S locus F-box brothers: multiple and pollen-specific F-box genes with S haplotype-specific polymorphisms in apple and Japanese pear

Although recent findings suggest that the F-box genes SFB/SLF control pollen-part S specificity in the S-RNase-based gametophytic self-incompatibility (GSI) system, how these genes operate in the system is unknown, and functional variation of pollen S genes in different species has been reported. Here, we analyzed the S locus of two species of Maloideae: apple (Malus domestica) and Japanese pear (Pyrus pyrifolia). The sequencing of a 317-kb region of the apple S9 haplotype revealed two similar F-box genes. Homologous sequences were isolated from different haplotypes of apple and Japanese pear, and they were found to be polymorphic genes derived from the S locus. Since each S haplotype contains two or three related genes, the genes were named SFBB for S locus F-box brothers. The SFBB genes are specifically expressed in pollen, and variable regions of the SFBB genes are under positive selection. In a style-specific mutant S haplotype of Japanese pear, the SFBB genes are retained. Apart from their multiplicity, SFBB genes meet the expected characteristics of pollen S. The unique multiplicity of SFBB genes as the pollen S candidate is discussed in the context of mechanistic variation in the S-RNase-based GSI system.     

Structural and transcriptional analysis of the self-incompatibility locus of almond: identification of a pollen-expressed F-box gene with haplotype-specific polymorphism

Gametophytic self-incompatibility in Rosaceae, Solanaceae, and Scrophulariaceae is controlled by the S locus, which consists of an S-RNase gene and an unidentified "pollen S" gene. An approximately 70-kb segment of the S locus of the rosaceous species almond, the S haplotype-specific region containing the S-RNase gene, was sequenced completely. This region was found to contain two pollen-expressed F-box genes that are likely candidates for pollen S genes. One of them, named SFB (S haplotype-specific F-box protein), was expressed specifically in pollen and showed a high level of S haplotype-specific sequence polymorphism, comparable to that of the S-RNases. The other is unlikely to determine the S specificity of pollen because it showed little allelic sequence polymorphism and was expressed also in pistil. Three other S haplotypes were cloned, and the pollen-expressed genes were physically mapped. In all four cases, SFBs were linked physically to the S-RNase genes and were located at the S haplotype-specific region, where recombination is believed to be suppressed, suggesting that the two genes are inherited as a unit. These features are consistent with the hypothesis that SFB is the pollen S gene. This hypothesis predicts the involvement of the ubiquitin/26S proteasome proteolytic pathway in the RNase-based gametophytic self-incompatibility system.     

Identification of the self-incompatibility locus F-box protein-containing complex in Petunia inflata

The polymorphic S-locus regulating self-incompatibility (SI) in Petunia contains the S-RNase gene and a number of S-locus F-box (SLF) genes. While penetrating the style through the stigma, a pollen tube takes up all S-RNases, but only self S-RNase inhibits pollen tube growth. Recent evidence suggests that SLFs produced by pollen collectively interact with and detoxify non-self S-RNases, but none can interact with self S-RNase. An SLF may be the F-box protein component of an SCF complex (containing Cullin1, Skp1 and Rbx1), which mediates ubiquitination of protein substrates for degradation by the 26S proteasome. However, the precise nature of the complex is unknown. We used pollen extracts of a transgenic plant over-expressing GFP-fused S₂-SLF1 (SLF1 of S ₂-haplotype) for co-immunoprecipitation (Co-IP) followed by mass spectrometry (MS). We identified PiCUL1-P (a pollen-specific Cullin1), PiSSK1 (a pollen-specific Skp1-like protein) and PiRBX1 (an Rbx1). To validate the results, we raised transgenic plants over-expressing PiSSK1:FLAG:GFP and used pollen extracts for Co-IP–MS. The results confirmed the presence of PiCUL1-P and PiRBX1 in the complex and identified two different SLFs as the F-box protein component. Thus, all but Rbx1 of the complex may have evolved in SI, and all SLFs may be the F-box component of similar complexes.     

