The male determinant of self-incompatibility in Brassica oleracea is located in the pollen coating

An in vitro bioassay has been developed to explore the role of the pollen coating in the pollen/stigma interaction in Brassica oleracea . In the assay, coating is removed from pollen grains, supplemented with protein fractions isolated from coatings of different S (self incompatibility) haplotypes, and then—using micromanipulation—interposed between individual pollen grains and the stigmatic surface. Normally, the coating used is of the same haplotype as the pollen in the experiment—thus constituting an 'extension' of its own coat—but carrying the supplemented protein fractions. Initial experiments confirmed preliminary data that the pollen coating contained the male determinant of self incompatibility (SI); not only did the addition of 'self' coating (i.e. that with the same S -haplotype as the stigma) prevent the success of a compatible cross pollination, but a 'cross' coating (i.e. that with a different S -haplotype from the stigma) could induce the germination and growth of self pollen. Protein supplementation experiments demonstrated that the pollen-held determinant is contained within the water soluble component of the pollen coat, while further analysis revealed that the active molecular species possesses an M_r10 kDa. More extensive fractionation by gel filtration and reverse phase HPLC was used to isolate a family of basic, cysteine-rich proteins (PCP-A: P ollen C oat P roteins-class A)—one of which is known to bind to stigmatically-expressed components of the S -locus in Brassica . Introduction of the PCP-A protein fraction into the bioassay confirmed the male determinant of SI as a protein, and probably a member of the PCP-A protein family.     

Immunohistochemical studies on translocation of pollen S-haplotype determinant in self-incompatibility of Brassica rapa

The self-incompatibility system in Brassica is controlled by the S-locus, which contains S-receptor kinase (SRK) and S-locus protein 11 (SP11). SRK and SP11 control stigma and pollen S-haplotype specificity, respectively. SP11 binding to SRK induces the autophosphorylation of SRK, which triggers the signaling cascade that results in the rejection of self-pollen. The localization of SP11 protein during pollen development and pollination, however, have never been demonstrated. In this study, we examined the localization of S(8)-SP11 protein in the anther or pollinated stigma by immuno-electron microscopy. The immunostaining suggested that S(8)-SP11 was secreted from the tapetal cell into the anther locule as a cluster and translocated to the pollen surface at the early developmental stage of the anther. During the pollination process, SP11 was translocated from the pollen surface to the papilla cell, and then penetrated the cuticle layer of the papilla cell to diffuse across the pectin cellulose layer. Furthermore, SP11 protein could only penetrate the cuticle layer of the papilla cell in the presence of pollen grains, and could not penetrate on its own. This suggests that another factor from the pollen grain is needed for SP11 protein to penetrate the papilla cell wall.     

Molecular mechanisms of self-incompatibility in Brassica

In Brassica species, self-incompatibility has been mapped genetically to a single chromosomal location. In this region several closely linked genes have been identified. One of them, S-locus receptor kinase (SRK), determines S haplotype specificity of the stigma and it's the key protein for SI reaction. The role of the S locus glycoprotein (SLG) gene remains unclear. In the last decade approximately 15 additional genes linked to S-locus have been found. Recently, a gene has been identified (SCR) that encodes a small cysteine-rich protein which is a candidate for the pollen ligand. In addition to S locus linked genes there are unlinked SLRgenes (S-locus related genes). In this review, we discuss the role of these genes and the current view on the self-incompatibility mechanism in Brassica.     

Molecular mechanisms of self-recognition in Brassica self-incompatibility

Plants have mechanisms to promote outbreeding and thereby to increase their genetic diversity. In species that are self-incompatible, self-pollen is rejected by the stigma. This mechanism has been the subject of intense study for many years and, in the past two years, significant progress has been made in identifying the genes involved in Brassica. Self-recognition involves two genes, one of which determines the male and the other the female specificity. Considerable progress has also been made on the mechanism by which self-recognition leads to pollen rejection, although the delineation of all the genes involved is still not complete.     

