Identification of major lysine residues of S(3)-RNase of Petunia inflata involved in ubiquitin-26S proteasome-mediated degradation in vitro

S-RNase-based self-incompatibility has been identified in three flowering plant families, including the Solanaceae, and this self/non-self recognition mechanism between pollen and pistil is controlled by two polymorphic genes at the S -locus, S-RNase and S-locus F-box ( SLF ). S-RNase is produced in the pistil and taken up by pollen tubes in a non- S- haplotype-specific manner. How an allelic product of SLF interacts with self and non-self S-RNases to result in growth inhibition of self pollen tubes is not completely understood. One model predicts that SLF targets non-self S-RNases for ubiquitin/26S proteasome-mediated degradation, thereby only allowing self S-RNase to exert cytotoxic activity inside a pollen tube. To test this model, we studied whether any of the 20 lysine residues in S₃-RNase of Petunia inflata might be targets for ubiquitination. We identified six lysines near the C-terminus for which mutation to arginine significantly reduced ubiquitination and degradation of the mutant S₃-RNase, GST:S₃-RNase (K141–164R) in pollen tube extracts. We further showed that GST:S₃-RNase (K141–164R) and GST:S₃-RNase had similar RNase activity, suggesting that their degradation was probably not caused by an ER-associated protein degradation pathway that removes mis-folded proteins. Finally, we showed that PiSBP1 ( P. inflata S-RNase binding protein 1), a potential RING-HC subunit of the PiSLF ( P. inflata SLF)-containing E3-like complex, could target S-RNase for ubiquitination in vitro . All these results suggest that ubiquitin/26S proteasome-dependent degradation of S-RNase may be an integral part of the S-RNase-based self-incompatibility mechanism.     

Functional characterization of two chimeric proteins between a <Emphasis Type="Italic">Petunia inflata</Emphasis><Emphasis Type="Italic">S</Emphasis>-locus F-box protein,PiSLF<Subscript>2</Subscript>, and a PiSLF-like protein,PiSLFLb-S<Subscript>2</Subscript>

Self-incompatible solanaceous species possess the S-RNase and SLF (S-locus F-box) genes at the highly polymorphic S-locus, and their products mediate S-haplotype-specific rejection of pollen tubes in the style. After a pollen tube grows into the style, the S-RNases produced in the style are taken up; however, only self S-RNase (product of the matching S-haplotype) can inhibit the subsequent growth of the pollen tube. Based on the finding that non-self interactions between PiSLF (Petunia inflata SLF) and S-RNase are stronger than self-interactions, and based on the biochemical properties of PiSLF, we previously proposed that a PiSLF preferentially interacts with its non-self S-RNases to mediate their ubiquitination and degradation, thereby only allowing self S-RNase to exert its cytotoxic function. We further divided PiSLF into three potential Functional Domains (FDs), FD1-FD3, based on sequence comparison of PiSLF and PiSLF-like proteins, and based on S-RNase-binding properties of these proteins and various truncated forms of PiSLF₂ (S ₂ allelic variant of PiSLF). In this work, we examined the in vivo function of FD2, which we proposed to be responsible for strong, general interactions between PiSLF and S-RNase. We swapped FD2 of PiSLF₂ with the corresponding region of PiSLFLb-S₂ (S ₂ allelic variant of a PiSLF-like protein), and expressed GFP-fused chimeric proteins, named b-2-b and 2-b-2, in S ₂ S ₃ transgenic plants. We showed that neither chimeric protein retained the SI function of PiSLF₂, suggesting that FD2 is necessary, but not sufficient, for the function of PiSLF. Moreover, since we previously found that b-2-b and 2-b-2 only interacted with S₃-RNase ~50 and ~30%, respectively, as strongly as did PiSLF₂ in vitro, their inability to function as PiSLF₂ is also consistent with our model predicating on strong interaction between a PiSLF and its non-self S-RNases as part of the biochemical basis for S-haplotype-specific rejection of pollen tubes.     

