Glycosylation of S-RNases may influence pollen rejection thresholds in Solanum chacoense

A survey of Solanum chacoense plants expressing an authenticS₁₁-RNase transgene identified a line with partial compatibilityto S₁₁ pollen. By comparing fruit set to the S-RNase levelsdetermined immunologically in single styles, the minimum levelof S₁₁-RNase required for full rejection of S₁₁ pollen was estimatedto be 18 ng per style. The S₁₁-RNase threshold levels are thusconsiderably lower than those previously reported for the S₁₂-RNase.Interestingly, these two allelic S-RNases differ dramaticallyin the extent of glycosylation, with the number of glycosylationsites varying from one (S₁₁-RNase) to four (S₁₂-RNase). It issuggested that reduced glycosylation of the S₁₁-RNase may berelated to the lower threshold for pollen rejection. Key words: Gametophytic self-incompatibility, glycosylation, pistil-by-pistil analysis, S-RNase, Solanum chacoense, threshold Received 13 August 2007; Revised 27 November 2007 Accepted 30 November 2007     

An S-RNase promoter from Nicotiana alata functions in transgenic N. alata plants but not Nicotiana tabacum

Nicotiana tabacum and Nicotiana alata plants were transformed with genomic clones of two S-RNase alleles from N. alata. Neither the S ₂ clone, with 1.6 kb of 5 sequence, nor the S ₆ clone, with 2.8 kb of 5 sequence, were expressed at detectable levels in transgenic N. tabacum plants. In N. alata, expression of the S ₂ clone was not detected, however the S ₆ clone was expressed (at low levels) in three out of four transgenic plants. An S ₆-promoter-GUS fusion gene was also expressed in transgenic N. alata but not N. tabacum. Although endogenous S-RNase genes are expressed exclusively in floral pistils, the GUS fusion was expressed in both styles and leaves.     

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.     

Identification of quantitative trait loci associated with self-compatibility in a Prunus species

Self-compatibility in Rosaceous fruit species is based on a single-locus qualitative trait. However, the evidence observed in different species has indicated the presence of modifier genes outside the S locus affecting the expression of self-compatibility/self-incompatibility. The study of a progeny obtained from the cross of the almond genotypes ‘Vivot’× ‘Blanquerna’ has allowed the construction of a genetic map based on microsatellite markers and the identification for the first time in the Rosaceae family of two additional loci located outside the S locus and affecting the expression of self-compatibility/self-incompatibility. A quantitative trait locus (QTL) was located relatively close to the S locus, on linkage group 6 (G6), whereas the second one was located on G8. These QTLs appear to be involved in conferring self-compatibility to genotypes not possessing the S _f allele. These results are consistent with almond being a self-incompatible species with a genetic background of pseudo-self-compatibility controlled by modifier genes. The effect of the S _f allele and the two QTLs may contribute to explain the wide range of fruit sets observed when self-pollinating different almond genotypes.     

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.     

A modifier locus affecting the expression of the S-RNase gene could be the cause of breakdown of self-incompatibility in almond

Self-compatibility has become the primary objective of most almond (Prunus amygdalus Batsch) breeding programmes in order to avoid the problems related to the gametophytic self-incompatibility system present in almond. The progeny of the cross ‘Vivot’ (S ₂₃ S _fa) × ‘Blanquerna’ (S ₈ S _fi) was studied because both cultivars share the same S _f allele but have a different phenotypic expression: active (S _fa) in ‘Vivot’ and inactive (S _fi) in ‘Blanquerna’. In addition, the microscopic observation of pollen tube growth after self-pollination over several years showed an unexpected self-incompatible behaviour in most seedlings of this cross. The genotypes of this progeny showed that the S _fi pollen from ‘Blanquerna’ was not able to grow down the pistils of ‘Vivot’ harbouring the S _fa allele, confirming the active function of this allele against the inactive form of the same allele, S _fi. As self-compatibility was observed in some S ₈ S ₂₃ and S ₈ S _fa individuals of this progeny, the S _f haplotype may not always be linked to the expression and transmission of self-compatibility in almond, suggesting that a modifier locus may be involved in the mechanism of self-incompatibility in plants.     

