The radish Ogura fertility restorer impedes translation elongation along its cognate CMS-causing mRNA.

The control of messenger RNA (mRNA) translation has been increasingly recognized as a key regulatory step for gene control, but clear examples in eukaryotes are still scarce. Nucleo-cytoplasmic male sterilities (CMS) represent ideal genetic models to dissect genetic interactions between the mitochondria and the nucleus in plants. This trait is determined by specific mitochondrial genes and is associated with a pollen sterility phenotype that can be suppressed by nuclear genes known as restorer-of-fertility (Rf). In this study, we focused on the Ogura CMS system in rapeseed and showed that reversion to male sterility by the PPR-B fertility restorer (also called Rfo) occurs through a specific translation inhibition of the mitochondria-encoded CMS-causing mRNA orf138 We also demonstrate that PPR-B binds within the coding sequence of orf138 and acts as a ribosome blocker to specifically impede translation elongation along the orf138 mRNA. Rfo is the first recognized fertility restorer shown to act this way. These observations will certainly facilitate the development of synthetic fertility restorers for CMS systems in which efficient natural Rfs are lacking.

A Case of Gene Fragmentation in Plant Mitochondria Fixed by the Selection of a Compensatory Restorer of Fertility-Like PPR Gene.

The high mutational load of mitochondrial genomes combined with their uniparental inheritance and high polyploidy favors the maintenance of deleterious mutations within populations. How cells compose and adapt to the accumulation of disadvantageous mitochondrial alleles remains unclear. Most harmful changes are likely corrected by purifying selection, however, the intimate collaboration between mitochondria- and nuclear-encoded gene products offers theoretical potential for compensatory adaptive changes. In plants, cytoplasmic male sterilities are known examples of nucleo-mitochondrial coadaptation situations in which nuclear-encoded restorer of fertility (Rf) genes evolve to counteract the effect of mitochondria-encoded cytoplasmic male sterility (CMS) genes and restore fertility. Most cloned Rfs belong to a small monophyletic group, comprising 26 pentatricopeptide repeat genes in Arabidopsis, called Rf-like (RFL). In this analysis, we explored the functional diversity of RFL genes in Arabidopsis and found that the RFL8 gene is not related to CMS suppression but essential for plant embryo development. In vitro-rescued rfl8 plantlets are deficient in the production of the mitochondrial heme-lyase complex. A complete ensemble of molecular and genetic analyses allowed us to demonstrate that the RFL8 gene has been selected to permit the translation of the mitochondrial ccmFN2 gene encoding a heme-lyase complex subunit which derives from the split of the ccmFN gene, specifically in Brassicaceae plants. This study represents thus a clear case of nuclear compensation to a lineage-specific mitochondrial genomic rearrangement in plants and demonstrates that RFL genes can be selected in response to other mitochondrial deviancies than CMS suppression.

The MTL1 Pentatricopeptide Repeat Protein Is Required for Both Translation and Splicing of the Mitochondrial NADH DEHYDROGENASE SUBUNIT7 mRNA in Arabidopsis.

Mitochondrial translation involves a complex interplay of ancient bacteria-like features and host-derived functionalities. Although the basic components of the mitochondrial translation apparatus have been recognized, very few protein factors aiding in recruiting ribosomes on mitochondria-encoded messenger RNA (mRNAs) have been identified in higher plants. In this study, we describe the identification of the Arabidopsis (Arabidopsis thaliana) MITOCHONDRIAL TRANSLATION FACTOR1 (MTL1) protein, a new member of the Pentatricopeptide Repeat family, and show that it is essential for the translation of the mitochondrial NADH dehydrogenase subunit7 (nad7) mRNA. We demonstrate that mtl1 mutant plants fail to accumulate the Nad7 protein, even though the nad7 mature mRNA is produced and bears the same 5' and 3' extremities as in wild-type plants. We next observed that polysome association of nad7 mature mRNA is specifically disrupted in mtl1 mutants, indicating that the absence of Nad7 results from a lack of translation of nad7 mRNA. These findings illustrate that mitochondrial translation requires the intervention of gene-specific nucleus-encoded PPR trans-factors and that their action does not necessarily involve the 5' processing of their target mRNA, as observed previously. Interestingly, a partial decrease in nad7 intron 2 splicing was also detected in mtl1 mutants, suggesting that MTL1 is also involved in group II intron splicing. However, this second function appears to be less essential for nad7 expression than its role in translation. MTL1 will be instrumental to understand the multifunctionality of PPR proteins and the mechanisms governing mRNA translation and intron splicing in plant mitochondria.

In vivo functional analysis of a nuclear restorer PPR protein.