Identification and characterization of components of a putative petunia S-locus F-box-containing E3 ligase complex involved in S-RNase-based self-incompatibility

Petunia inflata S-locus F-box (Pi SLF) is thought to function as a typical F-box protein in ubiquitin-mediated protein degradation and, along with Skp1, Cullin-1, and Rbx1, could compose an SCF complex mediating the degradation of nonself S-RNase but not self S-RNase. We isolated three P. inflata Skp1s (Pi SK1, -2, and -3), two Cullin-1s (Pi CUL1-C and -G), and an Rbx1 (Pi RBX1) cDNAs and found that Pi CUL1-G did not interact with Pi RBX1 and that none of the three Pi SKs interacted with Pi SLF₂. We also isolated a RING-HC protein, S-RNase Binding Protein1 (Pi SBP1), almost identical to Petunia hybrida SBP1, which interacts with Pi SLFs, S-RNases, Pi CUL1-G, and an E2 ubiquitin-conjugating enzyme, suggesting that Pi CUL1-G, SBP1, and SLF may be components of a novel E3 ligase complex, with Pi SBP1 playing the roles of Skp1 and Rbx1. S-RNases interact more with nonself Pi SLFs than with self Pi SLFs, and Pi SLFs also interact more with nonself S-RNases than with self S-RNases. Bacterially expressed S₁-, S₂-, and S₃-RNases are degraded by the 26S proteasomal pathway in a cell-free system, albeit not in an S-allele–specific manner. Native glycosylated S₃-RNase is not degraded to any significant extent; however, deglycosylated S₃-RNase is degraded as efficiently as the bacterially expressed S-RNases. Finally, S-RNases are ubiquitinated in pollen tube extracts, but whether this is mediated by the Pi SLF–containing E3 complex is unknown.     

Compatibility and incompatibility in S-RNase-based systems

Background: S-RNase-based self-incompatibility (SI) occurs in the Solanaceae, Rosaceae and Plantaginaceae. In all three families, compatibility is controlled by a polymorphic S-locus encoding at least two genes. S-RNases determine the specificity of pollen rejection in the pistil, and S-locus F-box proteins fulfill this function in pollen. S-RNases are thought to function as S-specific cytotoxins as well as recognition proteins. Thus, incompatibility results from the cytotoxic activity of S-RNase, while compatible pollen tubes evade S-RNase cytotoxicity. SCOPE: The S-specificity determinants are known, but many questions remain. In this review, the genetics of SI are introduced and the characteristics of S-RNases and pollen F-box proteins are briefly described. A variety of modifier genes also required for SI are also reviewed. Mutations affecting compatibility in pollen are especially important for defining models of compatibility and incompatibility. In Solanaceae, pollen-side mutations causing breakdown in SI have been attributed to the heteroallelic pollen effect, but a mutation in Solanum chacoense may be an exception. This has been interpreted to mean that pollen incompatibility is the default condition unless the S-locus F-box protein confers resistance to S-RNase. In Prunus, however, S-locus F-box protein gene mutations clearly cause compatibility. CONCLUSIONS: Two alternative mechanisms have been proposed to explain compatibility and incompatibility: compatibility is explained either as a result of either degradation of non-self S-RNase or by its compartmentalization so that it does not have access to the pollen tube cytoplasm. These models are not necessarily mutually exclusive, but each makes different predictions about whether pollen compatibility or incompatibility is the default. As more factors required for SI are identified and characterized, it will be possible to determine the role each process plays in S-RNase-based SI.     

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.     

Petunia Germinating Pollen S/D3 Interacts with S-RNases in Petunia hybrida Vilm.

