Structure and expression of AtS1, an Arabidopsis thaliana gene homologous to the S-locus related genes of Brassica

Summary Genetic and molecular analysis of the self-incompatibility locus (S-locus) of the crucifer Brassica has led to the characterization of a multigene family involved in pollen-stigma interactions. While the crucifer Arabidopsis thaliana does not have a self-incompatibility system, S-related sequences were detected in this species by cross-hybridization with Brassica DNA probes. In this paper, we show that an A. thaliana S-related sequence, designated AtS1, is expressed specifically in flower buds. Sequence analysis suggests that AtS1 encodes a secreted glycoprotein that is most similar to the Brassica S-locus related protein SLR1. As has been proposed for SLR1, this gene may be involved in determining some fundamental aspect of pollen-stigma interactions during pollination. The molecular and genetic advantages of the Arabidopsis system will provide many avenues for testing this hypothesis.     

Physical linkage of the SLG and SRK genes at the self-incompatibility locus of Brassica oleracea

Summary In Brassica oleracea, the pollen-stigma interaction of self-incompatibility is controlled by a single genetically defined locus designated S. Molecular studies have identified two genes that are tightly linked to the classically defined S locus: The S-Locus Glycoprotein (SLG) gene and the S-Receptor Kinase (SRK) gene. In previous RFLP linkage analyses with probes specific for SLG and SRK, we were unable to identify any recombination events between SLG, SRK, and self-incompatibility phenotype. In this paper, we use pulsed-field gel electrophoresis (PFGE) in conjunction with DNA blot analysis to characterize the S-locus region from two highly divergent self-incompatibility genotypes, S ₂ and S ₆. We establish the physical linkage of SLG and SRK in each genotype, and demonstrate that the two genes are separated by a maximum distance of 220 kb in the S ₆ genotype and 350 kb in the S ₂ genotype. Furthermore, a comparison of the data from the two genotypes reveals that a high level of polymorphism exists across the entire S-locus region.     

The S-locus of Nicotiana alata: genomic organization and sequence analysis of two S-RNase alleles

Genomic clones encoding the S ₂- and S ₆-RNases of Nicotiana alata Link and Otto, which are the allelic stylar products of the self-incompatibility (S) locus, were isolated and sequenced. Analysis of genomic DNA by pulsed-field gel electrophoresis and Southern blotting indicates the presence of only a single S-RNase gene in the N. alata genome. The sequences of the open-reading frames in the genomic and corresponding cDNA clones were identical. The organization of the genes was similar to that of other S-RNase genes from solanaceous plants. No sequence similarity was found between the DNA flanking the S ₂- and S ₆-RNase genes, despite extensive similarities between the coding regions. The DNA flanking the S ₆-RNase gene contained sequences that were moderately abundant in the genome. These repeat sequences are also present in other members of the Nicotianae.     

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.     

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.     

Sequence variability and gene structure at the self-incompatibility locus of Solanum tuberosum

Summary Allelic complexity is a key feature of self-incompatibility (S) loci in gametophytic plants. We describe in this report the allelic diversity and gene structure of the S locus in Solanum tuberosum revealed by the isolation and characterization of genomic and cDNA clones encoding S-associated major pistil proteins from three alleles (S ₁, S _r1, S ₂). Genomic clones encoding the S₁ and S₂ proteins provide evidence for a simple gene structure: Two exons are separated by a small intron of 113 (S ₁) and 117 by (S ₂). Protein sequences deduced from cDNA clones encoding S₁ and S_r1 proteins show 95% homology. 15 of the 25 residues that differ between these S ₁and S _r1alleles are clustered in a short hypervariable protein segment (amino acid positions 44–68), which corresponds in the genomic clones to DNA sequences flanking the single intron. In contrast, these alleles are only 66% homologous to the S ₂allele, with the residues that differ between the alleles being scattered throughout the sequence. DNA crosshybridization experiments identify a minimum of three classes of potato S alleles: one class contains the alleles S ₁, S _r1and S ₃, the second class S ₂and an allele of the cultivar Roxy, and the third class contains at present only S ₄. It is proposed that these classes reflect the origin of the S alleles from a few ancestral S sequence types.     

