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The mitochondrial genomes of flowering plants possess a promiscuous proclivity for taking up sequences from the chloroplast genome. All characterized chloroplast integrants exist apart from native mitochondrial genes, and only a few, involving chloroplast tRNA genes that have functionally supplanted their mitochondrial counterparts, appear to be of functional consequence. We developed a novel computational approach to search for homologous recombination (gene conversion) in a large number of sequences and applied it to 22 mitochondrial and chloroplast gene pairs, which last shared common ancestry some 2 billion years ago. We found evidence of recurrent conversion of short patches of mitochondrial genes by chloroplast homologs during angiosperm evolution, but no evidence of gene conversion in the opposite direction. All 9 putative conversion events involve the atp1/atpA gene encoding the alpha subunit of ATP synthase, which is unusually well conserved between the 2 organelles and the only shared gene that is widely sequenced across plant mitochondria. Moreover, all conversions were limited to the 2 regions of greatest nucleotide and amino acid conservation of atp1/atpA. These observations probably reflect constraints operating on both the occurrence and fixation of recombination between ancient homologs. These findings indicate that recombination between anciently related sequences is more frequent than previously appreciated and creates functional mitochondrial genes of chimeric origin. These results also have implications for the widespread use of mitochondrial atp1 in phylogeny reconstruction. gene conversion | gene transfer | recombination

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-[tRNA.sup.Gln] 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-[tRNA.sup.Gln] is also achieved by the indirect pathway in plant mitochondria. The mitochondrial-encoded [tRNA.sup.Gln], which is the only [tRNA.sup.Gln] present in mitochondria, is first charged with glutamate by a nondiscriminating GluRS, then is converted into GIn-[tRNA.sup.GIn] 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-[tRNA.sup.Gln] formation is shared with chloroplasts as both the GluRS, and the three AdT subunits are dual-imported into mitochondria and chloroplasts. amidation | aminoacylation | GatCAB | glutamyl-tRNA synthetase | protein trafficking

We have sequenced five distinct mitochondrial genomes in maize: two fertile cytotypes (NA and the previously reported NB) and three cytoplasmic-male-sterile cytotypes (CMS-C, CMS-S, and CMS-T). Their genome sizes range from 535,895 bp in CMS-T to 739,719 bp in CMS-C. Large duplications (0.5-190 kb) account for most of the size increases. Plastid DNA accounts for 2.3-4.6% of each mitochondrial genome. The genomes share a minimum set of 51 genes for 33 conserved proteins, three ribosomal RNAs, and 15 transfer RNAs. Numbers of duplicate genes and plastid-derived tRNAs vary among cytotypes. A high level of sequence conservation exists both within and outside of genes (1.65-7.04 substitutions/10 kb in pairwise comparisons). However, sequence losses and gains are common: integrated plastid and plasmid sequences, as well as noncoding 'native' mitochondrial sequences, can be lost with no phenotypic consequence. The organization of the different maize mitochondrial genomes varies dramatically; even between the two fertile cytotypes, there are 16 rearrangements. Comparing the finished shotgun sequences of multiple mitochondrial genomes from the same species suggests which genes and open reading frames are potentially functional, including which chimeric ORFs are candidate genes for cytoplasmic male sterility. This method identified the known CMS-associated ORFs in CMS-S and CMS-T, but not in CMS-C.

Stability of the mitochondrial genome is controlled by nuclear loci. In plants, nuclear genes suppress mitochondrial DNA rearrangements during development. One nuclear gene involved in this process is Msh1. Msh1 appears to be involved in the suppression of illegitimate recombination in plant mitochondria. To test the hypothesis that Msh1 disruption leads to the type of mitochondrial DNA rearrangements associated with naturally occurring cytoplasmic male sterility in plants, a transgenic approach for RNAi was used to modulate expression of Msh1 in tobacco and tomato. In both species, these experiments resulted in reproducible mitochondrial DNA rearrangements and a condition of male (pollen) sterility. The male sterility was, in each case, heritable, associated with normal female fertility, and apparently maternal in its inheritance. Segregation of the transgene did not reverse the male sterile phenotype, producing stable, non-transgenic male sterility. The reproducible transgenic induction of mitochondrial rearrangements in plants is unprecedented, providing a means to develop novel cytoplasmic male sterile lines for release as non-GMO or transgenic materials. MutS homolog | nonhomologous recombination | RNA interference | tobacco | tomato