An F-box gene linked to the self-incompatibility (S) locus of Antirrhinum is expressed specifically in pollen and tapetum

In many flowering plants, self-fertilization is prevented by an intraspecific reproductive barrier known as self-incompatibility (SI), that, in most cases, is controlled by a single multiallelic S locus. So far, the only known S locus product in self-incompatible species from the Solanaceae, Scrophulariaceae and Rosaceae is a class of ribonucleases called S RNases. Molecular and transgenic analyses have shown that S RNases are responsible for pollen rejection by the pistil but have no role in pollen expression of SI, which appears to be mediated by a gene called the pollen self-incompatibility or Sp gene. To identify possible candidates for this gene, we investigated the genomic structure of the S locus in Antirrhinum, a member of the Scrophulariaceae. A novel F-box gene, AhSLF-S2, encoded by the S2 allele, with the expected features of the Sp gene was identified. AhSLF-S2 is located 9 kb downstream of S2 RNase gene and encodes a polypeptide of 376 amino acids with a conserved F-box domain in its amino-terminal part. Hypothetical genes homologous to AhSLF-S2 are apparent in the sequenced genomic DNA of Arabidopsis and rice. Together, they define a large gene family, named SLF (S locus F-box) family. AhSLF-S2 is highly polymorphic and is specifically expressed in tapetum, microspores and pollen grains in an allele-specific manner. The possibility that Sp encodes an F-box protein and the implications of this for the operation of self-incompatibility are discussed.     

An F-box gene linked to the self-incompatibility (S) locus of Antirrhinum is expressed specifically in pollen and tapetum

In many flowering plants, self-fertilization is prevented by an intraspecific reproductive barrier known as self-incompatibility (SI), that, in most cases, is controlled by a single multiallelic S locus. So far, the only known S locus product in self-incompatible species from the Solanaceae, Scrophulariaceae and Rosaceae is a class of ribonucleases called S RNases. Molecular and transgenic analyses have shown that S RNases are responsible for pollen rejection by the pistil but have no role in pollen expression of SI, which appears to be mediated by a gene called the pollen self-incompatibility or Sp gene. To identify possible candidates for this gene, we investigated the genomic structure of the S locus in Antirrhinum, a member of the Scrophulariaceae. A novel F-box gene, AhSLF-S ₂, encoded by the S ₂ allele, with the expected features of the Sp gene was identified. AhSLF-S ₂ is located 9 kb downstream of S ₂ RNase gene and encodes a polypeptide of 376 amino acids with a conserved F-box domain in its amino-terminal part. Hypothetical genes homologous to AhSLF-S ₂ are apparent in the sequenced genomic DNA of Arabidopsis and rice. Together, they define a large gene family, named SLF (S locus F-box) family. AhSLF-S ₂ is highly polymorphic and is specifically expressed in tapetum, microspores and pollen grains in an allele-specific manner. The possibility that Sp encodes an F-box protein and the implications of this for the operation of self-incompatibility are discussed.     

The F-box protein AhSLF-S2 controls the pollen function of S-RNase-based self-incompatibility

