Search

Expression of Arabidopsis thaliana glutamate dehydrogenase genes is very low in the light and high in the dark. The molecular signals and mechanisms that provide the light-dependent glutamate dehydrogenase genes regulation remain unknown. The aim of this work was to study a role of redox signals which occur during light shifts in the regulation of the glutamate dehydrogenase genes expression. Using photosynthetic electron transport inhibitors 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl benzoquinone (DBMIB) we demonstrate that transcript levels of the GDH1 and GDH2 genes in Arabidopsis leaves change in accordance with a redox state of plastoquinone pool: they are low when it is highly reduced and high when it is oxidized. Hydrogen peroxide or high light treatment did not result in decreasing of GDH1 or GDH2 expression, so reactive oxygen species cannot be the signals that reduce expression of these genes during dark-to-light shifts. There was no significant difference between the glucose content in the leaves of plants treated with DCMU and the plants treated with DBMIB, so glucose is not the only or the main factor that regulates expression of the studied genes. We presume that expression of Arabidopsis GDH1 and GDH2 genes depends on the plastoquinone pool redox state. Expression of Arabidopsis glutamate dehydrogenase genes during light-to-dark and dark-to-light shifts depends on chloroplast electron transport chain redox state rather than reactive oxygen species or glucose content.

It is known that mitochondrial population of living organisms is heterogeneous. Using the density gradient of Percoll or sucrose, we obtained and characterized two distinct mitochondrial subfractions (named “light” and “heavy” according to their sedimentation) from three plant species (Brassica rapa, Zea mays, and Arabidopsis thaliana). Electron microscopy showed that mitochondria of a light subfraction have a poorly developed cristae structure. The respiratory control of these mitochondria was reduced; however the degree of their outer membrane integrity remained high. These data suggest that mitochondrial subfractions purified in the density gradient differ in their maturity. We carried out DNA import assays to study the ability of these two subfractions for DNA transport. We have shown that the mitochondria of light subfraction in all three plant species uptakes DNA more efficiently than the mitochondria of heavy subfraction. In addition, import of DNA with different sizes into the light and heavy mitochondrial subfractions has different sensitivity to specific inhibitors of the voltage-dependent anion channel and adenine nucleotide translocase, proteins playing a central role in DNA transport across the mitochondrial membrane. The obtained results suggest that DNA import depends on organization of the transport system in the double mitochondrial membrane influenced by the maturity of the mitochondria.

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.

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.

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.

Mitochondria are competent for DNA uptake in vitro, a mechanism which may support delivery of therapeutic DNA to complement organelle DNA mutations. We document here key aspects of the DNA import process, so as to further lay the ground for mitochondrial transfection in intact cells.We developed DNA import assays with isolated mitochondria from different organisms, using DNA substrates of various sequences and sizes. Further import experiments investigated the possible role of ATP and protein phosphorylation in the uptake process. The fate of adenine nucleotides and the formation of phosphorylated proteins were analyzed.We demonstrate that the efficiency of mitochondrial uptake depends on the sequence of the DNA to be translocated. The process becomes sequence-selective for large DNA substrates. Assays run with a natural mitochondrial plasmid identified sequence elements which promote organellar uptake. ATP enhances DNA import and allows tight integration of the exogenous DNA into mitochondrial nucleoids. ATP hydrolysis has to occur during the DNA uptake process and might trigger phosphorylation of co-factors.Our data contribute critical information to optimize DNA delivery into mitochondria and open the prospect of targeting whole mitochondrial genomes or complex constructs into mammalian organelles in vitro and in vivo.

Mitochondrial gene products are essential for the viability of eukaryote obligate aerobes. Consequently, mutations of the mitochondrial genome cause severe diseases in man and generate traits widely used in plant breeding. Pathogenic mutations can often be identified but direct genetic rescue remains impossible because mitochondrial transformation is still to be achieved in higher eukaryotes. Along this line, it has been shown that isolated plant and mammalian mitochondria are naturally competent for importing linear DNA. However, it has proven difficult to understand how such large polyanions cross the mitochondrial membranes. The genetic tractability of Saccharomyces cerevisae could be a powerful tool to unravel this molecular mechanism. Here we show that isolated S. cerevisiae mitochondria can import linear DNA in a process sharing similar characteristics to plant and mammalian mitochondria. Based on biochemical data, translocation through the outer membrane is believed to be mediated by voltage-dependent anion channel (VDAC) isoforms in higher eukaryotes. Both confirming this hypothesis and validating the yeast model, we illustrate that mitochondria from S. cerevisiae strains deleted for the VDAC-1 or VDAC-2 gene are severely compromised in DNA import. The prospect is now open to screen further mutant yeast strains to identify the elusive inner membrane DNA transporter.

