The effect of isolated components of Prunus avium L. styles on in vitro growth of pollen tubes

A number of components isolated from styles of P. avium cv. Napoleon (S ₃ S ₄) have been tested for their capacity to influence in vitro growth of pollen tubes from fresh and stored pollen (cv. Napoleon (S ₃ S ₄)). An antigenic glycoprotein (Antigen S) is a potent inhibitor of in-vitro pollen tube growth, causing a 65% reduction in tube length at a concentration of 20 g/ml. None of the other style components were effective inhibitors of pollen tube growth; neither were proteins of animal origin such as histone, serum albumin, cytochrome C, and the glycoproteins ovalbumin and thyroglobulin, effective inhibitors.     

Style antigens of Prunus avium L.

Antiserum to a protein fraction of an extract of mature styles of P. avium cv. Lambert (S ₃ S ₄) was raised in rabbits. Two major antigenic components of the style extracts were detected by immunoelectrophoresis and immunodiffusion. The presence of one antigen (S-antigen) correlated with a particular S-genotype (S ₃ S ₄). This antigen is restricted to mature styles of P. avium. The second antigen (P-antigen) was detected in styles of all Prunus species examined, but not in styles of other species of the Rosaceae. The S-antigen is positively charged and the P-antigen negatively charged at pH 8.8.     

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.     

Characterisation of novel S-alleles from cherry (Prunus avium L.)

In plant populations exhibiting gametophytic self-incompatibility, individuals harbouring rare S alleles are likely to have a reproductive advantage over individuals having more common alleles. Consequently, determination of the self-incompatibility haplotype of individuals is essential for genetic studies and the development of informed management strategies. This study characterises six new S alleles identified in wild cherry (Prunus avium L.). Investigations to determine the S genotype of individuals in recently planted woodland through length polymorphisms of introns associated with the stylar S-RNase gene and the pollen SFB gene revealed six S intron profiles which did not correspond to those of known S alleles. These are now attributed to S ₂₇ to S ₃₂ . Consensus primers, annealing in the S-RNase sequence coding for the signal peptide and C5 regions, were used to isolate the S-RNase alleles associated with the novel S intron profiles. The proteins corresponding to the new alleles were separated by isoelectric focusing from stylar extracts and their pI values determined. Similarities between the deduced amino acid sequence for the new alleles isolated and other cherry S-RNase sequences available on the databases ranged from 40% to 86%. Amplification products for SFB introns ranged from 172 to 208bp. New sequence regions exposed to positive selection were identified and the significance of the PS3 region reinforced. A phylogenetic relationship between P. avium S-RNases for S ₁₀ and S ₁₃ and between corresponding SFB alleles may indicate co-evolution of allele specificities of these two genes. The nucleotide sequences reported in this paper have been submitted to the EMBL/GenBank database under the following accession numbers: S ₂₇ (DQ266439), S ₂₈ (DQ266440), S ₂₉ (DQ266441), S ₃₀ (DQ266442), S ₃₁ (DQ266443), S ₃₂ (DQ266444).     

Tissue-specific antigens secreted by suspension-cultured callus cells of Prunus avium L.

A number of antigenic components are secreted into the medium by P. avium callus cells derived from different tissues and grown in suspension culture. These antigens have been detected using antiserum raised in rabbits to a protein fraction secreted by P. avium leaf callus. One antigen is specific to leaf tissue and is secreted by callus cells derived from stem, pistil and anthers as well as leaves. A second antigen is, in intact organs, restricted to styles of a particular self-incompatibility (S) genotype, but is also secreted by callus cells derived from the leaf. Another antigen, apparently not organ-specific, is secreted by all calli tested, including Rosa (cv. Paul's Scarlet).     