Knowledge of mitochondrial gene evolution in angiosperms has taken a dramatic shift within the past decade, from universal slow rates of nucleotide change to a growing realization of high variation in rates among lineages. Additionally, evidence of paternal inheritance of plant mitochondria and recombination among mitochondrial genomes within heteroplasmic individuals has led to speculation about the potential for independent evolution of organellar genes. We report intraspecific mitochondrial and chloroplast sequence variation in a cosmopolitan sample of 42 Silene vulgaris individuals. There was remarkably high variation in two mitochondrial genes (atp1, atp9) and additional variation within a third gene (cob). Tests for patterns of nonneutral evolution were significant for atp1 and atp9, indicative of the maintenance of balanced polymorphisms. Two chloroplast genes (matK, ndhF) possessed less, but still high, variation and no divergence from neutral expectations. Phylogenetic patterns of organelle genes in both the chloroplast and mitochondria were incongruent, indicating the potential for independent evolutionary trajectories. Evidence indicated reassociation among cytoplasmic genomes and recombination between mitochondrial genes and within atp1, implying transient heteroplasmy in ancestral lineages. Although the mechanisms for long-term maintenance of mitochondrial polymorphism are currently unknown, frequency-dependent selection on linked cytoplasmic male sterility genes is a potential candidate.

Because of their extremely low nucleotide mutation rates, plant mitochondrial genes are generally not expected to show variation within species. Remarkably, we found nine distinct cytochrome b sequence haplotypes in the gynodioecious alpine plant Silene acaulis, with two or more haplotypes coexisting locally in each of three sampled regions. Moreover, there is evidence for intragenic recombination in the history of the haplotype sample, implying at least transient heteroplasmy of mitochondrial DNA (mtDNA). Heteroplasmy might be achieved by one of two potential mechanisms, either continuous coexistence of subgenomic fragments in low stoichiometry, or occasional paternal leakage of mtDNA. On the basis of levels of synonymous nucleotide substitutions, the average divergence time between haplotypes is estimated to be at least 15 million years. Ancient coalescence of extant haplotypes is further indicated by the paucity of fixed differences in haplotypes obtained from related species, a pattern expected under trans-specific evolution. Our data are consistent with models of frequency-dependent selection on linked cytoplasmic male-sterility factors, the putative molecular basis of females in gynodioecious populations. However, associations between marker loci and the inferred male-sterility genes can be maintained only with very low rates of recombination. Heteroplasmy and recombination between divergent haplotypes imply unexplored consequences for the evolutionary dynamics of gynodioecy, a widespread plant breeding system.

Byline: I. B. Khvorostov (1), M. K. Ivanov (1), G. M. Dymshits (1) Keywords: mitochondrial genome; recombination; transcription; expression regulation Abstract: The multistep regulation of mitochondrial gene expression in higher plants is considered. Data are summarized that concern the structure and function of the transcription system, posttranscriptional changes in the mRNA structure, and other levels of expression regulation. Author Affiliation: (1) Institute of Cytology and Genetics, Siberian Division, Russian Academy of Sciences, Novosibirsk, 630090, Russia Article History: Registration Date: 09/10/2004

One-fifth of the tRNAs used in plant mitochondrial translation is coded for by chloroplast-derived tRNA genes. To understand how aminoacyl-tRNA synthetases have adapted to the presence of these tRNAs in mitochondria, we have cloned an Arabidopsis thaliana cDNA coding for a methionyl-tRNA synthetase. This enzyme was chosen because chloroplast-like elongator [tRNA.sup.Met] genes have been described in several plant species, including A. thaliana. We demonstrate here that the isolated cDNA codes for both the chloroplastic and the mitochondrial methionyl-tRNA synthetase (MetRS). The protein is transported into isolated chloroplasts and mitochondria and is processed to its mature form in both organelles. Transient expression assays using the green fluorescent protein demonstrated that the N-terminal region of the MetRS is sufficient to address the protein to both chloroplasts and mitochondria. Moreover, characterization of MetRS activities from mitochondria and chloroplasts of pea showed that only one MetRS activity exists in each organelle and that both are indistinguishable by their behavior on ion exchange and hydrophobic chromatographies. The high degree of sequence similarity between A. thaliana and Synechocystis MetRS strongly suggests that the A. thaliana MetRS gene described here is of chloroplast origin.