Characterization of the S locus genes, SLG and SRK, of the Brassica S3 haplotype: identification of a membrane-localized protein encoded by the S locus receptor kinase gene

The S locus, which controls the self-incompatibility response in Brassica, has been shown to contain at least two genes. SLG encodes a secreted S locus glycoprotein whilst SRK encodes a putative S locus receptor kinase. SRK has been shown potentially to encode a functional kinase and genetic evidence indicates that this gene is essential for the self-incompatibility response. Here the characterization of the SRK and SLG genes of a Brassica line homozygous for the S₃ haplotype is described. A 120 kDa glycoprotein was identified in stigmas and several lines of evidence indicated that this protein is encoded by the SRK₃ gene. First, the 120 kDa glycoprotein was recognized by antibodies raised against peptides based on the SRK₃ gene sequence. Secondly, this protein is polymorphic and, in an F₂ population segregating for the S₃ haplotype, was expressed only in plants possessing the S₃ haplotype. Thirdly, the 120 kDa protein was expressed specifically in stigmas. Finally, the 120 kDa protein was only extracted from stigmas in the presence of detergent indicating that it is anchored in the membrane. SRK has been predicted to encode a transmembrane glycoprotein based on the deduced amino acid sequence. Located on the membrane, SRK is in a position to interface between an extracellular recognition event between pollen and pistil and an intracellular signal transduction pathway which initiates the self-incompatibility response.     

RNase X2, a pistil-specific ribonuclease from Petunia inflata,shares sequence similarity with solanaceous S proteins

Petunia inflata, a species with gametophytic self-incompatibility, has previously been found to contain a large number of ribonucleases in the pistil. The best characterized of the pistil ribonucleases are the products of the S alleles, the S proteins, which are thought to be involved in self-incompatibility interactions. Here we report the characterization of a gene encoding another pistil ribonuclease of P. inflata, RNase X2. Degenerate oligonucleotides, synthesized based on the amino-terminal sequence of RNase X2, were used as probes to isolate cDNA clones, one of which was in turn used as a probe to isolate genomic clones containing the gene for RNase X2, rnx2. The deduced amino acid sequence of RNase X2 shows 42% to 71% identity to the 20 solanaceous S proteins reported so far, with the highest degree of similarity being to S₃ and S₆ proteins of Nicotiana alata. The cDNA sequence predicts a leader peptide of 22 amino acids, suggesting that RNase X2, like S proteins, is an extracellular ribonuclease. Also, similar to the S gene, rnx2 is expressed only in the pistil, and contains a single intron comparable in size and identical in location to that of the S gene. However, rnx2 is not linked to the S locus, and, in contrast to the highly polymorphic S gene, it is monomorphic. The possible biological function of RNase X2 is discussed.     

Use of immunochemical and SSCP analyses to test homozygosity at theS locus ofBrassica oleracea genotypes

The self-incompatibility reaction of cruciferous plants prevents self-fertilization and has been shown to be controlled by at least two genes situated at a single multiallelic locus, theS locus. One of these two genes, theS locus glycoprotein (SLG) gene, encodes an abundant glycoprotein secreted to the cell wall of stigma papillae. Identification of thoseS alleles present at theS locus is of prime interest when studying the self-incompatibility response and can be achieved by identifying the SLG of the stigma. Here, we show that using anti-SLG antibodies in an immunochemical analysis, combined with a SSCP (single-strand conformation polymorphism) approach to characterize the corresponding stigma-specific, SLG mRNA, allowed the identification of plants heterogeneous at theS locus among populations of plants that were thought to be homozygous for known SLG alleles. This analysis stresses the importance of testing the homozygosity at theS locus of lines considered inbred for a knownS allele as mix-up of seeds may occur during the breeding programme.     