Recently, we have provided evidence that the polymorphic self-incompatibility (S) locus-encoded F-box (SLF) protein AhSLF-S(2) plays a role in mediating a selective S-RNase destruction during the self-incompatible response in Antirrhinum hispanicum. To investigate its role further, we first transformed a transformation-competent artificial chromosome clone (TAC26) containing both AhSLF-S(2) and AhS(2)-RNase into a self-incompatible (SI) line of Petunia hybrida. Molecular analyses showed that both genes are correctly expressed in pollen and pistil in four independent transgenic lines of petunia. Pollination tests indicated that all four lines became self-compatible because of the specific loss of the pollen function of SI. This alteration was transmitted stably into the T1 progeny. We then transformed AhSLF-S(2) cDNA under the control of a tomato (Lycopersicon esculentum) pollen-specific promoter LAT52 into the self-incompatible petunia line. Molecular studies revealed that AhSLF-S(2) is specifically expressed in pollen of five independent transgenic plants. Pollination tests showed that they also had lost the pollen function of SI. Importantly, expression of endogenous SLF or SLF-like genes was not altered in these transgenic plants. These results phenocopy a well-known phenomenon called competitive interaction whereby the presence of two different pollen S alleles within pollen leads to the breakdown of the pollen function of SI in several solanaceaous species. Furthermore, we demonstrated that AhSLF-S(2) physically interacts with PhS(3)-RNase from the P. hybrida line used for transformation. Together with the recent demonstration of PiSLF as the pollen determinant in P. inflata, these results provide direct evidence that the polymorphic SLF including AhSLF-S(2) controls the pollen function of S-RNase-based self-incompatibility.     

Identification of a ubiquitin-binding structure in the S-locus F-box protein controlling S-RNase-based self-incompatibility

In flowering plants,self-incompatibility(SI) serves as an important intraspecific reproductive barrier to promote outbreeding.In species from the Solanaceae,Plantaginaceae and Rosaceae,S-RNase and SLF(S-locus F-box) proteins have been shown to control the female and male specificity of SI,respectively.However,little is known about structure features of the SLF protein apart from its conserved F-box domain.Here we show that the SLF C-terminal region possesses a novel ubiquitin-binding domain(UBD) structure conserved among the SLF protein family.By using an ex vivo system of Nicotiana benthamiana,we found that the UBD mediates the SLF protein turnover by the ubiquitin—proteasome pathway.Furthermore,we detected that the SLF protein was directly involved in S-RNase degradation.Taken together,our results provide a novel insight into the SLF structure and highlight a potential role of SLF protein stability and degradation in S-RNase-based self-incompatibility.     

The F-box protein AhSLF-S2 physically interacts with S-RNases that may be inhibited by the ubiquitin/26S proteasome pathway of protein degradation during compatible pollination in Antirrhinum

Self-incompatibility S-locus-encoded F-box (SLF) proteins have been identified in Antirrhinum and several Prunus species. Although they appear to play an important role in self-incompatible reaction, functional evidence is lacking. Here, we provide several lines of evidence directly implicating a role of AhSLF-S(2) in self-incompatibility in Antirrhinum. First, a nonallelic physical interaction between AhSLF-S(2) and S-RNases was demonstrated by both coimmunoprecipitation and yeast two-hybrid assays. Second, AhSLF-S(2) interacts with ASK1- and CULLIN1-like proteins in Antirrhinum, and together, they likely form an Skp1/Cullin or CDC53/F-box (SCF) complex. Third, compatible pollination was specifically blocked after the treatment of the proteasomal inhibitors MG115 and MG132, but they had little effect on incompatible pollination both in vitro and in vivo, indicating that the ubiquitin/26S proteasome activity is involved in compatible pollination. Fourth, the ubiquitination level of style proteins was increased substantially after compatible pollination compared with incompatible pollination, and coimmunoprecipitation revealed that S-RNases were ubiquitinated after incubating pollen proteins with compatible but not with incompatible style proteins, suggesting that non-self S-RNases are possibly degraded by the ubiquitin/26S proteasome pathway. Fifth, the S-RNase level appeared to be reduced after 36 h of compatible pollination. Taken together, these results show that AhSLF-S(2) interacts with S-RNases likely through a proposed SCF(AhSLF-S2) complex that targets S-RNase destruction during compatible rather than incompatible pollination, thus providing a biochemical basis for the inhibition of pollen tube growth as observed in self-incompatible response in Antirrhinum.     