The expression of a potato (Solanum tuberosum) S-RNase gene in Nicotiana tabacum pollen

Self-incompatibility in the Solanaceae is controlled by a single multiallelic genetic locus, the S locus. The stylar gene products of the S locus are abundant glycoproteins with ribonuclease activity, secreted in the transmitting tract tissue of the pistil. To investigate the structural and functional integrity and possible phenotypic effects of expression of the S-gene product in the male gametophyte, N. tabacum plants were transformed with a construct containing the genomic S ₂ -RNase coding sequence from S. tuberosum under the control of the promoter of the pollen-specific LAT52 gene from tomato. The expression pattern of the S ₂ RNase in the male gametophyte at both the protein and RNA level was found to be identical to that already reported for expression of the -glucuronidase (GUS) gene directed by the LAT52 promoter in transgenic tomato and tobacco. The S ₂ -RNase gene fusion led to a tissue-specific and developmentally regulated accumulation of the S ₂ polypeptide in pollen of transgenic tobacco plants. The transgenic protein product was of the same size and charge as the potato stylar product, had ribonuclease activity, and was glycosylated. The transgenic plants, however, did not show any morphological variations in their flower organs, and their fertility was not influenced by the accumulation of the S ₂ -RNase protein in pollen.     

Self-(in)compatibility of the almonds P. dulcis and P. webbii: detection and cloning of 'wild-type Sf ' and new self-compatibility alleles encoding inactive S-RNases

Prunus dulcis, the almond, is a predominantly self-incompatible (SI) species with a gametophytic self-incompatibility system mediated by S-RNases. The economically important allele S _f , which results in self-compatibility in P. dulcis, is said to have arisen by introgression from Prunus webbii in the Italian region of Apulia. We investigated the range of self-(in)compatibility alleles in Apulian material of the two species. About 23 cultivars of P. dulcis (14 self-compatible (SC) and nine SI) and 33 accessions of P. webbii (16 SC, two SI and 15 initially of unknown status), all from Apulia, were analysed using PCR of genomic DNA to amplify S-RNase alleles and, in most cases, IEF and staining of stylar protein extracts to detect S-RNase activity. Some amplification products were cloned and sequenced. The allele S _f was present in nearly all the SC cultivars of P. dulcis but, surprisingly, was absent from nearly all SC accessions of P. webbii. And of particular interest was the presence in many SI cultivars of P. dulcis of a new active allele, labelled S ₃₀ , the sequence of which showed it to be the wild-type of S _f so that S _f can be regarded as a stylar part mutant S ₃₀ °. These findings indicate S _f may have arisen within P. dulcis, by mutation. One SC cultivar of P. dulcis, ‘Patalina’, had a new self-compatibility allele lacking RNase activity, S _n5 , which could be useful in breeding programmes. In the accessions of P. webbii, some of which were known to be SC, three new alleles were found which lacked RNase activity but had normal DNA sequences.     

Pistil-function breakdown in a new <Emphasis Type="Italic">S</Emphasis>-allele of European pear, <Emphasis Type="Italic">S</Emphasis><Subscript><Emphasis Type="Italic">21</Emphasis></Subscript>°, confers self-compatibility

European pear exhibits RNase-based gametophytic self-incompatibility controlled by the polymorphic S-locus. S-allele diversity of cultivars has been extensively investigated; however, no mutant alleles conferring self-compatibility have been reported. In this study, two European pear cultivars, ‘Abugo’ and ‘Ceremeño’, were classified as self-compatible after fruit/seed setting and pollen tube growth examination. S-genotyping through S-PCR and sequencing identified a new S-RNase allele in the two cultivars, with identical deduced amino acid sequence as S ₂₁ , but differing at the nucleotide level. Test-pollinations and analysis of descendants suggested that the new allele is a self-compatible pistil-mutated variant of S ₂₁ , so it was named S ₂₁ °. S-genotypes assigned to ‘Abugo’ and ‘Ceremeño’ were S ₁₀ S ₂₁ ° and S ₂₁ °S ₂₅ respectively, of which S ₂₅ is a new functional S-allele of European pear. Reciprocal crosses between cultivars bearing S ₂₁ and S ₂₁ ° indicated that both alleles exhibit the same pollen function; however, cultivars bearing S ₂₁ ° had impaired pistil-S function as they failed to reject either S ₂₁ or S ₂₁ ° pollen. RT-PCR analysis showed absence of S ₂₁ °-RNase gene expression in styles of ‘Abugo’ and ‘Ceremeño’, suggesting a possible origin for S ₂₁ ° pistil dysfunction. Two polymorphisms found within the S-RNase genomic region (a retrotransposon insertion within the intron of S ₂₁ ° and indels at the 3′UTR) might explain the different pattern of expression between S ₂₁ and S ₂₁ °. Evaluation of cultivars with unknown S-genotype identified another cultivar ‘Azucar Verde’ bearing S ₂₁ °, and pollen tube growth examination confirmed self-compatibility for this cultivar as well. This is the first report of a mutated S-allele conferring self-compatibility in European pear.     

