Expression and trans-specific polymorphism of self-incompatibility RNases in coffea (Rubiaceae)

Self-incompatibility (SI) is widespread in the angiosperms, but identifying the biochemical components of SI mechanisms has proven to be difficult in most lineages. Coffea (coffee; Rubiaceae) is a genus of old-world tropical understory trees in which the vast majority of diploid species utilize a mechanism of gametophytic self-incompatibility (GSI). The S-RNase GSI system was one of the first SI mechanisms to be biochemically characterized, and likely represents the ancestral Eudicot condition as evidenced by its functional characterization in both asterid (Solanaceae, Plantaginaceae) and rosid (Rosaceae) lineages. The S-RNase GSI mechanism employs the activity of class III RNase T2 proteins to terminate the growth of "self" pollen tubes. Here, we investigate the mechanism of Coffea GSI and specifically examine the potential for homology to S-RNase GSI by sequencing class III RNase T2 genes in populations of 14 African and Madagascan Coffea species and the closely related self-compatible species Psilanthus ebracteolatus. Phylogenetic analyses of these sequences aligned to a diverse sample of plant RNase T2 genes show that the Coffea genome contains at least three class III RNase T2 genes. Patterns of tissue-specific gene expression identify one of these RNase T2 genes as the putative Coffea S-RNase gene. We show that populations of SI Coffea are remarkably polymorphic for putative S-RNase alleles, and exhibit a persistent pattern of trans-specific polymorphism characteristic of all S-RNase genes previously isolated from GSI Eudicot lineages. We thus conclude that Coffea GSI is most likely homologous to the classic Eudicot S-RNase system, which was retained since the divergence of the Rubiaceae lineage from an ancient SI Eudicot ancestor, nearly 90 million years ago.     

S-RNase complexes and pollen rejection

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

Self-incompatibility (S) alleles of the rosaceae encode members of a distinct class of the T2/S ribonuclease superfamily

Stylar riboncleases (RNases) are associated with gametophytic self-incompatibility in two plant families, the Solanaceae and the Rosaceae. The self-incompatibility-associated RNases (S-RNases) of both the Solanaceae and the Rosaceae were recently reported to belong to the T2 RNase gene family, based on the presence of two well-conserved sequence motifs. Here, the cloning and characterization of S-RNase genes from two species of Rosaceae, apple (Malus × domestica) and Japanese pear (Pyrus serotina) is described and these sequences are compared with those of other T2-type RNases. The S-RNases of apple specifically accumulated in styles following maturation of the flower bud. Two cDNA clones for S-RNases from apple, and PCR clones encoding a further two apple S-RNases as well as two Japanese pear S-RNases were isolated and sequenced. The deduced amino acid sequences of the rosaceous S-RNases contained two conserved regions characteristic of the T2/S-type RNases. The sequences showed a high degree of diversity, with similarities ranging from 60.4% to 69.2%. Interestingly, some interspecific sequence similarities were higher than those within a species, possibly indicating that diversification of S-RNase alleles predated speciation in the Rosaceae. A phylogenetic tree of members of the T2/S-RNase superfamily in plants was obtained. The rosaceous S-RNases formed a new lineage in the tree that was distinct from those of the solanaceous S-RNases and the S-like RNases. The findings suggested that self-incompatibility mechanisms in Rosaceae and Solanaceae are similar but arose independently in the course of evolution.     

Self-incompatibility (S) alleles of the rosaceae encode members of a distinct class of the T2/S ribonuclease superfamily

