbtogether are sufficient forS-haplotype specificity. However, since any domainswapping experiment can only address the role of those amino acids exchangedbetween the two proteins under study, it remains possible that amino acids outsideHVa and HVb and conserved between S11-RNase and S13-RNase are also involvedin S-haplotype specificity (Verica et al 1998). Nonetheless, it is clear from alldomain swapping experiments that the HVa and HVb regions play a key role inS-haplotype specificity.

Approaches to Identifying the Gene that Controls PollenS-Haplotype Specificity

Several lines of evidence have clearly suggested that a gene other than theS-RNase gene controls the pollen function in SI. First, some self-compatible mu-tations mapped to theS-locus affect only pollen function (pollen-part mutations),whereas others affect only pistil function (pistil-part mutations) (de Nettancourt1977). Second, when a new allele of the S-RNase gene was expressed in pollenof transgenic plants, no change in the SI phenotype of the pollen was observed(Dodds et al 1999). Third, when the antisense and sense S3-RNase genes drivenby the promoter of the S3-RNase gene were introduced into transgenic plants(as described above), the SI phenotype of the pistil but not pollen was affected(Lee et al 1994). Fourth, some pollen part mutants result from their pollen carry-ing two differentS-haplotypes, and analysis of such mutants has shown that somecarry a centric fragment containing part of theS-locus without the S-RNase gene(Golz et al 1999). Fifth, a chromosomal region (larger than 30 kb) containingthe S4-RNase gene was deleted from a self-compatible cultivar ofPyrus serotina(Japanese pear), a species of the Rosaceae that also employs S-RNases in SI, and

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this deletion affected the pistil function but not pollen function (Sassa et al 1997;H Sassa, personal communication).

The pollenS-gene is expected to exhibit the following characteristics. First, itis genetically very tightly linked to the S-RNase gene, as recombination betweenthe genes controlling pollen and pistil functions has never been observed andwould inevitably result in the breakdown of SI in the progeny (de Nettancourt1977). Second, it shows a high degree of allelic sequence diversity, at least in theregion(s) determiningS-haplotype specificity. Third, its allelic products interactwith S-RNases in anS-haplotype-specific manner. This has been demonstrated ina dominant-negative experiment (McCubbin et al 1997). When a mutant S3-RNasegene ofP. inflata, with the codon for one of the catalytic histidines replaced withan arginine codon, was introduced intoS2S3 plants, this mutant S3-RNase (withoutRNase activity) was found to render wild-type S3-RNase unable to completelyrejectS3 pollen, but not to affect the ability of wild-type S2-RNase to rejectS2pollen.

RNA differential display and subtractive hybridization have been used to iden-tify a number of pollen-expressed genes ofP. inflata that showS-haplotype-specific sequence polymorphism and are tightly linked to the S-RNase gene (Dowdet al 2000, McCubbin et al 2000). Whether any of these genes is the pollenS-gene remains to be determined. Regardless, they can serve as molecular mark-ers for the physical mapping of theS-locus and cloning of the DNA in thischromosomal region–an approach that has proven to be successful in the iden-tification of the gene encoding pollenS-haplotype specificity inBrassica(seebelow).

The Generation of New S-Haplotype Specificities

Several lines of evidence suggest that SI in the Solanaceae is controlled by twoseparate but tightly linked genes at theS-locus: the as yet unidentified pollenS-gene controlling pollen function and the S-RNase gene controlling pistil func-tion. Moreover, it has been clearly shown that SI inBrassicais controlled by twoseparate genes. This situation poses a conundrum in formulating models to explainhow newS-haplotypes are generated during the course of evolution. Accumulationof amino acid changes has been implicated in this process (Clark & Kao 1991,Saba-El-Leil et al 1994, Matton et al 1997); however, mutation in either the pollenor pistil gene thereby changing it to a newS-haplotype specificity would causebreakdown of SI.

A tantalizing possible solution to this problem recently emerged from domain-swapping experiments. Using the same pair of S-RNases fromS. chacoense(S11-and S13-RNases) described above, Matton et al (1999) showed that when three ofthe four amino acids in the HVa and HVb regions that differ between these twoRNases were swapped from S13-RNase into S11-RNase, the resulting S-RNasepossessed dualS-haplotype specificity. That is, this hybrid S-RNase rejectedboth S11 andS13 pollen. Based on this finding, Matton et al (1999) propose a

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Figure 2 Two models for explaining the generation of a newS-haplotype specificitywithout loss of self-incompatibility: (A) a three-mutational-step model; (B) a two-mutational-step model. F, pistil; M, pollen. See text for details.

three-mutational-step model for the generation of newS-haplotype specificities(Figure 2A ). Assume that a point mutation in an S-RNase gene ofSa-haplotyperesults in an S-RNase with bothSa- andSb-haplotypes. A subsequent point mutationin the pollenS-gene of the plant with dual pistilS-haplotypes would enable thepollen product to recognize the new (Sb) but not the original (Sa) haplotype speci-ficity. Finally, further mutation of the S-RNase gene with dual specificity resultsin the loss of the original specificity. This process would generate a newS-haplo-type specificity without the loss of SI at any intermediate stage. Conceivably,the initial mutation could occur in the pollenS-gene to result in a protein withdual specificity. Thus a reciprocal version of the above process would be equallypossible.

The domain swapping results described above can also be explained by an al-ternative model. As the sites ofS-RNases involved inS11-haplotype specificity donot appear to completely overlap with those involved inS13-haplotype specificity(Matton et al 1999), a given S-RNase or pollen S-protein may possess one or morelatent specificity(ies) aside from the one actively involved in the SI interaction. Forexample, an S-RNase ofSa-haplotype may have a latentSb-haplotype specificity,which would be uncovered if the pollenS-gene ofSa-haplotype mutates such thatit recognizes amino acids specifyingSb-haplotype, but not those involved in theoriginal haplotype specificity (Figure 2B ). Once this change in theS-haplotypespecificity of pollen has occurred, selection pressure to conserve amino acids ofSab-RNase involved in the original (Sa) haplotype specificity would be lost, sofurther mutation could occur, thereby causing the loss of the original specificity.According to this model, a newS-haplotype specificity could be generated inonly two mutational steps, and SI would remain intact throughout the mutationalprocess.

It should be noted that plants whose pistils or pollen possess dual specificity innature have never been reported. At present, we can do little more than hypothesizeabout the mechanism by which newS-haplotypes are generated. Identification ofthe pollenS-gene and subsequent sequence comparisons between the S-RNasegene and the pollenS-gene from differentS-haplotypes are likely to provide keyinformation about this process.