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     

Style proteins of a wild tomato (Lycopersicon peruvianum) associated with expression of self-incompatibility

The identification, isolation and aminoterminal sequencing of two S-genotype-associated proteins from style extracts of Lycopersicon peruvianum Mill. is reported. There is a high level of homology between these two sequences and with the amino-terminal sequences of other S-allele-associated glycoproteins isolated from Nicotiana alata Link et Otto. These sequences were obtained by a new high-sensitivity method of selected twodimensional gel analysis followed by electroelution and purification of proteins by inverse-gradient high-performance liquid chromatography before sequencing.Abbreviations HPLC high-performance liquid chromatography - Mr relative molecular mass - PTH phenylthiohydrantoin - SDS sodium dodecyl sulphate     

Biosynthesis of glycoproteins involved in the pollen-stigma interaction of incompatibility in developing flowers of Brassica oleracea L.

De-novo synthesis of the S-allele-specific glycoproteins of Brassica oleracea is demonstrated in stigmas at different developmental stages. Excised stigmas incorporate ¹⁴C-labeled amino acids into their S-glycoproteins early in development and before the self-incompatibility response is acquired, but the rate of synthesis accelerates prior to anthesis, resulting in the accumulation of high levels of the S-glycoproteins in the stigma and coinciding with the acquisition of the pollen-stigma incompatibility response. Since the self-compatible and self-incompatible zones of developing inflorescences are very sharply delineated, a threshold quantity of S-glycoproteins appears to be critical for the onset of self-incompatibility. Incorporation experiments in which [^35Smethionine was applied to intact stigma surfaces indicate that the papillae are the main sites of synthesis of the S-specific glycoproteins.Abbreviations IEF isoelectric focusing - SC self-compatibility - SDS sodium dodecyl sulfate - SI self-incompatibility     

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.     

Ultrastructural localization of acid phosphatase in the pollen tube of Prunus avium L. (sweet cherry)

The pollen tube of Prunus avium (cherry) consists of a growth zone of vesicles at the tip and an assemblage of organelles typical of an actively metabolizing cell. Electron opaque globules are closely associated with the plasma membrane and fibrillar cell wall layer at the tip. Acid phosphatase (EC 3.1.3.2) activity is localized in the membranes of 120 nm vesicles and ER system, the lumen of 50 nm vesicles, the plasma membrane and the tube nucleus.     

Isolation of S-alleles from a wild population of Brassica campestris L. at Balcesme,Turkey and their characterization by S-glycoproteins

Summary S-alleles of self-incompatibility were isolated from a wild population of Brassica campestris growing at Balcesme, Turkey. Out of 88 plants observed, 73 were self-incompatible and 4 were self-compatible. In certain families, selfed progenies from a self-incompatible plant segregated into fewer than three incompatibility classes, which is consistent with a one-locus sporophytic genetic control of self-incompatibility. Out of 25 combinations of S-alleles tested, dominance interactions were observed in 6 of them on the pollen side and on 5 of them on the stigma side. The 35 S-homozygotes thus isolated consisted of 18 independent S-alleles. The number of S-alleles in this population was estimated to be more than 30. The S-locus glycoproteins (SLGs) corresponding to the respective S-alleles were identified by iso-electric focusing (IEF)-gel immunoblotting with a polyclonal antiserum against SLG⁸. SLGs in a stigma were generally composed of several bands, one major and a few minor ones, whose molecular weight was similar to each other, and the major and minor bands were heritable in correlation with each other. SDS-PAGE analysis of SLGs differentiated a few juxtaposed bands between 50 and 60 kDa, and the variations in these bands were considered to be due to differences in the number of polysaccharide residues. General features of the variation of S-genes and their SLGs between the populations in Balcesme, Turkey and Oguni, Japan, were comparatively similar to one another, despite the different surroundings and history of these populations.     

