INSIGHT

Self-Incompatibility in Brassica: The Elusive Pollen S Gene Is Identified!

Correspondence to: Vernonica E. Franklin-Tong, V.E.Franklin-Tong{at}bham.ac.uk (E-mail), 44-121-414-5925 (fax)

Pollination in flowering plants is a complex process, demanding that pollen grains land on a stigma and undergo the steps of recognition, adherence, and hydration, followed by pollen germination and pollen tube growth through the pistil. The potential for molecular studies to reveal the mechanisms involved in fertility and reproduction in plants has generated considerable interest in the regulation of pollen tube growth in recent years. A number of studies have looked for important control points and signals involved (see Franklin-Tong 1999a and Franklin-Tong 1999b , for recent reviews). Although much progress has been made, many factors responsible for the control of pollination remain to be identified.

Many studies investigating the control of pollination have focused on the specific inhibition of pollen tube growth during self-incompatibility (SI), a response that has evolved to undermine the potential of hermaphroditic plants to self-fertilize. SI thus prevents problems such as inbreeding depression and reduced gene-pool variation. SI effects the recognition of "self" to the extent that a pollen grain and a stigmatic papilla cell that bear genetically identical S alleles interact so as to inhibit pollen tube growth. In contrast, "compatible" (i.e., genetically different) pollen landing on the same stigma grows normally and achieves fertilization.

The genetic basis for SI in many species has been determined by crossing experiments, and SI was consequently predicted to be controlled by a single multi-allelic S locus (East and Mangelsdorf 1925 ; Bateman 1955 ), under either gametophytic or sporophytic control. In a gametophytically controlled system, the pollen S phenotype is specified by its haploid S genotype. Sporophytic control means that the pollen displays the S phenotype of the sporophyte, so it behaves as if it were carrying two S alleles, even though it is haploid. It was therefore predicted that the pollen S component is present in the pollen coat, as this is derived from diploid tissues. Genes residing at the S locus were implicated in the recognition of incompatible pollen when pistil S gene products interacted with pollen carrying a corresponding S allele. SI behaves as a simple Mendelian trait, indicating that both the male and female S gene determinants must reside at a common locus. Although classical genetics predicts only two classes of SI systems, molecular studies have revealed that SI has apparently evolved independently several times and involves a range of different mechanisms.

Molecular studies of SI have generally addressed two issues. First, what are the pistil and pollen components at the S locus? Second, what is the mechanism of pollen tube inhibition? Three different SI systems have been studied in some considerable detail at the molecular level, and interestingly, all three have turned out to have rather different mechanisms (for a review, see Franklin et al. 1995 ). The Solanaceae appear to have recruited a ribonuclease to inhibit incompatible pollen. The pistil S gene (Anderson et al. 1986 ) is an S-linked ribonuclease (S RNase) that is secreted into the transmitting tract along the pathway where the pollen tubes will grow. These S RNases have been demonstrated to confer self-incompatibility through transformation experiments (Lee et al. 1994 ; Murfett et al. 1994 ). In Papaver, the stigmatic S gene encodes a small protein (Foote et al. 1994 ) that triggers a Ca²⁺-based signal transduction cascade resulting in the S-specific inhibition of incompatible pollen. A number of SI-induced events have been identified in incompatible pollen, including increases in cytosolic free Ca²⁺, protein phosphorylation, DNA fragmentation, and alterations to the actin cytoskeleton, all of which are thought to contribute to the irreversible inhibition of pollen tube growth (see Jordan et al. 2000 ; Snowman et al. 2000 ).

In Brassica, several pistil S-linked genes have been identified and studied by a number of groups. The first component identified was the S locus glycoprotein, SLG (Nasrallah et al. 1985 ). S locus–related (SLR) genes, thought to have evolved upon gene duplication of SLG, have also been identified. Although they are not linked to the S locus and so cannot play a role in determining S-specificity, SLR genes are thought to play a general role in pollination. Another pistil S-linked gene encodes a receptor kinase (SRK) with an extracellular domain with strong homology to SLG (Stein et al. 1991 ). The protein kinase activity of SRK has been verified, and a number of pieces of evidence indicate that a functional SRK is required for the SI response in Brassica. Recently, it has in fact been confirmed that the SRK determines the S haplotype specificity of the stigma (Takasaki et al. 2000 ; see also Kao et al., 2000).