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Models for S-RNase Mediated Self-Incompatibility Response

The intrinsic RNase activity of S-RNases is essential for their function in SI;however, the precise manner in which S-RNases inhibit the growth of self pollentubes is not clear. One question is on what type of RNA molecules do S-RNases act?S-RNases do not appear to have any substrate specificity in vitro (Singh et al 1991),but it is not known whether this is the case in vivo. It is thought that rRNA genesare not expressed in angiosperm pollen tubes (McClure et al 1990, Mascarenhas1993) and that protein synthesis during the growth of pollen tubes is carried out byribosome components that are fully formed at pollen maturity. Thus degradation ofrRNA by S-RNases would provide a plausible mechanism for inhibition of pollentube growth. Indeed, degradation of rRNA in incompatible pollen tubes has beenobserved (McClure et al 1990); however, whether degradation is a cause or an effectof the growth arrest of self pollen tubes cannot be clearly discerned. Experimentsthat involve grafting of incompatibly pollinated styles onto compatible ones haveshown that a certain percentage of arrested incompatible pollen tubes can resumegrowth in compatible styles (Lush & Clarke 1997). This finding suggests that ifrRNA is the target of S-RNases, the rRNA genes must be transcribed in pollentubes. Alternatively, the target of S-RNases may be a different class of RNAmolecules, perhaps mRNA.

The identity of the pollenS-gene is a key missing piece of the puzzle inunderstanding the mechanism of the S-RNase mediated SI response. There areconceivably two ways by which a pollenS-allele product can work together withS-RNases to elicitS-haplotype specific inhibition of pollen tube growth: it mayserve as a gatekeeper (Figure 3A ) or as an RNase inhibitor (Figure 3B ). An in vitropollen germination assay has been used to test the former possibility by determin-ing whether entry of S-RNases into pollen tubes isS-haplotype specific (Gray et al1991). The results showed that S-RNases enter the cytoplasm of both self andnon-self pollen tubes; however, because the growth of both kinds of pollen tubesis inhibited in this assay, these in vitro results may not reflect the in vivo situation.One theoretical problem in considering the pollen S-protein as an RNase inhibitor isthat, if this were the case, a pollen S-protein would have to recognize all S-RNasesexcept self S-RNase and inhibit the RNase activity of all non-self S-RNases. Giventhe high degrees of sequence diversity between S-RNases, this seems unlikely. Apossible solution to this problem would be to envisage that a pollen S-protein con-tains an RNase inhibitor domain and anS-haplotype specificity domain and that itinteracts with self and non-self S-RNases differently (Figure 3B ). The specificitydomain would interact with the matching specificity domain of self S-RNase andin so doing would physically occlude the RNase inhibitory interaction. In the caseof non-self S-RNases, the RNase inhibitor domain of a pollen S-protein wouldinteract with the active site of these S-RNases because there is no matching intheir specificity domains, and in so doing would inhibit the RNase activity.

Mutational studies have produced a number of self-compatible mutants thatare defective in pollen but not in pistil function in SI. Interestingly, the majority

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of the pollen-part mutants appear to have a centric fragment or a chromosomalduplication that contains part or all of theS-locus region. Moreover, only thosecarrying an extraS-locus region of anS-haplotype differing from that carried by theendogenous chromosomes lose pollen function in SI (de Nettancourt 1977, Golzet al 1999). This finding is consistent with the RNase inhibitor model because twodifferent pollen S-proteins produced in the same pollen tube would together inhibitthe RNase activity of allS-RNases. Ultimate understanding of the mechanism willhave to await the identification and characterization of the pollenS-gene.

Modifier Loci that Modulate the SI Response

The S-locus encodes all the determinants ofS-haplotype specificity. However,there is evidence for the existence of unlinked loci, termed modifier loci, thatmodulate the SI response. Early studies of the Solanaceae demonstrated that mul-tiple loci are required for a full manifestation of SI and that some of these can actdifferently on differentS-haplotypes (East 1932). An S-RNase gene that fails tofunction in a self-compatible line ofP. hybridahas been shown to be functionalwhen introgressed into a self-incompatibleP. inflatabackground, suggesting thatthe self-compatibleP. hybridabackground either lacks some factors that are re-quired for the function of the S-RNase gene or contains some factors that suppressthe function of the S-RNase gene (Ai et al 1991). Studies involving the intro-gression ofS-locus bearing chromosomal fragments from self-incompatibleL.hirsutum into self-compatibleL. esculentumlines have also demonstrated a re-quirement for additional non-S-linked factors in the SI response (Bernatzky et al1995).

Recent reports have begun to provide information on the mechanisms by whichsome of these modifier loci act. A modifier locus ofP. axillarishas been implicatedin the breakdown of pistil function in SI, and it specifically affects the expressionof one of the three alleles of theS-RNase gene examined (Tsukamoto et al 1999).The identity of the modifier gene(s) involved has not been determined. To identify

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−Figure 3 Models of the S-RNase mediated self-incompatibility response. (A) Gatekeepermodel. (B) RNase inhibitor model. A pistil cell synthesizes and secretes both S1- andS2-RNases into the transmitting tract of the pistil whereS1 andS3 pollen tubes are growing.In (A), pollenS-allele products are predicted to be membrane- or wall-bound receptors forS-RNases. The pollen S1-protein allows only the S1-RNase to enter the cytoplasm of theS1 pollen tube, which results in degradation of its RNA and cessation of tube growth; thepollen S3-protein allows neither S1- nor S2-RNase to enter the cytoplasm of theS3 pollentube. In (B), pollenS-allele products are predicted to be cytosolic RNase inhibitors. Both S1-and S2-RNases enter theS1 andS3 pollen tubes, but the pollen S1- and S3-proteins interactdifferently with self and non-self S-RNases, resulting in inhibition of RNase activity of allnon-self S-RNases (i.e. S1- and S2-RNases in theS3 pollen tube; S2-RNase in theS1 pollentube), but not that of self S-RNase (i.e. S1-RNase in theS1 pollen tube). Consequently, thegrowth of theS1 but notS3 pollen tube is inhibited.