Self-Incompatibility （SI） Is a genetic mechanism of self/non-self pollen recognition to prevent self-fertilization In many flowering plants and, In most cases, this is controlled by a multl-allellc S-locus. S-RNase and Slocus F box （SLF） proteins have been shown to be the female and male determinants of gametophytlc selfIncompatibility （GSI）, respectively, In the Solanaceae, Scrophulariaceae and Rosaceae. Nevertheless, It is thought that additional factors are required for the SI response. Herein, we constructed a mature anther cDNA library from a self-Incompatible Petunia hybrida Vllm. line of the S3S3 haplotype. Using AhS2-RNase from Antirrhinum hispanicum as a bait for yeast two-hybrid screening, we found that petunia germinating pollen （PGP） S/D3 was capable of Interacting physically with the bait. However, the Interaction lacked haplotype specificity. The PGPS/D3 gene Is a single copy gene that Is expressed In tissues such as the style, ovary, pollen, and leaf. The PGPS/D3：：GFP （green fluorescence protein） construct was detected In both the membrane and cytoplasm. The Implications of these findings In the operation of S-RNase-based SI are discussed.     

Identification of self-incompatibility (S-) locus linked pollen cDNA markers in Petunia inflata.

Solanaceous type self-incompatibility (SI) is controlled by a single polymorphic locus, termed the S-locus. The only gene at the S-locus that has been characterized thus far is the S-RNase gene, which controls pistil function, but not pollen function, in SI interactions between pistil and pollen. One approach to identifying additional genes (including the pollen S-gene, which controls pollen function in SI) at the S-locus and to study the structural organization of the S-locus is chromosome walking from the S-RNase gene. However, the presence of highly repetitive sequences in its flanking regions has made this approach difficult so far. Here, we used RNA differential display to identify pollen cDNAs of Petunia inflata, a self-incompatible solanaceous species, which exhibited restriction fragment length polymorphism (RFLP) for at least one of the three S-haplotypes (S1, S2, and S3) examined. We found that the genes corresponding to 10 groups of pollen cDNAs are genetically tightly linked to the S-RNase gene. These cDNA markers will expedite the mapping and cloning of the chromosomal region of the Solanaceae S-locus by providing multiple starting points.     

Ectopic expression of S-RNase of <Emphasis Type="Italic">Petunia inflata</Emphasis> in pollen results in its sequestration and non-cytotoxic function

The specificity of S-RNase-based self-incompatibility (SI) is controlled by two S-locus genes, the pistil S-RNase gene and the pollen S-locus-F-box gene. S-RNase is synthesized in the transmitting cell; its signal peptide is cleaved off during secretion into the transmitting tract; and the mature “S-RNase”, the subject of this study, is taken up by growing pollen tubes via an as-yet unknown mechanism. Upon uptake, S-RNase is sequestered in a vacuolar compartment in both non-self (compatible) and self (incompatible) pollen tubes, and the subsequent disruption of this compartment in incompatible pollen tubes correlates with the onset of the SI response. How the S-RNase-containing compartment is specifically disrupted in incompatible pollen tubes, however, is unknown. Here, we circumvented the uptake step of S-RNase by directly expressing S₂-RNase, S₃-RNase and non-glycosylated S₃-RNase of Petunia inflata, with green fluorescent protein (GFP) fused at the C-terminus of each protein, in self (incompatible) and non-self (compatible) pollen of transgenic plants. We found that none of these ectopically expressed S-RNases affected the viability or the SI behavior of their self or non-self-pollen/pollen tubes. Based on GFP fluorescence of in vitro-germinated pollen tubes, all were sequestered in both self and non-self-pollen tubes. Moreover, the S-RNase-containing compartment was dynamic in living pollen tubes, with movement dependent on the actin–myosin-based molecular motor system. All these results suggest that glycosylation is not required for sequestration of S-RNase expressed in pollen tubes, and that the cytosol of pollen is the site of the cytotoxic action of S-RNase in SI.     

