Laurence Maréchal-Drouard

Isolation of plant DNA: A fast, inexpensive, and reliable method

We describe here a simple method to isolate DNA of high molecular weight from a wide variety of plant materials, such as trees, herbaceous plants, cell suspension cultures, calli, seeds, dried embryos, ferns and lichens. The crucial step of the extraction is the use of an acidic extraction medium. When necessary, the sample was separated on a fast RPC-5 column providing us with highly purified DNA suitable not only for restriction endonuclease analyses but also for PCR experiments, RLFP analyses, or detection of adducts.

Mitochondrial tRNA import: are there distinct mechanisms?

Sequence information from an increasing number of complete mitochondrial genomes indicates that a large number of evolutionary distinct organisms import nucleus-encoded tRNAs. In the past five years, much research has been initiated on the features of imported tRNAs, the mechanism and the energetics of the process as well as on the components of the import machinery. In summary, these studies show that the import systems of different species exhibit some unique features, suggesting that more than one mechanism might exist to import tRNAs.

Recent advances in tRNA mitochondrial import

In many eukaryotes, tRNA import from the cytosol into mitochondria is essential for mitochondrial biogenesis and, consequently, for cell viability. Recent work has begun to unravel the molecular mechanisms involved in tRNA transport in yeast, trypanosomatids and plants. The mechanisms of tRNA targeting to, and translocation through, the double mitochondrial membrane in addition to how selectivity and regulation of these processes are achieved are the main questions that have been addressed. The characterization of both direct and co-import mechanisms involving distinct protein-import factors is in agreement with a polyphyletic origin of tRNA import. Moreover, our increased understanding of the tRNA-import pathway has been exploited recently to rescue dysfunctions associated with mitochondrial tRNA mutations.

Import of tRNAs and aminoacyl-tRNA synthetases into mitochondria

During evolution, most of the bacterial genes from the ancestral endosymbiotic α-proteobacteria at the origin of mitochondria have been either lost or transferred to the nuclear genome. A crucial evolutionary step was the establishment of macromolecule import systems to allow the come back of proteins and RNAs into the organelle. Paradoxically, the few mitochondria-encoded protein genes remain essential and must be translated by a mitochondrial translation machinery mainly constituted by nucleus-encoded components. Two crucial partners of the mitochondrial translation machinery are the aminoacyl-tRNA synthetases and the tRNAs. All mitochondrial aminoacyl-tRNA synthetases and many tRNAs are imported from the cytosol into the mitochondria in eukaryotic cells. During the last few years, their origin and their import into the organelle have been studied in evolutionary distinct organisms and we review here what is known in this field.

In vivo import of a normal or mutagenized heterologous transfer RNA into the mitochondria of transgenic plants: towards novel ways of influencing mitochondrial gene expression?

Evidence that nuclear‐encoded RNAs are present inside mitochondria has been reported from a wide variety of organisms, and is presumed to be due to import of specific cytosolic RNAs. In plants, the first examples were the mitochondrial leucine transfer RNAs of bean. In all cases, the evidence is circumstantial, based on hybridization of the mitochondrial RNAs to nuclear and not mitochondrial DNA. Here we show that transgenic potato plants carrying a leucine tRNA gene from bean nuclear DNA contain RNA transcribed from the introduced gene both in the cytosol and inside mitochondria, providing proof that the mitochondrial leucine tRNA is derived from a nuclear gene and imported into the mitochondria. The same bean gene carrying a 4 bp insertion in the anticodon loop was also expressed in transgenic potato plants and the transcript found to be present inside mitochondria, suggesting that this natural RNA import system could eventually be used to introduce foreign RNA sequences into mitochondria.

The voltage-dependent anion channel, a major component of the tRNA import machinery in plant mitochondria

In plants, as in most eukaryotic cells, import of nuclear-encoded cytosolic tRNAs is an essential process for mitochondrial biogenesis. Despite its broad occurrence, the mechanisms governing RNA transport into mitochondria are far less understood than protein import. This article demonstrates by Northwestern and gel-shift experiments that the plant mitochondrial voltage-dependent anion channel (VDAC) protein interacts with tRNA in vitro. It shows also that this porin, known to play a key role in metabolite transport, is a major component of the channel involved in the tRNA translocation step through the plant mitochondrial outer membrane, as supported by inhibition of tRNA import into isolated mitochondria by VDAC antibodies and Ruthenium red. However VDAC is not a tRNA receptor on the outer membrane. Rather, two major components from the TOM (translocase of the outer mitochondrial membrane) complex, namely TOM20 and TOM40, are important for tRNA binding at the surface of mitochondria, suggesting that they are also involved in tRNA import. Finally, we show that proteins and tRNAs are translocated into plant mitochondria by different pathways. Together, these findings identify unexpected components of the tRNA import machinery and suggest that the plant tRNA import pathway has evolved by recruiting multifunctional proteins.