Plant mitochondria are remarkable with respect to their content in foreign, alien and plasmid-like DNA, raising the question of the transfer of this information into the organelles. We demonstrate the existence of an active, transmembrane potential-dependent mechanism of DNA uptake into plant mitochondria. The process is restricted to double-strand DNA, but has no obvious sequence specificity. It is most efficient with linear fragments up to a few kilobase pairs. When containing appropriate information, imported sequences are transcribed within the organelles. The uptake likely involves the voltage-dependent anion channel and the adenine nucleotide translocator, i.e. the core components of the mitochondrial permeability transition pore complex in animal cells, but it does not rely on known mitochondrial membrane permeabilization processes. We conclude that DNA import into plant mitochondria might represent a physiological phenomenon with some functional relevance.

Aim. To understand the mechanism(s) underlying mitochondrial competence for DNA uptake and to exploit these pathways for the development of in vivo models of gene therapy. Methods. DNA uptake into isolated mitochondria from plant or from mutant Saccharomyces cerevisiae defective for mitochondrial proteins and carriers, biochemical approaches and transfection of mammalian cells with DNA bound to mitochondriotropic liposomes. Results. Special focus on the inner membrane showed the involvement of isoforms of the adenine nucleotide translocator and the contribution of proteins controlling mitochondrial morphology in DNA uptake into yeast organelles. Transfection assays led to significant incorporation of a mitochondrial construct into mammalian cells and expression of a marker gene. Conclusions. The data imply that there are multiple mitochondrial DNA import pathways. On the other hand, preliminary results suggest that mitochondriotropic liposomes can deliver DNA to mitochondria in live mammalian cells. Мета. Визначити механізми поглинання ДНК мітохондріями і використати їх для удосконалення існуючих моделей генної терапії in vivo. Методи. Поглинання ДНК ізольованими мітохондріями рослин або мітохондріями мутантних ліній Saccharomyces cerevisiae, дефектних за мітохондріальними білками та переносниками, біохімічні підходи і трансфекція в клітини ссавців ДНК, зв’язаної з мітохондріотропними ліпосомами. Результати. Основним підсумком вивчення внутрішньої мембрани виявилося встановлення того факта, що до процесу перенесення ДНК дріжджовими органелами залучені кілька ізоформ аденіннуклеотидтранслокази, а також білки, які контролюють мітохондріальну морфологію. В експериментах з трансфекції ДНК у клітини ссавців виявлено вбудовування в них мітохондріальної конструкції і експресію маркерного гена. Висновки. Отримані дані дозволяють припустити існування декількох механизмів імпорту ДНК у мітохондрії. Проте є попередні результати, які показують, що мітохондріотропні ліпосоми можуть бути використані для доставки ДНК у мітохондрії клітин ссавців in vivo. Цель. Выяснить механизмы поглощения ДНК митохондриями и использовать их для усовершенствования существующих моделей генной терапии in vivo. Методы. Поглощение ДНК изолированными митохондриями растений или митохондриями мутантных линий Saccharomyces cerevisiae, дефектных по митохондриальным белкам и переносчикам, биохимические подходы и трансфекция в клетки млекопитающих ДНК, связанной с митохондриотропными липосомами. Результаты. Основным итогом изучения внутренней мембраны оказалось установление того факта, что в процесс переноса ДНК дрожжевыми органеллами вовлечены несколько изоформ адениннуклеотидтранслоказы, а также белки, контролирующие митохондриальную морфологию. В экспериментах по трансфекции ДНК в клетки млекопитающих выявлены встраивание в них митохондриальной конструкции и экспрессию маркерного гена. Выводы. Полученные данные позволяют предположить существование нескольких механизмов импорта ДНК в митохондрии. Однако есть предварительные результаты, показывающие, что митохондриотропные липосомы могут быть использованы для доставки ДНК в митохондрии клеток млекопитающих in vivo.