Mitochondrial Genome Of Higher Plants Research Articles

The comparison of four mitochondrial genomes reveals cytoplasmic male sterility candidate genes in cotton

BackgroundThe mitochondrial genomes of higher plants vary remarkably in size, structure and sequence content, as demonstrated by the accumulation and activity of repetitive DNA sequences. Incompatibility between mitochondrial genome and nuclear genome leads to non-functional male reproductive organs and results in cytoplasmic male sterility (CMS). CMS has been used to produce F1 hybrid seeds in a variety of plant species.ResultsHere we compared the mitochondrial genomes (mitogenomes) of Gossypium hirsutum sterile male lines CMS-2074A and CMS-2074S, as well as their restorer and maintainer lines. First, we noticed the mitogenome organization and sequences were conserved in these lines. Second, we discovered the mitogenomes of 2074A and 2074S underwent large-scale substitutions and rearrangements. Actually, there were five and six unique chimeric open reading frames (ORFs) in 2074A and 2074S, respectively, which were derived from the recombination between unique repetitive sequences and nearby functional genes. Third, we found out four chimeric ORFs that were differentially transcribed in sterile line (2074A) and fertile-restored line.ConclusionsThese four novel and recombinant ORFs are potential candidates that confer CMS character in 2074A. In addition, our observations suggest that CMS in cotton is associated with the accelerated rates of rearrangement, and that novel expression products are derived from recombinant ORFs.

Horizontal Transfer of DNA from the Mitochondrial to the Plastid Genome and Its Subsequent Evolution in Milkweeds (Apocynaceae)

Horizontal gene transfer (HGT) of DNA from the plastid to the nuclear and mitochondrial genomes of higher plants is a common phenomenon; however, plastid genomes (plastomes) are highly conserved and have generally been regarded as impervious to HGT. We sequenced the 158 kb plastome and the 690 kb mitochondrial genome of common milkweed (Asclepias syriaca [Apocynaceae]) and found evidence of intracellular HGT for a 2.4-kb segment of mitochondrial DNA to the rps2–rpoC2 intergenic spacer of the plastome. The transferred region contains an rpl2 pseudogene and is flanked by plastid sequence in the mitochondrial genome, including an rpoC2 pseudogene, which likely provided the mechanism for HGT back to the plastome through double-strand break repair involving homologous recombination. The plastome insertion is restricted to tribe Asclepiadeae of subfamily Asclepiadoideae, whereas the mitochondrial rpoC2 pseudogene is present throughout the subfamily, which confirms that the plastid to mitochondrial HGT event preceded the HGT to the plastome. Although the plastome insertion has been maintained in all lineages of Asclepiadoideae, it shows minimal evidence of transcription in A. syriaca and is likely nonfunctional. Furthermore, we found recent gene conversion of the mitochondrial rpoC2 pseudogene in Asclepias by the plastid gene, which reflects continued interaction of these genomes.

Plastid Sequences Contribute to Some Plant Mitochondrial Genes

DNA of plastid (chloroplast) origin comprises between 1% and 10% of the mitochondrial genomes of higher plants, but functions are currently considered to be limited to rare instances where plastid tRNA genes have replaced their mitochondrial counterparts, where short patches of mitochondrial genes evolved using their homologous plastidic copies by gene conversion or where a new promoter region is created. Here, we show that, in some angiosperms, plastid-derived DNA in mitochondrial genomes (also called mtpt for mitochondrial plastid DNA) contributes codons to unrelated mitochondrial protein-coding sequences and may also have a role in posttranscriptional RNA processing. We determined that these transfers of plastid DNA occurred a few to 150 Ma and that mtpts can sometimes remain dormant many millions of years before contributing to the mitochondrial proteome.

