Hakim Mireau

Genome-Wide Analysis of Arabidopsis Pentatricopeptide Repeat Proteins Reveals Their Essential Role in Organelle Biogenesis

The complete sequence of the Arabidopsis thaliana genome revealed thousands of previously unsuspected genes, many of which cannot be ascribed even putative functions. One of the largest and most enigmatic gene families discovered in this way is characterized by tandem arrays of pentatricopeptide repeats (PPRs). We describe a detailed bioinformatic analysis of 441 members of the Arabidopsis PPR family plus genomic and genetic data on the expression (microarray data), localization (green fluorescent protein and red fluorescent protein fusions), and general function (insertion mutants and RNA binding assays) of many family members. The basic picture that arises from these studies is that PPR proteins play constitutive, often essential roles in mitochondria and chloroplasts, probably via binding to organellar transcripts. These results confirm, but massively extend, the very sparse observations previously obtained from detailed characterization of individual mutants in other organisms.

Two birds with one stone: genes that encode products targeted to two or more compartments.

Eukaryotic cells are divided into multiple membrane-bound compartments, all of which contain proteins. A large subset of these proteins perform functions that are required in more than one compartment. Although in most cases proteins carrying out the same function in different compartments are encoded by different genes, this is not always true. Numerous examples have now been found where a single gene encodes proteins (or RNAs) found in two (or more) cell organelles or membrane systems. Some particularly clear examples come from protein synthesis itself: plant cells contain three protein-synthesizing compartments, the cytosol, the mitochondrial matrix and the plastid stroma. All three compartments thus require tRNAs and aminoacyl-tRNA synthetases. Some mitochondrial tRNAs and their aminoacyl-tRNA synthetases are identical to their cytosolic counterparts and they are encoded by the same genes. Similarly, some mitochondrial and plastid aminoacyl-tRNA synthetases are encoded by the same nuclear genes. The various ways in which differentially targeted products can be generated from single genes is discussed.

Characterization of Raphanus sativus Pentatricopeptide Repeat Proteins Encoded by the Fertility Restorer Locus for Ogura Cytoplasmic Male Sterility

Cytoplasmic male sterility is a maternally inherited trait in higher plants that prevents the production of functional pollen. Ogura cytoplasmic male sterility in radish (Raphanus sativus) is regulated by the orf138 mitochondrial locus. Male fertility can be restored when orf138 accumulation is suppressed by the nuclear Rfo locus, which consists of three genes putatively encoding highly similar pentatricopeptide repeat proteins (PPR-A, -B, and -C). We produced transgenic rapeseed (Brassica napus) plants separately expressing PPR-A and PPR-B and demonstrated that both encoded proteins accumulated preferentially in the anthers of young flower buds. Immunodetection of ORF138 showed that, unlike PPR-B, PPR-A had no effect on the synthesis of the sterility protein. Moreover, immunolocalization experiments indicated that complete elimination of ORF138 from the tapetum of anthers correlated with the restoration of fertility. Thus, the primary role of PPR-B in restoring fertility is to inhibit ORF138 synthesis in the tapetum of young anthers. In situ hybridization experiments confirmed, at the cellular level, that PPR-B has no effect on the accumulation of orf138 mRNA. Lastly, immunoprecipitation experiments demonstrated that PPR-B, but not PPR-A, is associated with the orf138 RNA in vivo, linking restoration activity with the ability to directly or indirectly interact with the orf138 RNA. Together, our data support a role for PPR-B in the translational regulation of orf138 mRNA.

The same Arabidopsis gene encodes both cytosolic and mitochondrial alanyl-tRNA synthetases.