Loss of pollen-S function in two self-compatible selections of Prunus avium is associated with deletion/mutation of an S haplotype-specific F-box gene

Recently, an S haplotype-specific F-box (SFB) gene has been proposed as a candidate for the pollen-S specificity gene of RNase-mediated gametophytic self-incompatibility in Prunus (Rosaceae). We have examined two pollen-part mutant haplotypes of sweet cherry (Prunus avium). Both were found to retain the S-RNase, which determines stylar specificity, but one (S3' in JI 2434) has a deletion including the haplotype-specific SFB gene, and the other (S4' in JI 2420) has a frame-shift mutation of the haplotype-specific SFB gene, causing amino acid substitutions and premature termination of the protein. The loss or significant alteration of this highly polymorphic gene and the concomitant loss of pollen self-incompatibility function provides compelling evidence that the SFB gene encodes the pollen specificity component of self-incompatibility in Prunus. These loss-of-function mutations are inconsistent with SFB being the inactivator of non-self S-RNases and indicate the presence of a general inactivation mechanism, with SFB conferring specificity by protecting self S-RNases from inactivation.     

Sequence divergence and loss-of-function phenotypes of S locus F-box brothers genes are consistent with non-self recognition by multiple pollen determinants in self-incompatibility of Japanese pear (Pyrus pyrifolia)

The S-RNase-based gametophytic self-incompatibility (SI) of Rosaceae, Solanaceae, and Plantaginaceae is controlled by at least two tightly linked genes located at the complex S locus; the highly polymorphic S-RNase for pistil specificity and the F-box gene (SFB/SLF) for pollen. Self-incompatibility in Prunus (Rosaceae) is considered to represent a 'self recognition by a single factor' system, because loss-of-function of SFB is associated with self-compatibility, and allelic divergence of SFB is high and comparable to that of S-RNase. In contrast, Petunia (Solanaceae) exhibits 'non-self recognition by multiple factors'. However, the distribution of 'self recognition' and 'non-self recognition' SI systems in different taxa is not clear. In addition, in 'non-self recognition' systems, a loss-of-function phenotype of pollen S is unknown. Here we analyze the divergence of SFBB genes, the multiple pollen S candidates, of a rosaceous plant Japanese pear (Pyrus pyrifolia) and show that intrahaplotypic divergence is high and comparable to the allelic diversity of S-RNase while interhaplotypic divergence is very low. Next, we analyzed loss-of-function of the SFBB1 type gene. Genetic analysis showed that pollen with the mutant haplotype S(4sm) lacking SFBB1-S(4) is rejected by pistils with an otherwise compatible S(1) while it is accepted by other non-self pistils. We found that the S(5) haplotype encodes a truncated SFBB1 protein, even though S(5) pollen is accepted normally by pistils with S(1) and other non-self haplotypes. These findings suggest that Japanese pear has a 'non-self recognition by multiple factors' SI system, although it is a species of Rosaceae to which Prunus also belongs.     

Comparison of Petunia inflata S-Locus F-box protein (Pi SLF) with Pi SLF like proteins reveals its unique function in S-RNase based self-incompatibility

Petunia inflata possesses S-RNase-based self-incompatibility (SI), which prevents inbreeding and promotes outcrossing. Two polymorphic genes at the S-locus, S-RNase and P. inflata S-locus F-box (Pi SLF), determine the pistil and pollen specificity, respectively. To understand how the interactions between Pi SLF and S-RNase result in SI responses, we identified four Pi SLF-like (Pi SLFL) genes and used them, along with two previously identified Pi SLFLs, for comparative studies with Pi SLF(2). We examined the in vivo functions of three of these Pi SLFLs and found that none functions in SI. These three Pi SLFLs and two other Pi SLFs either failed to interact with S(3)-RNase (a non-self S-RNase for all of them) or interacted much more weakly than did Pi SLF(2) in vitro. We divided Pi SLF(2) into FD1 (for Functional Domain1), FD2, and FD3, each containing one of the Pi SLF-specific regions, and used truncated Pi SLF(2), chimeric proteins between Pi SLF(2) and one of the Pi SLFLs that did not interact with S(3)-RNase, and chimeric proteins between Pi SLF(1) and Pi SLF(2) to address the biochemical roles of these three domains. The results suggest that FD2, conserved among three allelic variants of Pi SLF, plays a major role in the strong interaction with S-RNase; additionally, FD1 and FD3 (each containing one of the two variable regions of Pi SLF) together negatively modulate this interaction, with a greater effect on interactions with self S-RNase than with non-self S-RNases. A model for how an allelic product of Pi SLF determines the fate of its self and non-self S-RNases in the pollen tube is presented.     