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non-S-RNase factors that are required for SI inNicotiana, McClure et al (1999)used two closely related species, a self-compatible line ofN. alata(which containsthe non-S-RNase factors) and self-compatibleN. plumbaginifolia(which does notcontain these factors) to perform a differential screen of pistil expressed genes.Among the cDNAs selected, one, designatedHT, has been shown by antisenseexperiments to be essential for SI; suppression of its expression did not affect thetranscript or protein level of the SC10-RNase gene but led to the inability of thepistil to rejectSC10 pollen. TheHT cDNA is predicted to encode a protein of 101amino acids, containing a stretch of 20 asparagine and aspartate residues near theC terminus. Unfortunately, database searches do not provide any insight into thecellular function of this protein, and no direct interaction of HT with S-RNases hasbeen detected. Nonetheless,HT represents the first modifier gene of gametophyticSI identified, and the elucidation of its role in SI and those of other modifier genes isof great importance for a full understanding of the mechanism of S-RNase based SI.

PAPAVERACEAE TYPE SELF-INCOMPATIBILITY

This type of SI mechanism has been studied almost exclusively inPapaver rhoeas(field poppy). This species is recalcitrant to transformation, making it difficultto use transgenic approaches to examine the function of genes implicated in SI.However, a reliable and efficient in vitro bioassay for the SI response has beendeveloped in which pollen germination or tube growth can be shown to be inhibitedby stigmatic extracts in anS-haplotype-specific manner (Franklin-Tong et al 1988).This assay has been used to identify S-proteins (stigma proteins that are involvedin pollen rejection), study the structure-function relationships of S-proteins, andcharacterize self-pollination specific biochemical events that occur in pollen.

The S-Gene Controls Stigma Function in Self-Incompatibility

Unlike S-RNases of the Solanaceae, S-proteins ofP. rhoeasare present at verylow levels in the pistil (nanograms per pistil as opposed to micrograms per pistilfor S-RNases). Nonetheless, the allelic sequence diversity of S-proteins is alsovery high, allowing identification of stigma proteins that are unique to particularS-haplotypes by isoelectric focusing. Proteins associated withS1- andS3-haplo-types have been identified and their N-terminal sequences have been determined.The sequence information has been used to design oligonucleotide probes forisolating their corresponding cDNAs.

Structural Features of S-Proteins

Five alleles of thePapaver S-gene have now been cloned and sequenced ( Foote et al1994, Walker et al 1996, Kurup et al 1998). The S-proteins ofP. rhoeasdo notshare any sequence similarity with S-RNases, despite the fact that both Solanaceaeand Papaveraceae possess GSI.PapaverS-proteins are small (∼15 kDa) secreted

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proteins, some of which are N-glycosylated. Because recombinant proteins pro-duced inEscherichia coliwere found to inhibit pollen tube growth in anS-haplo-type-specific manner in the in vitro bioassay (Franklin-Tong et al 1995), the glycanchains are unlikely to be required for the function of S-proteins. Like S-RNases,S-proteins are highly polymorphic, sharing between 51.3 and 63.7% amino acidsequence identity. However, they are predicted to possess a virtually identicalsecondary structure, which is made up of a series of sixβ strands followed bytwo α-helical regions located at the C terminus, all linked together by sevenhydrophilic loops ( Walker et al 1996, Kurup et al 1998). Significant sequencesimilarity has been found between S-proteins and a large number of open readingframes of theArabidopsisgenome (Ride et al 1999). Indeed it has been estimatedthat there are likely to be approximately one hundred S-protein homologues (SPHs)in Arabidopsis, but none are present in the current EST databases, suggesting thatthey may be expressed at very low levels, only at certain developmental stages,and /or only in response to certain environmental or biotic stimuli. The functionof theSPHgenes inArabidopsis, which is a self-compatible species, is unclear atthe present time.

Identification of Amino Acid Residues of S-ProteinsInvolved in Recognition

Because recombinant S-proteins produced inE. coli are functional in the in vitrobioassay, it has been possible to engineer mutant S-proteins for the identificationof amino acid residues that are required for the function of S-proteins in the SIresponse (Kakeda et al 1998). The majority of the mutant S1-proteins generatedcontain amino acid changes in the predicted surface loops. Most of these changeshave little or no effect on the ability of S1-protein to inhibit germination or tubegrowth ofS1 pollen. However, some residues in loop 6 were found to be essential.For example, changes of the only hypervariable amino acid residue in this loopand of several highly conserved amino acids adjacent to this residue resultedin complete loss of the ability of S1-protein to inhibitS1 pollen. These resultssuggest that loop 6 may be directly involved in recognition events essential for theSI response.

Biochemical Responses in Pollen Following Self-Recognition

The in vitro bioassay system has also enabled studies of the biochemical eventsthat occur in pollen during the SI response. These include changes in pollen geneexpression (Franklin-Tong et al 1990), protein phosphorylation (Franklin-Tonget al 1992), and cytosolic calcium levels (Franklin-Tong et al 1993) followingchallenge of pollen with self S-protein.

Actinomycin D, a transcription inhibitor, has no effect on pollen germination,presumably because mature pollen contains all the RNAs required for pollen tubegrowth. Application of actinomycin D does, however, partially alleviate polleninhibition by self S-protein (Franklin-Tong et al 1990), suggesting that de novo

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pollen gene expression is required for a full SI response. In vitro translation ofmRNAs isolated from pollen treated with self or non-self S-protein has revealedseveral proteins specific to an incompatible reaction (Franklin-Tong et al 1990).Several pollen cDNA clones have been identified whose expression appears to becorrelated with the SI response in pollen (Franklin-Tong et al 1992). However,it is not known if these genes are directly involved in the SI response or whetherthey are expressed as a result of the SI response.

The activation of pollen response genes in the SI response suggests that theinteraction between an S-protein and its receptor molecule in the pollen (presum-ably the product of the pollenS-gene with the sameS-haplotype) activates a signaltransduction mechanism that is responsible for the SI response. The involvementof cytosolic free calcium ([Ca2+]i) as a second messenger in many signal trans-duction processes in plants is well established (Hepler & Wayne 1985, Trewavas& Gilroy 1991), and a number of studies have implicated Ca2+ signaling in pollengermination and pollen tube growth (Franklin-Tong 1999). For example, growingpollen tubes have a [Ca2+]i gradient at their tips (Obermeyer & Weisenseel 1991,Miller et al 1992), whereas non-growing tubes lack this gradient. The role ofCa2+ signaling in the SI response ofP. rhoeashas been investigated using Ca2+

-selective dyes (Franklin-Tong et al 1993, 1995). Inhibition of pollen tube growthresulting from the addition of self S-protein is preceded by a transient increase in[Ca2+]i in pollen tubes. This elevation originates from the nuclear complex and theendoplasmic reticulum associated with this region, suggesting that [Ca2+]i may beinvolved in the regulation of gene expression (Franklin-Tong et al 1993).