Evolutionary dynamics of self-incompatibility alleles in Brassica

Self-incompatibility in Brassica entails the rejection of pollen grains that express specificities held in common with the seed parent. In Brassica, pollen specificity is encoded at the multipartite S-locus, a complex region comprising many expressed genes. A number of species within the Brassicaceae express sporophytic self-incompatibility, under which individual pollen grains bear specificities determined by one or both S-haplotypes of the pollen parent. Classical genetic and nucleotide-level analyses of the S-locus have revealed a dichotomy in sequence and function among S-haplotypes; in particular, all class I haplotypes show dominance over all class II haplotypes in determination of pollen specificity. Analysis of an evolutionary model that explicitly incorporates features of the Brassica system, including the class dichotomy, indicates that class II haplotypes may invade populations at lower rates and decline to extinction at higher rates than class I haplotypes. This analysis suggests convergence to an evolutionarily persistent state characterized by the maintenance in high frequency of a single class II haplotype together with many class I haplotypes, each in low frequency. This expectation appears to be consistent with empirical observations of high frequencies of relatively few distinct recessive haplotypes.     

Brassica self-incompatibility: A glimpse below the surface

Self-incompatibility (SI) has emerged as an evolutionary strategy to enhance the genetic variability of plant species. In Brassica, it is controlled by a single multiallelic locus, the S-locus, encoding a receptor kinase (SRK) expressed in the stigma papilla cells and its ligand, a small protein (SCR) located in the pollen coat. Pollen rejection is achieved only when the receptor recognizes SCR coming from the same S-allele. If a single papilla cell is simultaneously pollinated by a self- and a cross-pollen grain, it is capable of distinguishing between the two and responding accordingly, rejecting self while accepting cross pollen. This phenomenon reveals that SI response is strictly localized and does not involve the whole papilla cell. It also suggests that the distribution of SRK inside the cell may play an important role in regulating this dual response. We have recently demonstrated that SRK is mostly intracellular, only small amounts being present in distinct domains of the plasma membrane (PM), where interaction with SCR occurs. Following ligand recognition, the receptor-ligand complex is endocytosed and degraded. Based on this, we propose a model of the significance of SRK intracellular trafficking for the functioning and specificity of SI response.Key words: self-incompatibility, S-receptor kinase, internalization, SI domains     

Molecular mechanism of self-recognition in Brassica self-incompatibility

In most self-incompatible plant species, recognition of self-pollen is controlled by a single locus, termed the S-locus. In Brassica, genetic dissection of the S-locus has revealed the presence of three highly-polymorphic genes: S-receptor kinase (SRK), S-locus protein 11 (SP11) (also known as S-locus cysteine-rich protein; SCR) and S-locus glycoprotein (SLG). SRK encodes a membrane-spanning serine/threonine kinase that determines the S-haplotype specificity of the stigma. SP11 encodes a small cysteine-rich protein that determines the S-haplotype specificity of pollen. SLG encodes a secreted form of stigma protein similar to the extracellular domain of SRK. Recent biochemical studies have revealed that SP11 functions as the sole ligand for its cognate SRK receptor complex. Their interaction induces the autophosphorylation of SRK, which is expected to trigger the signalling cascade that results in the rejection of self-pollen. This so-called ligand-receptor complex interaction and receptor activation occur in an S-haplotype-specific manner, and this specificity is almost certainly the basis for self-pollen recognition.     

Recognition specificity of self-incompatibility maintained after the divergence of Brassica oleracea and Brassica rapa

The determinants of recognition specificity of self-incompatibility in Brassica are SRK in the stigma and SP11/SCR in the pollen, respectively. In the pair of S haplotypes BrS46 (S46 in B. rapa) and BoS7 (S7 in B. oleracea), which have highly similar SRK alleles, the SP11 alleles were found to be similar, with 96.1% identity in the deduced amino acid sequence. Two other pairs of S haplotypes, BrS47 and BoS12, and BrS8 and BoS32, having highly similar SRK and SP11 alleles between the two species were also found. The haplotypes in each pair are considered to have been derived from a single S haplotype in the ancestral species. The allotetraploid produced by interspecific hybridization between homozygotes of BrS46 and BoS15 showed incompatibility with a BoS7 homozygote and compatibility with other B. oleracea S haplotypes in reciprocal crossings. This result indicates that BrS46 and BoS7 have maintained the same recognition specificity after the divergence of the two species and that amino acid substitutions found in such cases in both SRK alleles and SP11 alleles do not alter the recognition specificity. DNA blot analysis of SRK, SP11, SLG and other S-locus genes showed different DNA fragment sizes between the interspecific pairs of S haplotypes. A much lower level of sequence similarity was observed outside the genes of SRK and SP11 between BrS46 and BoS7. These results suggest that the DNA sequences of the regions intervening between the S-locus genes were diversified after or at the time of speciation. This is the first report demonstrating the presence of common S haplotypes in different plant species and presenting definite evidence of the trans-specific evolution of self-incompatibility genes.     

