Evolutionary genetics of an S-like polymorphism in Papaveraceae with putative function in self-incompatibility

Background

Papaver rhoeas possesses a gametophytic self-incompatibility (SI) system not homologous to any other SI mechanism characterized at the molecular level. Four previously published full length stigmatic S-alleles from the genus Papaver exhibited remarkable sequence divergence, but these studies failed to amplify additional S-alleles despite crossing evidence for more than 60 S-alleles in Papaver rhoeas alone.

Methodology/Principal Findings

Using RT-PCR we identified 87 unique putative stigmatic S-allele sequences from the Papaveraceae Argemone munita, Papaver mcconnellii, P. nudicuale, Platystemon californicus and Romneya coulteri. Hand pollinations among two full-sib families of both A. munita and P. californicus indicate a strong correlation between the putative S-genotype and observed incompatibility phenotype. However, we also found more than two S-like sequences in some individuals of A. munita and P. californicus, with two products co-segregating in both full-sib families of P. californicus. Pairwise sequence divergence estimates within and among taxa show Papaver stigmatic S-alleles to be the most variable with lower divergence among putative S-alleles from other Papaveraceae. Genealogical analysis indicates little shared ancestral polymorphism among S-like sequences from different genera. Lack of shared ancestral polymorphism could be due to long divergence times among genera studied, reduced levels of balancing selection if some or all S-like sequences do not function in incompatibility, population bottlenecks, or different levels of recombination among taxa. Preliminary estimates of positive selection find many sites under selective constraint with a few undergoing positive selection, suggesting that self-recognition may depend on amino acid substitutions at only a few sites.

Conclusions/Significance

Because of the strong correlation between genotype and SI phenotype, sequences reported here represent either functional stylar S-alleles, tightly linked paralogs of the S-locus or a combination of both. The considerable complexity revealed in this study shows we have much to learn about the evolutionary dynamics of self-incompatibility systems.     

Cloning and sequencing of cDNAs encoding two self-incompatibility associated proteins in Solanum chacoense

Summary We have isolated and sequenced cDNAs for S₂- and S₃-alleles of the self-incompatibility locus (S-locus) in Solanum chacoense Bitt., a wild potato species displaying gametophytic self-incompatibility. The S₂-and S₃-alleles encode pistil-specific proteins of 30 kDa and 31 kDa, respectively, which were previously identified based on cosegregation with their respective alleles in genetic crosses. The amino acid sequence homology between the S₂- and S₃-proteins is 41.5%. This high degree of sequence variability between alleles is a distinctive feature of the S-gene system. Of the 31 amino acid residues which were previously found to be conserved among three Nicotiana alata S-proteins (S₂, S₃, and S₆) and two fungal ribonucleases (R Nase T₂ and R Nase Rh), 27 are also conserved in the S₂- and S₃-proteins of S. chacoense. These residues include two histidines implicated in the active site of the R Nase T₂, six cysteines, four of which form disulfide bonds in R Nase T₂, and hydrophobic residues which might form the core structure of the protein. The finding that these residues are conserved among S-proteins with very divergent sequences suggests a functional role for the ribonuclease activity of the S-protein in gametophytic self-incompatibility.     

S-alleles are retained and expressed in a self-compatible cultivar of Petunia hybrida.