Protein Kinase Activity Implicated in the SI Response

The discovery that [Ca2+]i is associated with the SI response has prompted exami-nation of differential protein phosphorylation in pollen tubes challenged with selfor non-self S-proteins. A number of proteins have been found to be either phos-phorylated or dephosphorylated when comparing pollen tubes that are challengedwith self S-protein with those that are challenged with non-self S-protein(s) (Ruddet al 1996). Two proteins whose phosphorylation is specifically increased as aconsequence of the SI response have been characterized in some detail. One, p26,is a 26-kDa protein with a pI of 6.2 and exhibits Ca2+-dependent phosphorylation.This phosphorylation occurs within 90 s of S-protein application to the in vitrobioassay, with further increase occurring at 400 s. This timing coincides with thetransient increase in [Ca2+]i on treatment with self S-protein (10–∼400 s), sug-gesting that phosphorylation of p26 may be stimulated directly by the elevation of[Ca2+]i, probably through activation of a CaM- or Ca2+-dependent protein kinase(Rudd et al 1996). The other protein, p68, is 68 kDa in size with pI in the rangeof 6.10 to 6.45. This protein is also phosphorylated in response to self S-protein,but in a Ca2+-independent manner. The timing of this phosphorylation is differ-ent from that of p26, i.e. barely detectable at 240 s but much increased at 400 s(Rudd et al 1997). The difference in the timing of phosphorylation of p26 and

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p68 suggests that p68 is likely to be downstream of p26 in the signal cascade.Given that growth inhibition of the pollen tube occurs very rapidly, within 1–2min (coinciding with the phosphorylation of p26), this might suggest that p68 isnot involved in growth inhibition, but perhaps in later events involving cell death.

S-Protein-Binding Proteins in Pollen

The technique of Western ligand blotting has identified a pollen protein, SBP(70–120 kDa in size), that binds to pistil S-proteins of allS-haplotypes examined(Hearn et al 1996). Phase partitioning has demonstrated that SBP is an integralplasma membrane protein, as one might expect of a cell surface receptor. Theinteraction between SBP and S-proteins appears to require glycan moieties as theinteraction was abolished by periodate treatment of the pollen protein blots priorto incubation with S-proteins (Hearn et al 1996). To examine the role of SBP inthe SI response, mutant S1-proteins were used in both in vitro bioassay for theSI response and the Western ligand blotting assay for SBP-binding activity. Inmost cases, there was a good correlation between SBP binding and the SI response(Jordan et al 1999). For example, amino acid changes in the predicted loop 6 ofS1-protein were found simultaneously to reduce greatly the ability of S1-proteinto bind SBP and to affect the in vitro SI response. Similar results were obtainedwith amino acid changes in loop 2, although the reduction was to a lesser degree.However, deletion of the first 16 amino acids of S1-protein did not affect the bindingactivity to SBP and yet completely abolished the ability of the protein to inhibitself pollen. The authors suggest that the lack of activity in the SI response is dueto the large deletion adversely affecting the protein structure–although binding toSBP was not affected. Of the first 16 amino acids only the first 4 are variablebetween differentS-proteins, and when these alone were deleted, the resultantmutant protein retained wild-type activity in both SBP binding and SI assays(cited in Jordan et al 1999). Because the remaining 12 amino acids are conservedbetween S-proteins, perhaps an alternative explanation for these results is that asecond non-S-specific interaction involving these amino acids and an unidentifiedprotein are essential for the SI response. Nonetheless, it would be interesting todetermine whether S1-protein lacking the first 16 amino acids has a dominant-negative effect over the wild-type S1-protein, which, if so, might be interpreted tobe a result of competition for SBP.

SBP has been proposed to function as an accessory receptor, whose role is tomodulate the interaction of S-proteins with the pollen S receptor (Jordanet al 1999). The authors suggest that SBP and S-proteins together would form arecognition complex analogous to those found in some mammalian systems, suchas the fibroblast growth factor (FGF ) signaling system, where a family of mem-brane proteins, the heparin sulfate proteoglycans (HSPG), function as accessoryreceptors (Spivak-Kroizman et al 1994). In this case, low-affinity high-capacitybinding of FGF to glycan side chains of HSPG facilitates oligomerization of theligand, which then interacts with the high-affinity FGF receptor triggering a signal.

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Model for Self-Incompatibility Interaction

The results reported to date suggest that SI inP. rhoeas, unlike SI in the Solanaceae,is controlled by a signal transduction mechanism (Figure 4 ). It can be envisagedthat the stigmatic S-protein acts as a signaling molecule, interacting with its re-ceptor on the plasma membrane of the pollen grain or tube. The identity of thepollen receptor (presumably encoded by the pollenS-gene) controlling the speci-ficity of this interaction is unknown, but there is evidence that one or more non

Figure 4 A model of S-protein mediated self-incompatibility response. S1- and S2-proteins are produced and secreted by the stigmatic surface cell. In the wall ofS1 andS3 pollen, both S-proteins interact with an accessory receptor protein, SBP; however, theinteraction with the pollen receptor (a pollen S-protein) can occur only between the S1-protein/SBP complex and the pollen S1-protein. This interaction leads to a cascade ofsignal transduction events in theS1 pollen, resulting in inhibition of germination or tubegrowth.

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S-specific accessory proteins are involved, one of which has been suggested tobe SBP discussed above. In an incompatible pollination, the S-protein (perhapscomplexed with SBP) would bind to its cognate receptor, triggering a cascade ofsignal transduction events. The first step in this cascade has been suggested to bea transient increase in [Ca2+]i in the pollen. This presumably activates CaM- orCa2+-dependent protein kinases and consequent phosphorylation of protein sub-strates of these enzymes. At the end of the cascade, changes in gene expressionare thought to occur. It is interesting to consider that there might be an amalga-mation of two separate processes at work in this system. One is growth inhibitionwhich results from a recognition event between the stigma and pollen proteins andinduction of a signal cascade, and the other is a cell death pathway not specificto SI, which is activated indirectly by the cessation of growth. Tentative evidencefor this comes in the form of phosphoprotein p68, the phosphorylation of whichoccurs later than the cessation of pollen tube growth and after the point at whichthe growth inhibition is reversible.

