Mitochondrial DNA Isolation from Plants.

For most eukaryotes, sequencing and assembly of the mitochondrial DNA (mtDNA) is possible by starting the analysis from total cellular DNA, but the exploration of the mtDNA of plants is more challenging because of the low copy number, limited sequence conservation, and complex structure of the mtDNA. The very large size of the nuclear genome of many plant species and the very high ploidy of the plastidial genome further complicate the analysis, sequencing, and assembly of plant mitochondrial genomes. An enrichment of mtDNA is therefore necessary.

RADA-dependent branch migration has a predominant role in plant mitochondria and its defect leads to mtDNA instability and cell cycle arrest.

Mitochondria of flowering plants have large genomes whose structure and segregation are modulated by recombination activities. The post-synaptic late steps of mitochondrial DNA (mtDNA) recombination are still poorly characterized. Here we show that RADA, a plant ortholog of bacterial RadA/Sms, is an organellar protein that drives the major branch-migration pathway of plant mitochondria.

Plant mitochondria import DNA via alternative membrane complexes involving various VDAC isoforms.

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.

Mitochondrial Transcriptome Control and Intercompartment Cross-Talk During Plant Development.

We address here organellar genetic regulation and intercompartment genome coordination. We developed earlier a strategy relying on a tRNA-like shuttle to mediate import of nuclear transgene-encoded custom RNAs into mitochondria in plants. In the present work, we used this strategy to drive -cleaving hammerhead ribozymes into the organelles, to knock down specific mitochondrial RNAs and analyze the regulatory impact.

Nucleic acid import into mitochondria: New insights into the translocation 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.

The plant mitochondrial genome: dynamics and maintenance.

Plant mitochondria have a complex and peculiar genetic system. They have the largest genomes, as compared to organelles from other eukaryotic organisms. These can expand tremendously in some species, reaching the megabase range.

Targeting nucleic acids into mitochondria: progress and prospects.

Given the essential functions of these organelles in cell homeostasis, their involvement in incurable diseases and their potential in biotechnological applications, genetic transformation of mitochondria has been a long pursued goal that has only been reached in a couple of unicellular organisms. The challenge led scientists to explore a wealth of different strategies for mitochondrial delivery of DNA or RNA in living cells. These are the subject of the present review.

DNA delivery to mitochondria: sequence specificity and energy enhancement.

Purpose: 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.

Methods: We developed DNA import assays with isolated mitochondria from different organisms, using DNA substrates of various sequences and sizes.

Mitochondrial transfection for studying organellar DNA repair, genome maintenance and aging.

Maintenance of the mitochondrial genome is a major challenge for cells, particularly as they begin to age. Although it is established that organelles possess regular DNA repair pathways, many aspects of these complex processes and of their regulation remain to be investigated. Mitochondrial transfection of isolated organelles and in whole cells with customized DNA synthesized to contain defined lesions has wide prospects for deciphering repair mechanisms in a physiological context.

DNA repair in organelles: Pathways, organization, regulation, relevance in disease and aging.

Both endogenous processes and exogenous physical and chemical sources generate deoxyribonucleic acid (DNA) damage in the nucleus and organelles of living cells. To prevent deleterious effects, damage is balanced by repair pathways. DNA repair was first documented for the nuclear compartment but evidence was subsequently extended to the organelles.

Developing a genetic approach to investigate the mechanism of mitochondrial competence for DNA import.

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.

Mitochondrial targeting and membrane anchoring of a viral replicase in plant and yeast cells.

Replication of the Carnation Italian ringspot virus genomic RNA in plant cells occurs in multivesicular bodies which develop from the mitochondrial outer membrane during infection. ORF1 in the viral genome encodes a 36-kDa protein, while ORF2 codes for the 95-kDa replicase by readthrough of the ORF1 stop codon. We have shown previously that the N-terminal part of ORF1 contains the information leading to vesiculation of mitochondria and that the 36-kDa protein localizes to mitochondria.

Biochemical and molecular studies on declining and decline-resistant spruce in the north-east of France.

In declining forests of the Vosges mountains (northeast of France), we previously observed that the yellowing of spruce (Picea abies L. cv. Karsten) needles was associated with impairment of the free radical scavenging capacity of the cells and coincided with chronic exposure to ozone.