Physical size of the <Emphasis Type="Italic">S</Emphasis> locus region defined by genetic recombination and genome sequencing in <Emphasis Type="Italic">Ipomoea trifida</Emphasis>, Convolvulaceae

Sporophytic self-incompatibility (SSI) in the genus Ipomoea (Convolvulaceae) is controlled by a single polymorphic S locus. We have previously analyzed genomic sequences of an approximately 300 kb region spanning the S locus of the S ₁ haplotype and characterized the genomic structure around this locus. Here, we further define the physical size of the S locus region by mapping recombination breakpoints, based on sequence analysis of PCR fragments amplified from the genomic DNA of recombinants. From the recombination analysis, the S locus of the S ₁ haplotype was delimited to a 0.23 cM region of the linkage map, which corresponds to a maximum physical size of 212 kb. To analyze differences in genomic organization between S haplotypes, fosmid contigs spanning approximately 67 kb of the S ₁₀ haplotype were sequenced. Comparison with the S ₁ genomic sequence revealed that the S haplotype-specific divergent regions (SDRs) spanned 50.7 and 34.5 kb in the S ₁ and S ₁₀ haplotypes, respectively and that their flanking regions showed a high sequence similarity. In the sequenced region of the S ₁₀ haplotype, five of the 12 predicted open reading frames (ORFs) were found to be located in the divergent region and showed co-linear organization of genes between the two S haplotypes. Based on the size of the SDRs, the physical size of the S locus was estimated to fall within the range 34–50 kb in Ipomoea.     

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.     

Expression of S-locus glycoprotein genes from Brassica oleracea and B. campestris in transgenic plants of self-compatible B. napus cv Westar

Summary Self-compatible Brassica napus var Westar was transformed with SLG, the S-locus-derived gene that encodes S-locus-specific glycoproteins (SLSG). Four allelic variants of SLG isolated from self-incompatible B. oleracea and B. campestris strains homozygous for different S alleles were used. We show that the transgenic plants synthesized SLSG with the same apparent charge, molecular weight, and antigenic properties as that produced by the corresponding self-incompatible strains from which the cloned SLG genes were isolated. In addition, transgene-encoded SLSG was detected specifically in the papillar cells of the stigma, and was correctly targeted to the papillar cell wall. However, SLSG was produced at reduced levels in transgenic plants relative to self-incompatible strains. The introduction of the SLG genes did not confer a self-incompatibility phenotype on the Westar cultivar.     

Characterization of proteins associated with self-incompatibility in Solanum tuberosum

Summary The gametophytic self-incompatibility system of Solarium tuberosum is controlled by a single locus, designated as the S-locus. Protein extracts from potato styles of defined S-genotypes have been analysed by two-dimensional gel electrophoresis, and found to contain a group of basic glycoproteins. Each genetically determined allele S ₁ to S ₄ was associated with the presence of one of a number of these polypeptides differing slightly in isoelectric points (in the range 8.3–>9.1) and/or apparent molecular weight (ranging from 23,000 to 29,000). Two abundant basic polypeptides, one of which is apparently not glycosylated, were present in all genotypes examined. Amino-terminal protein sequence determinations revealed homologies of the S. tuberosum stylar proteins S₂, S₃ and S₄ with SI-associated polypeptides from Nicotiana alata and Lycopersicon peruvianum. With an oligonucleotide generated to the potato-S₂ N-terminal protein sequence, it was possible to detect a style-specific RNA species of 920 nucleotides. The oligonucleotide also behaved as an allele-specific probe when hybridized to total RNA of different S-genotypes.     