Double-strand break repair processes drive evolution of the mitochondrial genome in Arabidopsis

BackgroundThe mitochondrial genome of higher plants is unusually dynamic, with recombination and nonhomologous end-joining (NHEJ) activities producing variability in size and organization. Plant mitochondrial DNA also generally displays much lower nucleotide substitution rates than mammalian or yeast systems. Arabidopsis displays these features and expedites characterization of the mitochondrial recombination surveillance gene MSH1 (MutS 1 homolog), lending itself to detailed study of de novo mitochondrial genome activity. In the present study, we investigated the underlying basis for unusual plant features as they contribute to rapid mitochondrial genome evolution.ResultsWe obtained evidence of double-strand break (DSB) repair, including NHEJ, sequence deletions and mitochondrial asymmetric recombination activity in Arabidopsis wild-type and msh1 mutants on the basis of data generated by Illumina deep sequencing and confirmed by DNA gel blot analysis. On a larger scale, with mitochondrial comparisons across 72 Arabidopsis ecotypes, similar evidence of DSB repair activity differentiated ecotypes. Forty-seven repeat pairs were active in DNA exchange in the msh1 mutant. Recombination sites showed asymmetrical DNA exchange within lengths of 50- to 556-bp sharing sequence identity as low as 85%. De novo asymmetrical recombination involved heteroduplex formation, gene conversion and mismatch repair activities. Substoichiometric shifting by asymmetrical exchange created the appearance of rapid sequence gain and loss in association with particular repeat classes.ConclusionsExtensive mitochondrial genomic variation within a single plant species derives largely from DSB activity and its repair. Observed gene conversion and mismatch repair activity contribute to the low nucleotide substitution rates seen in these genomes. On a phenotypic level, these patterns of rearrangement likely contribute to the reproductive versatility of higher plants.

DNA Recombination Activity in Soybean Mitochondria

Mitochondrial genomes in higher plants are much larger and more complex as compared to animal mitochondrial genomes. There is growing evidence that plant mitochondrial genomes exist predominantly as a collection of linear and highly branched DNA molecules and replicate by a recombination-dependent mechanism. However, biochemical evidence of mitochondrial DNA (mtDNA) recombination activity in plants has previously been lacking. We provide the first report of strand-invasion activity in plant mitochondria. Similar to bacterial RecA, this activity from soybean is dependent on the presence of ATP and Mg(2+). Western blot analysis using an antibody against the Arabidopsis mitochondrial RecA protein shows cross-reaction with a soybean protein of about 44 kDa, indicating conservation of this protein in at least these two plant species. mtDNA structure was analyzed by electron microscopy of total soybean mtDNA and molecules recovered after field-inversion gel electrophoresis (FIGE). While most molecules were found to be linear, some molecules contained highly branched DNA structures and a small but reproducible proportion consisted of circular molecules (many with tails) similar to recombination intermediates. The presence of recombination intermediates in plant mitochondria preparations is further supported by analysis of mtDNA molecules by 2-D agarose gel electrophoresis, which indicated the presence of complex recombination structures along with a considerable amount of single-stranded DNA. These data collectively provide convincing evidence for the occurrence of homologous DNA recombination in plant mitochondria.

The complete nucleotide sequence and multipartite organization of the tobacco mitochondrial genome: comparative analysis of mitochondrial genomes in higher plants

Tobacco is a valuable model system for investigating the origin of mitochondrial DNA (mtDNA) in amphidiploid plants and studying the genetic interaction between mitochondria and chloroplasts in the various functions of the plant cell. As a first step, we have determined the complete mtDNA sequence of Nicotiana tabacum. The mtDNA of N. tabacum can be assumed to be a master circle (MC) of 430,597 bp. Sequence comparison of a large number of clones revealed that there are four classes of boundaries derived from homologous recombination, which leads to a multipartite organization with two MCs and six subgenomic circles. The mtDNA of N. tabacum contains 36 protein-coding genes, three ribosomal RNA genes and 21 tRNA genes. Among the first class, we identified the genes rps1 and psirps14, which had previously been thought to be absent in tobacco mtDNA on the basis of Southern analysis. Tobacco mtDNA was compared with those of Arabidopsis thaliana, Beta vulgaris, Oryza sativa and Brassica napus. Since repeated sequences show no homology to each other among the five angiosperms, it can be supposed that these were independently acquired by each species during the evolution of angiosperms. The gene order and the sequences of intergenic spacers in mtDNA also differ widely among the five angiosperms, indicating multiple reorganizations of genome structure during the evolution of higher plants. Among the conserved genes, the same potential conserved nonanucleotide-motif-type promoter could only be postulated for rrn18-rrn5 in four of the dicotyledonous plants, suggesting that a coding sequence does not necessarily move with the promoter upon reorganization of the mitochondrial genome.