Stylar riboncleases (RNases) are associated with gametophytic self-incompatibility in two plant families, the Solanaceae and the Rosaceae. The self-incompatibility-associated RNases (S-RNases) of both the Solanaceae and the Rosaceae were recently reported to belong to the T2 RNase gene family, based on the presence of two well-conserved sequence motifs. Here, the cloning and characterization of S-RNase genes from two species of Rosaceae, apple (Malus × domestica) and Japanese pear (Pyrus serotina) is described and these sequences are compared with those of other T2-type RNases. The S-RNases of apple specifically accumulated in styles following maturation of the flower bud. Two cDNA clones for S-RNases from apple, and PCR clones encoding a further two apple S-RNases as well as two Japanese pear S-RNases were isolated and sequenced. The deduced amino acid sequences of the rosaceous S-RNases contained two conserved regions characteristic of the T2/S-type RNases. The sequences showed a high degree of diversity, with similarities ranging from 60.4% to 69.2%. Interestingly, some interspecific sequence similarities were higher than those within a species, possibly indicating that diversification of S-RNase alleles predated speciation in the Rosaceae. A phylogenetic tree of members of the T2/S-RNase superfamily in plants was obtained. The rosaceous S-RNases formed a new lineage in the tree that was distinct from those of the solanaceous S-RNases and the S-like RNases. The findings suggested that self-incompatibility mechanisms in Rosaceae and Solanaceae are similar but arose independently in the course of evolution.     

The system of flowering plants: synthesis of classical and molecular approaches

The proposed system of flowering plants (Angiospermae) is based on synthesis of cladistic researches from different authors, morphological data and "traditional" systematics as well. Classis Angiospermae is divided for 4 subclassis: Magloliidae, Liliidae, Rosidae and Asteridae. Thus, monocots belong to subclassis Liliidae, and so-called "dicots" to three other subclasses. Altogether there are 35 orders and 328 families of flowering plants in this system.     

Ensifer mexicanus sp. nov. a new species nodulating Acacia angustissima (Mill.) Kuntze in Mexico

A new lineage of Ensifer nodulating the American legume Acacia angustissima in the tropical forest of Chiapas and Morelos, Mexico is described. Bacteria were identified as Ensifer with ssb or nolR specific primers. Phylogenetic analysis with partial sequences of the five chromosomal genes gyrA, nolR, recA, rpoB and rrs revealed that this new lineage is related to African Ensifer terangae. The results of total DNA-DNA hybridization and selected phenotypic tests among the A. angustissima strains and E. terangae indicated that they belong to different species. The phylogeny with the symbiotic nifH gene also separates this group as a different clade but with close affinities to bacteria belonging to the genus Ensifer isolated from American hosts. ITTG R7(T) (=CFN ER1001, HAMBI 2910, CIP 109033, ATCC BAA-1312, DSM18446) is the type strain of a new species for which the name Ensifer mexicanus sp. nov. is proposed.     

Development of a CAPS marker system for genotyping European pear cultivars harboring 17 <Emphasis Type="Italic">S</Emphasis> alleles

The full-length cDNAs of eight S ribonucleases (S-RNases) were cloned from stylar RNA of European pear cultivars that could not be characterized by the cleaved amplified polymorphic sequences (CAPS) marker system for genotyping European pear cultivars harboring nine S alleles Sa, Sb, Sd, Se, Sh, Sk, Sl, Sq, and Sr. Comparison of the nucleotide sequences between these cDNAs and six putative S-RNase alleles previously amplified by genomic PCR revealed that five corresponded to the putative Sc-, Si-, Sm-, Sn-, and Sp-RNase alleles and the other three corresponded new S-RNase alleles (designated as putative Sg-, Ss-, and St-RNase alleles). Genomic PCR with a new set of primers was used to amplify 17 S-RNase alleles: 1906 bp (Sg), 1642 bp (St), 1414 bp (Sl), ca. 1.3 kb (Sk and Sq), 998 bp (Se), 440 bp (Sb), and ca. 350 bp (Sa, Sc, Sd, Sh, Si, Sm, Sn, Sp, Sr, and Ss). Among them, S-RNase alleles of similar size were discriminated by digestion with 11 restriction endo-nucleases. The PCR amplification of 17 S-RNase alleles following digestion with the restriction endonucleases provided a new CAPS marker system for rapid S-genotyping of European pear cultivars harboring 17 S alleles. Using the CAPS analysis, Sc, Sg, Si, Sm, Sn, Sp, Ss, and St alleles were found in 32 cultivars, which were classified into 23 S-genotypes.     

