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. For this, plant mitochondria are purified prior to mtDNA extraction and purification. The relative enrichment in mtDNA can be assessed by qPCR, while the absolute enrichment can be deduced from the proportion of NGS reads mapping to each of the three genomes of the plant cell. Here we present methods for mitochondrial purification and mtDNA extraction applied to different plant species and tissues, and compare the mtDNA enrichment obtained with the different procedures.

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. While RadA/Sms is dispensable in bacteria, RADA-deficient Arabidopsis plants are severely impacted in their development and fertility, correlating with increased mtDNA recombination across intermediate-size repeats and accumulation of recombination-generated mitochondrial subgenomes. The radA mutation is epistatic to recG1 that affects the additional branch migration activity. In contrast, the double mutation radA recA3 is lethal, underlining the importance of an alternative RECA3-dependent pathway. The physical interaction of RADA with RECA2 but not with RECA3 further indicated that RADA is required for the processing of recombination intermediates in the RECA2-depedent recombination pathway of plant mitochondria. Although RADA is dually targeted to mitochondria and chloroplasts we found little to no effects of the radA mutation on the stability of the plastidial genome. Finally, we found that the deficient maintenance of the mtDNA in radA apparently triggers a retrograde signal that activates nuclear genes repressing cell cycle progression.

Assessment of Mitochondrial DNA Copy Number, Stability, and Repair in Arabidopsis.

Mitochondrial functions depend on the proper maintenance and expression of the mitochondrial genome (mtDNA). Therefore, understanding mtDNA replication and repair requires methods to assess its integrity. Mutations or chemical treatments that affect processes involved in the maintenance or stability of the mtDNA can affect its global copy number, but also the relative abundance of different genomic regions or the frequency of illegitimate recombination across repeated sequences. These can be conveniently tested by quantitative PCR (qPCR). Arabidopsis thaliana offers several advantages for studying these processes, because of the extensive collections of mutants, natural accessions and other genetic resources available from stock centers. Here we describe protocols we routinely use to explore changes in mtDNA copy number and relative stoichiometry in Arabidopsis mutants of genes involved in the replication, repair and recombination of the mtDNA.

Sequence of the Mitochondrial Genome of Lactuca virosa Suggests an Unexpected Role in Lactuca sativa's Evolution.

The involvement of the different Lactuca species in the domestication and diversification of cultivated lettuce is not totally understood. Lactuca serriola is considered as the direct ancestor and the closest relative to Lactuca sativa, while the other wild species that can be crossed with L. sativa, Lactuca virosa, and Lactuca saligna, would have just contributed to the latter diversification of cultivated typologies. To contribute to the study of Lactuca evolution, we assembled the mtDNA genomes of nine Lactuca spp. accessions, among them three from L. virosa, whose mtDNA had not been studied so far. Our results unveiled little to no intraspecies variation among Lactuca species, with the exception of L. serriola where the accessions we sequenced diverge significantly from the mtDNA of a L. serriola accession already reported. Furthermore, we found a remarkable phylogenetic closeness between the mtDNA of L. sativa and the mtDNA of L. virosa, contrasting to the L. serriola origin of the nuclear and plastidial genomes. These results suggest that a cross between L. virosa and the ancestor of cultivated lettuce is at the origin of the actual mitochondrial genome of L. sativa.

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.

Reciprocal cybrids reveal how organellar genomes affect plant phenotypes.

Assessment of the impact of variation in chloroplast and mitochondrial DNA (collectively termed the plasmotype) on plant phenotypes is challenging due to the difficulty in separating their effect from nuclear-derived variation (the nucleotype). Haploid-inducer lines can be used as efficient plasmotype donors to generate new plasmotype-nucleotype combinations (cybrids)1. We generated a panel comprising all possible cybrids of seven Arabidopsis thaliana accessions and extensively phenotyped these lines for 1,859 phenotypes under both stable and fluctuating conditions. We show that natural variation in the plasmotype results in both additive and epistatic effects across all phenotypic categories. Plasmotypes that induce more additive phenotypic changes also cause more epistatic effects, suggesting a possible common basis for both additive and epistatic effects. On average, epistatic interactions explained twice as much of the variance in phenotypes as additive plasmotype effects. The impact of plasmotypic variation was also more pronounced under fluctuating and stressful environmental conditions. Thus, the phenotypic impact of variation in plasmotypes is the outcome of multi-level nucleotype-plasmotype-environment interactions and, as such, the plasmotype is likely to serve as a reservoir of variation that is predominantly exposed under certain conditions. The production of cybrids using haploid inducers is a rapid and precise method for assessment of the phenotypic effects of natural variation in organellar genomes. It will facilitate efficient screening of unique nucleotype-plasmotype combinations to both improve our understanding of natural variation in these combinations and identify favourable combinations to enhance plant performance.