Recombination and selection at Brassica self-incompatibility loci

In Brassica species, self-incompatibility is controlled genetically by haplotypes involving two known genes, SLG and SRK, and possibly an as yet unknown gene controlling pollen incompatibility types. Alleles at the incompatibility loci are maintained by frequency-dependent selection, and diversity at SLG and SRK appears to be very ancient, with high diversity at silent and replacement sites, particularly in certain "hypervariable" portions of the genes. It is important to test whether recombination occurs in these genes before inferences about function of different parts of the genes can be made from patterns of diversity within their sequences. In addition, it has been suggested that, to maintain the relationship between alleles within a given S-haplotype, recombination is suppressed in the S-locus region. The high diversity makes many population genetic measures of recombination inapplicable. We have analyzed linkage disequilibrium within the SLG gene of two Brassica species, using published coding sequences. The results suggest that intragenic recombination has occurred in the evolutionary history of these alleles. This is supported by patterns of synonymous nucleotide diversity within both the SLG and SRK genes, and between domains of the SRK gene. Finally, clusters of linkage disequilibrium within the SLG gene suggest that hypervariable regions are under balancing selection, and are not merely regions of relaxed selective constraint.     

Towards an in vitro bioassay for the self-incompatibility response in Brassica oleracea

Summary Eluates of stigmas of Brassica oleracea that were known to contain S locus-specific glycoproteins (SLSG) discriminated between self and cross pollen in vitro in three different media. Discrimination was equally evident in experiments that were the in vitro equivalents of reciprocal pollinations. In a TAPS-buffered medium, self eluates depressed pollen germination in a dose-dependent manner. TAPS medium allowed a bioassay of the effects of SLSG in eluates because it optimized germination in a way that eliminated the complicating features of the stimulatory substances in the eluates. Stigma eluates affected percentage pollen germination and optimum calcium concentrations in vitro whether or not SLSG were present in the eluates, but differently in different media, and depending on whether the eluates were cross or self with respect to the pollen tested. Thus, the effect of stigma eluates on the in vitro germination of pollen in Brassica depends on the balance of stimulatory versus inhibitory substances in the eluates.     

Molecular aspects of self-incompatibility in Brassica species.

Many flowering plants possess self-incompatibility (SI) systems to prevent inbreeding. SI in Brassica species is controlled by a single S locus with multiple alleles. In recent years, much progress has been made in determining the male and female S determinant in Brassica species. In the female, a gain-of-function experiment clearly demonstrated that SRK was the sole S determinant, and that SLG enhanced the SI recognition process. By contrast, the male S determinant (termed SP11/SCR) was identified in the course of genome analysis of S locus to be a small cysteine-rich protein, which was classified as a pollen coat protein. This SP11/SCR may function as a ligand for the S domain of SRK in the SI recognition reaction of Brassica species.     