Phylogenetic relationships in Saxifragaceae sensu lato: A comparison of topologies based on 18S rDNA and rbcL sequences

Relationships among the morphologically diverse members of Saxifragaceae sensu lato were inferred using 130 18S rDNA sequences. Phylogenetic analyses were conducted using representatives of all 17 subfamilies of Saxifragaceae sensu lato, as well as numerous additional taxa traditionally assigned to subclasses Magnoliidae, Caryophyllidae, Hamamelidae, Dilleniidae, Rosidae, and Asteridae. This analysis indicates that Saxifragaceae should be narrowly defined (Saxifragaceae sensu stricto) to consist of ~30 herbaceous genera. Furthermore, Saxifragaceae s. s. are part of a well-supported clade (referred to herein as Saxifragales) that also comprises lteoideae, Pterostemonoideae, Ribesioideae, Penthoroideae, and Tetracarpaeoideae, all traditional subfamilies of Saxifragaceae sensu lato, as well as Crassulaceae and Haloragaceae (both of subclass Rosidae). Paeoniaceae (Dilleniideae), and Hamamelidaceae, Cercidiphyllaceae, and Daphniphyllaceae (all of Hamamelidae). The remaining subfamilies of Saxifragaceae sensu lato fall outside this clade. Francoa (Francooideae) and Bauera (Baueroideae) are allied, respectively, with the rosid families Greyiaceae and Cunoniaceae. Brexia (Brexioideae), Parnassia (Parnassioideae), and Lepuropetolon (Lepuropetaloideae) appear in a clade with Celastraceae. Representatives of Phyllonomoideae, Eremosynoideae, Hydrangeoideae, Escallonioideae, Montinioideae, and Vahlioideae are related to taxa belonging to an expanded asterid clade (Asteridae sensu lato). The relationships suggested by analysis of 18S rDNA sequences are highly concordant with those suggested by analysis of rbcL sequences. Furthermore, these relationships are also supported in large part by other lines of evidence, including embryology. serology, and iridoid chemistry.     

Identification and evolutionary analysis of a relic S-RNase in<Emphasis Type="Italic"> Antirrhinum</Emphasis>

In several gametophytic self-incompatible species of the Solanaceae, a group of RNases named relic S-RNase has been identified that belong to the S-RNase lineage but are no longer involved in self-incompatibility. However, their function, evolution and presence in the Scrophulariaceae remained largely unknown. Here, we analyzed the expression of S-RNase and its related genes in Antirrhinum, a member of the Scrophulariacaeae, and identified a pistil-specific RNase gene; AhRNase29 encodes a predicted polypeptide of 235 amino acids with an estimated molecular weight of 26 kDa. Sequence and phylogenetic analyses indicated that AhRNase29 forms a monophyletic clade with Antirrhinum S-RNases, similar to that observed for other relic S-RNases. Possible evolution and function of relic S-RNases are discussed.     

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

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

Identification of <Emphasis Type="Italic">S</Emphasis>-haplotype-specific F-box gene in Japanese plum (<Emphasis Type="Italic">Prunus salicina</Emphasis> Lindl.)

Self-incompatibility has been studied extensively at the molecular level in Solanaceae, Rosaceae and Scrophulariaceae, all of which exhibit gametophytic self-incompatibility controlled by a single polymorphic locus containing at least two linked genes, i.e., the S-RNase gene and the pollen-expressed SFB/SLF (S-haplotype-specific F-box/S-locus F-box) gene. However, the SFB gene in Japanese plum (Prunus salicina Lindl.) has not yet been identified. We determined eight novel sequences homologous to the SFB genes of other Prunus species and named these sequences PsSFB. The gene structure of the SFB genes and the characteristic domains in deduced amino acid sequences were conserved. Three sequences from 410 to 2,800 bp of the intergenic region between the PsSFB sequences and the S-RNase alleles were obtained. The eight identified PsSFB sequences showed S-haplotype-specific polymorphism, with 74–83% amino acid identity. These alleles were exclusively expressed in the pollen. These results suggest that the PsSFB alleles are the pollen S-determinants of GSI in Japanese plum. Nucleotide sequence data reported are available in the NCBI database under the accession numbers DQ849084–DQ849090 and DQ849118.     