Summary We identified two S-allele-associated proteins (S-proteins) in a self-compatible cultivar of Petunia hybrida based on their segregation in F₁ hybrids between P. hybrida and its self-incompatible relative, Petunia inflata (with S₂S₂ genotype), and in selfed progeny of P. hybrida. These two S-proteins, designated S_x-protein (24 kDa) and S_o- protein (31 kDa), are pistil specific, and their expression follows a temporal and spatial pattern similar to that of S-proteins characterized in self-incompatible solanaceous species. Their amino-terminal sequences also share a high degree of similarity with those of solanaceous S-proteins. Selfing of P. hybrida yielded plants with S_oS_o, SxSo, and S_xS_x genotypes in an approximately 1:2:1 ratio, indicating that the S_x- and S_o-alleles, though expressed in the pistil, failed to elicit a self-incompatibility response. The S₂-allele of P. inflata is expressed in all the F₁ hybrids, rendering them capable of rejecting pollen bearing the S₂-allele. The S_o-allele is not functional in the F1 hybrids, because all the F₁ progeny with S₂S_o genotype are self-compatible. However, in F₁ hybrids with S₂S_x genotype, approximately half are self-incompatible and half are self-compatible, indicating that the function of the S_x-allele depends on the genetic background. These results strongly suggest that the presence of functional S-alleles alone is not sufficient for expression of a self-incompatibility phenotype, and reaffirm the multigenic nature of gametophytic self-incompatibility suggested by earlier genetic studies.     

Sequence of an S-protein of Lycopersicon peruvianum and comparison with other solanaceous S-proteins

Summary cDNA clones for an S-allele, designated S₅, of the self-incompatibility locus (S-locus) of Lycopersicon peruvianum have been isolated by probing a pistil cDNA library with cDNAs for S-alleles of Petunia inflata and Solanum chacoense. The longest S₅-cDNA is 869 bp and contains an open reading frame of 217 amino acids. An alignment of the deduced amino acid sequence of S₅-protein with that of the 18 S-proteins from five other solanaceous species is presented. Sequence comparison further refines the primary structural features of the S-proteins previously revealed from comparison of subsets of these sequences. Based on this comparison and evidence presented elsewhere, it is proposed that accumulation of point mutations, and not intragenic recombination, is responsible for the generation of new allelic specificities.     

Molecular analysis of two functional homologues of the S 3 allele of the Papaver rhoeas self-incompatibility gene isolated from different populations

The S ₃ allele of the S gene has been cloned from Papaver rhoeas cv. Shirley. The sequence predicts a hydrophilic protein of 14.0 kDa, showing 55.8% identity with the previously cloned S ₁ allele, preceded by an 18 amino acid signal sequence. Expression of the S ₃ coding region in Escherichia coli produced a form of the protein, denoted S₃e, which specifically inhibited S₃ pollen in an in vitro bioassay. The recombinant protein was ca. 0.8 kDa larger than the native stigmatic form, indicating post-translational modifications in planta, as was previously suggested for the S₁ protein. In contrast to other S proteins identified to date, S₃ protein does not appear to be glycosylated. Of particular significance is the finding that despite exhibiting a high degree of sequence polymorphism, secondary structure predictions indicate that the S₁ and S₃ proteins may adopt a virtually identical conformation. Sequence analysis also indicates that the P. rhoeas S alleles share some limited homology with the SLG and SRK genes from Brassica oleracea. Previously, cross-classification of different populations of P. rhoeas had revealed a number of functionally identical alleles. Probing of western blots of stigma proteins from plants derived from a wild Spanish population which contained an allele functionally identical to the Shirley S ₃ allele with antiserum raised to S₃e, revealed a protein (S ₃ s) which was indistinguishable in pI and M _r from that in the Shirley population. A cDNA encoding S ₃ s was isolated, nucleotide sequencing revealing a coding region with 99.4% homology with the Shirley-derived clone at the DNA level, and 100% homology at the amino acid level.     

Comparative macroarray analysis of morphine containing Papaver somniferum and eight morphine free Papaver species identifies an O-methyltransferase involved in benzylisoquinoline biosynthesis

