The pentatricopeptide repeat protein MTSF2 stabilizes a nad1 precursor transcript and defines the 3΄ end of its 5΄-half intron

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 3΄ extremity with high affinity, suggesting a protective action by blocking exoribonuclease progression. By defining the 3΄ end of nad1 exons 2-3 precursor, MTSF2 concomitantly determines the 3΄ 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 3΄ 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 3΄ end concerns also mitochondrial precursor transcripts

Variability for nitrogen management in genetically-distant maize (Zea mays L.) lines: impact of post-silking nitrogen limiting conditions

The impact of nitrogen (N)-limiting conditions after silking on kernel yield (KY)-related traits and whole plant N management was investigated using fifteen maize lines representative of plant genetic diversity in Europe and America. A large level of genetic variability of these traits was observed in the different lines when post-silking fertilization of N was strongly reduced. Under such N-fertilization conditions, four different groups of lines were identified on the basis of KY and kernel N content. Although the pattern of N management, including N uptake and N use was variable in the four groups of lines, a number of them were able to maintain both a high yield and a high kernel N content by increasing shoot N remobilization. No obvious relationship between the genetic background of the lines and their mode of N management was found. When N was limiting after silking, N remobilization appeared to be a good predictive marker for identifying maize lines that were able to maintain a high yield and a high kernel N content irrespective of their female flowering date. The use of N remobilization as a trait to select maize genotypes adapted to low N input is discussed

The translational landscape of Arabidopsis mitochondria

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

Three new pentatricopeptide repeat proteins facilitate the splicing of mitochondrial transcripts and complex I biogenesis in Arabidopsis

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 mitochondria! group II introns and thereby required for complex I biogenesis

Translational control by PPR proteins in plant mitochondria

International audienceThe control of translation in plant mitochondria is still not understood at the molecular level. A growing body of evidence indicates though that mitochondrial translation differs from its bacterial counterpart in many key aspects. Moreover, recent cryo-EM structural analyses have revealed highly divergent composition and structures of mitoribosomes in eukaryotes, suggesting specific adaptations of the translational apparatus between fila that may notably impact translation initiation. Our recent analysis of plant translatomes by ribosome profiling analysis revealed highly variable ribosome loads per mitochondrial mRNA, suggesting tight control of translation initiation or elongation in plant mitochondria (Planchard et al, 2018). Furthermore, downstream orfs of di-citronic transcripts have higher ribosomal loads than their upstream cognate orfs, clearly indicating an ability of plant ribosomes to initiate translation internally in mRNAs. To better understand the basis of this control, we have identified and characterized mitochondria-targeted pentatricopeptide repeat (PPR) proteins specifically involved in translation. I will present our most recent advances on the characterization of these PPR proteins.Planchard, N., Bertin, P., Quadrado, M., Dargel-Graffin, C., Hatin, I., Namy, O. and Mireau, H. (2018) The translational landscape of Arabidopsis mitochondria. Nucleic Acids Res., 283, 1476

The translational landscape of Arabidopsis mitochondria

International audienceMessenger RNA translation is a complex process that is still poorly understood in eukaryotic or-ganelles 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 mitochon-drial 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 ribo-some 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 ribo-somal protein encoding transcripts and a reduction in translation of a few complex V mRNAs. Taken together , our observations reveal that plant mitochon-drial translation is a dynamic process and that trans-lational 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

The translational landscape of plant mitochondria and control by PPR proteins.

International audienceTranslation in plant mitochondria is a complex process that is still poorly understood at the molecular level. Growing evidence indicates though that mitochondrial translation differs from its bacterial counterpart in many key aspects. To better understand how mitochondrial translation is orchestrated and regulated in plants, we have used the ribosome profiling technology to generate genome-wide views of the mitochondrial translatome in different plant genotypes. This approach led us to reveal that most plant mitochondrial ribosome footprints measure 27 and 28 bases. Quantification of ribosome footprints along transcripts revealed that mRNAs have highly divergent ribosome densities, suggesting a tight control of translation initiation or elongation in plant mitochondria. To better understand the basis of this control, we identified and have been characterizing mitochondria-targeted pentatricopeptide repeat (PPR) proteins specifically involved in translation. I will present our most recent advances on the characterization of these PPR proteins

An integrated “omics” approach to the characterization of maize (Zea mays L.) mutants deficient in the expression of two genes encoding cytosolic glutamine synthetase

International audienceBackgroundTo identify the key elements controlling grain production in maize, it is essential to have an integrated view of the responses to alterations in the main steps of nitrogen assimilation by modification of gene expression. Two maize mutant lines (gln1.3 and gln1.4), deficient in two genes encoding cytosolic glutamine synthetase, a key enzyme involved in nitrogen assimilation, were previously characterized by a reduction of kernel size in the gln1.4 mutant and by a reduction of kernel number in the gln1.3 mutant. In this work, the differences in leaf gene transcripts, proteins and metabolite accumulation in gln1.3 and gln1.4 mutants were studied at two key stages of plant development, in order to identify putative candidate genes, proteins and metabolic pathways contributing on one hand to the control of plant development and on the other to grain production.ResultsThe most interesting finding in this study is that a number of key plant processes were altered in the gln1.3 and gln1.4 mutants, including a number of major biological processes such as carbon metabolism and transport, cell wall metabolism, and several metabolic pathways and stress responsive and regulatory elements. We also found that the two mutants share common or specific characteristics across at least two or even three of the “omics” considered at the vegetative stage of plant development, or during the grain filling period.ConclusionsThis is the first comprehensive molecular and physiological characterization of two cytosolic glutamine synthetase maize mutants using a combined transcriptomic, proteomic and metabolomic approach. We find that the integration of the three “omics” procedures is not straight forward, since developmental and mutant-specific levels of regulation seem to occur from gene expression to metabolite accumulation. However, their potential use is discussed with a view to improving our understanding of nitrogen assimilation and partitioning and its impact on grain production

A case of gene fragmentation in plant mitochondria fixed by the selection of a compensatory restorer of fertility-like PPR gene

International audienceAbstract The high mutational load of mitochondrial genomes combined with their uniparental inheritance and high polyploidy favor 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 co-adaptation 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 (PPR) 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