BRASSICACEAE TYPE SELF-INCOMPATIBILITY

For the families that possess SSI, so far the Brassicaceae is the only one studiedat the molecular level. The species that have been used in most of the studies areB. campestris(Chinese cabbage),B. oleracea(cabbage, cauliflower, kale, Brusselssprout, broccoli, etc), andB. napus(oil seed rape). More than 100 haplotypes havebeen identified in these species.

The SLG Gene–a Polymorphic Stigma-ExpressedGene at the S-Locus

As recognition and rejection of self pollen occurs on the stigmatic surface, searchesfor the female component of SI focused on stigmatic proteins that co-segregate withS-haplotypes. The first such proteins identified were SLGs (S-locus glycoproteins).These proteins are abundant in the stigma and exhibit a number of characteristicsexpected of proteins that would determine theS-haplotype specificity of the stigma.First, they are predominantly produced in the stigma and located in the wall ofthe epidermal cells (papillae) of the stigma, which comes into direct contact withpollen. Second, they are present in very small amounts in the stigmas of immaturebuds which are self-compatible, and the timing of the sharp increase in amountjust prior to flower opening coincides with the timing of the acquisition of SI bythe stigma. Third, their sequences are highly divergent, with pair-wise sequenceidentity ranging from 65 to 97.5% (Kusaba et al 1997). Thus for a number ofyears since its identification theSLGgene has been thought to be required for theS-haplotype specificity of the stigma.

However, early attempts to demonstrate the function of SLG by gain- and loss-of-function approaches did not yield conclusive results. For the loss-of-function

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experiments, introduction of an antisenseSLG gene into transgenic plants ledto the breakdown of SI, but the transcript levels of bothSLGandSRK(S-locusreceptor kinase) were reduced (Shiba et al 1995). Analysis of a naturally occurringself-compatible mutant (scf1) whose stigma, but not pollen, function was defec-tive showed that the transcript and protein levels of bothSLGandSLRs(S-locusrelated) were reduced (Nasrallah et al 1992). The simultaneous reduction in theexpression ofSLGand other related genes made it difficult to draw any conclusionabout the function of SLG from these experiments. In the gain-of-function exper-iments, introduction of anSLGgene of a newS-haplotype did not confer the newS-haplotype specificity on the stigma of the transgenic plants, and instead, causedthe breakdown of SI. This phenotype was attributed to a phenomenon termedhomology-dependent gene silencing, as the expression of the transgene and theendogenousSLG, SLRs, andSRKwas suppressed (Conner et al 1997). In a fewcases where SLG of a newS-haplotype was produced in transgenic plants be-cause the transgene and its recipient plants were from differentBrassicaspecies,the analysis of the SI behavior was complicated by interspecific incompatibility(Toriyama et al 1991, Nishio et al 1992).

Several lines of evidence obtained recently cast doubt on the requirement ofSLGfor S-haplotype specificity of the stigma. First, mature flowers of a line ofself-incompatibleB. oleraceawere found to produce lower levels of SLG thanthose produced by immature buds (which are unable to reject self pollen) of otherself-incompatible lines (Gaude et al 1995). Second, from sequence analysis of alarge number of SLGs, it was found that several pairs of SLGs from geneticallydistinctS-haplotypes are virtually identical in their sequences (Kusaba et al 1997,Kusaba & Nishio 1999). For example, SLG23 and SLG29 of B. oleraceadifferonly in three amino acids and none are in the three hypervariable regions thoughtto be involved inS-haplotype specificity. Third,SLGwas found to be deletedin a self-incompatible line ofB. oleracea(Okazaki et al 1999). Fourth,SLG43 ofB. campestriswas introduced intoS52S60plants ofB. campestris, and the transgenicplants producing SLG43 at a normal level failed to acquire the ability to rejectS43 pollen (Takasaki et al 1999). However, the results from this transformationexperiment were interpreted to mean thatSLGalone is not sufficient, rather thannot required, forS-haplotype specificity.

Recent transformation experiments by Takasaki et al (2000) have clearly shownthat SLG is not required for theS-haplotype specificity of the stigma, but maynonetheless play a role in the enhancement of the SI response. These experimentsalso involveSRKand are discussed below.

The SRK Gene Encodes Stigma S-Haplotype Specificity

SRK is the second highly polymorphic gene identified at theBrassica S-locus;this was accomplished by virtue of the sequence similarity betweenSRKandSLG(Stein et al 1991). SRK has the hallmarks of a receptor kinase: it consistsof an extracellular domain, a single transmembrane domain, and a cytoplasmic

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domain that has serine/threonine kinase activity (Goring & Rothstein 1992). Theextracellular domain shares extensive sequence similarity with SLG, and is calledthe S-domain. LikeSLG, SRKalso exhibits properties expected of the gene thatdetermines theS-haplotype specificity of the stigma. First,SRKis predominantlyexpressed in the stigmatic papillae (albeit at a level at least 100 times lower thanthe expression level ofSLG). Second, SRK spans the plasma membrane of thepapillar cell (Delorme et al 1995) and might serve as a receptor for transducing apollen signal to the cytoplasm of the papillar cell. Third,SRKshows a high degreeof allelic sequence polymorphism, as doesSLG.

Results from analyses of self-compatible lines ofBrassicaare consistent withthe notion thatSRK is essential for the stigma function in SI. First,SRKof aself-compatible line ofB. napushas a 1-bp deletion, resulting in a truncated SRK(Goring et al 1993). Second, a chromosomal region of theS-locus containingSRKis deleted from a self-compatible mutant ofB. oleraceawhose stigma function isdefective (Nasrallah et al 1994). Third, anSRKgene engineered to encode a kinase-deficient mutant has anS-haplotype-specific dominant-negative effect on wild-typeSRK, which results in partial breakdown of SI in transgenic plants that producedthe wild-type SRK and mutant SRK of the sameS-haplotype (Stahl et al 1998).

The most direct means to ascertain the function of SRK in SI is by gain-of-function experiments. However, initial attempts to determine whetherSRKof anewS-haplotype, when expressed in transgenic plants, could confer on them thenew S-haplotype specificity also had the same co-suppression problem encoun-tered in the gain-of-function experiments withSLG. Only very recently has thefunction ofSRKin SI been definitively established. Takasaki et al (2000) were ableto overcome the problem of homology-dependent gene silencing by introducingSLG28 andSRK28 of B. campestrisinto S52S60 andS60S60 plants, respectively, be-cause theSLGandSRKof S52- andS60-haplotypes have a low degree of sequencesimilarity with SLG28 andSRK28. They found that production of SRK28 alone, butnot of SLG28 alone, in the stigmas of the transgenic plants conferred the abilityto rejectS28 pollen. These results provide the first direct evidence that SRK isthe sole determinant of theS-haplotype specificity of the stigma and rule out theinvolvement of SLG in this function.