Dry stigmas,water and self-incompatibility in Brassica

The evolution of dry stigmas has been accompanied by the development — in the pollen — of mechanisms for accessing water from the stigmatic epidermis. Development of self- and cross-pollen on the stigmatic surface has been examined in Brassica oleracea, focusing on the hydration of the grains. Unlike self-compatible (SC) Arabidopsis thaliana, pollen hydration of self-incompatible (SI) Brassica oleracea is preceded by a latent period of between 30–90 min, which is significantly shortened by inhibition of protein synthesis in the stigma. Physiological experiments, some with isolated pollen coatings, indicate that during the latent period signals passing from the pollen to the sigma are responsible for readying the stigmatic surface for penetration and — after self-pollination — activation of the SI system. The changes at the stigma surface include the expansion of the outer layer of the cell wall beneath the grain. This expansion does not occur following self-pollination, when coating-derived signals stimulate a stigmatic response which interrupts hydration and arrests grain development. Cell manipulation studies suggest that self grains are not inhibited metabolically, but are physiologically isolated from the subjacent stigmatic papilla. This focusing of the SI response at the pollen-stigma interface ensures that a single papilla can simultaneously accept cross-pollen and reject self-grains. The evolution of this highly efficient SI system is disussed in the perspective of pathogen-defence mechanisms known also to be located in epidermal cells.     

Control of self-incompatibility by CO2 gas in Brassica

Pollen tubes penetrated stigma papilla cells in flowers thatwere illegitimately (self) pollinated, after CO₂ treatment ofthese flowers. This shows that the self-incompatible reactionin Brassica can be removed by CO₂ gas. Ethylene gas was noteffective. (Received May 16, 1969; )     

Conservation of S-locus for self-incompatibility in Brassica napus (L.) and Brassica oleracea (L.)

 Self-incompatibility (SI) in Brassica is a sporophytic system, genetically determined by alleles at the S-locus, which prevents self-fertilization and encourages outbreeding. This system occurs naturally in diploid Brassica species but is introduced into amphidiploid Brassica species by interspecific breeding, so that in both cases there is a potential for yield increase due to heterosis and the combination of desirable characteristics from both parental lines. Using a polymerase chain reaction (PCR) based analysis specific for the alleles of the SLG (S-locus glycoprotein gene) located on the S-locus, we genetically mapped the S-locus of B. oleracea for SI using a F₂ population from a cross between a rapid-cycling B. oleracea line (CrGC-85) and a cabbage line (86-16-5). The linkage map contained both RFLP (restriction fragment length polymorphism) and RAPD (random amplified polymorphic DNA) markers. Similarly, the S-loci were mapped in B. napus using two different crosses (91-SN-5263×87-DHS-002; 90-DHW-1855-4×87-DHS-002) where the common male parent was self-compatible, while the S-alleles introgressed in the two different SI female parents had not been characterized. The linkage group with the S-locus in B. oleracea showed remarkable homology to the corresponding linkage group in B. napus except that in the latter there was an additional locus present, which might have been introgressed from B. rapa. The S-allele in the rapid-cycling Brassica was identified as the S₂₉ allele, the S-allele of the cabbage was the S ₅ allele. These same alleles were present in our two B. napus SI lines, but there was evidence that it might not be the active or major SI allele that caused self-incompatibility in these two B. napus crosses. Received: 7 June 1996/Accepted: 6 September 1996     

Visualization of the Brassica self-incompatibility S-locus on identified oilseed rape chromosomes

Seventy years after Karpechenko [15] first reported the accurate chromosome number of oilseed rape (Brassica napus L., 2n=38), we have developed a quantitative chromosome map of rape using computer imaging technology. The capacity to identify individual rape chromosomes will facilitate a wide range of genetic studies. Here we demonstrate the use of imaging methods in combination with fluorescence in situ hybridization to localize, on identified chromosomes, the single copy S-locus glycoprotein and S-locus-related genes involved in the self-incompatibility system of Brassica. These techniques have a broader application in plant genome research involving the mapping of single-copy genes and markers, irrespective of the plant species.     

Isolation of a cDNA for a nucleoside diphosphate kinase capable of phosphorylating the kinase domain of the self-incompatibility factor SRK of Brassica campestris

SRK is a plant receptor kinase involved in the self-incompatibility system of Brassica species. During a cDNA screening for the phosphoproteins from a stigma expression library, a clone encoding the nucleoside diphosphate kinase III (Bc-NDPK III) was obtained. After in vitro phosphorylation assays with recombinant proteins, Bc-NDPK III contained mostly phosphoserine. By contrast, the kinase domain of SRK contained phosphoserine and phosphothreonine, both of which were significantly increased by the addition of Bc-NDPK III in the presence of an SRK inhibitor KN-62. The result suggested the possible involvement of Bc-NDPK III in the signal transduction pathway through SRK.