ABSTRACT
Group II introns in plant organelles have lost splicing autonomy and require the assistance of nuclear-encoded trans-factors whose roles remain to be elucidated. These factors can be mono- or poly-specific with respect to the number of introns whose splicing they facilitate. Poly-acting splicing factors are often essential and their genetic identification may benefit from the use of conditional mutations. Temperature-sensitive (TS) mutations in the ROOT PRIMORDIUM DEFECTIVE 1 (RPD1) gene were initially selected for their inhibitory effect on root formation in Arabidopsis. Further analysis revealed that RPD1 encodes a mitochondria-targeted RNA-binding protein family member, suggesting a role in mitochondrial gene expression and making its role in root formation enigmatic. We analysed the function of RPD1 and found that it is required for the removal of 9 mitochondrial group II introns and that the identified TS mutations affect the splicing function of RPD1. These results support that the inhibition of adventitious root formation at non-permissive temperature results from a reduction in RPD1 activity and thus mitochondrial activity. We further show that RPD1 physically associates in vivo with the introns whose splicing it facilitates. Preliminary mapping indicates that RPD1 may not bind to the same regions within all of its intron targets, suggesting potential variability in its influence on splicing activation.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Introns , Mitochondria , Mutation , Arabidopsis/genetics , Arabidopsis/growth & development , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Gene Expression Regulation, Plant , Introns/genetics , Mitochondria/genetics , Mitochondria/metabolism , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Plant Roots/genetics , Plant Roots/growth & development , Plant Roots/metabolism , RNA Splicing , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , TemperatureABSTRACT
Initiation and termination of plant mitochondrial transcription are poorly controlled steps. Precursor transcripts are thus often longer than necessary, and 3'-end processing as well as control of RNA stability are essential to produce mature mRNAs in plant mitochondria. Plant mitochondrial 3' ends are determined by 3'-to-5' exonucleolytic trimming until the progression of mitochondrial exonucleases along transcripts is stopped by stable RNA structures or RNA binding proteins. In this analysis, we investigated the function of the endonucleolytic mitochondrial stability factor 1 (EMS1) pentatricopeptide repeat (PPR) protein and showed that it is essential for the production and the stabilization of the mature form of the nad2 exons 1-2 precursor transcript, whose 3' end corresponds to the 5' half of the nad2 trans-intron 2. The accumulation of an extended rather than a truncated form of this transcript in ems1 mutant plants suggests that the role of EMS1 in 3' end formation is not strictly limited to blocking the passage of 3'-5' exonucleolytic activity, but that 3' end formation of the nad2 exons 1-2 transcript involves an EMS1-dependent endonucleolytic cleavage. This study demonstrates that the formation of the 3' end of mitochondrial transcripts may involve an interplay of endonucleolytic and exonucleolytic processing mediated by PPR proteins.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Mitochondria/genetics , Mitochondria/metabolism , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , RNA Precursors/genetics , RNA Precursors/metabolism , RNA, Mitochondrial/genetics , RNA, Mitochondrial/metabolismABSTRACT
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.
