Sequence analysis of two alleles reveals that intra-and intergenic recombination played a role in the evolution of the radish fertility restorer (Rfo)

Background \ud Land plant genomes contain multiple members of a eukaryote-specific gene family encoding proteins with pentatricopeptide repeat (PPR) motifs. Some PPR proteins were shown to participate in post-transcriptional events involved in organellar gene expression, and this type of function is now thought to be their main biological role. Among PPR genes, restorers of fertility (Rf) of cytoplasmic male sterility systems constitute a peculiar subgroup that is thought to evolve in response to the presence of mitochondrial sterility-inducing genes. Rf genes encoding PPR proteins are associated with very close relatives on complex loci. \ud Results \ud We sequenced a non-restoring allele (L7rfo) of the Rfo radish locus whose restoring allele (D81Rfo) was previously described, and compared the two alleles and their PPR genes. We identified a ca 13 kb long fragment, likely originating from another part of the radish genome, inserted into the L7rfo sequence. The L7rfo allele carries two genes (PPR-1 and PPR-2) closely related to the three previously described PPR genes of the restorer D81Rfo allele (PPR-A, PPR-B, and PPR-C). Our results indicate that alleles of the Rfo locus have experienced complex evolutionary events, including recombination and insertion of extra-locus sequences, since they diverged. Our \ud analyses strongly suggest that present coding sequences of Rfo PPR genes result from intragenic recombination. We found that the 10 C-terminal PPR repeats in Rfo PPR gene encoded proteins result from the tandem duplication of a 5 PPR repeat block. \ud Conclusions \ud The Rfo locus appears to experience more complex evolution than its flanking \ud sequences. The Rfo locus and PPR genes therein are likely to evolve as a result of \ud intergenic and intragenic recombination. It is therefore not possible to determine which genes on the two alleles are direct orthologs. Our observations recall some \ud previously reported data on pathogen resistance complex loci. \u

Etude des relations structure-fonction de la protéine ORF 138 responsable de la stérilité mâle cytosplasmique Ogura

La protéine ORF138 (codée par le génome mitochondrial) est une protéine membranaire mitochondriale responsable de la stérilité mâle cytoplasmique (SMC) Ogura. Le phénotype des plantes stériles se manifeste par une dégénérescence prématurée du tapis de l'anthère selon un mécanisme encore inconnu alors que la protéine ORF138 est présente dans tous les tissus. Les objectifs du travail de thèse étaient de développer des outils moléculaires et biochimiques pour obtenir des informations pertinentes sur cette protéine, et permettre l'élaboration d'hypothèses solides quant au mécanisme de stérilité mâle.Nous avons montré que l'association des domaines hydrophobe et hydrophile de la protéine est indispensable à son effet. Un domaine de 15 acides aminés dont la structure secondaire est conservée entre 3 protéines de SMC non apparentées a été mis en évidence et joue également un rôle dans cet effet.Nous avons montré que l'ORF138 est enchâssée dans la membrane interne mitochondriale de colza sous des formes homooligomériques. Un complexe protéique de taille comprise entre 750 et 900 kDa impliquant l'ORF138 a également été identifié. Nous avons aussi prouvé que l'ORF138 peut être co-purifiée avec des composants nucléoprotéiques mitochondriaux de plantes mâle stériles.Une plus forte accumulation de l'alternative oxydase mitochondriale probablement accompagnée d'une activité accrue ont été observées chez les plantes stériles. La liaison entre stress oxydant et activité alternative oxydase est bien connue, et un lien possible entre un stress oxydant et la mort prématurée du tapis constitue une des pistes les plus prometteuses pour l'élucidation du mécanisme de stérilité.The ORF138 protein (encoded by the mitochondrial genome) is a mitochondrial membrane protein responsible of Ogura cytoplasmic male sterility (CMS) in Brassicaceae. In sterile plants microspore abortion is apparently due to premature degeneration of the anther tapetum, by a still unknown mechanism. This is the only induced phenotype whereas ORF138 is present in all tissues. The objectives of this work were to develop molecular and biochemical tools for the study of this protein, and to obtain relevent information for setting up hypotheses about the mechanism of male sterility.We showed that the association of the hydrophobic and hydrophilic domains of the protein is essential for its effect. A domain of 15 amino acids, sharing predicted secondary structure with two other proteins of unrelated CMS's, was highlighted and showed to play a role in this effect. We showed that ORF138 was integrated in the mitochondrial inner membrane of rapeseed and formed homooligomers. A proteic complex of size ranging between 750 and 900 kDa implying ORF138 was also identified. We also proved that ORF138 can be copurified with mitochondrial nucleoproteic components of male sterile plants. An increased accumulation of mitochondrial alternative oxydase probably accompanied by an increased activity, was observed in the sterile plants compared to fertiles. The connection between oxidative stress and alternative oxydase activity is well-known, and a possible bond between an oxidizing stress and the premature death of the tapetum constitutes one of the most promising tracks for the elucidation of the sterility mechanism.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Data from: A meta-analysis of the strength and nature of cytoplasmic genetic effects