Trans-splicing in angiosperm plant mitochondria connects exons from independent RNA molecules by means of group II intron fragments. Homologues of trans-splicing introns in the angiosperm mitochondrial nad2 and nad5 genes are now identified as uninterrupted group II introns in the ferns Asplenium nidus and Marsilea drummondii. These fern introns are correctly spliced from the pre-mRNA at the sites predicted from their well-conserved secondary structures. The flanking exon sequences of the nad2 and nad5 genes in the ferns require RNA editing, including the removal of in.frame stop codons by U-to-C changes for correct expression of the genetic information. We conclude that cis-splicing introns like the ones now identified in ferns are the ancestors of trans-splicing introns in angiosperm mitochondria. Intron disruption is apparently due to a size increase of the structurally variable group II intron domain IV followed by DNA recombination in the plant mitochondrial genome.

Previous studies have shown that the mitochondrial orf224/atp6 gene region is correlated with the Polima (pol) cytoplasmic male sterility (CMS) of Brassica napus. We now extend this correlation by showing that the effects of nuclear fertility restoration on orf224/atp6 transcripts cosegregate with the pol restorer gene Rfp1 in genetic crosses. We also show, however, that the recessive rfp1 allele, or a very tightly linked gene, acts as a dominant gene, designated Mmt (modifier of mitochondrial transcripts), in controlling the presence of additional smaller transcripts of the nad4 gene and a gene possibly involved in cytochrome c biogenesis. A common sequence, TTGTGG, maps immediately downstream of the 5[prime] termini of both of the transcripts specific to plants with the Mmt gene and may serve as a recognition motif in generation of these transcripts. A similar sequence, TTGTTG, that may be recognized by the product of the alternate allele (or haplotype), Rfp1, is found within orf224 just downstream of the major 5[prime] transcript terminus specific to fertility restored plants. Our results suggest that Rfp1/Mmt is a novel nuclear genetic locus that affects the expression of multiple mitochondrial gene regions, with different alleles or haplotypes exerting specific effects on different mitochondrial genes.

The central part of the mitochondrial coxII gene was amplified from 38 different dicots and two monocots using polymerase chain reaction. In 30 of the 40 plants studied, the amplified coxII gene-fragment contains an intron, ranging from 930 bp in Capsicum (pepper) in Solanaceae to 1,635 bp in Ampelamus albidans (climbing milkweed) in Asclepiadaceae. The composition of this intron varies as revealed by Southern hybridizations using oligonucleotide probes specific to the coxII intron-regions in maize, wheat, and rice. In the Apocynaceae, Catharanthus roseus (Madagascar periwinkle) and Vinca minor (common periwinkle) lack the coxII intron, while other members of the same family (various Mandevilla species, Nerium oleander and Apocynum cannabinum) and members of the closely related Asclepiadaceae (Asclepias incarnata, Ampelamus albidans and Asclepias tuberosa) retain the intron. Analysis of these data suggest a selective loss of the coxII intron from a plant, ancestral to both Catharanthus and Vinca, after the divergence of the Asclepiadaceae and Apocynaceae. The remaining eight plants from the Brassicaceae, Cucurbitaceae, Fabaceae, and Onagraceae lacking the intron fall into a single group or clade using the phylogenetic tree proposed by Chase et al. (Annals of the Missouri Botanical Garden: 80: 528-580, 1993) based on sequence of the chloroplast rbcL gene.

A model to simulate the evolution of various frequencies of the different type of molecules over successive cell cycles was developed. The model views the mitochondrial genome as a population of circular molecules developed through recombination, replication and random segregation. The parameters of the model are the rates of recombination of each sequence, the frequency of each type of recombination, the replication rates of the circles and the total number of mitochondrial DNA per cell. It was proved that high recombination rates leads to rapid deletions of sequences, in the absence of selection.