So, what might the function(s) of SLG be? In the transgenic experimentscarried out by Takasaki et al (2000), the strength of self pollen rejection bySRK28

(as measured by the average seed set per flower) in plants that carried theSLGgene of differentS-haplotypes was also examined. A good correlation was foundbetween the strength of self pollen rejection and the degree of amino acid sequenceidentity between the S-domain of SRK28and the SLG produced in the same stigma.The higher the identity, the stronger the rejection. The authors propose that SLG,although not required for theS-haplotype specificity, interacts with theS-domainof SRK to facilitate the process of the recognition reaction between SRK and thepollen determinant of theS-haplotype specificity. The strength of the associationbetween SLG and SRK might then decrease as the sequence identity between themdecreases. However, since the level of SRK28 produced in the transgenic plants

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was only approximately one third of the normal level in plants carrying one copy oftheSRK28gene, it remains possible that SLG is not required for a full manifestationof the SI response when SRK is produced at normal levels.

SLG has also been shown to be required for adhesion of pollen to the surfaceof the papillar cell, the first step in the process of compatible pollination (Luu et al1999). The force of pollen-stigma adhesion was found to be reduced when stigmasof S2- and S5-haplotypes were pretreated with an anti-SLG2 antibody (which cross-reacted with SLG5), and this reduction was attributed to the masking of the bindingsites of SLG for pollen surface molecules during adhesion. A good candidate forsuch a molecule is a pollen coat protein, PCP-A1 (pollen coat protein-A1), whichhas been shown to interact with SLG in vitro in anS-haplotype-independent manner(Doughty et al 1993).

The SCR/SP11 Gene Encodes Pollen S-Haplotype Specificity

As in the Solanaceae SI system, several lines of evidence suggest that stigma andpollen functions inBrassicaSI are controlled by different genes at theS-locus.First, some self-compatible mutants are defective only in pollen function or onlyin stigma function (Hinata & Okasaki 1986, Nasrallah et al 1992). Second, down-regulation of the expression ofSRK, or production of a dominant-negative formof SRK, in transgenic plants affects their stigma but not pollen function (Conneret al 1997, Stahl et al 1998). Third, expression ofSRKof a newS-haplotype intransgenic plants confers the newS-haplotype specificity to the stigma but not topollen (Takasaki et al 2000).

The pollen coat has long been suspected to be the site where the determinantof pollen S-haplotype specificity is located (for a review of pollen coating, seeDoughty et al 1992) for these reasons: (a) The SI interaction occurs at the stigmaticsurface, which makes direct contact with the pollen coat; (b) the pollen coatcontains sporophytically derived materials (released from the tapetum), consistentwith the sporophytic control of the pollen SI behavior. A pollination bioassay wasused to ascertain whether the pollen coating indeed contained theS-determinant(Stephenson et al 1997). In this assay, self or cross pollen coating was first appliedto the papillar cell of a stigma, and the ability of cross pollen to hydrate andgerminate on the stigma was subsequently examined. The results showed thatpretreatment of self pollen coating, but not cross pollen coating, of a stigma ledto a significant reduction of hydration/germination of cross pollen on its surface.Thus a stigma either accepts or rejects a pollen grain based on theS-haplotype ofthe pollen coating applied to the papillar surface. The implication is that the pollencoating contains molecule(s) that can induce the SI response in the stigma. HPLCfractionation of the pollen coating coupled with the bioassay led to the suggestionthat the pollenS-determinant is a basic, cysteine-rich protein of the PCP familywith a molecular mass less than 10 kDa (Stephenson et al 1997).

One approach toward identifying the gene encoding the pollenS-determinantis to examine the region of theS-locus that containsSRK and SLG in order

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to identify genes that exhibit the properties expected of such a gene. The firstpotential candidate,SLA(S-locus anther), was identified from theS2-haplotype ofB. oleracea(Boyes & Nasrallah 1995).SLAis located just downstream ofSLG2

and is expressed exclusively in the anther and microspores of theS2-haplotype,but not in a self-compatible line ofB. napusof theS2-haplotype, which contains aretrotransposon-like insertion inSLA.However, subsequent examination of someself-incompatible lines ofB. oleracearevealed that they also carried an alleleof the SLAgene with a similar retrotransposon insertion (Pastuglia et al 1997).Therefore,SLAis unlikely to be the gene encoding the pollenS-determinant. TwoadditionalS-linked genes,SLL1andSSL2(S-locus linked gene 1 and 2), havealso been ruled out as potential candidates because they exhibit little or no allelicsequence polymorphism (Yu et al 1996).

A 76-kb region of theS-locus containingSRK and SLG of the S9-haplo-type ofB. campestriswas completely sequenced, and found to contain 11 genes(in addition toSRK, SLG, andSLL2) that are expressed in anther and/or pistil(Suzuki et al 1999). The authors postulate that one of these genes,SP11, is a poten-tial candidate for encoding the pollenS-determinant because (a) it encodes a proteincharacteristic of PCP family of proteins (small, basic cysteine-rich proteins witheight cysteines), (b) it is located in the immediate 3′ flanking region ofSRK9, and(c) it is expressed predominantly in the anther (Suzuki et al 1999). Another allele ofSP11was identified independently from the sequencing of a 13-kb region betweenSRKandSLGof theS8-haplotype ofB. campestris(Schopfer et al 1999); however,this gene was given a different name,SCR(S-locus cysteine-rich). cDNA clones forS6-, S12-, S13-, andS52-alleles ofSCR /SP11have also been isolated and sequenced(Schopfer et al 1999, Takayama et al 2000). SCRs/SP11s are small proteins 74–77amino acids in length and are hydrophilic except for an N-terminal stretch of 19amino acids. The mature protein is predicted to be a secreted protein with 8.4–8.6kDa and a pI of 8.1–8.4. Sequence comparison of these SCRs/SP11s has revealedthat while the putative signal peptide is highly conserved, the mature protein ishighly divergent, with conservation limited to just 12 amino acids, 8 of which arecysteines. Overall amino acid sequence identities are between 26 and 46%.