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.

Role of pyrimidine salvage pathway in the maintenance of organellar and nuclear genome integrity.

Nucleotide biosynthesis proceeds through a de novo pathway and a salvage route. In the salvage route, free bases and/or nucleosides are recycled to generate the corresponding nucleotides. Thymidine kinase (TK) is the first enzyme in the salvage pathway to recycle thymidine nucleosides as it phosphorylates thymidine to yield thymidine monophosphate. The Arabidopsis genome contains two TK genes -TK1a and TK1b- that show similar expression patterns during development. In this work, we studied the respective roles of the two genes during early development and in response to genotoxic agents targeting the organellar or the nuclear genome. We found that the pyrimidine salvage pathway is crucial for chloroplast development and genome replication, as well as for the maintenance of its integrity, and is thus likely to play a crucial role during the transition from heterotrophy to autotrophy after germination. Interestingly, defects in TK activity could be partially compensated by supplementation of the medium with sugar, and this effect resulted from both the availability of a carbon source and the activation of the nucleotide de novo synthesis pathway, providing evidence for a compensation mechanism between two routes of nucleotide biosynthesis that depend on nutrient availability. Finally, we found differential roles of the TK1a and TK1b genes during the plant response to genotoxic stress, suggesting that different pools of nucleotides exist within the cells and are required to respond to different types of DNA damage. Altogether, our results highlight the importance of the pyrimidine salvage pathway, both during plant development and in response to genotoxic stress.

Efficient Replication of the Plastid Genome Requires an Organellar Thymidine Kinase.

Thymidine kinase (TK) is a key enzyme of the salvage pathway that recycles thymidine nucleosides to produce deoxythymidine triphosphate. Here, we identified the single TK of maize (Zea mays), denoted CPTK1, as necessary in the replication of the plastidial genome (cpDNA), demonstrating the essential function of the salvage pathway during chloroplast biogenesis. CPTK1 localized to both plastids and mitochondria, and its absence resulted in an albino phenotype, reduced cpDNA copy number and a severe deficiency in plastidial ribosomes. Mitochondria were not affected, indicating they are less reliant on the salvage pathway. Arabidopsis (Arabidopsis thaliana) TKs, TK1A and TK1B, apparently resulted from a gene duplication after the divergence of monocots and dicots. Similar but less-severe effects were observed for Arabidopsis tk1a tk1b double mutants in comparison to those in maize cptk1 TK1B was important for cpDNA replication and repair in conditions of replicative stress but had little impact on the mitochondrial phenotype. In the maize cptk1 mutant, the DNA from the small single-copy region of the plastidial genome was reduced to a greater extent than other regions, suggesting preferential abortion of replication in this region. This was accompanied by the accumulation of truncated genomes that resulted, at least in part, from unfaithful microhomology-mediated repair. These and other results suggest that the loss of normal cpDNA replication elicits the mobilization of new replication origins around the rpoB (beta subunit of plastid-encoded RNA polymerase) transcription unit and imply that increased transcription at rpoB is associated with the initiation of cpDNA replication.

Plant Mitochondrial Genomes: Dynamics and Mechanisms of Mutation.

The large mitochondrial genomes of angiosperms are unusually dynamic because of recombination activities involving repeated sequences. These activities generate subgenomic forms and extensive genomic variation even within the same species. Such changes in genome structure are responsible for the rapid evolution of plant mitochondrial DNA and for the variants associated with cytoplasmic male sterility and abnormal growth phenotypes. Nuclear genes modulate these processes, and over the past decade, several of these genes have been identified. They are involved mainly in pathways of DNA repair by homologous recombination and mismatch repair, which appear to be essential for the faithful replication of the mitogenome. Mutations leading to the loss of any of these activities release error-prone repair pathways, resulting in increased ectopic recombination, genome instability, and heteroplasmy. We review the present state of knowledge of the genes and pathways underlying mitochondrial genome stability.

Arabidopsis Seed Mitochondria Are Bioenergetically Active Immediately upon Imbibition and Specialize via Biogenesis in Preparation for Autotrophic Growth.