Subject(s)
Gene Expression Regulation, Plant , Plant Breeding/methods , Plant Infertility , Plant Proteins/genetics , Protein Biosynthesis , RNA, Messenger/genetics , Raphanus/physiology , Cytoplasm/metabolism , Plant Proteins/metabolism , RNA, Messenger/metabolismABSTRACT
Gene expression in plant mitochondria is predominantly governed at the post-transcriptional level and relies mostly on nuclear-encoded proteins. However, the protein factors involved and the underlying molecular mechanisms are still not well understood. Here, we report on the function of the MITOCHONDRIAL STABILITY FACTOR 3 (MTSF3) protein, previously named EMBRYO DEFECTIVE 2794 (EMB2794), and show that it is essential for accumulation of the mitochondrial NADH dehydrogenase subunit 2 (nad2) transcript in Arabidopsis (Arabidopsis thaliana) but not for splicing of nad2 intron 2 as previously proposed. The MTSF3 gene encodes a pentatricopeptide repeat protein that localizes in the mitochondrion. An MTSF3 null mutation induces embryonic lethality, but viable mtsf3 mutant plants can be generated through partial complementation with the developmentally regulated ABSCISIC ACID INSENSITIVE3 promoter. Genetic analyses revealed growth retardation in rescued mtsf3 plants owing to the specific destabilization of mature nad2 mRNA and a nad2 precursor transcript bearing exons 3 to 5. Biochemical data demonstrate that MTSF3 protein specifically binds to the 3' terminus of nad2. Destabilization of nad2 mRNA induces a substantial decrease in complex I assembly and activity and overexpression of the alternative respiratory pathway. Our results support a role for MTSF3 protein in protecting two nad2 transcripts from degradation by mitochondrial exoribonucleases by binding to their 3' extremities.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Embryonic Development , Gene Expression Regulation, Plant , Introns/genetics , Mitochondria/genetics , Mitochondria/metabolism , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Plant Proteins/metabolism , RNA Splicing , RNA, Messenger/metabolism , RNA, Mitochondrial/genetics , RNA, Mitochondrial/metabolismABSTRACT
Production and expression of RNA requires the action of multiple RNA-binding proteins (RBPs). New RBPs are most often created by novel combinations of dedicated RNA-binding modules. However, recruiting existing genes to create new RBPs is also an important evolutionary strategy. In this report, we analyzed the eight-member uL18 ribosomal protein family in Arabidopsis uL18 proteins share a short structurally conserved domain that binds the 5S ribosomal RNA (rRNA) and allows its incorporation into ribosomes. Our results indicate that Arabidopsis uL18-Like proteins are targeted to either mitochondria or chloroplasts. While two members of the family are found in organelle ribosomes, we show here that two uL18-type proteins function as factors necessary for the splicing of certain mitochondrial and plastid group II introns. These two proteins do not cosediment with mitochondrial or plastid ribosomes but instead associate with the introns whose splicing they promote. Our study thus reveals that the RNA-binding capacity of uL18 ribosomal proteins has been repurposed to create factors that facilitate the splicing of organellar introns.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/genetics , Chloroplasts/metabolism , Mitochondria/metabolism , RNA Splicing , Ribosomal Proteins/metabolism , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Introns/genetics , Mutation , Plants, Genetically Modified , RNA, Ribosomal, 5S/metabolism , Ribosomal Proteins/geneticsABSTRACT
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.
Subject(s)
Arabidopsis/genetics , Genome, Mitochondrial , Selection, Genetic , Arabidopsis/embryology , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Cytochrome c Group/metabolism , Embryonic Development , Protein Biosynthesis , RNA SplicingABSTRACT
Mitochondria play key roles in cellular energy metabolism in eukaryotes. Mitochondria of most organisms contain their own genome and specific transcription and translation machineries. The expression of angiosperm mtDNA involves extensive RNA-processing steps, such as RNA trimming, editing, and the splicing of numerous group II-type introns. Pentatricopeptide repeat (PPR) proteins are key players in plant organelle gene expression and RNA metabolism. In the present analysis, we reveal the function of the MITOCHONDRIAL SPLICING FACTOR 2 gene (MISF2, AT3G22670) and show that it encodes a mitochondria-localized PPR protein that is crucial for early embryo development in Arabidopsis. Molecular characterization of embryo-rescued misf2 plantlets indicates that the splicing of nad2 intron 1, and thus respiratory complex I biogenesis, are strongly compromised. Moreover, the molecular function seems conserved between MISF2 protein in Arabidopsis and its orthologous gene (EMP10) in maize, suggesting that the ancestor of MISF2/EMP10 was recruited to function in nad2 processing before the monocot-dicot divergence ~200 million years ago. These data provide new insights into the function of nuclear-encoded factors in mitochondrial gene expression and respiratory chain biogenesis during plant embryo development.
Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis , Electron Transport Complex I/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/metabolism , Embryonic Development , Gene Expression Regulation, Plant , Introns/genetics , Mitochondria/genetics , Mitochondria/metabolism , Mutation , Plant Proteins/genetics , RNA/metabolism , RNA Splicing , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
Messenger RNA translation is a complex process that is still poorly understood in eukaryotic organelles like mitochondria. Growing evidence indicates though that mitochondrial translation differs from its bacterial counterpart in many key aspects. In this analysis, we have used ribosome profiling technology to generate a genome-wide snapshot view of mitochondrial translation in Arabidopsis. We show that, unlike in humans, most Arabidopsis mitochondrial ribosome footprints measure 27 and 28 bases. We also reveal that respiratory subunits encoding mRNAs show much higher ribosome association than other mitochondrial mRNAs, implying that they are translated at higher levels. Homogenous ribosome densities were generally detected within each respiratory complex except for complex V, where higher ribosome coverage corroborated with higher requirements for specific subunits. In complex I respiratory mutants, a reorganization of mitochondrial mRNAs ribosome association was detected involving increased ribosome densities for certain ribosomal protein encoding transcripts and a reduction in translation of a few complex V mRNAs. Taken together, our observations reveal that plant mitochondrial translation is a dynamic process and that translational control is important for gene expression in plant mitochondria. This study paves the way for future advances in the understanding translation in higher plant mitochondria.
Subject(s)
Arabidopsis/genetics , Mitochondria/genetics , Protein Biosynthesis , Electron Transport Complex I/genetics , Genes, Mitochondrial , Mutation , RNA Editing , RNA, Messenger/metabolism , Ribosomes/metabolismABSTRACT
RNA expression in plant mitochondria implies a large number of post-transcriptional events in which transcript processing and stabilization are essential. In this study, we analyzed the function of the Arabidopsis mitochondrial stability factor 2 gene (MTSF2) and show that the encoded pentatricopeptide repeat protein is essential for the accumulation of stable nad1 mRNA. The production of mature nad1 requires the assembly of three independent RNA precursors via two trans-splicing reactions. Genetic analyses revealed that the lack of nad1 in mtsf2 mutants results from the specific destabilization of the nad1 exons 2-3 precursor transcript. We further demonstrated that MTSF2 binds to its 3Î extremity with high affinity, suggesting a protective action by blocking exoribonuclease progression. By defining the 3Î end of nad1 exons 2-3 precursor, MTSF2 concomitantly determines the 3Î extremity of the first half of the trans-intron found at the end of the transcript. Therefore, binding of the MTSF2 protein to nad1 exons 2-3 precursor evolved both to stabilize the transcript and to define a 3Î extremity compatible with the trans-splicing reaction needed to reconstitute mature nad1. We thus reveal that the range of transcripts stabilized by association with protective protein on their 3Î end concerns also mitochondrial precursor transcripts.
Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/genetics , Mitochondria/metabolism , NADH Dehydrogenase/genetics , RNA Precursors/metabolism , RNA, Plant/metabolism , RNA-Binding Protein EWS/physiology , RNA/metabolism , Amino Acid Sequence , Arabidopsis/growth & development , Arabidopsis Proteins/metabolism , Arabidopsis Proteins/physiology , Base Sequence , Binding Sites , CRISPR-Cas Systems , Electron Transport Complex I/metabolism , Exons , Introns/genetics , Mitochondria/genetics , Plants, Genetically Modified , Protein Binding , RNA Splicing , RNA Stability , RNA, Mitochondrial , RNA-Binding Protein EWS/genetics , Recombinant Fusion Proteins/metabolism , Sequence Alignment , Sequence Homology, Nucleic AcidABSTRACT
Group II introns are common features of most angiosperm mitochondrial genomes. Intron splicing is thus essential for the expression of mitochondrial genes and is facilitated by numerous nuclear-encoded proteins. However, the molecular mechanism and the protein cofactors involved in this complex process have not been fully elucidated. In this study, we characterized three new pentatricopeptide repeat (PPR) genes, called MISF26, MISF68, and MISF74, of Arabidopsis and showed they all function in group II intron splicing and plant development. The three PPR genes encode P-type PPR proteins that localize in the mitochondrion. Transcript analysis revealed that the splicing of a single intron is altered in misf26 mutants, while several mitochondrial intron splicing defects were detected in misf68 and misf74 mutants. To our knowledge, MISF68 and MISF74 are the first two PPR proteins implicated in the splicing of more than one intron in plant mitochondria, suggesting that they may facilitate splicing differently from other previously identified PPR splicing factors. The splicing defects in the misf mutants induce a significant decrease in complex I assembly and activity, and an overexpression of mRNAs of the alternative respiratory pathway. These results therefore reveal that nuclear encoded proteins MISF26, MISF68, and MISF74 are involved in splicing of a cohort of mitochondrial group II introns and thereby required for complex I biogenesis.
