RESUMO
The transcription of Arabidopsis organellar genes is performed by three nuclear-encoded RNA polymerases: RPOTm, RPOTmp, and RPOTp. The RPOTmp protein possesses ambiguous transit peptides, allowing participation in gene expression control in both mitochondria and chloroplasts, although its function in plastids is still under discussion. Here, we show that the overexpression of RPOTmp in Arabidopsis, targeted either to mitochondria or chloroplasts, disturbs the dormant seed state, and it causes the following effects: earlier germination, decreased ABA sensitivity, faster seedling growth, and earlier flowering. The germination of RPOTmp overexpressors is less sensitive to NaCl, while rpotmp knockout is highly vulnerable to salt stress. We found that mitochondrial dysfunction in the rpotmp mutant induces an unknown retrograde response pathway that bypasses AOX and ANAC017. Here, we show that RPOTmp transcribes the accD, clpP, and rpoB genes in plastids and up to 22 genes in mitochondria.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Cloroplastos , Regulação da Expressão Gênica de Plantas , Germinação , Mitocôndrias , Transcriptoma , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Mitocôndrias/metabolismo , Mitocôndrias/genética , Cloroplastos/metabolismo , Cloroplastos/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Germinação/genética , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Plântula/genética , Plântula/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/genéticaRESUMO
Short interrupted repeat cassette (SIRC)-a novel DNA element found throughout the A. thaliana nuclear genome. SIRCs are represented by short direct repeats interrupted by diverse DNA sequences. The maxima of SIRC's distribution are located within pericentromeric regions. We suggest that originally SIRC was a special case of the complex internal structure of the miniature inverted repeat transposable element (MITE), and further MITE amplification, transposition, and loss of terminal inverted repeats gave rise to SIRC as an independent DNA element. SIRC sites were significantly enriched with several histone modifications associated with constitutive heterochromatin and mobile genetic elements. The majority of DNA-binding proteins, strongly associated with SIRC, are related to histone modifications for transcription repression. A part of SIRC was found to overlap highly inducible protein-coding genes, suggesting a possible regulatory role for these elements, yet their definitive functions need further investigation.
Assuntos
Arabidopsis , Arabidopsis/genética , Sequências Repetitivas de Ácido Nucleico/genética , Sequência de Bases , Elementos de DNA Transponíveis/genética , Sequências Repetidas TerminaisRESUMO
Recombinant proteins are the most important product of current industrial biotechnology. They are indispensable in medicine (for diagnostics and treatment), food and chemical industries, and research. Plant cells combine advantages of the eukaryotic protein production system with simplicity and efficacy of the bacterial one. The use of plants for the production of recombinant proteins is an economically important and promising area that has emerged as an alternative to traditional approaches. This review discusses advantages of plant systems for the expression of recombinant proteins using nuclear, plastid, and mitochondrial genomes. Possibilities, problems, and prospects of modifications of the three parts of the genome in light of obtaining producer plants are examined. Examples of successful use of the nuclear expression platform for production of various biopharmaceuticals, veterinary drugs, and technologically important proteins are described, as are examples of a high yield of recombinant proteins upon modification of the chloroplast genome. Potential utility of plant mitochondria as an expression system for the production of recombinant proteins and its advantages over the nucleus and chloroplasts are substantiated. Although these opportunities have not yet been exploited, potential utility of plant mitochondria as an expression system for the production of recombinant proteins and its advantages over the nucleus and chloroplasts are substantiated.
RESUMO
Mitochondria possess transport mechanisms for import of RNA and DNA. Based on import into isolated Solanum tuberosum mitochondria in the presence of competitors, inhibitors or effectors, we show that DNA fragments of different size classes are taken up into plant organelles through distinct channels. Alternative channels can also be activated according to the amount of DNA substrate of a given size class. Analyses of Arabidopsis thaliana knockout lines pointed out a differential involvement of individual voltage-dependent anion channel (VDAC) isoforms in the formation of alternative channels. We propose several outer and inner membrane proteins as VDAC partners in these pathways.