Since the identification of pistil S genes, all of the major research groups investigating SI have been searching for the male counterpart, the pollen S gene. Because Mendelian genetics predicts that both the male and female S gene determinants must be tightly linked to the S locus, it should be feasible to identify the pollen S gene by analyzing nucleotide sequences at the S locus. The search, however, has proved to be difficult due to the complex structure of the S locus. Nevertheless, it is this approach that has enabled the group led by June and Mikhail Nasrallah to successfully identify the pollen S gene from Brassica oleracea, as reported recently in Science (Schopfer et al. 1999 ).

Before discussing this seminal paper in SI research, it is worth mentioning another strategy that provided important insights on the pollen component. The group led by Hugh Dickinson at Oxford has used an ingenious bioassay for pollen coat proteins. Unfortunately, a protein fraction identified by the group has turned out to be a complex mixture of related pollen coat proteins (designated PCPs) of ~7 kD. The gene encoding one member of the PCPs, PCP-A1, was cloned and analyzed, which revealed that the PCPs are related to a family of cysteine-rich proteins, the defensins, that are char-acterized by the presence of eight conserved cysteine residues (Doughty et al. 1998 ). Interestingly, PCP-A1 was found to interact with SLG proteins, although this interaction was not S-allele specific and the PCP-A1 gene proved to be unlinked to the S locus. It was therefore ruled out as a candidate for the pollen S gene. Instead, the authors speculated that PCP-A1 might act as a cofactor in the SI reaction. A further finding was that PCP-A1 and another member of this family, PCP1, were gametophytically expressed, prompting the authors to suggest that gametophytic expression might also prove to be the case for the pollen S genes, despite the fact that these exhibit a sporophytic-like phenotype.

The chief problem confronting the labs attempting to identify the pollen S gene through a genetic linkage approach was to define the actual physical size of the S locus. Extensive genetic analysis has failed to detect recombination between the SRK and SLG stigmatic genes, and initial mapping studies demonstrated that the physical distance between these two genes is 200 to 400 kb in some haplotypes. Although a minimum size for the S locus has thus been elaborated, recombination in the region around the S locus would need to be studied to identify breakpoints and confirm the actual extent of the locus. Molecular studies aimed at identifying S-linked, anther-expressed genes have resulted in the identification of several candidates for the pollen S gene, including SLA (Boyes and Nasrallah 1995 ), SLL₁, and SLL₂ (Yu et al. 1996 ). Each of these genes, however, failed to exhibit the features expected of the pollen S component, despite the fact that they are all linked to the S locus. Transformation experiments suggested that a functional SLA was not required for an SI phenotype, SLL₁ did not show any allelic sequence variation, and SLL₂ did not exhibit pollen-specific expression. Nevertheless, this general strategy of identifying S-linked genes was to prove successful in the identification of the pollen S gene. Eventually, S haplotypes were identified in which the SRK/SLG genes were substantially close (within 20 to 25 kb; Yu et al. 1996 ; Boyes et al. 1997 ).

Most recently, the Nasrallah lab has published the results of detailed recombination analysis of the S₈ haplotype (Casselman et al. 2000 ). Analysis of a region encompassing between 740 and 1000 kb and 1.46 centimorgans of the S locus region using a large segregating F₂ population enabled the identification of recombination breakpoints and thereby established that the size of the S locus in the S₈ haplotype was ~50 kb. This delineation of the S locus was undoubtedly an important factor in the group's identification of the pollen S gene. Another important observation of this study was an apparent lack of recombination suppression in the region immediately flanking the S locus (at least in this particular haplotype). Previously, it had been assumed that recombination suppression played an important role in maintenance of the S locus complex.