Subject(s)
Arabidopsis/genetics , Introns/genetics , Mitochondria/metabolism , RNA Splicing/genetics , Arabidopsis/metabolismABSTRACT
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.
Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , NADH Dehydrogenase/genetics , RNA Splicing , Arabidopsis/genetics , Arabidopsis/growth & development , Arabidopsis/metabolism , Gene Expression Regulation, Plant , Introns , Mitochondria/genetics , Mitochondria/metabolism , Mutation , NADH Dehydrogenase/metabolism , Plants, Genetically Modified , Polyribosomes/genetics , Polyribosomes/metabolism , Protein Biosynthesis , RNA, Messenger/genetics , RNA, MitochondrialABSTRACT
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.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/genetics , Gene Expression Regulation, Plant , RNA, Messenger/metabolism , Arabidopsis Proteins/genetics , Chromosome Mapping , Evolution, Molecular , Genes, Reporter , Mitochondria/metabolism , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Mutation , Phylogeny , Plant Infertility/genetics , Plants, Genetically Modified , RNA Cleavage , RNA Processing, Post-Transcriptional , RNA, Messenger/genetics , RNA, Plant/genetics , RNA, Plant/metabolism , Recombinant Fusion Proteins , Ribosomal Proteins/geneticsABSTRACT
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.
Subject(s)
Arabidopsis/enzymology , Electron Transport Complex IV/genetics , Gene Expression Regulation, Plant , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Cell Respiration , Electron Transport , Electron Transport Complex I/genetics , Electron Transport Complex I/metabolism , Electron Transport Complex IV/metabolism , Gene Expression Regulation, Enzymologic , Metabolomics , Mitochondria/enzymology , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , NADH Dehydrogenase/genetics , NADH Dehydrogenase/metabolism , Oxidation-Reduction , Oxidoreductases/genetics , Oxidoreductases/metabolism , Oxygen Consumption , Plant Proteins/genetics , Plant Proteins/metabolism , Plant Roots/enzymology , Plant Roots/geneticsABSTRACT
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.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/genetics , Electron Transport Complex I/genetics , Mitochondrial Proteins/metabolism , RNA 3' End Processing , RNA Stability , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , Arabidopsis/growth & development , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Binding Sites , Cell Respiration , Electron Transport Complex I/metabolism , Gene Expression Regulation, Plant , Mitochondria/metabolism , Mitochondrial Proteins/genetics , Mutation , Photosynthesis , RNA Splicing , RNA-Binding Proteins/geneticsABSTRACT
In recent years Arabidopsis thaliana has become a model species for genomic variability and adaptation studies. Although impressive quantities of data have been gathered on the nuclear genomic diversity of this species, little has been published regarding its cytoplasmic diversity. We analyzed the diversity of plastid (pt) and mitochondrial (mt) genomes among 95 accessions, covering most Arabidopsis geographic origins. Four intergenic regions of the pt genome were sequenced, and a total of 68 polymorphisms and 65 pt haplotypes were identified. Several strategies were developed to identify mt polymorphisms among a subset of 14 accessions. Fifteen polymorphisms were further developed as PCR-based markers and used to analyze the whole set of 95 accessions. Using statistical parsimony, we built pt and mt phylogenetic networks of haplotype groups. To root the pt network, the pt intergenic regions of two related Arabidopsis species, Arabidopsis lyrata and Arabidopsis arenosa, were also sequenced. The mt and pt phylogenies are highly congruent and could be combined into a single cytoplasmic phylogeny. To estimate whether co-adaptation between nuclear and cytoplasmic genomes exists in A. thaliana, we tested the germination capacity in challenging conditions of 27 pairs of reciprocal F(2) families. We found that the cytoplasm donor had a significant effect on the germination capacity of some F(2) families.