基于S-核酸酶的自交不亲和性的分子机制

自交不亲和性是一种广泛存在于显花植物中的种内生殖障碍,可以抑制近亲繁殖而促进异交。其中,以茄科、玄参科和蔷薇科为代表的配子体自交不亲和性是最常见的类型。这类自交不亲和性是由单一的多态性S-位点所控制。目前的研究发现这一位点至少包含两个自交不亲和反应特异性决定因子：花柱中的S-核酸酶和花粉中的SLF（S-Locus F-box）蛋白。该文将主要介绍并讨论基于S-核酸酶的自交不亲和性分子机制的研究进展。     

基于S- 核酸酶的自交不亲和性的分子机制

自交不亲和性是一种广泛存在于显花植物中的种内生殖障碍, 可以抑制近亲繁殖而促进异交。其中, 以茄科、玄参科和蔷薇科为代表的配子体自交不亲和性是最常见的类型。这类自交不亲和性是由单一的多态性S-位点所控制。目前的研究发现这一位点至少包含两个自交不亲和反应特异性决定因子: 花柱中的S-核酸酶和花粉中的SLF(S-Locus F-box)蛋白。该文将主要介绍并讨论基于S-核酸酶的自交不亲和性分子机制的研究进展。     

S-RNase-based self-incompatibility in Petunia inflata

Background

For the Solanaceae-type self-incompatibility, also possessed by Rosaceae and Plantaginaceae, the specificity of self/non-self interactions between pollen and pistil is controlled by two polymorphic genes at the S-locus: the S-locus F-box gene (SLF or SFB) controls pollen specificity and the S-RNase gene controls pistil specificity.

Scope

This review focuses on the work from the authors'' laboratory using Petunia inflata (Solanaceae) as a model. Here, recent results on the identification and functional studies of S-RNase and SLF are summarized and a protein-degradation model is proposed to explain the biochemical mechanism for specific rejection of self-pollen tubes by the pistil.

Conclusions

The protein-degradation model invokes specific degradation of non-self S-RNases in the pollen tube mediated by an SLF, and can explain compatible versus incompatible pollination and the phenomenon of competitive interaction, where SI breaks down in pollen carrying two different S-alleles. In Solanaceae, Plantaginaceae and subfamily Maloideae of Rosaceae, there also exist multiple S-locus-linked SLF/SFB-like genes that potentially function as the pollen S-gene. To date, only three such genes, all in P. inflata, have been examined, and they do not function as the pollen S-gene in the S-genotype backgrounds tested. Interestingly, subfamily Prunoideae of Rosaceae appears to possess only a single SLF/SFB gene, and competitive interaction, observed in Solanaceae, Plantaginaceae and subfamily Maloideae, has not been observed. Thus, although the cytotoxic function of S-RNase is an integral part of SI in Solanaceae, Plantaginaceae and Rosaceae, the function of SLF/SFB may have diverged. This highlights the complexity of the S-RNase-based SI mechanism. The review concludes by discussing some key experiments that will further advance our understanding of this self/non-self discrimination mechanism.     

AhSSK1, a novel SKP1-like protein that interacts with the S-locus F-box protein SLF

The S-locus F-box (SLF/SFB) protein, recently identified as the pollen determinant of S-RNase-based self-incompatibility (SI) in Solanaceae, Scrophulariaceae and Rosaceae, has been proposed to serve as the subunit of an SCF (SKP1-CUL1-F-box) ubiquitin ligase and to target its pistil counterpart S-RNase during the SI response. However, the underlying mechanism is still in dispute, and the putative SLF-binding SKP1-equivalent protein remains unknown. Here, we report the identification of AhSSK1, Antirrhinum hispanicumSLF-interacting SKP1-like1, using a yeast two-hybrid screen against a pollen cDNA library. GST pull-down assays confirmed the SSK1-SLF interaction, and showed that AhSSK1 could connect AhSLF to a CUL1-like protein. AhSSK1, despite having a similar secondary structure to other SKP1-like proteins, appeared quite distinctive in sequence and unique in a phylogenetic analysis, in which no SSK1 ortholog could be predicted in the sequenced genomes of Arabidopsis and rice. Thus, our results suggest that the pollen-specific SSK1 could be recruited exclusively as the adaptor of putative SCF(SLF) in those plants with S-RNase-based SI, providing an important clue to dissecting the function of the pollen determinant.     