The recombination and copy number shifting activities of the plant mitochondrial genome are widely documented across plant genera, but these genome processes have not been as well examined with regard to their roles in plant evolution. Because of the extensive plant collections of Phaseolus spp and the degree to which cytoplasmic male sterility (cms) has been characterized in the common bean, this system would be valuable for investigating mitochondrial genome dynamics in natural populations. We have used the cms-associated sequence pvs-orf239 as a mitochondrial genetic marker for these studies and have demonstrated its universal presence throughout a diversity of undomesticated Phaseolus lines. Within these populations, the pvs-orf239 sequence is present in high copy number in ~10% of the lines, but substoichiometric in all others. This mitochondrial sequence, derived apparently by at least two recombination events, is well conserved with two point mutations identified that are both apparently silent with regard to the sterility phenotype. A putative progenitor sequence was identified in Phaseolus glabelus in substoichiometric levels, suggesting that the present-day pvs-orf239 sequence was likely introduced substoichiometrically. Copy number shifting within the mitochondrial genome results in a 1000- to 2000-fold change, so that substoichiometric forms are estimated at less than one copy per every 100 cells. On the basis of PCR analysis of root tips, we postulate that a mitochondrial 'transmitted form' resides within the meristem to assure transmission of a complete genetic complement to progeny.

The replication and partitioning of chloroplast and mitochondrial genomes to daughter cells at cell division occur in a flexible manner, unlike in the case of nuclear genomes. Many of the genes present in the prokaryotic endosymbionts that became chloroplasts and mitochondria have been lost or transferred to the nucleus of the host, where they have joined the stringent genetic system of the nuclear genome, including also sexual recombination and more efficient DNA repair. However, genes retained within the cytoplasmic organelles can be involved in selection processes both within and among individuals. In the case of heteroplasmy, which is attributed to mutations or biparental inheritance, within-individual selection on cytoplasmic DNA may provide a mechanism by which to adapt rapidly. The persistence of genes in cytoplasmic genomes indicates that chloroplast and mitochondrial genomes can be maintained in the process of evolution. The inheritance of cytoplasmic genomes is not universally maternal (e.g. about one-third of the angiosperm genera seem to display biparental chloroplast inheritance to some degree) and, therefore, unlikely to be a mere consequence of the asymmetry in gamete sizes. The presence of inheritance patterns other than the strictly maternal pattern indicates that different strategies have been adopted among different organisms.

The plant mitochondrial genome is unique among all mitochondrial genomes in that it exhibits rapid genome rearrangements and a wide range of size variation. In our investigation of mitochondrial genome evolution in land plants, we discovered several genomic rearrangements that are of phylogenetic and molecular evolutionary interest. The gain of two introns in the gene coxII and one intron in the gene nad1 suggests that liverworts are the basal-most land plant lineage. The intron gained in nad1 also became trans-spliced several times independently in angiosperms. Trans-splicing of this intron confirms a previously identified basal angiosperm clade in rbcL sequence analysis, which comprises Chloranthaceae, Austrobaileyaceae, Amborellaceae, Illiciales, and Nymphaeales s.s. Trans-splicing of this intron in Dioscoreales, Liliales, Asparagales, and commelinoids, and loss of the gene rps10 in Araceae and alismatids define two major lineages of monocots. Acorus and Tofieldieae lack both genomic changes, and thus represent the basal monocots. The gene rps 10 has been lost [approximately]25 times, probably due to an ancient transfer before the evolution of angiosperms. The two introns gained in coxII have been lost 27 and 38 times, respectively, with one ancient loss in the common ancestor of rosid I and II. These results also highlight the fluid nature of plant mitochondrial genome.

The first intron of the cox2 gene and its bordering exon sequences have been cloned and sequenced from a variety of distantly related land plants: Gymnostomum recurvirostrum, Equisetum arvense, Cycas revoluta, Nymphaea sp., Calycanthus floridus, Austrobaileya scandens, Aristolochia macrophylla, Ceratophyllum demersum, Tetracentron sinensis, Asimina triloba and Clivia miniata. Each gene contains a clearly homologous intron, located at exactly the same position and sharing high sequence similarity. The introns vary greatly in size; the smallest is 474 bp (C. revoluta) and the largest one is 2805 bp (Nymphaea sp.). Most of this variation is due to the presence of different insertion elements in domain IV of this group II intron. With regard to the angiosperms analyzed, the size variations are most often found in the plants from basal groups. Monocots and eudicots tend to h ave a stable rice-like intron of about 1.3 kb in size. The introns from A. scandens, C. floridus, Nymphaea sp., A. triloba and A. macrophylla share a unique insertion. The first three plants share a very large insertion which is missing in the latter two. These results suggest the possibility of establishing phylogenetic relationships among the basal angiosperms using mitochondrial intron insertion/deletion elements.