Inheritance and interactions of incompatibility alleles in the tetraploid sour cherry

Three progenies of sour cherry (Prunus cerasus) were analysed to correlate self-(in)compatibility status with S-RNase phenotype in this allotetraploid hybrid of sweet and ground cherry. Self-(in)compatibility was assessed in the field and by monitoring pollen tube growth after selfing. The S-RNase phenotypes were determined by isoelectric focusing of stylar proteins and staining for RNase activity and, for the parents, confirmed by PCR. Seedling phenotypes were generally consistent with disomic segregation of S-RNase alleles. The genetic arrangements of the parents were deduced to be ‘Köröser’ (self-incompatible) S ₁ S ₄ .S _B S _D , ‘Schattenmorelle’ (self-compatible) S ₆ S ₁₃ .S _B S _B , and clone 43.87 (self-compatible) S ₄ S ₁₃ .S _B S _B , where “.” separates the two homoeologous genomes. The presence of S ₄ and S ₆ alleles at the same locus led to self-incompatibility, whereas S ₁₃ and S _B at homoeologous loci led to self-compatibility. The failure of certain heteroallelic genotypes in the three crosses or in the self-incompatible seedlings indicates that S ₄ and S ₆ are dominant to S _B . However, the success of S ₁₃ S _B pollen on styles expressing corresponding S-RNases indicates competitive interaction or lack of pollen-S components. In general, the universal compatibility of S ₁₃ S _B pollen may explain the frequent occurrence of S ₁₃ and S _B together in sour cherry cultivars. Alleles S _B and S _D , that are presumed to derive from ground cherry, and S ₁₃ , presumably from sweet cherry, were sequenced. Our findings contribute to an understanding of inheritance of self-(in)compatibility, facilitate screening of progenies for self-compatibility and provide a basis for studying molecular interactions in heteroallelic pollen.     

The inheritance of partial self-compatibility in Brassica oleracea L. inbreds homozygous for different S-alleles

Summary In a study of partial self-compatibility in Brassica oleracea, flower number, seeded siliqua and seed production were recorded on self and cross-pollinated inflorescences of 32 progenies obtained by inter-crossing and selfing 8 plants homozygous for the incompatibility alleles S₂, S₅, S₁₅ and S₄₅.Progeny differences for both self-and out-cross seed production could be largely attributed to G.C.A. effects which were essentially uncorrelated. For cross-pollinated inflorescences heterosis was also important. Significant differences were found for selfed seed set and its two components, the proportion of flowers producing seeded siliquae and the numbers of seed per seeded siliqua, between parents with the same S-allele which could not be attributed to S-genotype alone. No evidence of increased self-compatibility in particular S-allele heterozygotes (mutual weakening) could be found.Outcross seed production depended primarily on the numbers of seeds set per seeded siliqua while self seed production was largely determined by the proportion of flowers which produced seeded siliqua. It is suggested that this is a key character for the production of inbred lines with reduced partial self-compatibility.     

Identification of new S-RNase self-incompatibility alleles and characterization of natural mutations in Iranian almond cultivars

The two main objectives of this research were to identify new S-RNase alleles in Iranian almond cultivars and to characterize naturally occurring mutations in these alleles that may cause self-compatibility. We investigated S genotypes of 22 Iranian almond cultivars using stylar RNase electrophoresis, PCR and DNA sequencing. We report six previously unidentified P. dulcis S-RNase alleles (S ₄₅ , S ₄₆ , S ₄₇ , S ₄₈ , S ₄₉ and S ₅₀ ). Four of 12 tested S-RNases were found to be non-functional in vitro: S ₄₉ , S ₅₀ , S ₂₄ /S _na and S ₂₅ /S ₄₇ . Detected point mutations in the C3 coding region of S ₄₉ - and S ₅₀ -RNase, leading to the replacement of a highly conserved cysteine and histidine residues, are with the highest probability the reason of these S-RNases inactivity. Results also suggested that ten Iranian almond cultivars display unique S genotype. All presented data confirm Iranian cultivars as valuable almond sources which are of interest to almond breeding and conservation programs.     

Expression of a fusion protein of Pyrus pyrifolia S-RNase with glutathione-S-transferase in E. coli

S-RNase is a style-specific ribonuclease which is associated with gametophytic self-incompatibility. An expression vector of a fusion protein of Pyrus pyrifolia(Japanese pear) S3-RNase with glutathione-S-transferase (GST) was constructed and transformed into E. coli. Using this system, the fusion protein, GST-S3-RNase, was expressed as an active form and can be used for screening pollen S-gene product(s).     

