DNA Repair and the Stability of the Plant Mitochondrial Genome.

The mitochondrion stands at the center of cell energy metabolism. It contains its own genome, the mtDNA, that is a relic of its prokaryotic symbiotic ancestor. In plants, the mitochondrial genetic information influences important agronomic traits including fertility, plant vigor, chloroplast function, and cross-compatibility. Plant mtDNA has remarkable characteristics: It is much larger than the mtDNA of other eukaryotes and evolves very rapidly in structure. This is because of recombination activities that generate alternative mtDNA configurations, an important reservoir of genetic diversity that promotes rapid mtDNA evolution. On the other hand, the high incidence of ectopic recombination leads to mtDNA instability and the expression of gene chimeras, with potential deleterious effects. In contrast to the structural plasticity of the genome, in most plant species the mtDNA coding sequences evolve very slowly, even if the organization of the genome is highly variable. Repair mechanisms are probably responsible for such low mutation rates, in particular repair by homologous recombination. Herein we review some of the characteristics of plant organellar genomes and of the repair pathways found in plant mitochondria. We further discuss how homologous recombination is involved in the evolution of the plant mtDNA.

Complete Sequence, Multichromosomal Architecture and Transcriptome Analysis of the Solanum tuberosum Mitochondrial Genome.

Mitochondrial genomes (mitogenomes) in higher plants can induce cytoplasmic male sterility and be somehow involved in nuclear-cytoplasmic interactions affecting plant growth and agronomic performance. They are larger and more complex than in other eukaryotes, due to their recombinogenic nature. For most plants, the mitochondrial DNA (mtDNA) can be represented as a single circular chromosome, the so-called master molecule, which includes repeated sequences that recombine frequently, generating sub-genomic molecules in various proportions. Based on the relevance of the potato crop worldwide, herewith we report the complete mtDNA sequence of two S. tuberosum cultivars, namely Cicero and Désirée, and a comprehensive study of its expression, based on high-coverage RNA sequencing data. We found that the potato mitogenome has a multi-partite architecture, divided in at least three independent molecules that according to our data should behave as autonomous chromosomes. Inter-cultivar variability was null, while comparative analyses with other species of the Solanaceae family allowed the investigation of the evolutionary history of their mitogenomes. The RNA-seq data revealed peculiarities in transcriptional and post-transcriptional processing of mRNAs. These included co-transcription of genes with open reading frames that are probably expressed, methylation of an rRNA at a position that should impact translation efficiency and extensive RNA editing, with a high proportion of partial editing implying frequent mis-targeting by the editing machinery.

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 trans-cleaving hammerhead ribozymes into the organelles, to knock down specific mitochondrial RNAs and analyze the regulatory impact. In a similar approach, the tRNA mimic was used to import into mitochondria in Arabidopsis thaliana the orf77, an RNA associated with cytoplasmic male sterility in maize and possessing sequence identities with the atp9 mitochondrial RNA. In both cases, inducible expression of the transgenes allowed to characterise early regulation and signaling responses triggered by these respective manipulations of the organellar transcriptome. The results imply that the mitochondrial transcriptome is tightly controlled by a "buffering" mechanism at the early and intermediate stages of plant development, a control that is released at later stages. On the other hand, high throughput analyses showed that knocking down a specific mitochondrial mRNA triggered a retrograde signaling and an anterograde nuclear transcriptome response involving a series of transcription factor genes and small RNAs. Our results strongly support transcriptome coordination mechanisms within the organelles and between the organelles and the nucleus.

Plastidic P2 glucose-6P dehydrogenase from poplar is modulated by thioredoxin m-type: Distinct roles of cysteine residues in redox regulation and NADPH inhibition.