The use of the S haplotype-specific F-box protein gene,SFB, as a molecular marker for S-haplotypes and self-compatibility in Japanese apricot (Prunus mume)

Japanese apricot (Prunus mume) exhibits the S-RNase-based gametophytic self-incompatibility system as do other self-incompatible Prunus species. This report identifies the S haplotype-specific F-box protein gene (SFB), a candidate gene for pollen-S, of Japanese apricot, which leads to the development of a molecular typing system for S-haplotype in this fruit species. Both 5- and 3-RACE (rapid amplification of cDNA ends) were performed with SFB gene-specific oligonucleotide primers to clone Pm-SFB¹ and Pm-SFB⁷ of 'Nanko (S¹S⁷)'. As in the case of SFB of other Prunus species, Pm-SFB¹ and Pm-SFB⁷ showed a high level of S-haplotype-specific sequence polymorphism and their expression was specific to pollen. Genomic DNA-blot analyses of 11 Japanese apricot cultivars with the Pm-SFB probes under low stringency conditions yielded RFLP bands specific to the S¹- to S⁸-haplotypes as well as a self-compatible S^f-haplotype. A practical usage of SFB as a molecular marker for S-haplotypes and self-compatibility in Japanese apricot is discussed.Communicated by H.F. LinskensThe nucleotide sequences reported in this paper have been submitted to the EMBL/GenBank/DDBJ database under accession numbers, AB101440 and AB101441, for SFB¹ and SFB⁷, respectively     

Genomic organization of the Papaver rhoeas self-incompatibility S(1) locus

The self-incompatibility (SI) response in Papaver rhoeas depends upon the cognate interaction between a pollen-expressed receptor and a stigmatically expressed ligand. The genes encoding these components are situated within the S-locus. In order for SI to be maintained, the genes encoded by the S-locus must be co-inherited with no recombination between them. Several hypotheses, including sequence heterogeneity and chromosomal position, have been put forward to explain the maintenance of the S-locus in the SI systems of the Brassicaceae and the Solanaceae. A region of the Papaver rhoeas genome encompassing part of the self-incompatibility S(1) locus has been cloned and sequenced. The clone contains the gene encoding the stigmatic component of the response, but does not contain a putative pollen S-gene. The sequence surrounding the S(1) gene contains several diverse repetitive DNA elements. As such, the P. rhoeas S-locus bears similarities to the S-loci of other SI systems. An attempt to localize the P. rhoeas S-locus using fluorescence in situ hybridization (FISH) has also been made. The potential relevance of the findings to mechanisms of recombination suppression is discussed.     

Genetic mapping and molecular characterization of the self-incompatibility (S) locus in Petunia inflata

Gametophytic self-incompatibility (SI) possessed by the Solanaceae is controlled by a highly polymorphic locus called the S locus. The S locus contains two linked genes, S-RNase, which determines female specificity, and the as yet unidentified pollen S gene, which determines male specificity in SI interactions. To identify the pollen S gene of Petunia inflata, we had previously used mRNA differential display and subtractive hybridization to identify 13 pollen-expressed genes that showed S -haplotype-specific RFLP. Here, we carried out recombination analysis of 1205 F2 plants to determine the genetic distance between each of these S -linked genes and S-RNase. Recombination was observed between four of the genes (3.16, G211, G212, and G221) and S-RNase, whereas no recombination was observed for the other nine genes (3.2, 3.15, A113, A134, A181, A301, G261, X9, and X11). A genetic map of the S locus was constructed, with 3.16 and G221 delimiting the outer limits. None of the observed crossovers disrupted SI, suggesting that all the genes required for SI are contained in the chromosomal region defined by 3.16 and G221. These results and our preliminary chromosome walking results suggest that the S locus is a huge multi-gene complex. Allelic sequence diversity of G221 and 3.16, as well as of 3.2, 3.15, A113, A134 and G261, was determined by comparing two or three alleles of their cDNA and/or genomic sequences. In contrast to S-RNase, all these genes showed very low degrees of allelic sequence diversity in the coding regions, introns, and flanking regions.     