Competitive interaction between two functional S-haplotypes confer self-compatibility on tetraploid Chinese cherry (Prunus pseudocerasus Lindl. CV. Nanjing Chuisi)

Self-incompatibility (SI) has been studied extensively at the molecular level in Solanaceae, Rosaceae and Scrophulariaceae, all of which exhibit gametophytic self-incompatibility (GSI). In the present study, four PpsS-haplotypes (Prunus pseudocerasus S-haplotypes) comprising at least two genes, i.e., PpsS-RNase (P. pseudocerasus S-RNase) and PpsSFB (P. pseudocerasus S-haplotype-specific F-box) have been successfully isolated in tetraploid P. pseudocerasus Lindl. CV. Nanjing Chuisi ("NC") which exhibited self-compatibility (SC), and its S-genotype was determined as S-1/S-3'/S-5/S-7. These PpsS-RNases, which were expressed exclusively in style, shared the typical structural features with S-RNases from other Prunus species exhibiting GSI. All PpsSFBs showed similar structure characteristics of SFBs from other Prunus species, and matched with the necessary conditions for pollen S-determinant. No mutations leading to dysfunction of S-haplotype were found in their full-length c-DNA sequences, except for PpsS-3'-haplotype which was not amplified by PCR. These four S-haplotypes complied with tetrasomic inheritance. Diploid pollen grains with S-genotypes S-7/S-1, S-7/S-5 and S-1/S-5 can grow the full length of the style after self-pollination, while pollen grains with S-3'/S-7, S-3'/S-1 and S-3'/S-5 cannot. These results suggest that PpsS-haplotypes-1, -5 and -7 are functional, and that competitive interaction between two of them confer self-compatibility on cultivar "NC". Furthermore, in terms of recognition specificity, diploid pollen grains carrying PpsS-3'-haplotype are equal to monoploid pollen grains carrying the other functional S-haplotype.     

Convergent Evolution at the Gametophytic Self-Incompatibility System in Malus and Prunus

S-RNase-based gametophytic self-incompatibility (GSI) has evolved once before the split of the Asteridae and Rosidae. This conclusion is based on the phylogenetic history of the S-RNase that determines pistil specificity. In Rosaceae, molecular characterizations of Prunus species, and species from the tribe Pyreae (i.e., Malus, Pyrus, Sorbus) revealed different numbers of genes determining S-pollen specificity. In Prunus only one pistil and pollen gene determine GSI, while in Pyreae there is one pistil but multiple pollen genes, implying different specificity recognition mechanisms. It is thus conceivable that within Rosaceae the genes involved in GSI in the two lineages are not orthologous but possibly paralogous. To address this hypothesis we characterised the S-RNase lineage and S-pollen lineage genes present in the genomes of five Rosaceae species from three genera: M. × domestica (apple, self-incompatible (SI); tribe Pyreae), P. persica (peach, self-compatible (SC); Amygdaleae), P. mume (mei, SI; Amygdaleae), Fragaria vesca (strawberry, SC; Potentilleae), and F. nipponica (mori-ichigo, SI; Potentilleae). Phylogenetic analyses revealed that the Malus and Prunus S-RNase and S-pollen genes belong to distinct gene lineages, and that only Prunus S-RNase and SFB-lineage genes are present in Fragaria. Thus, S-RNase based GSI system of Malus evolved independently from the ancestral system of Rosaceae. Using expression patterns based on RNA-seq data, the ancestral S-RNase lineage gene is inferred to be expressed in pistils only, while the ancestral S-pollen lineage gene is inferred to be expressed in tissues other than pollen.     