Seed germination is a vital developmental transition for production of progeny by sexual reproduction in spermatophytes. Quiescent cells in nondormant dry embryos are reawakened first by imbibition and then by perception of germination triggers. Reanimated tissues enter into a germination program requiring energy for expansion growth. However, germination requires that embryonic tissues develop to support the more energy-demanding processes of cell division and organogenesis of the new seedling. Reactivation of mitochondria to supply the required energy is thus a key process underpinning germination and seedling survival. Using live imaging, we investigated reactivation of mitochondrial bioenergetics and dynamics using Arabidopsis thaliana as a model. Bioenergetic reactivation, visualized by presence of a membrane potential, is immediate upon rehydration. However, reactivation of mitochondrial dynamics only occurs after transfer to germination conditions. Reactivation of mitochondrial bioenergetics is followed by dramatic reorganization of the chondriome (all mitochondrial in a cell, collectively) involving massive fusion and membrane biogenesis to form a perinuclear tubuloreticular structure enabling mixing of previously discrete mitochondrial DNA nucleoids. The end of germination coincides with fragmentation of the chondriome, doubling of mitochondrial number, and heterogeneous redistribution of nucleoids among the mitochondria, generating a population of mitochondria tailored to seedling growth.

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.

Insights into ascorbate regeneration in plants: investigating the redox and structural properties of dehydroascorbate reductases from Populus trichocarpa.

Dehydroascorbate reductases (DHARs), enzymes belonging to the GST superfamily, catalyse the GSH-dependent reduction of dehydroascorbate into ascorbate in plants. By maintaining a reduced ascorbate pool, they notably participate to H2O2 detoxification catalysed by ascorbate peroxidases (APXs). Despite this central role, the catalytic mechanism used by DHARs is still not well understood and there is no supportive 3D structure. In this context, we have performed a thorough biochemical and structural analysis of the three poplar DHARs and coupled this to the analysis of their transcript expression patterns and subcellular localizations. The transcripts for these genes are mainly detected in reproductive and green organs and the corresponding proteins are expressed in plastids, in the cytosol and in the nucleus, but not in mitochondria and peroxisomes where ascorbate regeneration is obviously necessary. Comparing the kinetic properties and the sensitivity to GSSG-mediated oxidation of DHAR2 and DHAR3A, exhibiting 1 or 3 cysteinyl residues respectively, we observed that the presence of additional cysteines in DHAR3A modifies the regeneration mechanism of the catalytic cysteine by forming different redox states. Finally, from the 3D structure of DHAR3A solved by NMR, we were able to map the residues important for the binding of both substrates (GSH and DHA), showing that DHAR active site is very selective for DHA recognition and providing further insights into the catalytic mechanism and the roles of the additional cysteines found in some DHARs.

The RECG1 DNA Translocase Is a Key Factor in Recombination Surveillance, Repair, and Segregation of the Mitochondrial DNA in Arabidopsis.

The mitochondria of flowering plants have considerably larger and more complex genomes than the mitochondria of animals or fungi, mostly due to recombination activities that modulate their genomic structures. These activities most probably participate in the repair of mitochondrial DNA (mtDNA) lesions by recombination-dependent processes. Rare ectopic recombination across short repeats generates new genomic configurations that contribute to mtDNA heteroplasmy, which drives rapid evolution of the sequence organization of plant mtDNAs. We found that Arabidopsis thaliana RECG1, an ortholog of the bacterial RecG translocase, is an organellar protein with multiple roles in mtDNA maintenance. RECG1 targets to mitochondria and plastids and can complement a bacterial recG mutant that shows defects in repair and replication control. Characterization of Arabidopsis recG1 mutants showed that RECG1 is required for recombination-dependent repair and for suppression of ectopic recombination in mitochondria, most likely because of its role in recovery of stalled replication forks. The analysis of alternative mitotypes present in a recG1 line and of their segregation following backcross allowed us to build a model to explain how a new stable mtDNA configuration, compatible with normal plant development, can be generated by stoichiometric shift.

Organellar non-coding RNAs: emerging regulation mechanisms.

Originally focused on the nuclear and cytosolic compartments, the concept of regulation driven by non-coding RNAs (ncRNAs) is extending to mitochondria and chloroplasts. These organelles have distinct genetic systems that need coordination with cellular demands. In mammals, nuclear-encoded microRNAs were found associated with the mitochondria. Some of these contribute to the regulation of mitochondrial transcription and translation. Others were proposed to be stored in the organelles and to be released for regulation of nuclear transcripts. Further ncRNAs of various sizes derive from the mitochondrial genome and it was speculated that organelles host antisense or RNA interference pathways. Long ncRNAs mapping to the mitochondrial DNA seem to operate in the nucleus. Altogether, the origin and trafficking of ncRNAs categorized as mitochondrial in mammals raise questions far beyond the current knowledge. In protozoa, hundreds of guide RNAs specify editing events needed to generate functional messenger RNAs. Only few ncRNAs have been reported in plant mitochondria, but editing sites were revealed in non-coding regions of the organellar genome, suggesting that the corresponding transcripts have a function. Conversely, numerous ncRNA candidates were identified in chloroplasts, essentially mapping to the plastid genome. A synthetic view of the data with their functional implications is given here.