An Arabidopsis homologue of bacterial RecA that complements an E. coli recA deletion is targeted to plant mitochondria.

Homologous recombination results in the exchange and rearrangement of DNA, and thus generates genetic variation in living organisms. RecA is known to function in all bacteria as the central enzyme catalyzing strand transfer and has functional homologues in eukaryotes. Most of our knowledge of homologous recombination in eukaryotes is limited to processes in the nucleus. The mitochondrial genomes of higher plants contain repeated sequences that are known to undergo frequent rearrangements and recombination events. However, very little is known about the proteins involved or the biochemical mechanisms of DNA recombination in plant mitochondria. We provide here the first report of an Arabidopsis thaliana homologue of Escherichia coli RecA that is targeted to mitochondria. The mt recA gene has a putative mitochondrial presequence identified from the A. thaliana genome database. This nuclear gene encodes a predicted product that shows highest sequence homology to chloroplast RecA and RecA proteins from proteobacteria. When fused to the GFP coding sequence, the predicted presequence was able to target the fusion protein to isolated mitochondria but not to chloroplasts. The mitochondrion-specific localization of the mt recA gene product was confirmed by Western analysis using polyclonal antibodies raised against a synthetic peptide from a unique region of the mature mtRecA. The Arabidopsis mt recA gene partially complemented a recA deletion in E. coli, enhancing survival after exposure to DNA-damaging agents. These results suggest a possible role for mt recA in homologous recombination and/or repair in Arabidopsis mitochondria.

The gene encoding Arabidopsis thaliana mitochondrial ribosomal protein S13 is a recent duplication of the gene encoding plastid S13.

A gene encoding mitochondrial S13 is generally present in the mitochondrial genome of higher plants, but is lacking from the Arabidopsis thaliana mitochondrial genome. Previous research has failed to identify a nuclear gene capable of encoding a mitochondrial S13 protein or the protein itself. Doubts have even been raised as to whether a mitochondrial S13 exists in Arabidopsis. Here, we show that the nuclear gene encoding the plastid S13 has been partially duplicated in A. thaliana, such that the copy has lost the exon encoding the plastid transit peptide and acquired a sequence capable of encoding a mitochondrial targeting sequence. The two S13 sequences were fused to green fluorescent protein and shown to be targeted to plastids and mitochondria respectively.

The mitochondrial genome of higher plants: gene expression regulation

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.

Small interspersed sequences that serve as recombination sites at the cox2 and atp6 loci in the mitochondrial genome of soybean are widely distributed in higher plants.

Mitochondrial DNA (mtDNA) fragments that contain cox2 and atp6 were cloned from a wild soybean (Glycine soja, accession 'B09002') and from a cultivated soybean (G. max, 'Harosoy'). Comparison of these DNAs revealed that two sets of repeated sequences, namely, 299 bp and 23 bp, were present in the 5' regions of cox2 and atp6. The 299-bp and 23-bp repeats were present close to each other on the 5' flanking region and the 5' part of the coding region of cox2 in both 'Harosoy' and 'B09002', as well as on the 5' flanking region of atp6 in 'Harosoy', while these two repeats were separated by a 706-bp nucleotide sequence that contained a truncated sequence of nad3 at the 5' flanking region of atp6 in 'B09002'. The mtDNA configurations upstream from atp6 and cox2 found in 'Harosoy' appeared to have been generated from configurations of cox2 and atp6 found in 'B09002' via recombination across the 299-bp or 23-bp repeated sequences, or vice versa, in the mitochondrial genome of the hypothetical progenitor of these plants. The 299-bp sequence was found to be interspersed in the mitochondrial genome. Eight loci were identified that include mtDNA configurations that are inter-convertible with each other via recombination across this sequence in 'B09002'. Various loci on the mitochondrial genomes of higher plants that harbor segments of the 299-bp repeats in Glycine were identified.

Methylation of mitochondrial DNA in carrot (Daucus carota L.).