Schopfer et al. 1999 identified the pollen S gene in Brassica oleracea by sequencing the 13-kb region between the S₈ SRK and SLG genes, and was designated as the S locus cysteine rich protein (SCR), due to its encoding a short open reading frame with an unusually high frequency of cysteine residues. Analysis revealed that this gene was present as a single copy and was anther-expressed, accumulating postmeiotically. SCR is secreted as a mature hydrophilic protein of 8.4 to 8.6 kD containing eight cysteine residues. Clearly, these features are reminiscent of the PCP family, but the positioning of the cysteine residues within SCR is nevertheless distinctive.

A number of lines of evidence confirmed that SCR corresponds to the pollen S gene. First, a B. oleracea self-compatible mutant in which the breakdown of SI was confined to the male determinant was found to lack detectable SCR expression. Second, comparisons of the deduced amino acid sequence of SCR proteins from three S haplotypes, S₆, S₈,and S₁₃, revealed a high degree of polymorphism. Only 11 amino acids (eight of which are the cysteine residues) are conserved, giving an overall similarity of only 30 to 42% for these alleles. This high degree of variation is consistent with a role as allele-specific ligands in the SI response. The final and most compelling piece of evidence was obtained by transforming a B. oleracea S₂S₂ homozygote with the SCR₆ coding region under control of the SCR₈ promoter. Pollen from resultant SCR₆⁺ transformants had acquired S₆ specificity. This result unequivocally demonstrated that SCR encodes the pollen SI specificity determinant (Schopfer et al. 1999 ). One of the paradoxes that the identification of SCR has revealed is that it is expressed gametophytically, despite the genetic prediction that it should be sporophytically determined. We commented earlier that both the Dickinson and Hinata labs also had noted the perplexing property of PCP and SP11 to be gametophytic-ally determined. Dickinson and colleagues have provided a possible explanation for this. They propose that the proteins determined by the two S alleles might be secreted during pollen development and then incorporated into the pollen coat as a mixture, thereby giving the apparent sporophytic phenotype (Doughty et al. 1998 ).

Thus, well over a decade since the Nasrallah lab reported the first cloning of a female SI determinant, the same group has again met with success by identifying for the first time the elusive male S gene. Ironically, just prior to the publication of this paper, Hinata's group in Japan reported the molecular analysis of the B. campestris S₉ haplotype (Suzuki et al. 1999 ). The authors commented that one of the open reading frames (SP₁₁) was similar to PCP-A1, but differed slightly in the predicted position of the 8 cysteine residues. In fact, comparison of this gene with the SCR allele reveals a striking similarity, suggesting that SP₁₁ is, in fact, a pollen SI determinant in B. campestris.

So, how do the S locus components in Brassica interact to reject incompatible pollen? Fig 1 attempts to indicate the current model. It seems likely that interaction of SCR with SRK or an SRK–SLG complex results in the activation of a signal transduction pathway leading to the arrest of incompatible pollen very early in pollination. Evidence suggests that at the end of the signaling pathway, the regulation of a specific aquaporin-like gene may inhibit incompatible pollen hydration (Ikeda et al. 1997 ). Coupling of the recognition event to the rejection response is assumed to be dependent on components within the stigmatic papillae cells. Daphne Goring and colleagues have used a two-hybrid approach to identify three proteins that interact with the kinase domain of SRK. One of these, ARC1, was specifically phosphorylated by SRK (Gu et al. 1998 ). However, whether ARC1 plays a role in the SI response in Brassica had not, until recently, been established.

View larger version (61K): [in this window] [in a new window]   Figure 1. A Model for the Brassica SI Reaction. The SI response occurs within the stigmatic papilla cells. When a pollen grain alights on the papilla surface, the pollen coat, containing pollen coat proteins such as PCPs (black trapezoids) and the recently identified pollen S ligand, SCR (black circles), flows to form a layer (shown in light gray) between the pollen and stigma. If, as shown in this case, the SCR carried within this coating is allelic with the recipient stigma, an incompatible reaction is triggered. S allele–specific recognition of SCR by the extracellular region of the S receptor kinase (SRK) results in activation of its intracellular Ser-Thr protein kinase domain (shown as a black star). Although the role of the S locus glycoprotein (SLG), which has the same structure as the extracellular domain of SRK, is unclear, it may function as an accessory receptor, or alternatively, it may have a more general role in the pollen–stigma interactions. After activation, SRK phosphorylates ARC1, presumably initiating an intracellular signaling cascade within the papilla cell. Although a detailed analysis remains to be undertaken, evidence suggests that the signaling pathway may ultimately regulate the activity of aquaporins in the stigmatic papillae to limit the availability of water to the incompatible pollen.