Construction of a binary bacterial artificial chromosome library of Petunia inflata and the isolation of large genomic fragments linked to the self-incompatibility (S-) locus.

The Solanaceae family of flowering plants possesses a type of self-incompatibility mechanism that enables the pistil to reject self pollen but accept non-self pollen for fertilization. The pistil function in this system has been shown to be controlled by a polymorphic gene at the S-locus, termed the S-RNase gene. The pollen function is believed to be controlled by another as yet unidentified polymorphic gene at the S-locus, termed the pollen S-gene. As a first step in using a functional genomic approach to identify the pollen S-gene, a genomic BAC (bacterial artificial chromosome) library of the S2S2 genotype of Petunia inflata, a self-incompatible solanaceous species, was constructed using a Ti-plasmid based BAC vector, BIBAC2. The average insert size was 136.4 kb and the entire library represented a 7.5-fold genome coverage. Screening of the library using cDNAs for the S2-RNase gene and 13 pollen-expressed genes that are linked to the S-locus yielded 51 positive clones, with at least one positive clone for each gene. Collectively, at least 2 Mb of the chromosomal region was spanned by these clones. Together, three clones that contained the S2-RNase gene spanned approximately 263 kb. How this BAC library and the clones identified could be used to identify the pollen S-gene and to study other aspects of self-incompatibility is discussed.     

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.     

Action of the Style Product of the Self-Incompatibility Gene of Nicotiana alata (S-RNase) on in Vitro-Grown Pollen Tubes

The products of the S-locus expressed in female tissues of Nicotiana alata are ribonucleases (S-RNases). The arrest of growth of incompatible pollen tubes in styles may result from entry of the S-RNase into the pollen tube and degradation of pollen tube RNA. We investigated the action of isolated S-RNases on pollen tubes grown in vitro and found that S-RNase is taken up by the pollen without substantial alteration. The S-RNases inhibit incorporation of exogenously added radioactive amino acids into protein by the germinated pollen. The S-RNases also inhibit in vitro translation of pollen tube RNA in a wheat germ cell-free extract. We found no evidence for a specific mRNA substrate for the S-RNases, which implies that if RNase activity is involved in the control of self-incompatibility, allelic specificity is more likely to depend on the selective uptake of S-RNases into pollen tubes or their selective activation or inactivation by pollen factors, rather than cleavage of a specific substrate. Heat treating S2-RNase largely destroys its RNase activity but increases its inhibitory effect on in vitro pollen tube growth. This effect is not due to an increased uptake of S2-RNase by the pollen but is associated with a greatly enhanced accumulation of S2-RNase on the outer surface of the pollen grains.     

Exchanging sequence domains between S-RNases from Nicotiana alata disrupts pollen recognition

In self-incompatible plants of the Solanaceae, the specificity of pollen rejection is controlled by a single multiallelic S-locus. Pollen tube growth is inhibited in the style when its single S-allele matches either S-allele present in the diploid pistil. Each S-allele encodes an S-RNase with a unique sequence. S-RNases are secreted into the extracellular matrix of the transmitting tract which guides pollen tubes toward the ovary. Although it is known that S-RNases are the determinants of S-allele specificity in the pistil, it is not known how allele-specific information is encoded in the sequence. Therefore, we exchanged domains between S-RNases with different recognition specificities and expressed the chimeric proteins in transgenic plants to determine their effects on pollination behavior. Nine chimeric constructs were prepared in which domains from Nicotiana alata S_A2- and S_C10-RNases were exchanged. Among these nine constructs, the entire S-RNase sequence was sampled by exchanging single variable domains as well as larger blocks of contiguous sequences. The chimeric S-RNases retained enzymatic activity and were expressed at levels comparable to control transformants expressing S_A2- and S_C10-RNase. However, none of the chimeric S-RNases caused rejection of either S_A2- or S_C10-pollen. We conclude that the recognition function of S-RNases can be disrupted by alterations in many parts of the sequence. It appears that the recognition function of S-RNase is not localized to a specific domain.     