ThatSCR/SP11determines theS-haplotype specificity of pollen has been defini-tively established by a gain-of-function experiment (Schopfer et al 1999) and apollination bioassay (Takayama et al 2000). TheSCR8 promoter was used to drivethe expression ofSCR6 cDNA in transgenicB. oleraceaplants ofS2S2 genotype,and transgenic plants expressing the transgene were used to pollinate wild-typeS6S6 andS22S22 plants. In all cases, the pollen of the transgenic plants expressingtheSCR6 cDNA was rejected byS6S6 pistils but accepted byS22S22pistils (Schopferet al 1999). In the pollination bioassay much like that used by Stephenson et al(1997) described above, recombinant SCR/SP11 of theS9-haplotype was appliedto papillar cells of stigmas of theS9-haplotype and theS8-haplotype, and the pro-tein was found to elicit the SI response only in the former (self ) stigmas, whichresulted in inhibition of hydration of cross pollen (Takayama et al 2000). More-over, in situ hybridization of anther sections showed thatSCR/SP11is expressed

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in the tapetum of the anther (Takayama et al 2000). This sporophytic expressionpattern explains why inBrassicathe SI phenotype of pollen is determined by thegenotype of the pollen parent.

Structural Organization of the S-Locus

TheBrassica S-locus, by definition, is composed of all the components that specifyanS-haplotype. For SI to be maintained, these components must remain as a tightlylinked genetic unit. In recent years, a number of studies have shed light on howthis might be accomplished. Physical maps have been constructed of theS-locusregion of severalS-haplotypes fromB. oleracea(Boyes et al 1997),B. campestris(Boyes et al 1997, Suzuki et al 1999), andB. napus(Cui et al 1999). Considerablestructural divergence has been found in this region of differentS-haplotypes. Forexample, the distance betweenSLGandSRKvaries from as short as 6 kb (Cui et al1999) to as long as over 200 kb (Boyes & Nasrallah 1993). Using direct sequencing,cDNA selection techniques, and RNA differential display, a number of genes havebeen identified in theS-locus region (Cui et al 1999, Suzuki et al 1999, Casselmanet al 2000). Retroelements (deletion derivatives of transposable elements) havealso been found in this region (Cui et al 1999). The structural heteromorphismbetweenS-haplotypes appears to be a result of chromosomal rearrangements(duplications and inversions), compounded by the accumulation of transposonsand retroelements, and haplotype-specific genes. Recombination is believed to besuppressed in theS-locus region owing to this structural heteromorphism, whichallows the male and female genes that determine theS-haplotype specificity toremain linked as a genetic unit.

The genes downstream ofSLGhave been found to be co-linear between dif-ferentS-haplotypes, and this finding has led to the speculation that one border ofthe S-locus may lie in this region (Cui et al 1999). This has been confirmed bycomprehensive recombinational and physical mapping analyses of theS-locusof the S8-haplotype ofB. campestris(Casselman et al 2000). The entire re-gion examined constitutes about 740–1000 kb and 1.46 cM. The two recombi-nation breakpoints flanking a sub-region containingSLG, SCR/SP11, andSRKare separated by approximately 50 kb, with one of the breakpoints located 2 to4 kb downstream ofSLG. Surprisingly, kilobase to centimorgan ratios across theS-locus region analyzed are comparable to those found for the entire genome ofBrassica(500–700/cM). These results might suggest that there is no suppressionof recombination in theS-locus region. Casselman et al (2000) point out, how-ever, that recombination has never been observed betweenSRKandSLGeven intheS-haplotypes where these two genes are separated by>200 kb. They suggestthat recombination suppression in a physically compressedS-haplotype such asS8, whereSLG, SCR/SP11, andSRKall lie within 13 kb, operates only within thisregion. In other haplotypes where the essential components of the SI system arespread over a larger region, suppression of recombination would necessarily covera much larger region.

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For the fourS-haplotypes reported so far,SCR/SP11is located betweenSLGandSRK, although its distance toSLGor SRKis not conserved (Schopfer et al 1999,Suzuki et al 1999, Takayama et al 2000). The necessity of the presence ofSLGinthisS-locus region has been brought into question by the recent finding of Takasakiet al (2000) thatSRKalone is sufficient to determineS-haplotype specificity of thestigma. However, their results also show thatSLGmay be necessary for a full SIresponse. Moreover, in most cases, for a givenS-haplotype, SLG shows the highestdegree of sequence similarity with the S-domain of SRK of the sameS-haplotype,suggesting thatSLGandSRKmay have co-evolved. Lastly, no recombination hasever been detected betweenSLGandSRK. ThusSLGshould be considered anintegral part of theS-locus.

A comparative mapping study has also been conducted between theB. campestris S8-haplotype and theArabidopsisgenome (Conner et al 1998).Both species are members of the same family (Brassicaceae), althoughArabidop-sis is self-compatible. A region ofArabidopsischromosome 1 (in the immediatevicinity of the ethylene response geneETR1) was found to be homologous to theBrassica S-locus. A high degree of synteny was found at the sub-megabase scalebetween the two homologous regions, but all theBrassica S-locus genes are absentfrom Arabidopsis, both in the homologous region and in the rest of the genome.The authors propose that this is likely to be the result of deletion of theS-locusgenes fromArabidopsis.

Model for Self-Incompatibility Interaction

A working model for SI inBrassica, like Papaver, envisages a recognition eventfollowed by a signal transduction cascade (Figure 5). UnlikePapaver, how-ever, the recognition event occurs at the stigma surface, and the cascade takesplace in the stigmatic papillar cell rather than in the pollen grain. TheBrassicamodel dictates that the pollenS-haplotype determinant, SCR/SP11, interacts in anS-haplotype-specific manner with the extracellular (S-) domain of SRK, whichspans the plasma membrane of the papillar cell. Although this interaction is as yetto be biochemically demonstrated, it seems likely. What is uncertain is whetherSLG interacts with the S-domain of SRK and/or with SCR/SP11. By analogy withanimal receptor kinases, SRK is generally thought to function as a dimer, althoughthis has not been shown experimentally. Some animal receptor kinases are activeas monomers, which remains a possibility with SRK. One possible explanationfor the enhancement of the SRK-mediated SI response by SLG as observed byTakasaki et al (2000) could be that SRK is active as a homodimer but is alsoactive as a heterodimer in combination with an SLG molecule. Given the highdegree of sequence similarity between SLG and the S-domain of SRK of the sameS-haplotype, it is possible that SLG also interacts with SCR/SP11. However, ifso, one would expect this interaction to compete with the binding of SCR/SP11to SRK, potentially having a dominant-negative effect, as SLG is>100-fold moreabundant than SRK.