Benzylisoquinoline alkaloids constitute a group of about 2,500 structures and are mainly produced by plants of the order Ranunculales. But only the opium poppy, Papaver somniferum, and Papaver setigerum are able to produce morphine. In this study, we started to investigate by gene expression analysis the molecular basis for this exceptional biosynthetic ability. A sequencing project from P. somniferum seedlings was initiated using a method based on the amplified fragment length polymorphism technique that resulted in 849 UniGenes. These cDNAs were analysed on macroarrays for differential expression between morphine-containing P. somniferum plants and eight other Papaver species, which accumulate other benzylisoquinolines instead of morphine. Three cDNAs showing increased expression in P. somniferum compared to all the other Papaver species were identified. Whereas two showed no significant homology to any known protein, one putatively encoded an O-methyltransferase. Analysis of substrate specificity of the heterologously expressed protein and mass spectrometric identification of the enzymatic products identified this protein as S-adenosyl-L-methionine:(R,S)-3-hydroxy-N-methylcoclaurine 4-O-methyltransferase (EC 2.1.1.116). Unlike other O-methyltransferases of different positional specificities implicated in benzylisoquinoline metabolism, the enzyme only accepted tetrahydroxylated tetrahydrobenzylisoquinolines as substrates; methylation was tolerated only at the 6-hydroxy position.     

Sequence diversity of pistil S-proteins associated with gametophytic self-incompatibility in Nicotiana alata

Summary In order to study the extent and nature of differences among various S-allele-associated proteins in N. alata, we carried out comparative studies of seven such proteins. We first isolated and sequenced cDNA clones for the S_z-, S_F11-, S₁-, and S_a-alleles, and then we compared the deduced amino acid sequences both of these four S-proteins and of three previously published S₂-, S₃-, and S₆-proteins. This comparison revealed (1) an average homology of 53.8% among the seven proteins and (2) two homology classes, with S_z and S_F11 in one class and S₁, S₂, S₃, and S₆ in the other class. There are 60 conserved residues, including 9 cysteines. Of the 144 variable residues, 50 were identified as hypervariable based on a calculation of their Similarity Indices. Although conserved, variable, and hypervariable residues are dispersed throughout the protein, some are clustered to form five conserved, five hypervariable, and a number of variable regions. Those variable sites which contain residues conserved within one class of S-proteins but different between classes might provide a clue to the evolutionary relationship of these two classes of S-proteins. The hypervariable residues, which account for sequence variability, may contribute to allelic specificity.     

Petunia hybrida S-proteins: ribonuclease activity and the role of their glycan side chains in self-incompatibility

Summary Self-incompatibility in flowering plants is controlled by the S-gene, encoding stylar S (allele-specific) glycoproteins. In addition to three previously characterized Petunia hybrida S-proteins, we identified by N-terminal sequence analysis another stylar S-protein, co-segregating with the S _b-allele. Purified S-proteins reveal biological activity, as is demonstrated for two of them by the allele-specific inhibition of pollen tube growth in vitro. Moreover, the four isolated S-proteins are ribonucleases (S-RNases). Specific activities vary from 30 (S₁) to 1000 (S₂) units per min per mg protein. We attempted to investigate the functionality of the carbohydrate portion of the S-RNases. Deglycosylation studies with the enzyme peptide-N-glycosidase F (PNGase F) reveals differences in the number of N-linked glycan chains present on the four S-RNases. Variability in the extent of glycosylation accounts for most of the molecular weight differences observed among these proteins. By amino acid sequencing, the positions of two of the three N-glycosylation sites on the S₂-RNase could be located near the N-terminus. Enzymic removal of the glycan side chains has no effect on the RNase activity of native S-RNases. This suggests another role of the glycan moiety in the self-incompatibility mechanism.     

Cloning and sequencing of cDNAs encoding two SSS

A defective S-allele, S ₀, and a functional S-allele, S _x, have previously been found to be retained in an F₁ hybrid of a self-compatible commercial cultivar of Petunia hybrida. Pistil proteins associated with these two alleles have also been identified. Their amino-terminal sequences have been found to share a high degree of similarity with those of S-proteins characterized from self-incompatible solanaceous species. Here we report the isolation and sequencing of cDNAs encoding S ₀- and S _x-proteins. Their deduced amino acid sequences contain all the consensus primary structural features of S-proteins from self-incompatible solanaceous species. Both proteins also have ribonuclease activity. The implications of these findings are discussed in relation to the presumed function of the S-protein in the self-incompatibility interaction.     