In plants, all aminoacyl-tRNA synthetases are nuclearly encoded, despite the fact that their activities are required in the three protein-synthesizing cell compartments (cytosol, mitochondria, and chloroplasts). To investigate targeting of these enzymes, we cloned cDNAs encoding alanyl-tRNA synthetase (AlaRS) and the corresponding nuclear gene, ALATS, from Arabidopsis by using degenerate polymerase chain reaction primers based on highly conserved regions shared between known AlaRSs from other organisms. Analysis of the transcription of the gene showed the presence of two potential translation initiation codons in some ALATS mRNAs. Translation from the upstream AUG would generate an N-terminal extension with features characteristic of mitochondrial targeting peptides. A polyclonal antibody raised against part of the Arabidopsis AlaRS revealed that the Arabidopsis cytosolic and mitochondrial AlaRSs are immunologically similar, suggesting that both isoforms are encoded by the ALATS gene. In vitro experiments confirmed that two polypeptides can be translated from AlATS transcripts, with most ribosomes initiating on the downstream AUG to give the shorter polypeptide corresponding in size to the cytosolic enzyme. The ability of the presequence encoded between the two initiation codons to direct polypeptides to mitochondria was demonstrated by expression of fusion proteins in tobacco protoplasts and in yeast. We conclude that the ALATS gene encodes both the cytosolic and the mitochondrial forms of AlaRS, depending on which of the two AUG codons is used to initiate translation.

The Rf and Rf-like PPR in higher plants, a fast-evolving subclass of PPR genes.

In the last years, a number of nuclear genes restoring cytoplasmic male sterility (CMS) have been cloned in various crop species. The majority of these genes have been shown to encode pentatricopeptide repeat proteins (PPR) that act by specifically suppressing the expression of sterility-causing mitochondrial transcripts. Functional analysis of these proteins has indicated that the inhibitory effects of restoring PPR (Rf-PPR) proteins involve various mechanisms, including RNA cleavage, RNA destabilization, or translation inhibition. Cross-species sequence comparison of PPR protein complements revealed that most plant genomes encode 10–30 Rf-like (RFL) proteins sharing high-sequence similarity with the identified Rf-PPRs from crops. Evolutionary analyses further showed that they constitute a monophyletic group apart in the PPR family, with peculiar evolution dynamic and constraints. Here we review recent data on RF-PPRs and present the latest discoveries on the RFL family, with prospects on the functionality and evolution of this peculiar subclass of PPR.

Disruption of the CYTOCHROME C OXIDASE DEFICIENT1 Gene Leads to Cytochrome c Oxidase Depletion and Reorchestrated Respiratory Metabolism in Arabidopsis

Depletion of the mitochondrial respiratory complex IV activates the alternative respiratory pathway and important switches in metabolic flux. 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.

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.

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

BackgroundLand 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.ResultsWe 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 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.ConclusionsThe Rfo locus appears to experience more complex evolution than its flanking sequences. The Rfo locus and PPR genes therein are likely to evolve as a result of intergenic and intragenic recombination. It is therefore not possible to determine which genes on the two alleles are direct orthologs. Our observations recall some previously reported data on pathogen resistance complex loci.

Expression of Barstar as a selectable marker in yeast mitochondria

We describe a new and potentially universal selection system for mitochondrial transformation based on bacterial genes, and demonstrate its feasibility in Saccharomyces cerevisiae. We first found that cytoplasmically synthesized Barnase, an RNase, interferes with mitochondrial gene expression when targeted to the organelle, without causing lethality when expressed at appropriate levels. Next, we synthesized a gene that uses the yeast mitochondrial genetic code to direct the synthesis of the specific Barnase inhibitor Barstar, and demonstrated that expression of this gene, BARSTM, integrated in mtDNA protects respiratory function from imported barnase. Finally, we showed that screening for resistance to mitochondrially targeted barnase can be used to identify rare mitochondrial transformants that had incorporated BARSTM in their mitochondrial DNA. The possibility of employing this strategy in other organisms is discussed.

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

A mitochondria-targeted pentatricopeptide repeat protein specifically facilitates the translation of a respiratory chain transcript without inducing any cleavage in its upstream noncoding region. 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.