In the same issue of Science in which the identification of SCR was reported, another major breakthrough in the Brassica SI system was reported. Daphne Goring's group provided definitive evidence that ARC1 is required for the SI response in Brassica. Transgenic B. napus plants in which ARC1 expression was downregulated by an antisense approach exhibited a reduced ability to reject self-pollen and set a significant amount of self-seed (Stone et al. 1999 ). This work clearly suggests a role for ARC1 in the SI response. However, as the authors point out, knocking out ARC1 expression does not result in full self-compatibility because the level of seed set is not equivalent to that obtained from cross-pollination. Other components, still unidentified, must also participate in this complex cell recognition and rejection mechanism.

Nevertheless, considerable progress has been made with respect to our understanding of the components and processes involved in SI since the cloning of the first Brassica pistil S gene some 15 years ago. The SI systems have held some surprises, and additional components that were not anticipated when these studies were first undertaken have been identified. The hunt for the pollen S gene has been difficult and often frustrating. However, persistence has paid off. So, what next? With the "holy grail" found for the Brassica SI system, other aspects of SI become all the more intriguing. The nature of how S specificity is encoded, not only in the pollen, but also the pistil component, remains to be elucidated. There still remain two other well-characterized SI systems for which the pollen component has not been identified. Furthermore, the broad differences between the SI systems already studied beg the question as to how many other mechanisms have evolved to prevent self-pollination.

	REFERENCES

TOP REFERENCES

Anderson, M.A., Cornish, E.C., Mau, S-L., Williams, E.G., Hoggart, R., Atkinson, A., Bonig, I., Grego, B., Simpson, R., Roche, P.J., Haley, J.D., Penschow, J.D., Niall, H.D., Tregear, G.W., Cochlan, J.P., Crawford, R.J., and Clarke, A.E. (1986) Cloning of a cDNA for a stylar glycoprotein associated with expression of self-incompatibility in Nicotiana alata.. Nature 321:38-44[CrossRef].

Bateman, A.J. (1955) Self-incompatibility systems in angiosperms. III. Cruciferae. Heredity 9:51-68.

Boyes, D.C., and Nasrallah, J.B. (1995) An anther-specific gene encoded by an S locus haplotype of Brassica produces complementary and differentially regulated transcripts. Plant Cell 7:1283-1294[Abstract].

Boyes, D.C., Nasrallah, M.E., Vrebalov, J., and Nasrallah, J.B. (1997) The self-incompatibility (S) haplotypes of Brassica contain highly divergent and rearranged sequences of ancient origin. Plant Cell 9:237-247[Abstract].

Casselman, A.L., Vrebalov, J., Conner, J.A., Singal, A., Giovanni, J., Nasrallah, M.E., and Nasrallah, J.B. (2000) Determining the physical limits of the Brassica S locus by recombinational analysis. Plant Cell 12:23-33[Abstract/Free Full Text].

Doughty, J., Dixon, S., Hiscock, S.J., Willis, A.C., Parkin, I.A., and Dickinson, H.G. (1998) PCP-A1, a defensin-like Brassica pollen coat protein that binds the S locus glycoprotein, is the product of gametophytic gene expression. Plant Cell 10:1333-1347[Abstract/Free Full Text].

East, E.H., and Mangelsdorf, A.J. (1925) A new interpretation of the hereditary behaviour of self-sterile plants. Proc. Natl. Acad. Sci. USA 11:166-183[Free Full Text].

Foote, H.C.C., Ride, J.P., Franklin-Tong, V.E., Walker, E.A., Lawrence, M.J., and Franklin, F.C.H. (1994) Cloning and expression of a distinctive class of self-incompatibility (S-) gene from Papaver rhoeas L. Proc. Natl. Acad. Sci USA 91:2265-2269[Abstract/Free Full Text].