Comparison of S-alleles and S-glycoproteins between two wild populations of Brassica campestris in Turkey and Japan

Summary The number of identical S-alleles between two wild populations of B. campestris, one in Turkey, the other in Japan, that have been independent of one another for a long time was investigated. Diallel pollination tests between 38 S-allele homozygotes, i.e., 16 S-allele homozygotes from Turkey and 22 from Japan, revealed that these were 29 different S-alleles only 4 common ones. These S-alleles were differentiated by the iso-electric focusing (IEF) analysis of S-locus glycoproteins (SLGs) stained with an antiserum against SLG⁸. All identical S-alleles had the major SLG band at the same pI value without exception, even though they were collected from different populations. However, the number of minor bands of SLGs varied between the two populations; the S-alleles in Balcesme had generally fewer minor bands than those in Oguni. The 29 independent S-alleles were numbered from S ²¹ to S ⁴⁹ according to the pI value of the major SLG band. The major bands whose pI values were 7.5–8.5 were most common. Blot-hybridization patterns of genomic DNA hybridized with SLG ⁸ cDNA were not always the same among the strains of identical S-alleles obtained from different populations. Because about 20% of the S-alleles were shared between the two populations, it can be inferred that more than hundreds of S-alleles have been accumulated by mutation in B. campestris throughout the world.     

Identification of self-incompatibility genotypes of almond by allele-specific PCR analysis

In almond, gametophytic self-incompatibility is controlled by a single multiallelic locus (S-locus). In styles, the products of S-alleles are ribonucleases, the S-RNases. Cultivated almond in California have four predominant S-alleles (S ^a, S ^b, S ^c, S ^d). We previously reported the cDNA cloning of three of these alleles, namely S ^b, S ^c and S ^d. In this paper we report the cloning and DNA sequence analysis of the S ^a allele. The S^a-RNase displays approximately 55% similarity at the amino-acid level with other almond S-RNases (S^b, S^c, and S^d) and this similarity was lower than that observed among the S^b, S^c and S^d-RNases. Using the cDNA sequence, a PCR-based identification system using genomic DNA was developed for each of the S-RNase alleles. Five almond cultivars with known self-incompatibility (SI) geno-types were analyzed. Common sequences among four S-alleles were used to create four primers, which, when used as sets, amplify DNA bands of unique size that corresponded to each of the four almond S-alleles; S ^a (602 bp), S ^b (1083 bp), S ^c (221 bp) and S ^d (343 bp). All PCR products obtained from genomic DNA isolated from the five almond cultivars were cloned and their DNA sequence obtained. The nucleotide sequence of these genomic DNA fragments matched the corresponding S-allele cDNA sequence in every case. The amplified products obtained for the S ^a- and S ^b-alleles were both longer than that expected for the coding region, revealing the presence of an intron of 84 bp in the S ^a-allele and 556 bp in the S ^b-allele. Both introns are present within the site of the hypervariable region common in S-RNases from the Rosaceae family and which may be important for S specificity. The exon portions of the genomic DNA sequences were completely consistent with the cDNA sequence of the corresponding S-allele. A useful application of these primers would be to identify the S-genotype of progeny in a breeding program, new varieties in an almond nursery, or new grower selections at the seedling stage. Received: 21 June 1999 / Accepted: 15 November 1999     

Ability of the endangered species Papaver fauriei to produce hybrids with a cultivated poppy (Papaver sp.)