The methylation status of carrot (Daucus carota L.) mitochondrial DNA (mtDNA) was studied using isoschizomeric restriction enzymes MspI/HpaII (CCGG) and MvaI/EcoRII [CC(A/T)GG]. Southern hybridisations with probes for mitochondrial genes coxII and atpA were performed. MtDNAs isolated from non-embryogenic cell suspensions and roots were analysed. No differences were found using MspI/HpaII but after digesting the mtDNA with MvaI and EcoRII, some qualitative and quantitative differences between the restriction patterns appeared. Distinction was also revealed after Southern hybridisation with the coxII probe. These data indicate that the mtDNA of carrot is methylated in CNG trinucleotides and unmethylated in CG dinucleotides in CCGG sequences. The results were reproducible for cell suspensions of various genotypes and even cultivars but the extent of methylation was different in the root. The possible role of methylation in the mitochondrial genome of higher plants is discussed.

The rapeseed mitochondrial gene for subunit 2 of the NADH dehydrogenase complex: a trans-spliced structure is conserved in one of the smallest plant mitochondrial genomes.

The single-copy gene encoding NADH dehydrogenase subunit 2 (nad2) has been identified in the mitochondrial genome of rapeseed (Brassica napus L.). The rapeseed nad2 gene has the same gene organization in Oenothera and wheat: it consists of five exons located in two loci encoding the two first exons and the last three exons respectively. All exons are separated by group-II introns. A trans-splicing event is required to join exons B and C. Putative splicing intermediates were identified by transcriptional analysis of the nad2 gene. The complexity of organization of the nad2 gene is completely conserved even in one of the smallest mitochondrial genomes of higher plants.

Rolling-circle replication of mitochondrial DNA in the higher plant Chenopodium album (L.).

The mitochondrial genomes of higher plants are larger and more complex than those of all other groups of organisms. We have studied the in vivo replication of chromosomal and plasmid mitochondrial DNAs prepared from a suspension culture and whole plants of the dicotyledonous higher plant Chenopodium album (L.). Electron microscopic studies revealed sigma-shaped, linear, and open circular molecules (subgenomic circles) of variable size as well as a minicircular plasmid of 1.3 kb (mp1). The distribution of single-stranded mitochondrial DNA in the sigma structures and the detection of entirely single-stranded molecules indicate a rolling-circle type of replication of plasmid mp1 and subgenomic circles. About half of the sigma-like molecules had tails exceeding the lengths of the corresponding circle, suggesting the formation of concatemers. Two replication origins (nicking sites) could be identified on mpl by electron microscopy and by a new approach based on the mapping of restriction fragments representing the identical 5' ends of the tails of sigma-like molecules. These data provide, for the first time, evidence for a rolling-circle mode of replication in the mitochondria of higher plants.

Mitochondrial nucleic acid purification and analysis.

During the last years the structure and gene content of the mitochondrial genome of land plants have been subject of intensive molecular investigations. Because of the large size (200–2500 kb), and the complex, multipartite organization, the actual configuration of the mitochondrial genome and its coding capacity is still unclear (). A currently accepted model suggests that the mitochondrial genome of higher plants is organized as multiple circular molecules. A master chromosome is assumed to represent the entire mitochondrial genome, although its existence in vivo has not been shown. However, subgenomic circles, which are formed by intra- and/or intergenomic homologous recombination via direct and/or inverted repeats, have been found in almost all land plants (,). In addition, circular or linear DNA plasmids of low molecular weight have been reported in plant mitochondria (,).

Nucleotide sequence of a 28-kbp portion of rice mitochondrial DNA: the existence of many sequences that correspond to parts of mitochondrial genes in intergenic regions.

The nucleotide sequence of a 27,588-bp region of rice mitochondrial DNA was determined. This sequence contains putative genes that encode initiator methionine tRNA (trnfM), subunits III (nad3) and IV (nad4) of the NADH dehydrogenase complex, and ribosomal proteins S3 (rps3), S12 (rps12) and L16 (rpl16). An open reading frame that contains sequences homologous to parts of rps2 and atpA is also present. In addition to these regions, there are many short sequences with homology to fragments of mitochondrial DNAs from rice or other plants. These sequences may be remnants of multiple rearrangements of the genome and their presence seems to explain, in part, the large sizes of the mitochondrial genomes of higher plants.