Patterns of variation within self-incompatibility loci

Diverse self-incompatibility (SI) mechanisms permit flowering plants to inhibit fertilization by pollen that express specificities in common with the pistil. Characteristic of at least two model systems is greatly reduced recombination across large genomic tracts surrounding the S-locus, which regulates SI. In three angiosperm families, including the Solanaceae, the gene that controls the expression of gametophytic SI in the pistil encodes a ribonuclease (S-RNase). The gene that controls pollen SI expression is currently unknown, although several candidates have recently been proposed. Although each candidate shows a high level of polymorphism and complete allelic disequilibrium with the S-RNase gene, such properties may merely reflect tight linkage to the S-locus, irrespective of any functional role in SI. We analyzed the magnitude and nature of nucleotide variation, with the objective of distinguishing likely candidates for regulators of SI from other genes embedded in the S-locus region. We studied the S-RNase gene of the Solanaceae and 48A, a candidate for the pollen gene in this system, and we also conducted a parallel analysis of the regulators of sporophytic SI in Brassica, a system in which both the pistil and pollen genes are known. Although the pattern of variation shown by the pollen gene of the Brassica system is consistent with its role as a determinant of pollen specificity, that of 48A departs from expectation. Our analysis further suggests that recombination between 48A and S-RNase may have occurred during the interval spanned by the gene genealogy, another indication that 48A may not regulate SI expression in pollen.     

S-RNase complexes and pollen rejection

Biochemical interactions between the pollen and the pistil allow plants fine control over fertilization. S-RNase-based pollen rejection is among the most widespread and best understood of these interactions. At least three plant families have S-RNase-based self-incompatibility (SI) systems, and S-RNases have also been implicated in interspecific pollen rejection. Although S-RNases determine the specificity of SI, other genes are required for the pollen rejection system to function. Progress is being made toward identifying these non-S-RNase factors. HT-protein, first identified as a non-S-RNase factor that was required for SI in Nicotiana alata, has now been implicated in other species as well. In addition, several pistil proteins bind to S-RNase in vitro. One hypothesis is that S-RNase forms a complex with these proteins in vivo that is the active form of S-RNase in pollen rejection.     

Expression of 10 S-class SLF-like genes in Nicotiana alata pollen and its implications for understanding the pollen factor of the S locus

The S locus of Nicotiana alata encodes a polymorphic series of ribonucleases (S-RNases) that determine the self-incompatibility (SI) phenotype of the style. The pollen product of the S locus (pollen S) in N. alata is unknown, but in species from the related genus Petunia and in self-incompatible members of the Plantaginaceae and Rosaceae, this function has been assigned to an F-box protein known as SLF or SFB. Here we describe the identification of 10 genes (designated DD1-10) encoding SLF-related proteins that are expressed in N. alata pollen. Because our approach to cloning the DD genes was based on sequences of SLFs from other species, we presume that one of the DD genes encodes the N. alata SLF ortholog. Seven of the DD genes were exclusively expressed in pollen and a low level of sequence variation was found in alleles of each DD gene. Mapping studies confirmed that all 10 DD genes were linked to the S locus and that at least three were located in the same chromosomal segment as pollen S. Finally, the different topologies of the phylogenetic trees produced using available SLF-related sequences and those produced using S-RNase sequences suggests that pollen S and the S-RNase have different evolutionary histories.