Subject(s)
Arabidopsis/genetics , Chloroplasts/genetics , DNA, Mitochondrial/genetics , Phylogeny , Adaptation, Physiological/genetics , Arabidopsis/classification , Cell Nucleus/genetics , DNA, Mitochondrial/chemistry , DNA, Mitochondrial/classification , DNA, Plant/chemistry , DNA, Plant/genetics , Genome, Mitochondrial/genetics , Genotype , Geography , Haplotypes , INDEL Mutation , Molecular Sequence Data , Polymorphism, Genetic , Sequence Analysis, DNA , Species SpecificityABSTRACT
Mitochondria are responsible for energy production through aerobic respiration, and represent the powerhouse of eukaryotic cells. Their metabolism and gene expression processes combine bacterial-like features and traits that evolved in eukaryotes. Among mitochondrial gene expression processes, translation remains the most elusive. In plants, while numerous pentatricopeptide repeat (PPR) proteins are involved in all steps of gene expression, their function in mitochondrial translation remains unclear. Here we present the biochemical characterization of Arabidopsis mitochondrial ribosomes and identify their protein subunit composition. Complementary biochemical approaches identified 19 plant-specific mitoribosome proteins, of which ten are PPR proteins. The knockout mutations of ribosomal PPR (rPPR) genes result in distinct macroscopic phenotypes, including lethality and severe growth delay. The molecular analysis of rppr1 mutants using ribosome profiling, as well as the analysis of mitochondrial protein levels, demonstrate rPPR1 to be a generic translation factor that is a novel function for PPR proteins. Finally, single-particle cryo-electron microscopy (cryo-EM) reveals the unique structural architecture of Arabidopsis mitoribosomes, characterized by a very large small ribosomal subunit, larger than the large subunit, bearing an additional RNA domain grafted onto the head. Overall, our results show that Arabidopsis mitoribosomes are substantially divergent from bacterial and other eukaryote mitoribosomes, in terms of both structure and protein content.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/cytology , Mitochondrial Proteins/metabolism , Mitochondrial Ribosomes/chemistry , Arabidopsis/genetics , Arabidopsis/growth & development , Arabidopsis Proteins/genetics , Cryoelectron Microscopy , Gene Knockout Techniques , Mitochondrial Proteins/genetics , Mitochondrial Ribosomes/metabolism , Mitochondrial Ribosomes/ultrastructure , Plant Cells , Proteomics/methods , RNA, Plant , RNA, Ribosomal/chemistryABSTRACT
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.
Subject(s)
Arabidopsis Proteins/metabolism , Mitochondria/metabolism , Mitochondrial Proteins/metabolism , Plant Proteins/metabolism , Polyribosomes/metabolism , Amino Acid Motifs , Amino Acid Substitution , Arabidopsis/cytology , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/genetics , Binding Sites , Escherichia coli/genetics , Homozygote , Mitochondrial Membranes/chemistry , Mitochondrial Proteins/chemistry , Mitochondrial Proteins/genetics , Molecular Weight , Phylogeny , Protein Binding , Repetitive Sequences, Amino Acid/geneticsABSTRACT
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.