Expression level of the SLG gene is not correlated with the self-incompatibility phenotype in the class II S haplotypes of Brassica oleracea

In Brassica, the S-locus glycoprotein (SLG) gene has been strongly implicated in the self-incompatibility reaction. Several alleles of this locus have been sequenced, and accordingly grouped as class I (corresponding to dominant S-alleles) and class II (recessive). We recently showed that a self-compatible (Sc) line of Brassica oleracea expressed a class II-like SLG (SLG-Sc) gene. Here, we report that the SLG-Sc glycoprotein is electrophoretically and immunochemically very similar to the recessive SLG-S15 glycoprotein, and is similarly expressed in stigmatic papillae. Moreover, by seed yield analysis, we observe that both alleles are associated with a self-compatibility response, in contrast with the other known recessive S haplotypes (S2 and S5). By genomic DNA blot analysis, we show the existence of molecular homologies between the Sc and S15 haplotypes, but demonstrate that they are not identical. On the other hand, we also report that the S2 haplotype expresses very low amounts of SLG glycoproteins, although it exhibits a self-incompatible phenotype. These results strongly question the precise role of the SLG gene in the molecular mechanisms that control the self-incompatibility reaction of Brassica.     

Molecular diversity at the self-incompatibility locus is a salient feature in natural populations of wild tomato (Lycopersicon peruvianum)

A cDNA encoding a stylar protein was cloned from flowers of self-incompatible wild tomato (Lycopersicon peruvianum). The corresponding gene was mapped to the S locus, which is responsible for self-incompatibility. The nucleotide sequence was determined for this allele, and compared to other S-related sequences in the Solanaceae. The S allele was used to probe DNA from 92 plants comprising 10 natural populations of Lycopersicon peruvianum. Hybridization was conducted under moderate and permissive stringencies in order to detect homologous sequences. Few alleles were detected, even under permissive conditions, underscoring the great sequence diversity at this locus. Those alleles that were detected are highly homologous. Sequences could not be detected in self-incompatible Nicotiana alata, self-compatible L. esculentum (cultivated tomato) or self-compatible L. hirsutum. However, hybridization to an individual of self-incompatible L. hirsutum revealed a closely related sequence that maps to the S locus in this reproductively isolated species. This supports the finding that S locus polymorphism predates speciation. The extraordinarily high degree of sequence diversity present in the gametophytic self-incompatibility system is discussed in the context of other highly divergent systems representing several kingdoms.     

The S11 and S13 self incompatibility alleles in Solanum chacoense Bitt. are remarkably similar

A genomic clone of the S11 allele from the self-incompatibility locus (S locus) in Solanum chacoense Bitt. has been isolated by cross-hybridization to the S. chacoense S13 allele and sequenced. The sequence of the S11 allele contains all the features expected for S genes of the Solanaceae, and S11 expression, as assessed by northern blots and RNA-PCR, was similar to that of other S. chacoense S alleles. The S11 protein sequence shares 95% identity with the phenotypically distinct S13 protein of S. chacoense and is the gametophytic S allele with the highest similarity to an existing allele so far discovered. Only 10 amino acid changes differentiate the mature proteins from these two alleles, which sets a new lower limit to the number of changes that can produce an altered S allele specificity. The amino acid substitutions are not clustered, suggesting that an accumulation of random point mutations can generate S allele diversity. The S11 intron is unusual in that it could be translated in frame with the coding sequence, thus suggesting an additional mechanism for the generation of new S alleles.     

SLR1 function is dispensable for both self-incompatible rejection and self-compatible pollination processes inBrassica

TheSLR1 gene inBrassica is related both in DNA sequence and in pattern of expression to theS-locus glycoprotein (SLG) gene involved in the self-incompatibility mechanism which recognises and arrests the germination of self pollen. However,SLR1 shows minimal allelic variation and is expressed in both self-incompatible and compatibleBrassica lines and in related, self-compatible cruciferous plants. The function of the SLR1 protein is unknown. TheSLR1 gene was specifically ablated in self-incompatible and self-compatibleBrassica plants byAgrobacterium-mediated transformation with an antisense construct. Primary transformants and homozygous T2 progeny of both self-incompatibleB. oleracea and self-compatibleB. napus recipients were found to exhibit normal pollination responses despite having no detectable SLR1 glycoprotein. This shows that the high, wild-type level of SLR1 protein is not required to sustain the self-incompatibility reaction, nor is it necessary for successful intra-specific cross-pollination between compatible lines.