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

The P-type pentatricopeptide repeat protein DWEORG1 is a non-previously reported rPPR protein of Arabidopsis mitochondria

Gene expression in plant mitochondria is mainly regulated by nuclear-encoded proteins on a post-transcriptional level. Pentatricopeptide repeat (PPR) proteins play a major role by participating in mRNA stability, splicing, RNA editing, and translation initiation. PPR proteins were also shown to be part of the mitochondrial ribosome (rPPR proteins), which may act as regulators of gene expression in plants. In this study, we focus on a mitochondrial-located P-type PPR protein-DWEORG1-from Arabidopsis thaliana. Its abundance in mitochondria is high, and it has a similar expression pattern as rPPR proteins. Mutant dweorg1 plants exhibit a slow-growth phenotype. Using ribosome profiling, a decrease in translation efficiency for cox2, rps4, rpl5, and ccmFN2 was observed in dweorg1 mutants, correlating with a reduced accumulation of the Cox2 protein in these plants. In addition, the mitochondrial rRNA levels are significantly reduced in dweorg1 compared with the wild type. DWEORG1 co-migrates with the ribosomal proteins Rps4 and Rpl16 in sucrose gradients, suggesting an association of DWEORG1 with the mitoribosome. Collectively, this data suggests that DWEORG1 encodes a novel rPPR protein that is needed for the translation of cox2, rps4, rpl5, and ccmFN2 and provides a stabilizing function for mitochondrial ribosomes

An mTRAN-mRNA interaction mediates mitochondrial translation initiation in plants

Plant mitochondria represent the largest group of respiring organelles on the planet. Plant mitochondrial messenger RNAs (mRNAs) lack Shine-Dalgarno-like ribosome-binding sites, so it is unknown how plant mitoribosomes recognize mRNA. We show that “mitochondrial translation factors” mTRAN1 and mTRAN2 are land plant–specific proteins, required for normal mitochondrial respiration chain biogenesis. Our studies suggest that mTRANs are noncanonical pentatricopeptide repeat (PPR)–like RNA binding proteins of the mitoribosomal “small” subunit. We identified conserved Adenosine (A)/Uridine (U)-rich motifs in the 5′ regions of plant mitochondrial mRNAs. mTRAN1 binds this motif, suggesting that it is a mitoribosome homing factor to identify mRNAs. We demonstrate that mTRANs are likely required for translation of all plant mitochondrial mRNAs. Plant mitochondrial translation initiation thus appears to use a protein-mRNA interaction that is divergent from bacteria or mammalian mitochondria

Caractérisation biochimique de protéines de la famille Pentatricopeptide repeat chez Arabidopsis thaliana et le colza

Les protéines PPR (PentatricoPeptide Repeat), particulièrement nombreuses chez les plantes supérieures, se révèlent être des acteurs majeurs de l expression des gènes des chloroplastes et des mitochondries. Leur fonctionnement à l échelle moléculaire est encore inconnu ; il est cependant proposé qu elles puissent servir d adaptateurs moléculaires pour recruter des activités enzymatiques spécifiques sur des cibles ARN précises. Afin de tester la validité de cette hypothèse, l objet de ce travail de thèse a consisté à rechercher les partenaires protéiques et les cibles ARN de certaines protéines PPR d Arabidopsis à l aide d approches biochimiques. La fonction de la protéine restauratrice PPRB, codée au locus Rfo, a également été étudiée chez le colza, afin de mieux comprendre son incidence sur l expression du gène mitochondrial orf138 inducteur de la stérilité mâle cytoplasmique Ogura et contribuer à l élucidation du mécanisme de restauration de la fertilité. Nous avons tout d abord produit des transformants stables d Arabidopsis permettant de détecter 9 protéines PPR différentes grâce à leur fusion à de courts épitopes peptidiques. Ce matériel a permis de révéler la présence de quatre d entre elles dans des complexes multiprotéiques in vivo. Le faible rendement du protocole de double purification choisi pour l isolement ultérieur des complexes protéiques a limité l identification de partenaires protéiques. Les cibles ARN, ont été recherchées à l aide de la méthode du RIP-Chip. Cette analyse révèle pour la protéine PPR336, une capacité à lier de nombreux ARN mitochondriaux, avançant ainsi une nouvelle fonction pour une protéine PPR. Enfin, nous avons pu montrer que parmi les différentes protéines PPR fortement similaires également codées au locus Rfo, et accumulées dans les anthères de jeunes boutons floraux, seule la protéine PPRB induit la disparition de l ORF138 de façon quasi-totale et homogène. Nous avons révélé la corrélation de l activité de restauration de PPRB avec sa capacité à lier le transcrit orf138, dont l accumulation et la maturation ne sont pas affectées, en ciblant très probablement sa région 5 UTR. Nous proposons donc que cette liaison soit à l origine d un blocage de l initiation de la traduction du transcrit orf138 empêchant ainsi la synthèse de la protéine responsable de la SMC-Ogura.PentatricoPeptide Repeats proteins, whose family has literally exploded in higher plants, are key factors of mitochondria and chloroplast gene expression. Their molecular functions remain to be elucidated. The PPR proteins are proposed to constitute molecular adaptors that could recruit catalytic proteins to a specific site on RNA targets. In order to test the validity of this assumption, the aim of this work was to try to determine their protein partners and their RNA targets with biochemical approaches. The role of the restorer protein, PPRB, encoded at the Rfo locus, was also studied in rapeseed in order to gain insight into its role during the expression of the orf138 mitochondrial gene which induces Ogura male sterility. First, Arabidopsis stable transformants expressing nine PPR proteins fused to small tags were produced. Analysis of stromal and mitochondrial extracts by size exclusion chromatography allowed us to observe that 4 of the detected PPR proteins were involved in large multi-protein complexes. We used a tandem affinity purification strategy in order to identify their putative partners but its poor yield, limited the identification of partners. We used a RIP-Chip strategy for the characterization of PPR proteins RNA targets. This strategy revealed that PPR336 was able to bind a lot of RNA targets, thus suggesting a new function for PPR proteins. Finally we showed that the PPRB protein accumulates in young buds anthers, like other PPR proteins encoded at the Rfo locus. However, it was the only PPR protein to induce the disappearance of ORF138 protein in these tissues. We revealed the correlation of restoration activity with an ability to bind the orf138 transcript, whose accumulation and maturation are unchanged in restored plants, probably by binding the 5 UTR region of the mRNA. We proposed that the binding could impair the translation initiation and inhibit ORF138 protein synthesis.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

The propensity of Pentatricopeptide Repeat genes to evolve into restorers of cytoplasmic male sterility

Cytoplasmic male sterility (CMS) is a widespread phenotype in plants, which present a defect in the production of functional pollen. The male sterilizing factors usually consist of unusual genes or open reading frames (ORFs) encoded by the mitochondrial genome. CMS can be suppressed by specific nuclear genes called restorers of fertility (Rfs). In the majority of cases, Rf genes produce proteins that act directly on the CMS conferring mitochondrial transcripts by binding them specifically and promoting processing events. In this review, we explore the wide array of mechanisms guiding fertility restoration. PPR proteins represent the most frequent protein class among identified Rfs and they exhibit ideal characteristics to evolve into restorer of fertility when the mechanism of restoration implies a post-transcriptional action. Here, we review the literature that highlights those characteristics and help explain why PPR proteins are ideal for the roles they play as restorers of fertility

Partial sequences of Alanyl-tRNA Synthetase cDNAs from A. thaliana

Partial sequences of Alanyl-tRNA Synthetase cDNAs from A. thaliana. 3. International ISPMB Congres

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