A family of RRM-type RNA-binding proteins specific to plant mitochondria

Expression of higher plant mitochondrial (mt) genes is regulated at the transcriptional, posttranscriptional, and translational levels, but the vast majority of the mtDNA and RNA-binding proteins involved remain to be identified. Plant mt single-stranded nucleic acid-binding proteins were purified by affinity chromatography, and corresponding genes have been identified. A majority of these proteins belong to a family of RNA-binding proteins characterized by the presence of an N-terminal RNA-recognition motif (RRM) sequence. They diverge in their C-terminal sequences, suggesting that they can be involved in different plant mt regulation processes. Mitochondrial localization of the proteins was confirmed both in vitro and in vivo and by immunolocalization. Binding experiments showed that several proteins have a preference for poly(U)-rich sequences. This mt protein family contains the ubiquitous RRM motif and has no known mt counterpart in non-plant species. Phylogenetic and functional analysis suggest a common ancestor with RNA-binding glycine-rich proteins (GRP), a family of developmentally regulated proteins of unknown function. As with several plant, cyanobacteria, and animal proteins that have similar structures, the expression of one of the Arabidopsis thaliana mt RNA-binding protein genes is induced by low temperatures.

Striking differences in mitochondrial tRNA import between different plant species

A systematic comparison of the tRNAs imported into the mitochondria of larch, maize and potato reveals considerable differences among the three species. Larch mitochondria import at least eleven different tRNAs (more than half of those tested) corresponding to ten different amino acids. For five of these tRNAs [tRNAPhe(GAA), tRNALys(CUU), tRNAPro(UGG), tRNASer(GCU) and tRNASer(UGA)] this is the first report of import into mitochondria in any plant species. There are also differences in import between relatively closely related plants; wheat mitochondria, unlike maize mitochondria import tRNAHis, and sunflower mitochondria, unlike mitochondria from other angiosperms tested, import tRNASer(GCU) and tRNASer(UGA). These results suggest that the ability to import each tRNA has been acquired independently at different times during the evolution of higher plants, and that there are few apparent restrictions on which tRNAs can or cannot be imported. The implications for the mechanisms of mitochondrial tRNA Import in plants are discussed.

In vitro import of a nuclearly encoded tRNA into mitochondria of Solanum tuberosum.

ABSTRACT Some of the mitochondrial tRNAs of higher plants are nuclearly encoded and imported into mitochondria. The import of tRNAs encoded in the nucleus has been shown to be essential for proper protein translation within mitochondria of a variety of organisms. Here, we report the development of an in vitro assay for import of nuclearly encoded tRNAs into plant mitochondria. This in vitro system utilizes isolated mitochondria from Solanum tuberosum and synthetic tRNAs transcribed from cloned nuclear tRNA genes. Although incubation of radioactively labeled in vitro-transcribed tRNAAla, tRNAPhe, and tRNAMet-e with isolated potato mitochondria resulted in importation, as measured by nuclease protection, the amount of tRNA transcripts protected at saturation was at least five times higher for tRNAAla than for the two other tRNAs. This difference in in vitro saturation levels of import is consistent with the in vivo localization of these tRNAs, since cytosolic tRNAAla is naturally imported into potato mitochondria whereas tRNAPhe and tRNAMet-e are not. Characterization of in vitro tRNA import requirements indicates that mitochondrial tRNA import proceeds in the absence of any added cytosolic protein fraction, involves at least one protein component on the surface of mitochondria, and requires ATP-dependent step(s) and a membrane potential.

Dual-targeted tRNA-dependent amidotransferase ensures both mitochondrial and chloroplastic Gln-tRNAGln synthesis in plants

Aminoacyl-tRNAs are generally formed by direct attachment of an amino acid to tRNAs by aminoacyl-tRNA synthetases, but Gln-tRNA is an exception to this rule. Gln-tRNAGln is formed by this direct pathway in the eukaryotic cytosol and in protists or fungi mitochondria but is formed by an indirect transamidation pathway in most of bacteria, archaea, and chloroplasts. We show here that the formation of Gln-tRNAGln is also achieved by the indirect pathway in plant mitochondria. The mitochondrial-encoded tRNAGln, which is the only tRNAGln present in mitochondria, is first charged with glutamate by a nondiscriminating GluRS, then is converted into Gln-tRNAGln by a tRNA-dependent amidotransferase (AdT). The three subunits GatA, GatB, and GatC are imported into mitochondria and assemble into a functional GatCAB AdT. Moreover, the mitochondrial pathway of Gln-tRNAGln formation is shared with chloroplasts as both the GluRS, and the three AdT subunits are dual-imported into mitochondria and chloroplasts.