Genetic variation in cytoplasmic genomes (i.e. the mitochondrial genome in animals, and the combined mitochondrial and chloroplast genomes in plants) was traditionally assumed to accumulate under a neutral equilibrium model. This view has, however, come under increasing challenge from studies that have experimentally linked cytoplasmic genetic effects to the expression of life history phenotypes. Such results suggest that genetic variance located within the cytoplasm might be of evolutionary importance and potentially involved in shaping population evolutionary trajectories. As a step towards assessing this assertion, here we conduct a formal meta-analytic review to quantitatively assess the extent to which cytoplasmic genetic effects contribute to phenotypic expression across animal and plant kingdoms. We report that cytoplasmic effect sizes are generally moderate in size and associated with variation across a range of factors. Specifically, cytoplasmic effects on morphological traits are generally larger than those on life history or metabolic traits. Cytoplasmic effect sizes estimated at the between-species scale (via interspecies mix-and-matching of cytoplasmic and nuclear genomes) are larger than those at the within-species scale. Furthermore, cytoplasmic effects tied to epistatic interactions with the nuclear genome tend to be stronger than additive cytoplasmic effects, at least when restricting the data set to gonochorous animal species. Our results thus confirm that cytoplasmic genetic variation is commonly tied to phenotypic expression across plants and animals, implicate the cytoplasmic–nuclear interaction as a key unit on which natural selection acts and generally suggest that the genetic variation that lies within the cytoplasm is likely to be entwined in adaptive evolutionary processes

Evolutionary Dynamics of the Restorer and a non-restorer Allelic Rfo Locus.

National audienceThe Rfo locus, first described in Asian radish cultivars and later introduced into Brassica, is involved in the restoration of fertility in the Ogura cytoplasmic male sterility system (CMS). CMS systems are constituted of two actors: a mitochondrial protein that causes male sterility and a nuclear gene that encodes a mitochondria-targeted protein able to impair the expression of the sterility gene. The nuclear restorer gene in the Rfo-Ogura system, as all identified restorer genes (with the only exception of the Rf2 Texas maize restorer), encodes a protein belonging to the pentatricopeptide repeat (PPR) family. The PPR gene family is a very large family in plants, with about 450 members in A. thaliana, and their function is largely unknown, although some members have proven to play roles in posttranscriptional organelle gene regulation. Recent studies analyzing chromosomes regions with duplicated PPR genes (allelic variants of a restorer locus in rice and two chromosome regions in A. thaliana) suggest revealed high levels of recombination in these regions. In addition, restorer genes (because of their ability to adapt to specificity changing/fast evolving targets) are evolved in a similar way as “resistant genes”. Anyway, evolutionary dynamics of these loci is largely unknown so far. In the Rfo region, the restorer gene seems to be duplicated too: three genes encoding highly related PPR proteins are present in the Rfo locus (even if just one of the proteins is able to restorer fertility). We recently sequenced a European non-restorer allelic locus. Analysis of this sequence shows the presence of only two PPR genes in an apparently very dynamic region. In this study, we analyzed the European allelic variant sequence of the Rfo locus and the possible evolutionary events that occurred in both loci, in order to give insight into the dynamics and evolution of the Rfo locus