A glycoprotein associated with the acquisition of the self-incompatibility system by maturing stigmas of Brassica oleracea

Iso-electric focusing of extracts derived from stigmatic homogenates of Brassica oleracea reveals that the mature stigma possesses large quantities of a glycoprotein not present in earlier stages of development in the bud. Pollen germination experiments carried out in parallel with the biochemical tests suggest that the appearance of this glycoprotein, which has an isoelectric point of pH 5.8, is coincident with the development of the self-incompatibility response. The site of this protein, and the role it may play in pollen-stigma interactions are discussed in terms of current models of the self-incompatibility system in Brassica.Abbreviations PAS periodic acid-schiffs - PEG polyethylene glycol - SI system self-incompatibility system     

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.     

Genomic characterization of self-incompatibility ribonucleases (S-RNases) in loquat (Eriobotrya japonica Lindl.) (Rosaceae, Pyrinae)

Loquat (Eriobotrya japonica Lindl.) is a fruit tree species of the Pyrinae subtribe of the Rosaceae that behaves as self incompatible. Since self-incompatibility in the Rosaceae is of the gametophytic type where a stylar ribonuclease (S-RNase) controls the female function of pollen–pistil recognition, consensus primers derived from the alignment of S-RNase sequences from other Pyrinae species were used to search for S-RNases in loquat. As a result, the first four S-RNases were sequenced for this species. The genomic sequences obtained showed the structural features of Pyrinae S-RNases. Moreover, microscopic observations of pollen tube growth in the style confirmed the inter-(in)compatibility relationships predicted from the molecular analyses. Phylogenetic analysis of the deduced amino acid sequences with other Pyrinae S-RNases confirmed that divergence of S-alleles in loquat and the Pyrinae predated speciation. This study reports for the first time the genomic characterization of S-RNases in loquat, providing a sound basis for an appropriate selection of pollinator cultivars and an adequate design of breeding programs.     

Isolation and characterization of glycoprotein lectins from the bark of three species of elder,Sambucus ebulus,S. nigra and S. racemosa

Lectins have been isolated from the bark of three members of the family Caprifoliaceae, Sambucus nigra (elder), S. racemosa (red-berried elder) and S. ebulus (dwarf elder), by affinity chromatography on fetuin-agarose, ion-exchange and gel-filtration chromatography. They are all glycoproteins of M _r 140 000 made up of at least four subunits. The lectin have similar but not identical amino-acid compositions and the carbohydrate content varies between 12% and 19% (w/w), the main sugars being (N-acetyl)glucosamine, mannose, fucose and xylose. Inhibition studies of hemagglutination with various mono- and oligosaccharides have shown that N-acetylgalactosamine and galactose together with galactose-containing oligosaccharides are the most effective inhibitors. There are some differences in specificity, in particular S. ebulus agglutinin is inhibited to the same degree by galactosamine, N-acetylgalactosamine and by galactose.Abbreviations PBS phosphate-buffered saline - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - SEA S. ebulus agglutinin - SNA S. nigra agglutinin - SRA S. racemosa agglutinin     

Isolation and characterization of an extracellular hydroxyproline-rich glycoprotein and a mannose-rich polysaccharide from Eudorina californica (Shaw)

Mucilage and colony walls of E. californica were separated from the cells by homogenization, filtration, and differential centrifugation. The chief components of the mucilage were a high-molecular-weight (MW) hydroxyproline-rich glycoprotein and a very high-MW polysaccharide in the proportions 47% and 34%, respectively. The glycoprotein consisted of galactose, arabinose, xylose and an unidentified neutral sugar; and the amino acids cysteine, aspartic acid, glutamic acid, arginine, lysine, glycine, serine, methionine, histidine, alanine, proline, hydroxyproline, tyrosine, threonine, valine, phenylalanine, isoleucine and leucine. The principal sugar of the polysaccharide was mannose. The chemical composition of the colony walls was essentially the same as that of the glycoprotein in the mucilage except that there was almost twice as much hydroxyproline. Also the protein content of the colony walls was 34% while that of the glycoprotein in the mucilage was 22%. No glucose, sugar acids or nucleic acids were found in the extracellular matrix.     