Ribosomal protein gene rpl5 is cotranscribed with the nad3 gene in Oenothera mitochondria

The rpl5 ribosomal protein gene was identified in the mitochondrial genome of the higher plant Oenothera berteriana. The gene is present in a unique genomic location upstream of the gene encoding subunit 3 of the NADH dehydrogenase (nad3). Both genes are cotranscribed, and the mRNA is modified at several cytidine residues by RNA editing. Analysis of the editing profiles of both genes by direct cDNA analysis and polymerase chain reaction (PCR) revealed that not all transcripts are fully edited at all sites. Eight of the nine C to U conversions in the rpl5 reading frame are non-silent and change the deduced amino acid sequence. The genes of the prokaryotic-like cistron that includes the rpsl9, rps3, rpl16, rpl5, and rpsl4 genes, which is at least partially conserved in the mitochondrial genomes of other higher and lower plants, are dispersed in the Oenothera mitochondrial genome.

Fluorescence microscopic study of the formation of giant mitochondrial nuclei in the young ovules ofPelargonium zonale

The size of mitochondrial genomes in higher plants are known to range from 200 to 2400 kilobase pairs. However, we failed to identify cytochemically any mitochondria that contain an identifiable master mitochondrial genome. In the present experiments, we have found the giant mitochondrial nuclei which have the capacity for including the master mitochondrial genome in the young ovaries ofPelargonium zonale by use of a 4′-6-diamidino-2-phenylindole (DAPI) epifluorescence microscopy, a Technovit embedding, and a video-intensified photon counting system.

The plant mitochondrial genome: homologous recombination as a mechanism for generating heterogeneity

The mitochondrial genomes of higher plants are among the largest and most complex organelle genomes described. They are generally multicircular or partly linear; in some species, extrachromosomal plasmids are present. It is proposed that inter- and intramolecular homologous recombination can account for the diversity of the observed genome organizations. The ability of mitochondria to fuse establishes a panmictic mitochondrial DNA population which is in recombinational equilibrium. It is suggested that this suppresses the base mutation rate, and unequal partitioning of the cytoplasm during cell division can lead to the rapid evolution of mitochondrial genome structure. This contrasts with the observed rates of base-sequence and genome evolution in chloroplasts. This difference can be accounted for solely by the inability of chloroplasts to fuse, thereby preventing chloroplast genome panmixis.

On a model for the structure of circular mitochondrial genomes in higher plants.

The mitochondrial genome of some higher plants consists of a discrete set of circular molecules. The heterogeneity found in the number of circles and their size is puzzling. In some plants evidence exists that indicates that these circular DNA molecules undergo intermolecular and intramolecular recombination events. The result of these events is that two small circles can combine to give one large circle or one large circle can break up into two small circles. Here we pursue the idea that such recombination events are responsible for the variability in genome composition. Treating the recombination events as chemical reactions we derive the equilibrium size distribution of circles. In Brassica campestris there are two basic subunits, circles A and B, containing 135 and 83 kilobases, respectively. By restriction-enzyme mapping one can determine in a multimeric circle, the number of interfaces between A- and B-derived DNA. Using a combinatorial argument we predict the frequency of such interfaces and compare our predictions with published data. A number of suggestions are made for additional experimental tests of the recombinational theory of genomic diversity.

The mitochondrial genome of higher plants

The mitochondrial genome of higher plants appears to be the largest known of any life form. Plant mitochondrial (mt) DNA displays substantial intermolecular heterogeneity with respect to size and physical organization. Electron microscopic studies reveal both linear and circular DNA molecules of varying size. Mapping data suggest that a circular master chromosome representing the entire sequence complexity of the genome gives rise to a series of discrete DNA molecules which are generated via recombination processes within certain repeats. Additionally to the large mtDNA, mitochondria of higher plants contain small plasmid-like DNAs behaving, at least in certain cases, like transpositional elements. Plant mtDNA codes for 18S, 26S and 5S rRNAs and mitochondrial tRNAs as well as for at least 18–20 polypeptides, mainly membrane-bound components of the oxidative phosphorylation system. Substantial evidence has been accumulated that cytoplasmic male sterility CMS, a maternally inherited trait is controlled by mtDNA. Male fertile and CMS cytoplasms differ in their mtDNA restriction patterns, in the polypeptides synthesized by their mitochondria, and in the appearance and distribution of the plasmid-like small mtDNAs.