BACKGROUND: Nuclear restorers of cytoplasmic male fertility (CMS) act to suppress the male sterile phenotype by down-regulating the expression of novel CMS-specifying mitochondrial genes. One such restorer gene is Rfo, which restores fertility to the radish Ogura or ogu CMS. Rfo, like most characterized restorers, encodes a pentatricopeptide repeat (PPR) protein, a family of eukaryotic proteins characterized by tandem repeats of a 35 amino acid motif. While over 400 PPR genes are found in characterized plant genomes and the importance of this gene family in organelle gene expression is widely recognized, few detailed in vivo assessments of primary structure-function relationships in this protein family have been conducted. RESULTS: In contrast to earlier studies, which identified 16 or 17 PPR domains in the Rfo protein, we now find, using a more recently developed predictive tool, that Rfo has 18 repeat domains with the additional domain N-terminal to the others. Comparison of transcript sequences from pooled rfo/rfo plants with pooled Rfo/Rfo plants of a mapping population led to the identification of a non-restoring rfo allele with a 12 bp deletion in the fourth domain. Introduction into ogu CMS plants of a genetic construct in which this deletion had been introduced into Rfo led to a partial loss in the capacity to produce viable pollen, as assessed by vital staining, pollen germination and the capacity for seed production following pollination of CMS plants. The degree of viable pollen production among different transgenic plants roughly correlated with the copy number of the introduced gene and with the reduction of the levels of the ORF138 CMS-associated protein. All other constructs tested, including one in which only the C-terminal PPR repeat was deleted and another in which this repeat was replaced by the corresponding domain of the related, non-restoring gene, PPR-A, failed to result in any measure of fertility restoration. CONCLUSIONS: The identification of the additional PPR domain in Rfo indicates that the protein, apart from its N-terminal mitochondrial targeting presequence, consists almost entirely of PPR repeats. The newly identified rfo allele carries the same 4 amino acid deletion as that found in the neighboring, related, non-restoring PPR gene, PPR-A. Introduction of this four amino acid deletion into a central domain the Rfo protein, however, only partially reduces its restoration capacity, even though this alteration might be expected to alter the spacing between the adjoining repeats. All other tested alterations, generated by deleting specific PPR repeats or exchanging repeats with corresponding domains of PPR-A, led to a complete loss of restorer function. Overall we demonstrate that introduction of targeted alterations of Rfo into ogu CMS plants provides a sensitive in vivo readout for analysis of the relationship between primary structure and biological function in this important family of plant proteins.

Disruption of the CYTOCHROME C OXIDASE DEFICIENT1 gene leads to cytochrome c oxidase depletion and reorchestrated respiratory metabolism in Arabidopsis.

Cytochrome c oxidase is the last respiratory complex of the electron transfer chain in mitochondria and is responsible for transferring electrons to oxygen, the final acceptor, in the classical respiratory pathway. The essentiality of this step makes it that depletion in complex IV leads to lethality, thereby impeding studies on complex IV assembly and respiration plasticity in plants. Here, we characterized Arabidopsis (Arabidopsis thaliana) embryo-lethal mutant lines impaired in the expression of the CYTOCHROME C OXIDASE DEFICIENT1 (COD1) gene, which encodes a mitochondria-localized PentatricoPeptide Repeat protein. Although unable to germinate under usual conditions, cod1 homozygous embryos could be rescued from immature seeds and developed in vitro into slow-growing bush-like plantlets devoid of a root system. cod1 mutants were defective in C-to-U editing events in cytochrome oxidase subunit2 and NADH dehydrogenase subunit4 transcripts, encoding subunits of respiratory complex IV and I, respectively, and consequently lacked cytochrome c oxidase activity. We further show that respiratory oxygen consumption by cod1 plantlets is exclusively associated with alternative oxidase activity and that alternative NADH dehydrogenases are also up-regulated in these plants. The metabolomics pattern of cod1 mutants was also deeply altered, suggesting that alternative metabolic pathways compensated for the probable resulting restriction in NADH oxidation. Being the first complex IV-deficient mutants described in higher plants, cod1 lines should be instrumental to future studies on respiration homeostasis.

A restorer-of-fertility like pentatricopeptide repeat gene directs ribonucleolytic processing within the coding sequence of rps3-rpl16 and orf240a mitochondrial transcripts in Arabidopsis thaliana.