Assuntos
Arabidopsis/genética , DNA Mitocondrial/genética , DNA de Plantas/genética , Mitocôndrias/genética , Membranas Mitocondriais/fisiologia , Solanum tuberosum/genética , Arabidopsis/metabolismo , Transporte Biológico/genética , Deleção de Genes , Solanum tuberosum/metabolismoRESUMO
BACKGROUND: Plant mitochondrial genomes (mitogenomes) can be structurally complex while their size can vary from ~ 222 Kbp in Brassica napus to 11.3 Mbp in Silene conica. To date, in comparison with the number of plant species, only a few plant mitogenomes have been sequenced and released, particularly for conifers (the Pinaceae family). Conifers cover an ancient group of land plants that includes about 600 species, and which are of great ecological and economical value. Among them, Siberian larch (Larix sibirica Ledeb.) represents one of the keystone species in Siberian boreal forests. Yet, despite its importance for evolutionary and population studies, the mitogenome of Siberian larch has not yet been assembled and studied. RESULTS: Two sources of DNA sequences were used to search for mitochondrial DNA (mtDNA) sequences: mtDNA enriched samples and nucleotide reads generated in the de novo whole genome sequencing project, respectively. The assembly of the Siberian larch mitogenome contained nine contigs, with the shortest and the largest contigs being 24,767 bp and 4,008,762 bp, respectively. The total size of the genome was estimated at 11.7 Mbp. In total, 40 protein-coding, 34 tRNA, and 3 rRNA genes and numerous repetitive elements (REs) were annotated in this mitogenome. In total, 864 C-to-U RNA editing sites were found for 38 out of 40 protein-coding genes. The immense size of this genome, currently the largest reported, can be partly explained by variable numbers of mobile genetic elements, and introns, but unlikely by plasmid-related sequences. We found few plasmid-like insertions representing only 0.11% of the entire Siberian larch mitogenome. CONCLUSIONS: Our study showed that the size of the Siberian larch mitogenome is much larger than in other so far studied Gymnosperms, and in the same range as for the annual flowering plant Silene conica (11.3 Mbp). Similar to other species, the Siberian larch mitogenome contains relatively few genes, and despite its huge size, the repeated and low complexity regions cover only 14.46% of the mitogenome sequence.
Assuntos
Tamanho do Genoma , Genoma Mitocondrial , Larix/genética , Mapeamento de Sequências Contíguas , Anotação de Sequência Molecular , Proteínas de Plantas/genética , RNA Ribossômico/genética , RNA de Transferência/genética , Sequências Repetitivas de Ácido NucleicoRESUMO
BACKGROUND: The efficacy of treating acute myocardial ischemic damages depends, to a large extent, on the development of technologies for predicting their course and outcome. The aim of this paper was to explore whether it would be possible to consider the content of free circulating mitochondrial DNA as a danger-associated molecular pattern for assessing the probability of death from myocardial infarction. METHODS: We have analyzed the clinical outcomes based on discharge summaries and autopsy reports obtained in the course of the PROTOCOL observational trial. This study was approved by the Irkutsk Scientific Center of Surgery and Traumatology ethics committee (protocol No. 3, 10.08.2015). To examine whether the assessment of the level of free circulating mtDNA in acute coronary syndrome can help predicting clinical outcomes, all patients were divided into two groups: group 1, involving those who survived during 30 days after hospitalization, and group 2, involving those who died during this time. A quantitative analysis of the free circulating mtDNA was conducted using the PCR method in situ. RESULTS: The analysis showed that in patients who survived the level of freely circulating mtDNA (36.0 copies/ml) was 164 times lower than in those who died (5900 copies/ml, p = 0.049). It should be mentioned that according to the logistic regression analysis, the probability of death of patients with the increased level of blood plasma mtDNA (more than 4000 copies/ml) is 50%. CONCLUSIONS: Thus, the PROTOCOL observational trial proved that the increase in the content of free circulating mtDNA in blood is a predictor of lethal outcome in patients with acute coronary syndrome. Trial registration The observational studies (those in which the assignment of the medical intervention is not at the discretion of the investigator) do not require registration.