Genomic characterization of self-incompatibility ribonucleases (S-RNases) in European pear cultivars and development of PCR detection for 20 alleles

Sexual self-incompatibility in European pear (Pyrus communis L.) is controlled by a single locus (S-locus) encoding a polymorphic stylar ribonuclease (S-RNase) that is responsible for the female function in pollen–pistil recognition. In this study, genomic DNA sequences corresponding to five new S-RNase alleles (named S ₂₀ , S ₂₁ , S ₂₂ , S ₂₃ , and S ₂₄ ) and to S _m were characterized in European pear cultivars. Re-sequencing S _q from ‘General Le Clerc’ showed this S-RNase to encode the same protein as S ₁₂ . Based on these findings, a polymerase chain reaction (PCR)-based method was developed for the molecular typing of cultivars bearing 20 S-RNases (S ₁ –S ₁₄ , S _m , and S ₂₀ –S ₂₄ ) using consensus and allele-specific primers. Genomic PCR with consensus primers amplified product sizes characteristic of the S-RNases S ₁ , S ₂ , S ₄ , S ₁₀ , S ₁₃ , and S ₂₀ . However, the allele groups S ₃ /S ₁₂ , S ₆ /S ₈ /S ₁₁ /S ₂₂ and S ₅ /S ₇ /S ₉ /S ₁₄ /S _m /S ₂₁ /S ₂₃ /S ₂₄ amplified PCR products of similar size. To discriminate between alleles within these groups, primers to specifically amplify each S-RNase were developed. Application of this approach in 19 cultivars with published S-alleles allowed re-evaluation of one of the alleles of ‘Passe Crassane,’ ‘Conference,’ and ‘Condo.’ Finally, this method was used to assign S-genotypes to 37 cultivars. Test crosses confirmed molecular results. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cloning and characterization of genomic DNA sequences of four self-incompatibility alleles in sweet cherry (<Emphasis Type="Italic">Prunus avium</Emphasis> L.)

Gametophytic self-incompatibility (GSI) in sweet cherry is determined by a locus S with multiple alleles. In the style, the S-locus codifies for an allele-specific ribonuclease (S-RNase) that is involved in the rejection of pollen that carries the same S allele. In this work we report the cloning and genomic DNA sequence analysis including the 5 flanking regions of four S-RNases of sweet cherry (Prunus avium L., Rosaceae). DNA from the cultivars Ferrovia, Pico Colorado, Taleguera Brillante and Vittoria was amplified through PCR using primers designed in the conserved sequences of sweet cherry S-RNases. Two alleles were amplified for each cultivar and three of them correspond to three new S-alleles named S ₂₃ , S ₂₄ and S ₂₅ present in 'Pico Colorado', 'Vittoria' and 'Taleguera Brillante' respectively. To confirm the identity of the amplified fragments, the genomic DNA of these three putative S-RNases and the allele S ₁₂ amplified in the cultivar Ferrovia were cloned and sequenced. The nucleotide and deduced amino-acid sequences obtained contained the structural features of rosaceous S-RNases. The isolation of the 5-flanking sequences of these four S-RNases revealed a conserved putative TATA box and high similarity among them downstream from that sequence. However, similarity was low compared with the 5-flanking regions of S-RNases from the Maloideae. S ₆ - and S ₂₄ -RNase sequences are highly similar, and most amino-acid substitutions among these two RNases occur outside the rosaceous hypervariable region (RHV), but within another highly variable region. The confirmation of the different specificity of these two S-RNases would help elucidate which regions of the S-RNase sequences play a role in S-pollen specific recognition.Communicated by H.F. Linskens     

Analysis of <Emphasis Type="Italic">Malus</Emphasis><Emphasis Type="Italic">S</Emphasis>-RNase gene diversity based on a comparative study of old and modern apple cultivars and European wild apple

Malus S-RNase genetic diversity was analyzed in Malus × domestica cultivars and compared to European wild apple (Malus sylvestris). Using PCR-based approaches, the S-RNase genotype of 140 M. × domestica cultivars, 196 M. sylvestris trees and 27 M. sylvestris—M. × domestica hybrids was determined. S-RNase allelic richness in M. sylvestris was much higher than in M. × domestica, indicating the negative influence of domestication on S-RNase diversity. Heterogeneity of the S-RNase allelic distribution is much higher in cultivated apple than in wild apple, which shows that breeding leads to strong departure from the expected homogeneity of genes under negative frequency-dependent selection. The majority of the M. × domestica S-alleles has been found in M. sylvestris as well, which points to strong conservation of the S-locus gene structure. Based on the sequence of all different SCAR-fragments, which comprise both the hypervariable PS1 region and the single intron, S-RNase genetic diversity was further explored. It provided some clues to the occurrence of new S-alleles among the multitude of novel S-RNase sequences that have been identified, which were mostly unique for the group of M. sylvestris individuals. The determination of the S-RNase genotypes of old cultivars and M. sylvestris will enable their introduction into new breeding strategies. As M. sylvestris has become an endangered species in Belgium, the knowledge gathered in this study will be an important tool for selecting useful genotypes for a core collection.