Diversity of LTR-retrotransposons and <Emphasis Type="Italic">Enhancer/Suppressor</Emphasis><Emphasis Type="Italic">Mutator</Emphasis>-like transposons in cassava (<Emphasis Type="Italic">Manihot esculenta</Emphasis> Crantz)

Cassava (Manihot esculenta Crantz), though a major world crop with enormous potential, is very under studied. Little is known about its genome structure and organisation. Transposable elements have a key role in the evolution of genome structure, and can be used as important tools in applied genetics. This paper sets out to survey the diversity of members of three major classes of transposable element within the cassava genome and in relation to similar elements in other plants. Members of two classes of LTR-retrotransposons, Ty1/copia-like and Ty3/gypsy-like, and of Enhancer/Suppressor Mutator (En/Spm)-like transposons were isolated and characterised. Analyses revealed 59 families of Ty1/copia, 26 families of Ty3/gypsy retrotransposons, and 40 families of En/Spm in the cassava genome. In the comparative analyses, the predicted amino acid sequences for these transposon classes were compared with those of related elements from other plant species. These revealed that there were multiple lineages of Ty1/copia-like retrotransposons in the genome of cassava and suggested that vertical and horizontal transmission as the source of cassava Mecops may not be mutually exclusive. For the Ty3/gypsy elements network, two groups of cassava Megyps were evident including the Arabidopsis Athila lineage. However, cassava En/Spm-like elements (Meens) constituted a single group within a network of plant En/Spm-like elements. Hybridisation analysis supported the presence of transposons in the genome of cassava in medium (Ty3/gypsy and En/Spm) to high (Ty1/copia) copy numbers. Thus the cassava genome was shown to contain diverse members of three major classes of transposable element; however, the different classes exhibited contrasting evolutionary histories.     

Molecular evidence for the endosymbiont <Emphasis Type="BoldItalic">Wolbachia</Emphasis> in a non-filaroid nematode, <Emphasis Type="BoldItalic">Angiostrongylus cantonensis</Emphasis>

Wolbachia harbored by most filarial parasites, is critical to both embryogenesis and microfilarial development, and may lead to inflammation and pathogenesis in infected hosts. Based on alignment of the sequences from the wsp, ftsZ, and 16S rRNA genes, Wolbachia was demonstrated to exist in Angiostrongylus cantonensis, a non-filaroid nematode. Although the wsp gene may not be the best candidate for evolutionary analysis of Wolbachia, this gene has been sequenced from a broader coverage of the host species, making it feasible to be used for phylogenetic analysis in this study. The results from both Neighbor-joining and Maximum parsimony methods showed that this novel Wolbachia does not belong to any of the known groups (C or D) of nematode-derived Wolbachia. In addition, the wsp gene sequence of this newly identified endosymbiont revealed a high degree of identity (98%) with that from Diaea circumlita c2, tentatively classified into the putative group G. This suggests that Wolbachia from A. cantonensis could represent a deeply branched lineage in Wolbachia evolution or the occurrence of horizontal transfer between infected hosts. In conclusion, the findings provide some insights into our understanding of the evolution of Wolbachia, particularly the isolate from A. cantonensis.     

Introns in,introns out in plant gene families: a genomic approach of the dynamics of gene structure

Gene duplication is considered to be a source of genetic information for the creation of new functions. The Arabidopsis thaliana genome sequence revealed that a majority of plant genes belong to gene families. Regarding the problem of genes involved in the genesis of novel organs or functions during evolution, the reconstitution of the evolutionary history of gene families is of critical importance. A comparison of the intron/exon gene structure may provide clues for the understanding of the evolutionary mechanisms underlying the genesis of gene families. An extensive study of A. thaliana genome showed that families of duplicated genes may be organized according to the number and/or density of intron and the diversity in gene structure. In this paper, we propose a genomic classification of several A. thaliana gene families based on introns in an evolutionary perspective. abbreviations BGAL, -galactosidases; PCMP, plant combinatorial and modular protein     

Systematic distribution and ecological function of the secondary metabolites of the rosidae-asteridae

The systematic distribution of the secondary metabolities of angiosperms has been shaped by the role of these substances as plant defence in the continual chemical werfare between plants and their pests and predators. In the Rosidae-Asteridae, these coevolutionary interactions have caused the gradual replacement of a defence based on tannin and crystals (primitive Rosidae) by defences based on a variety of toxic and repellent substances (advanced Asteridae). Coevolutionary considerations support the concept that within related taxa, biosynthetic sequences of secondary metabolites can correspond directly with chemotaxonomic advancement.