A cDNA coding for a plastidic P2-type G6PDH isoform from poplar (Populus tremula x tremuloides) has been used to express and purify to homogeneity the mature recombinant protein with a N-terminus His-tag. The study of the kinetic properties of the recombinant enzyme showed an in vitro redox sensing modulation exerted by reduced DTT. The interaction with thioredoxins (TRXs) was then investigated. Five cysteine to serine variants (C145S - C175S - C183S - C195S - C242S) and a variant with a double substitution for Cys175 and Cys183 (C175S/C183S) have been generated, purified and biochemically characterized in order to investigate the specific role(s) of cysteines in terms of redox regulation and NADPH-dependent inhibition. Three cysteine residues (C145, C194, C242) are suggested to have a role in controlling the NADP+ access to the active site, and in stabilizing the NADPH regulatory binding site. Our results also indicate that the regulatory disulfide involves residues Cys175 and Cys183 in a position similar to those of chloroplastic P1-G6PDHs, but the modulation is exerted primarily by TRX m-type, in contrast to P1-G6PDH, which is regulated by TRX f. This unexpected specificity indicates differences in the mechanism of regulation, and redox sensing of plastidic P2-G6PDH compared to chloroplastic P1-G6PDH in higher plants.

Atypical thioredoxins in poplar: the glutathione-dependent thioredoxin-like 2.1 supports the activity of target enzymes possessing a single redox active cysteine.

Plant thioredoxins (Trxs) constitute a complex family of thiol oxidoreductases generally sharing a WCGPC active site sequence. Some recently identified plant Trxs (Clot, Trx-like1 and -2, Trx-lilium1, -2, and -3) display atypical active site sequences with altered residues between the two conserved cysteines. The transcript expression patterns, subcellular localizations, and biochemical properties of some representative poplar (Populus spp.) isoforms were investigated. Measurements of transcript levels for the 10 members in poplar organs indicate that most genes are constitutively expressed. Using transient expression of green fluorescent protein fusions, Clot and Trx-like1 were found to be mainly cytosolic, whereas Trx-like2.1 was located in plastids. All soluble recombinant proteins, except Clot, exhibited insulin reductase activity, although with variable efficiencies. Whereas Trx-like2.1 and Trx-lilium2.2 were efficiently regenerated both by NADPH-Trx reductase and glutathione, none of the proteins were reduced by the ferredoxin-Trx reductase. Only Trx-like2.1 supports the activity of plastidial thiol peroxidases and methionine sulfoxide reductases employing a single cysteine residue for catalysis and using a glutathione recycling system. The second active site cysteine of Trx-like2.1 is dispensable for this reaction, indicating that the protein possesses a glutaredoxin-like activity. Interestingly, the Trx-like2.1 active site replacement, from WCRKC to WCGPC, suppresses its capacity to use glutathione as a reductant but is sufficient to allow the regeneration of target proteins employing two cysteines for catalysis, indicating that the nature of the residues composing the active site sequence is crucial for substrate selectivity/recognition. This study provides another example of the cross talk existing between the glutathione/glutaredoxin and Trx-dependent pathways.

Structure-function relationship of the chloroplastic glutaredoxin S12 with an atypical WCSYS active site.

Glutaredoxins (Grxs) are efficient catalysts for the reduction of mixed disulfides in glutathionylated proteins, using glutathione or thioredoxin reductases for their regeneration. Using GFP fusion, we have shown that poplar GrxS12, which possesses a monothiol (28)WCSYS(32) active site, is localized in chloroplasts. In the presence of reduced glutathione, the recombinant protein is able to reduce in vitro substrates, such as hydroxyethyldisulfide and dehydroascorbate, and to regenerate the glutathionylated glyceraldehyde-3-phosphate dehydrogenase. Although the protein possesses two conserved cysteines, it is functioning through a monothiol mechanism, the conserved C terminus cysteine (Cys(87)) being dispensable, since the C87S variant is fully active in all activity assays. Biochemical and crystallographic studies revealed that Cys(87) exhibits a certain reactivity, since its pK(a) is around 5.6. Coupled with thiol titration, fluorescence, and mass spectrometry analyses, the resolution of poplar GrxS12 x-ray crystal structure shows that the only oxidation state is a glutathionylated derivative of the active site cysteine (Cys(29)) and that the enzyme does not form inter- or intramolecular disulfides. Contrary to some plant Grxs, GrxS12 does not incorporate an iron-sulfur cluster in its wild-type form, but when the active site is mutated into YCSYS, it binds a [2Fe-2S] cluster, indicating that the single Trp residue prevents this incorporation.