Evolutionary dynamics of a locus of fertility restoration in plants

Abstract: In higher plants, hermaphrodites may genetically loose their male fertility through the cytoplasmic male sterility (CMS) system. In radish, a nuclear locus, denoted Rfo, has evolved that is able to counteract the effect of CMS and restore the fertility. This locus encodes three similar genes in tandem that belong to the pentatricopeptide repeat (PPR) family and each gene encloses a tandem repeat of PPR motif. Among the hundreds of members of this family, some play a role in the post-transcriptional gene regulation in organelles (mitochondria and chloroplasts). In this study, we recently sequenced a European non-restorer allelic locus and compare it to the original Rfo restorer allele to investigate its evolutionary dynamics. We conducted bioinformatic analysis to determine the putative border of tandem duplications both at protein motif level and at gene level. Our results present the picture of complex evolution with multiple gene duplications at a fast evolving locus

Agrobacterium-mediated gene transfer results mainly in transgenic plants transmitting T-DNA as a single mendelian factor

Forty-four independent transformed tobacco plants were obtained from a cocultivation experiment with Agrobacterium tumefaciens strains carrying modified Ti-plasmids. The transformed plants were either self-fertilized or crossed with nontransformed plants or with other transformed plants. The segregation of a phenotypic marker (kanamycin resistance) in the progenies of these plants was determined. In 40 cases out of 44, the segregation of the kanamycin resistance marker is consistent with Mendelian genetics. Among these 40 clones, 35 contain a single kanamycin resistance locus. The five others segregate two independent resistance loci. In two of the single insert clones, the segregation ratio after selfing indicates that the T-DNA insertion may have caused a recessive lethal mutation

pur4 Mutations Are Lethal to the Male, But Not the Female, Gametophyte and Affect Sporophyte Development in Arabidopsis[C][W]

Purine metabolism is crucial in living cells and involves three complex pathways in plants: the de novo synthesis, the salvage, and the degradation pathways. The relative importance of each pathway in plant development and reproduction, however, is still unclear. We identified two T-DNA insertions in the Arabidopsis (Arabidopsis thaliana) PUR4 gene (At1g74260) that encodes formylglycinamidine ribonucleotide synthase (EC 6.3.5.3), the fourth enzyme in the de novo purine biosynthesis pathway. The mutated alleles were never transmitted through the pollen of heterozygous plants but could be inherited through the female gametophyte, indicating that de novo purine synthesis is specifically necessary for pollen development. Because the pur4 mutations were lethal to the male gametophyte, homozygous pur4 plants could not be obtained. However, the reproductive phenotype of hetererozygous plants carrying the pur4-2 mutated allele was more severe than that carrying the pur4-1 mutated allele, and pur4-2/+ plants showed slightly delayed early development. We showed that the pur4-2 allele produces an antisense transcript and that the amount of PUR4 mRNA is reduced in these plants. Transient expression of a translational fusion with the green fluorescent protein in Arabidopsis plantlets showed that the formylglycinamidine ribonucleotide synthase protein is dually targeted to chloroplast and mitochondria, suggesting that at least some steps of the de novo purine biosynthesis pathway can take place in both organelles in Arabidopsis, a dual location previously thought to be a peculiarity of ureide-forming tropical legumes

Genetic evidence that the tryptophan 2-mono-oxygenase gene of Pseudomonas savastonoi is functionally equivalent to one of the T-DNA genes involved in plant tumour formation by Agrobacterium tumefaciens

The combined activities of the Agrobacterium tumefaciens T-DNA genes 1 and 2 are sufficient to induce tumorous growth on several plants, by introducing a new auxin biosynthetic pathway in infected cells. We have isolated Nicotiana tabacum plants containing only gene 1 or gene 2. These plants, respectively called rG1 and rG2, grow and develop in a normal fashion, indicating that neither the gene 1 nor the gene 2 activity by itself interferes with the endogenous auxin metabolism in plants. Previous evidence indicated that the auxin biosynthetic pathway of Pseudomonas savastanoi and that proposed to be encoded by the T-DNA of Agrobacterium tumefaciens are similar. When rG2 plants were infected with non-oncogenic A. tumefaciens or Escherichia coli strains that harbour the P. savastanoi iaaM gene (responsible for indole-3-acetamide synthesis) root and callus formation at the infection site was readily observed. This shows that the product of iaaM, indole-3-acetamide, is an in vivo substrate for the gene 2 encoded enzyme and supports the proposal that the gene 1-encoded enzyme is involved in the synthesis of indole-3-acetamide in transformed plants. This result offers new insights in evolution of bacteria and plants involved in pathogenic and symbiotic interactions