The self-incompatibility phenotype in brassica is altered by the transformation of a mutant S locus receptor kinase

The self-incompatible (SI) Brassica napus line W1, which carries the 910 S allele, was transformed with an inactive copy of the 910 S locus receptor kinase (SRK) gene. Two transformed lines were analyzed based on their heritable ability to set self-seed. The first line was virtually completely self-compatible (SC), and reciprocal pollinations with the original W1 line demonstrated that only the stigma side of the SI phenotype was altered. An analysis of the expression of endogenous SRK-910 demonstrated that the mechanism of transgene action is via gene suppression. Furthermore, the expression of the S locus glycoprotein gene present in the 910 allele (SLG-910), SLG-A10, which is derived from a nonfunctional S allele, and an S locus-related gene were also suppressed. When the transgene was crossed into another SI line carrying the A14 S allele, it was also capable of suppressing the expression of the endogenous genes and of making this line SC. The second transgenic line studied was only partly SC. In this case as well, only the stigma phenotype was affected, although no gene suppression was detected for endogenous SRK-910 or SLG-910. In this line, the expression of the transgene most likely was causing the change in phenotype, and no effect was observed when this transgene was crossed into the other SI line. Therefore, this work reinforces the hypothesis that the SRK gene is required, but only for the stigma side of the SI phenotype, and that a single transgene can alter the SI phenotype of more than one S allele.     

Identification of a gene linked to the Brassica S (self-incompatibility) locus by differential display

Self-incompatibility in Brassica is controlled by a complex locus, the S locus, that includes several expressed genes. Two S locus genes, SLG and SRK, are expressed in the stigma and have been implicated in self-pollen recognition. The male component of this recognition system is also predicted to be encoded by a gene at the S locus but this gene has not been identified to date. In this study, we have used differential display to screen for polymorphic, S-locus-linked genes that are expressed in anthers. This approach has allowed the identification of a gene, named S5J, which was shown to segregate completely with the S locus. We discuss the possible role of this gene in the self-incompatibility response and evaluate the utility of differential display for the identification of genes at specific genetic loci.     

Presence of asparagine-linked N-acetylglucosamine and chitobiose in Pyrus pyrifolia S-RNases associated with gametophytic self-incompatibility.

S-RNases encoded by the S-locus of rosaceous and solanaceous plants discriminate between the S-alleles of pollen in gametophytic self-incompatibility reactions, but it is not clear how. We report the structures of N-glycans attached to each of the N-glycosylation sites of seven S-RNases in Pyrus pyrifolia of the Rosaceae. The structures were identified by chromatographic analysis of pyridylaminated sugar chains prepared from S4-RNase and by liquid chromatography/electrospray ionization-mass spectrometric analysis of the protease digests of reduced and S-carboxymethylated S-RNases. S4-RNase carries various types of sugar chains, including plant-specific ones with beta1-->2-linked xylose and alpha1-->3-linked fucose residues. More than 70% of the total N-glycans of S4-RNase are, however, an N-acetylglucosamine or a chitobiose (GlcNAcbeta1-->4GlcNAc), which has not been found naturally. The N-acetylglucosamine and chitobiose are mainly present at the N-glycosylation sites within the putative recognition sites of the S-RNase, suggesting that these sugar chains may interact with pollen S-product(s).