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Figure 5 A model of SRK-mediated self-incompatibility response. A pollen grain withthe SI phenotype ofS1S3 and a pollen grain with the SI phenotype ofS4S5 land on thepapillar cell of a stigma withS1S2-genotype. All four allelic forms of SCR/SP11 producedby the pollen grains are taken up by the papillar cell, but the only interaction with theS-domain of an SRK is between SCR1 and SRK1. This interaction sets off a cascade ofsignal transduction events, including phosphorylation of ARC1, in the papillar cell, whichresults in inhibition of germination of the pollen grain carryingS1- andS3-haplotypes.

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Activation of SRK by self SCR/SP11 presumably causes autophosphorylationof SRK and subsequent phosphorylation of intracellular substrates of SRK. One ofthese substrates has been positively identified as ARC1, a stigma protein that showsa phosphorylation-dependent interaction with the cytosolic domain of SRK in vitro(Gu et al 1998). Suppression of ARC1 production in the stigma of transgenic plantscauses breakdown of SI (Stone et al 1999). Unfortunately, the sequence of ARC1does not give any clues as to what its cellular function may be. The identity ofother SRK substrates, if any, and downstream components of the signaling cascadeis unknown.

Another key question is how does this sequence of signaling events ultimatelylead to the inhibition of pollen germination or tube growth? To date, there has beenonly one report concerning a potential mechanism for pollen inhibition. Ikeda et al(1997) studied a self-compatible line ofB. campestris, which has mutations in boththeS-locus (Nasrallah et al 1994) and an unlinked locus, calledMOD. Comparingthis line with its self-incompatible counterpart, the authors determined that themutation (recessive) in theMOD locus results from a chromosomal deletion andidentified a putative aquaporin (water channel) gene within this deleted region. Theauthors suggest that this aquaporin-like protein is encoded by theMOD gene andpropose that it regulates the availability of water at the stigma surface. Specifically,in the SI response, SRK activation would lead to activation of this aquaporin-likeprotein and an increase in the water flow away from the pollen, thus preventingpollen hydration. As attractive as this model may appear, further experimentsare necessary to substantiate its validity. The reasons are as follows. First, sincethe full extent of the chromosomal deletion in themodbackground has not beenreported, it is not known whether the aquaporin-like gene is the only gene deletedin this region, or whether there are other unidentified genes that are also deleted.Second, the aquaporin-like protein has not been reported to indeed possess water(or any other molecule) channel activity, as its sequence seems to suggest. Third,the aquaporin-like gene has not been shown to be able to restore SI tomod/modplants. Nonetheless, even if the aquaporin-like gene turns out not to be theMODgene, its linkage to theMOD locus will allow its use as a molecular marker for theeventual identification of theMOD gene.

CONCLUSIONS AND FUTURE PERSPECTIVES

Considerable knowledge about the gene controlling pistil function in SI has beenobtained for all three types of SI mechanisms discussed here. The identificationof these genes was greatly facilitated by the vast amount of information obtainedfrom genetic studies carried out in the first half of the last century. The recentuse of transgenic approaches or in vitro bioassay not only has provided conclusiveevidence that these genes encode the pistilS-haplotype determinant in their re-spective SI systems, but also allows dissection of structure/function relationshipsof their gene products.

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However, despite the impressive progress that has been made, our understandingof the mechanism of any of these SI systems is still far from complete. Forthe Solanaceae and Papavaraceae types, a key missing piece of the puzzle is thepollen S-gene. For the Brassicaceae type, as well as the Papaveraceae type, theintricacies of the signal cascades triggered in the SI response are largely unknownas is how these cascades lead to inhibition of germination and/or tube growthof self pollen. However, recent identification of SCR/SP11, the determinant ofpollenS-haplotype specificity, and ARC1, one of the most upstream componentsof the signal cascade, in Brassicaceae represents a significant advancement towardaddressing these questions.

It is clear that even though theS-haplotype specificity of these three types of SIis determined by theS-locus alone, protein factors encoded by unlinked loci arerequired for a full manifestation of the SI response. If these proteins are involvedonly in SI, and not essential for other developmental processes, mutations in theirgenes would not be lethal but would cause breakdown of SI. These mutant allelesmay constitute the modifier genes that have been identified from studies of self-compatible lines of self-incompatible species (e.g., theMOD gene ofBrassicaandthe HT gene ofNicotiana). Many cultivated species, although self-compatible,are derived from their SI ancestors, with the SI trait having been selected out inthe domestication process. In some cases, the cause of the breakdown of SI islikely to be a result of mutations in one or more of the modifier genes. Thuscomparative study of self-compatible species and their wild self-incompatiblerelatives may prove to be an effective means for the identification of non-S-linkedgenes that encode components of an SI system. One of the goals of SI researchis to introduce SI into self-compatible crop plant species to facilitate hybrid seedproduction. Identification of modifier genes will make this goal more attainablebecause transfer of theS-locus genes alone into these species is unlikely to besufficient to confer SI.

To date, the RNase-based SI mechanism employed by the Solanaceae is theonly SI mechanism discussed here that has been found in more than one family,i.e. in the Rosaceae and Scrophulariaceae. An interesting but unresolved questionis whether S-RNase-based SI evolved before the divergence of these three familiesor evolved independently in these families. An important gap in our knowledge ofSI systems comes in the form of the two-locus gametophytic SI system found inthe Poaceae (grass) family. This family contains economically important species(maize, rice, rye, etc), but efforts to identify the genes involved in SI have not beensuccessful. (Initial reports of the cloning of the pollenS-gene in a grass specieshave now been retracted; see Langridge et al 1999). SI systems, while performingthe same reproductive function, are proving to be diverse in their mechanisms. Itis interesting to note that DNA sequences with similarity to genes that determineS-haplotype specificity in the three types of SI mechanisms discussed here havebeen identified through theArabidopsisgenome sequencing projects, and it seemslikely that the study of SI mechanisms will provide information relevant to otherdevelopmental processes such as cell-cell recognition and cell death. The extent

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of crossover between SI systems and other recognition processes involved in plantgrowth, development, or defense is also likely to be revealed during the next fewyears.

ACKNOWLEDGMENTS

The research in self-incompatibility carried out in our laboratory was supportedby grants from the National Science Foundation.

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