Isolation and partial characterization of an opioid-like 88 kDa hibernation-related protein

Previous studies show that infusion of hibernating woodchuck albumin (HWA) induces hibernation in summer-active ground squirrels and results in profound behavioral and physiological depression in primates. These effects are reversed by the administration of opiate antagonists, suggesting that the putative hibernation induction trigger (HIT) may act through opioid receptors. We have demonstrated that both HIT-containing plasma and the synthetic α opioid -Ala²- -Leu⁵-enkephalin (DADLE), which mimics the activity of HIT in hibernators, extend tissue survival time of a multi-organ autoperfusion system by 3-fold. In this study we present the first data showing biological activity with a much more highly purified plasma fraction from hibernating woodchucks, identified as the hibernation-related factor (HRF). Both the HRF and DADLE show opiate-like contractile inhibition in the mouse vas deferens (Mvd) bioassay. We also have preliminary evidence in an isolated rabbit heart preparation indicating that the HRF and DADLE act similarly to restore left ventricular function following global myocardial ischemia. Furthermore, we have partially sequenced an α 1-glycoprotein-like 88 kDa hibernation-related protein (p88 HRP) present in this fraction, which may prove to be the blood-borne HIT molecule.     

Characterization of SLFL1, a pollen-expressed F-box gene located in the Prunus S locus

The S locus and its flanking regions in the genus Prunus (Rosaceae) contain four pollen-expressed F-box genes. These genes contain the S locus F-box genes with low allelic sequence polymorphism genes 1, 2, and 3 (SLFL1, SLFL2, and SLFL3) as well as the putative pollen S gene, named the S haplotype-specific F-box protein gene (SFB). As much less information is available on the function of SLFLs than that of SFB, we analyzed the SLFLs of six S haplotypes of sweet cherry (Prunus avium) in this study. Genomic DNA blot analysis and the isolation of SLFL1 showed that the SLFL1 gene in a functional self-incompatible S ³ haplotype is deleted and only a partial sequence resembling SLFL1 is left in the S ³ locus region, suggesting that SLFL1 by itself is not directly involved in either the GSI reaction or pollen-tube growth. Genomic DNA blot analysis showed that there was no substantial modification or mutation in SLFL2 and SLFL3. A phylogenic analysis of F-box genes in the rosaceous S locus and its border regions showed that Prunus SLFLs were more closely related to maloid S locus F-box brothers than to Prunus SFBs. The functions of SLFLs and the evolution of self-incompatibility in Prunus are discussed based on these results. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. The nucleotide sequence data reported appear in the DDBJ, EMBL, and GenBank Nucleotide Sequence Databases under the accession numbers, AB360339, AB360340, AB360341, and AB360342, for SLFL1-S ¹ , SLFL1-S ² , SLFL1-S ⁵ , and SLFL1-S ⁶ , respectively.     

Somatic hybridization of sexually incompatible top-fruit tree rootstocks,wild pear (Pyrus communis var. pyraster L.) and Colt cherry (Prunus avium x pseudocerasus)

Summary Mesophyll protoplasts of wild pear (Pyrus communis var. pyraster L., Pomoideae) were chemically fused with cell suspension protoplasts of cherry rootstock Colt (Prunus avium x pseudocerasus, Prunoideae), following an electroporation treatment of the separate parental protoplast systems. Fusion-treated protoplasts were cultured, on modified K8P medium, where it had been previously established that neither parental protoplasts were capable of division. Somatic hybrid calli were recovered and, following caulogenesis on MS medium with zeatin and after rooting of regenerated shoots, complete trees were obtained and grown in vivo. Hybridity of these trees was confirmed based on morphological characters, chromosome complement and isozyme analysis. Two separate cloned lines of this intersubfamilial rootstock somatic hybrid (wild pear (+) Colt cherry) were produced. This is the first report of the production of somatic hybrid plants of two woody species, of agronomic value, within the order Rosales.