Assuntos
Síndrome Coronariana Aguda/sangue , DNA Mitocondrial/sangue , Síndrome Coronariana Aguda/mortalidade , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Feminino , Humanos , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Estudos ProspectivosRESUMO
In a number of dicotyledonous plants, including Arabidopsis, the transcription of organellar genes is performed by three nuclear-encoded RNA polymerases, RPOTm, RPOTmp, and RPOTp. RPOTmp is a protein with a dual targeting, which is presumably involved in the control of gene expression in both mitochondria and chloroplasts. A previous study of the Arabidopsis insertion rpotmp mutant showed that it has retarded growth and development, altered leaf morphology, changed expression of mitochondrial and probably some chloroplast genes, and decreased activities of the mitochondrial respiratory complexes. To date, there is no clear evidence as to which of these disorders are associated with a lack of RPOTmp in each of the two organelles. The aim of this study was to elucidate the role that this RNA polymerase specifically plays in mitochondria and chloroplasts. Two sets of Arabidopsis transgenic lines with complementation of RPOTmp function in either mitochondria or chloroplasts were obtained. It was found that the recovery of RPOTmp RNA polymerase activity in chloroplasts, although restoring the transcription from the RPOTmp-specific PC promoter, did not lead to compensation of the mutant growth defects. In contrast, the rpotmp plants expressing RPOTmp with mitochondrial targeting restored the level of mitochondrial transcripts and exhibit a phenotype resembling that of the wild-type plants. We conclude that despite its localization in two cell compartments, Arabidopsis RPOTmp plays an important role in mitochondria, but not in chloroplasts.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/enzimologia , Cloroplastos/enzimologia , RNA Polimerases Dirigidas por DNA/fisiologia , Mitocôndrias/enzimologia , Arabidopsis/metabolismo , Arabidopsis/fisiologia , Cloroplastos/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Mitocôndrias/metabolismo , Plantas Geneticamente ModificadasRESUMO
Mitochondria have retained indispensable but limited genetic information and they import both proteins and nucleic acids from the cytosol. RNA import is essential for gene expression and regulation, whereas competence for DNA uptake is likely to contribute to organellar genome dynamics and evolution. Contrary to protein import mechanisms, the way nucleic acids cross the mitochondrial membranes remains poorly understood. Using proteomic, genetic and biochemical approaches with both plant and yeast organelles, we develop here a model for DNA uptake into mitochondria. The first step includes the voltage-dependent anion channel and an outer membrane-located precursor fraction of a protein normally located in the inner membrane. To proceed, the DNA is then potentially recruited in the intermembrane space by an accessible subunit of one of the respiratory chain complexes. Final translocation through the inner membrane remains the most versatile but points to the components considered to make the mitochondrial permeability transition pore. Depending on the size, DNA and RNA cooperate or compete for mitochondrial uptake, which shows that they share import mechanisms. On the other hand, our results imply the existence of more than one route for nucleic acid translocation into mitochondria.
Assuntos
Mitocôndrias/metabolismo , Ácidos Nucleicos/metabolismo , Arabidopsis/metabolismo , Transporte Biológico , Saccharomyces cerevisiae/metabolismoRESUMO
Mitochondrial transcription rate and RNA steady-state levels were examined in shoots of Arabidopsis seedlings. The shoots were treated with inhibitors of complex III and IV of the cytochrome pathway (CP) and with an inhibitor of the alternative oxidase (AOX) of the mitochondrial electron transport chain. The inhibition of AOX and CP complexes III and IV affected transcription and transcript levels in different ways. CP and AOX inhibitors had opposite effects. Our data support the idea that the redox state of the electron transport chain is involved in the regulation of mitochondrial gene expression at transcriptional and post-transcriptional levels.