Papaver fauriei is an endemic and endangered species that grows only on the gravelly alpine slopes of Mt. Rishiri, Japan. Cultivated poppy (Papaver sp.), the species name of which is unknown, has been introduced to the natural habitats of P. fauriei through human activities. Because the appearance and internal transcribed spacer (ITS) sequences of these two poppies are highly similar, it is of concern that they could produce hybrids in their natural habitats. Thus, first, the ability of these two poppies to produce hybrids was analyzed by artificial fertilization in this study. A large number of seeds were produced by reciprocal crosses between P. fauriei and the cultivated poppy, comparable with the number of seeds obtained by self‐ or cross‐fertilization of P. fauriei or the cultivated poppy. In addition, high germination was observed for seeds obtained from crosses between the two poppies, and deleterious phenotypes, such as albinism and dwarfism, were not detected in the F₁ generation. These results indicate that after pollination, there is no reproductive isolation between the two poppies. Second, we sequenced the internal transcribed spacer (ITS) region of 240 poppy individuals collected from the gravelly alpine slopes of Mt. Rishiri, and 66 showed the sequence of P. fauriei, whereas 174 showed the sequence of the cultivated poppy. However, the ITS sequence that confirms hybridism between the two poppies was not detected in these individuals, indicating that hybridization of P. fauriei and the cultivated poppy rarely occurs under natural conditions. Unknown mechanism(s) appear to prevent cross‐pollination between the two poppies.     

Characterization of Ribonuclease Activity of Three S-Allele-Associated Proteins of Petunia inflata

Three S-allele-associated proteins (S-proteins) of Petunia inflata, a species with gametophytic self-incompatibility, were previously found to share sequence similarity with two fungal ribonucleases, RNase T₂ and RNase Rh. In this study, the S-proteins from P. inflata plants of S₁S₂ and S₂S₃ genotypes were purified to homogeneity by gel filtration and cation-exchange chromatography, and their enzymatic properties were characterized. The three S-proteins (S₁, S₂, and S₃), with pairwise sequence identity ranging from 73.1 to 80.5%, were similar in most of the enzymatic properties characterized. The ribonuclease activity had a pH optimum of 7.0 and a temperature optimum of 50°C. Diethylpyrocarbonate at 1 millimolar almost completely abolished the ribonuclease activity; cupric sulfate and zinc sulfate at 1 millimolar reduced the ribonuclease activity of the three S-proteins by 50 to 75%. EDTA and RNasin had no inhibitory effect. All three S-proteins hydrolyzed polycytidylic acid preferentially, but varied in their nucleolytic activity toward polyadenylic acid and polyuridylic acid.     

Selective inhibition of the growth of incompatible pollen tubes by S-protein in the Japanese pear

The S-allele-associated proteins (S-proteins) in the styles of the Japanese pear (Pyrus serotina Rehd. var. culta Rehd.) were purified by cation exchange chromatography. Their inhibitory action on the growth of incompatible pollen tubes (pollen tubes bearing the same S- allele as in the style from which the S-proteins were prepared) was characterized in vitro. Germination and tube growth of self-pollen (pollen from the same cultivar from which the S-proteins were prepared) decreased dose-dependently when the S-protein was added to the medium. Tube length was reduced to 10% that of compatible pollen tubes (pollen tubes bearing the S-allele different from that in the style from which the S-proteins were prepared) at 1.5 μg μl¹. S-proteins from Shinsui (S ₄ S ₅ ) also inhibited growth of cross-incompatible Kosui (S ₄ S ₅ ) pollen tubes, but not of compatible Chojuro (S ₂ S ₃ ) pollen tubes. After inactivation of RNase of the S- protein, the inhibitory action of the S-protein disappeared. These results indicate that the S-protein acts directly to inhibit growth of incompatible pollen tubes in Japanese pear styles, and that the RNase activity of the protein is essential for the biological function. However, small amounts of proteins that co-migrated with the S-protein may also play some roles in the inhibition. This is the first report on the selective inhibitory action of S-proteins in Rosaceae. Received: 11 April 2000 / Revision accepted: 28 September 2000     

Self-incompatibility in Petunia inflata: isolation and characterization of cDNAs encoding three S-allele-associated proteins