The RAD52-like protein ODB1 is required for the efficient excision of two mitochondrial introns spliced via first-step hydrolysis.

Transcript splicing in plant mitochondria involves numerous nucleus-encoded factors, most of which are of eukaryotic origin. Some of these belong to protein families initially characterised to perform unrelated functions. The RAD52-like ODB1 protein has been reported to have roles in homologous recombination-dependent DNA repair in the nuclear and mitochondrial compartments in Arabidopsis thaliana. We show that it is additionally involved in splicing and facilitates the excision of two cis-spliced group II introns, nad1 intron 2 and nad2 intron 1, in Arabidopsis mitochondria. odb1 mutants lacking detectable amounts of ODB1 protein over-accumulated incompletely spliced nad1 and nad2 transcripts. The two ODB1-dependent introns were both found to splice via first-step hydrolysis and to be released as linear or circular molecules instead of lariats. Our systematic analysis of the structures of excised introns in Arabidopsis mitochondria revealed several other hydrolytically spliced group II introns in addition to nad1 intron 2 and nad2 intron 1, indicating that ODB1 is not a general determinant of the hydrolytic splicing pathway.

Glutathionyl-hydroquinone reductases from poplar are plastidial proteins that deglutathionylate both reduced and oxidized glutathionylated quinones.

Glutathionyl-hydroquinone reductases (GHRs) catalyze the deglutathionylation of quinones via a catalytic cysteine. The two GHR genes in the Populus trichocarpa genome, Pt-GHR1 and Pt-GHR2, are primarily expressed in reproductive organs. Both proteins are localized in plastids. More specifically, Pt-GHR2 localizes in nucleoids. At the structural level, Pt-GHR1 adopts a typical GHR fold, with a dimerization interface comparable to that of the bacterial and fungal GHR counterparts. Pt-GHR1 catalyzes the deglutathionylation of both reduced and oxidized glutathionylated quinones, but the enzyme is more catalytically efficient with the reduced forms.

Structural and enzymatic insights into Lambda glutathione transferases from Populus trichocarpa, monomeric enzymes constituting an early divergent class specific to terrestrial plants.

GSTs represent a superfamily of multifunctional proteins which play crucial roles in detoxification processes and secondary metabolism. Instead of promoting the conjugation of glutathione to acceptor molecules as do most GSTs, members of the Lambda class (GSTLs) catalyse deglutathionylation reactions via a catalytic cysteine residue. Three GSTL genes (Pt-GSTL1, Pt-GSTL2 and Pt-GSTL3) are present in Populus trichocarpa, but two transcripts, differing in their 5' extremities, were identified for Pt-GSTL3. Transcripts for these genes were primarily found in flowers, fruits, petioles and buds, but not in leaves and roots, suggesting roles associated with secondary metabolism in these organs. The expression of GFP-fusion proteins in tobacco showed that Pt-GSTL1 is localized in plastids, whereas Pt-GSTL2 and Pt-GSTL3A and Pt-GSTL3B are found in both the cytoplasm and the nucleus. The resolution of Pt-GSTL1 and Pt-GSTL3 structures by X-ray crystallography indicated that, although these proteins adopt a canonical GST fold quite similar to that found in dimeric Omega GSTs, their non-plant counterparts, they are strictly monomeric. This might explain some differences in the enzymatic properties of both enzyme types. Finally, from competition experiments between aromatic substrates and a fluorescent probe, we determined that the recognition of glutathionylated substrates is favoured over non-glutathionylated forms.

DNA-binding proteins in plant mitochondria: implications for transcription.

The structural complexity of plant mitochondrial genomes correlates with the variety of single-strand DNA-binding proteins found in plant mitochondria. Most of these are plant-specific and have roles in homologous recombination and genome maintenance. Mitochondrial nucleoids thus differ fundamentally between plants and yeast or animals, where the principal nucleoid protein is a DNA-packaging protein that binds double-stranded DNA. Major transcriptional cofactors identified in mitochondria of non-plant species are also seemingly absent from plants. This article reviews current knowledge on plant mitochondrial DNA-binding proteins and discusses that those may affect the accessibility and conformation of transcription start sites, thus functioning as transcriptional modulators without being dedicated transcription factors.