The pentatricopeptide repeat (PPR) proteins represent a large family of RNA-binding proteins that have many roles in post-transcriptional RNA processes within plant organelles. Among the PPR proteins that target plant mitochondria, the restorer-of-fertility (Rf) proteins are characterized by their inhibitory action on mitochondrion-localized cytoplasmic male sterility (CMS) genes in various crop species. Close homologs to known Rfs from radish, petunia, and rice can be identified in most higher plant species and these proteins define the recognized subgroup of Rf-like (RFL) PPR proteins. In this paper we describe the function of the RFL9 gene from Arabidopsis thaliana, and show that it is associated with ribonucleolytic cleavages within the coding sequences of rps3-rpl16 and orf240a mitochondrial transcripts in the Col-0 accession. RFL9 therefore represents an Rf-like PPR gene that has the potential to compromise the function of an essential mitochondrial gene and whose function is also associated with a mitochondrial orf sharing significant homology with a proven CMS-causing orf. We observe that RFL9 is active in only a few Arabidopsis accessions genetically close to Col-0, which supports the idea that the genetic fixation of this gene represents a regional event in the recent evolution of Arabidopsis. Additionally, RFL9 counts among the RFL genes that are probably controlled by short regulatory RNAs, and our results provides a potential explanation for such control, which in the case of RFL9 might have evolved to limit its detrimental effect on rps3 expression.

The pentatricopeptide repeat MTSF1 protein stabilizes the nad4 mRNA in Arabidopsis mitochondria.

Gene expression in plant mitochondria involves a complex collaboration of transcription initiation and termination, as well as subsequent mRNA processing to produce mature mRNAs. In this study, we describe the function of the Arabidopsis mitochondrial stability factor 1 (MTSF1) gene and show that it encodes a pentatricopeptide repeat protein essential for the 3'-processing of mitochondrial nad4 mRNA and its stability. The nad4 mRNA is highly destabilized in Arabidopsis mtsf1 mutant plants, which consequently accumulates low amounts of a truncated form of respiratory complex I. Biochemical and genetic analyses demonstrated that MTSF1 binds with high affinity to the last 20 nucleotides of nad4 mRNA. Our data support a model for MTSF1 functioning in which its association with the last nucleotides of the nad4 3' untranslated region stabilizes nad4 mRNA. Additionally, strict conservation of the MTSF1-binding sites strongly suggests that the protective function of MTSF1 on nad4 mRNA is conserved in dicots. These results demonstrate that the mRNA stabilization process initially identified in plastids, whereby proteins bound to RNA extremities constitute barriers to exoribonuclease progression occur in plant mitochondria to protect and concomitantly define the 3' end of mature mitochondrial mRNAs. Our study also reveals that short RNA molecules corresponding to pentatricopeptide repeat-binding sites accumulate also in plant mitochondria.

PPR336 is associated with polysomes in plant mitochondria.

The function of pentatricopeptide repeat (PPR) proteins has been associated with various post-transcriptional steps of organelle gene expression. Among them, translation and its regulation are essential processes. However, in plant mitochondria, they are also the steps of gene expression that are the least understood. In this study, PPR336 was identified as part of a high-molecular-weight complex in Arabidopsis mitochondria. PPR336 is an unusual representative of the large PPR family because it is relatively short and is characterised by a high expression level compared with other PPR proteins. PPR336 defines a small subgroup of eight class P PPR proteins that are similar in terms of motif organization. Among them, PPR336-like is the closest homolog of PPR336. Biochemical analysis has indicated that PPR336 is a strictly mitochondrial protein, extrinsically attached to the inner mitochondrial membrane and part of an RNase-sensitive complex. Sucrose gradients and polysome destabilisation experiments show that PPR336 is associated with ribosomes in plant mitochondria. Moreover, in Ppr336/336-like mutants, mitochondrial polysomes of lower molecular weight accumulate compared with wild-type plants. Polysome association and these unusual features suggest that PPR336 could be involved in a distinctive process, possibly translation in plant mitochondria.

The Arabidopsis Bio2 protein requires mitochondrial targeting for activity.

Mitochondria are involved in the production of various vitamins, such as biotin, in plants. It is unclear why these biosynthetic pathways have been maintained partly or entirely within the mitochondria throughout evolution. The last step in biotin biosynthesis occurs within the mitochondria and is catalyzed by the biotin synthase complex containing the BIO2 gene product. We investigated whether the Arabidopsis Bio2 enzyme could function outside mitochondria, by trying to complement a bio2 mutant with a truncated version of BIO2 lacking the region encoding the mitochondrial targeting sequence. We describe the characterization of a new T-DNA allele of bio2, with the sole phenotype of an absence of biotin production, in contrast to the previously characterized EMS bio2 allele (Patton et al. 1998, Plant Physiol 116(3):935-946). We found that a cytosolic version of the Bio2 protein could not complement this mutant. Supplementation with the substrate dethiobiotin (DTB) also failed to rescue the mutant phenotype. Thus, the lack of availability of DTB in the cytosol is not the only factor preventing this reaction from occurring outside mitochondria. Bio2 requires mitochondrial targeting for activity, enabling it to fulfill its role in biotin synthesis. The reaction catalyzed by Bio2 may be subject to biochemical constraints, and the apparent close connection with the mitochondrial Fe-S machinery may account for the reaction being retained within the organelle.