Summary We have identified three alleles of the S-locus controlling self-incompatibility and their associated pistil proteins in Petunia inflata, a species that displays monofactorial gametophytic self-incompatibility. These S-allele-associated proteins (S-proteins) are pistil specific, and their levels are developmentally regulated. The amino-terminal sequences determined for the three S-proteins are highly conserved and show considerable homology to those of S-proteins from Petunia hybrida, Nicotiana alata and Lycopersicon peruvianum, three other species of the Solanaceae that also exhibit gametophytic self-incompatibility. cDNA clones encoding the three S-proteins were isolated and sequenced. Comparison of their deduced amino acid sequences reveals an average homology of 75.6%, with conserved and variable residue interspersed throughout the protein. Of the 137 conserved residues, 53 are also conserved in the N. alata S-proteins studies so far; of the 64 variable residues, 29 were identified as hypervariable based on calculation of the Similarity Index. There is only one hypervariable region of significant length, and it consists of eight consecutive hypervariable residues. This region correspond approximately to the hypervariable region HV2 identified in N. alata S-proteins. Of the two classes of N. alata S-proteins previously identified, one class exhibits greater homology to the three P. inflata S-proteins reported here than to the other class of N. alata S-proteins.     

Evaluation of seed and leaf epidermis characters in the taxonomy of some annual species of the genus Papaver (Papaveraceae)

To get a better understanding of their taxonomic importance, we examined morphological, leaf epidermal and seed micromorphological characters of 19 taxa of annual Papaver L. belonging to the sections Papaver, Carinatae Fedde and Rhoeadium Spach. Among the studied taxa, the pattern of the coat of the seed testa cells and seed epidermal cell showed large variation. The granulate type of testa sculpturing was most common, but other types were represented by one or two species. In contrast, most of the taxa have broadly similar leaf epidermal characteristics. We conclude that variation in seed characters are significant for the taxonomy at the specific and generic levels, whereas leaf epidermis morphology, with few exceptions, is mostly only useful for separation of taxa within species. Based on the results, P. lacerum Popov is regarded as a synonym of P. commutatum Fisch. & C. A. Mey, while P. piptostigma Bienert ex Fedde, P. halophilum (Fedde) Cullen and P. tubuliferum Fedde are regarded as synonyms of P. macrostomum Boiss. et Huet ex Boiss. var. macrostomum. Further, Papaver gaubae Cullen & Rech. f. and P. bipinnatum C. A. Mey. are included into the synonymy list of P. glaucum Boiss. et Hausskn. and P. arenarium M.‐Bieb., respectively. In contrast, Papaver chelidoniifolium Fisch. & C. A. Mey. var. pinnatum Tavakkoli & Assadi from Gilan province is described as a new variety, and P. rhoeas L. var. strigosum Boenn. and var. glabrum Fedde and P. commutatum subsp. euxinum Kadereit are introduced as new records to Iran. The evolutionary and phylogenetic implications of the observed variation in seed characteristics are also discussed.     

3属4种罂粟科植物幼苗初生维管系统的结构

罂粟科属间演化关系一直存在争议。本文以罂粟科罂粟属的虞美人（Papaver rhoeas）和东方罂粟（Papaver orientale）、白屈菜属的白屈菜（Chelidonium majus）以及紫堇属的地丁草（Corydalis bungeana）为材料,应用整体透明和石蜡连续切片的方法,在明场显微镜和偏光显微镜下对其幼苗期的结构及发育进行了观察。结果表明,3属植物幼苗子叶节区下部存在的单中柱类型各异：罂粟属为外始式二原型圆形单中柱,白屈菜属为外始式二原型近圆形单中柱,紫堇属为外始式二原型“S”形单中柱。3属植物的子叶脉序也存在区别：虞美人和东方罂粟具单一叶脉,无叶隙;白屈菜为环结曲行羽状脉;地丁草为基出完全顶聚脉。根据上述幼苗初生维管系统的结构,认为3属中罂粟属较为原始,白屈菜属较为进化,紫堇属更为进化。     

Pelargonidin 3-malonylsophoroside from the red Iceland poppy,Papaver nudicaule

Pelargonidin 3-malonylsophoroside was characterized as a flower pigment in a red form of the Iceland poppy, Papaver nudicaule.