Chloroplast monothiol glutaredoxins as scaffold proteins for the assembly and delivery of [2Fe-2S] clusters.

Glutaredoxins (Grxs) are small oxidoreductases that reduce disulphide bonds or protein-glutathione mixed disulphides. More than 30 distinct grx genes are expressed in higher plants, but little is currently known concerning their functional diversity. This study presents biochemical and spectroscopic evidence for incorporation of a [2Fe-2S] cluster in two heterologously expressed chloroplastic Grxs, GrxS14 and GrxS16, and in vitro cysteine desulphurase-mediated assembly of an identical [2Fe-2S] cluster in apo-GrxS14. These Grxs possess the same monothiol CGFS active site as yeast Grx5 and both were able to complement a yeast grx5 mutant defective in Fe-S cluster assembly. In vitro kinetic studies monitored by CD spectroscopy indicate that [2Fe-2S] clusters on GrxS14 are rapidly and quantitatively transferred to apo chloroplast ferredoxin. These data demonstrate that chloroplast CGFS Grxs have the potential to function as scaffold proteins for the assembly of [2Fe-2S] clusters that can be transferred intact to physiologically relevant acceptor proteins. Alternatively, they may function in the storage and/or delivery of preformed Fe-S clusters or in the regulation of the chloroplastic Fe-S cluster assembly machinery.

Functional, structural, and spectroscopic characterization of a glutathione-ligated [2Fe-2S] cluster in poplar glutaredoxin C1.

When expressed in Escherichia coli, cytosolic poplar glutaredoxin C1 (CGYC active site) exists as a dimeric iron-sulfur-containing holoprotein or as a monomeric apoprotein in solution. Analytical and spectroscopic studies of wild-type protein and site-directed variants and structural characterization of the holoprotein by using x-ray crystallography indicate that the holoprotein contains a subunit-bridging [2Fe-2S] cluster that is ligated by the catalytic cysteines of two glutaredoxins and the cysteines of two glutathiones. Mutagenesis data on a variety of poplar glutaredoxins suggest that the incorporation of an iron-sulfur cluster could be a general feature of plant glutaredoxins possessing a glycine adjacent to the catalytic cysteine. In light of these results, the possible involvement of plant glutaredoxins in oxidative stress sensing or iron-sulfur biosynthesis is discussed with respect to their intracellular localization.

Plant mitochondrial genes can be expressed from mRNAs lacking stop codons.

The mRNAs of the nad6 and ccmC genes of Arabidopsis and cauliflower were found to be processed upstream of the inframe stop codons. This result was confirmed by northern hybridization and by RT-PCR. There is no evidence that an alternative stop codon is created post-transcriptionally, either by RNA editing or by polyadenylation. The non-stop mRNAs are found in the high molecular weight polysomal fractions, suggesting that they are translated. Using antibodies directed against CcmC, the corresponding protein was detected in Arabidopsis mitochondrial extracts. These observations raise the question of how the plant mitochondrial translation system deals with non-stop mRNAs.

Plant mitochondrial rps2 genes code for proteins with a C-terminal extension that is processed.

A gene (rps2) coding for ribosomal protein S2 (RPS2) is present in the mitochondrial (mt) genome of several monocot plants, but absent from the mtDNA of dicots. Confirming that in dicot plants the corresponding gene has been transferred to the nucleus, a corresponding Arabidopsis thaliana nuclear gene was identified that codes for mitochondrial RPS2. As several yeast and mammalian genes coding for mt ribosomal proteins, the Arabidopsis RPS2 apparently has no N-terminal targeting sequence. In the maize mt genome, two rps2 genes were identified and both are transcribed, although at different levels. As in wheat and rice, the maize genes code for proteins with long C-terminal extensions, as compared to their bacterial counterparts. These extensions are not conserved in sequence. Using specific antibodies against one of the maize proteins we found that a large protein precursor is indeed synthesized, but it is apparently processed to give the mature RPS2 protein which is associated with the mitochondrial ribosome.