Franklin, F.C.H., Lawrence, M.J., and Franklin-Tong, V.E. (1995) Cell and molecular biology of self-incompatibility in flowering plants. Int. Rev. Cytol. 158:1-64.

Franklin-Tong, V.E. (1999a) Signalling and the modulation of pollen tube growth. Plant Cell 11:727-738[Free Full Text].

Franklin-Tong, V.E. (1999b) Signaling in pollination. Curr. Opin. Plant Biol. 2:490-495[CrossRef][ISI][Medline].

Gu, T.S., Mazzurco, M., Sulaman, W., Matias, D.D., and Goring, D.R. (1998) Binding of an arm repeat protein to the kinase do-main of the S-locus receptor kinase. Proc. Natl. Acad. Sci. USA 95:382-387[Abstract/Free Full Text].

Ikeda, S., Nasrallah, J.B., Dixit, R., Preiss, S., and Nasrallah, M.E. (1997) An aquaporin-like gene required for the Brassica self-incompatibility response. Science 276:1564-1566[Abstract/Free Full Text].

Jordan, N.D., Ride, J.P., Rudd, J.J., Davies, E.M., Franklin-Tong V.E., and Franklin F.C.H. (2000). Inhibition of self-incompatible pollen in Papaver rhoeas involves a complex series of cellular events. Ann. Bot., in press.

Kao, T.-h., and McCubbin, A.G. (2000) A social stigma. Nature 403:840-841[Medline].

Lee, H.-S., Huang, S., and Kao, T.-h. (1994) S proteins control rejection of incompatible pollen in Petunia inflata. Nature 367:560-563[CrossRef][Medline].

Murfett, J., Atherton, T.L., Mou, B., Gasser, C.S., and McClure, B.A. (1994) S-RNase expressed in transgenic Nicotiana causes S-allele-specific pollen rejection. Nature 367:563-566[CrossRef][Medline].

Nasrallah, J.B., Kao, T.-h., Goldberg, M.L., and Nasrallah, M.E. (1985) A cDNA encoding an S-locus-specific glycoprotein from Brassica oleracea.. Nature 318:263-267[CrossRef].

Schopfer, C.R., Nasrallah, M.E., and Nasrallah, J.B. (1999) The male determinant of self-incompatibility in Brassica. Science 286:1697-1700[Abstract/Free Full Text].

Snowman, B.N., Geitmann, A., Clarke, S.R., Staiger, C.J., Franklin, F.C.H., Emons, A.M.C., and Franklin-Tong, V.E. (2000) Signalling and the cytoskeleton in pollen tubes of Papaver rhoeas.. Ann. Bot. in press.

Stein, J.C., Howlett, B., Boyes, D.C., Nasrallah, M.E., and Nasrallah, J.B. (1991) Molecular cloning of a putative receptor kinase gene encoded by the self-incompatibility locus of Brassica oleracea.. Proc. Natl. Acad. Sci. USA 88:8816-8820[Abstract/Free Full Text].

Stone, S.L., Arnoldo, M., and Goring, D.R. (1999) A breakdown of self-incompatibility in ARC1 antisense transgenic plants. Science 286:1729-1731[Abstract/Free Full Text].

Suzuki, G., Kai, N., Hirose, T., Fukui, K., Nihio, T., Takayama, S., Isogai, A., Watanabe, M., and Hinata, K. (1999) Genomic organization of the S locus: Identification and characterization of genes in SLG/SRK region of S⁹ haplotype of Brassica campestris (syn.) rapa. Genetics 153:391-400[Abstract/Free Full Text].

Takasaki, T., Hatakeyama, K., Suzuki, G., Watanabe, M., Isogai, A., and Hinata, K. (2000) The S receptor kinase determines self-incompatibility in Brassica stigma. Nature 403:913-916[CrossRef][Medline].

Yu, K., Scafer, U., Glavin, T.L., Goring, D.R., and Rothstein, S.J. (1996) Molecular characterization of the S locus in two self-incompatible Brassica napus lines. Plant Cell 8:2369-2380[Abstract].

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