Plant Mitochondrial Genomes Research Articles

Multichromosomal structure of the onion mitochondrial genome and a transcript analysis

The structures of plant mitochondrial genomes are more complex than those of animals. One of the reasons for this is that plant mitochondrial genomes typically have many long and short repeated sequences and intra- and intermolecular recombination may create various DNA molecules in this organelle. Recombination may sometimes create a novel gene that causes cytoplasmic male sterility (CMS).The onion has several cytoplasm types, with some causing CMS while others do not. The complete mitochondrial genome sequence of the onion was reported for an inbred line with CMS-S cytoplasm; however, the number of differences between onion strains remains unclear, and studies on purified mitochondrial DNA (mtDNA) have not yet been performed. Furthermore, analyses of transcripts in the mitochondrial genome have not been conducted.In the present study, we examined the mitochondrial genome of the onion variety “Momiji-3” (Allium cepa L.) possessing CMS-S-type cytoplasm using next-generation sequencing (NGS). The “Momiji-3” mitochondrial genome mainly exists as three circles as a result of recombination through repeated sequences and we herein succeeded for the first time in visualizing its structure using pulsed field gel electrophoresis (PFGE). The ability to clarify the structure of the mitochondrial genome is rare in plant mitochondria; therefore, “Momiji-3” represents a good example for elucidating complex plant mitochondrial genomes.We also mapped transcript data to the mitochondrial genome in order to identify the RNA-editing positions in all gene-coding regions and estimate the expression levels of genes. We identified 635 editing positions in gene-coding regions. Start and stop codons were created by RNA editing in six genes (nad1, nad4L, atp6, atp9, ccmFC, and orf725). The transcript amounts of novel open reading frames (ORFs) were all markedly lower than those of functional genes. These results suggest that a new functional gene was not present in the mitochondrial genome of “Momiji-3”, and that the candidate gene for CMS is orf725, as previously reported.

Whole-genome sequencing of Brassica oleracea var. capitata reveals new diversity of the mitogenome.

Plant mitochondrial genomes (mtDNAs) vary in sequence structure. We assembled the Brassica oleracea var. capitata mtDNA using a mean coverage depth of 25X whole genome sequencing (WGS) and confirmed the presence of eight contigs/fragments by BLASTZ using the previously reported KJ820683 and AP012988 mtDNA as reference. Assembly of the mtDNA sequence reads resulted in a circular structure of 219,975 bp. Our assembled mtDNA, NCBI acc. no. KU831325, contained 34 protein-coding genes, 3 rRNA genes, and 19 tRNA genes with similarity to the KJ820683 and AP012988 reference mtDNA. No large repeats were found in the KU831325 assembly. However, KU831325 showed differences in the arrangement of bases at different regions compared to the previously reported mtDNAs. In the reference mtDNAs KJ820683 and AP012988, contig/fragment number 4 is partitioned into two contigs/fragments, 4a and 4b. However, contig/fragment number 4 was a single contig/fragment with 29,661 bp in KU831325. PCR and qRT-PCR using flanking markers from separate parts of contig/fragment number 4 confirmed it to be a single contig/fragment. In addition, genome re-alignment of the plastid genome and mtDNAs supported the presence of heteroplasmy and reverse arrangement of the heteroplasmic blocks within the other mtDNAs compared to KU831325 that might be one of the causal factors for its diversity. Our results thus confirm the existence of different mtDNAs in diverse B. oleracea subspecies.

Identification of a unique insertion in plant organellar DNA polymerases responsible for 5′-dRP lyase and strand-displacement activities: Implications for Base Excision Repair

Plant mitochondrial and chloroplast genomes encode essential proteins for oxidative phosphorylation and photosynthesis. For proper cellular function, plant organelles must ensure genome integrity. Although plant organelles repair damaged DNA using the multi-enzyme Base Excision Repair (BER) pathway, the details of this pathway in plant organelles are largely unknown. The initial enzymatic steps in BER produce a 5′-deoxyribose phosphate (5′-dRP) moiety that must be removed to allow DNA ligation and in plant organelles, the enzymes responsible for the removal of a 5′-dRP group are unknown. In metazoans, DNA polymerases (DNAPs) remove the 5′-dRP moiety using their intrinsic lyase and/or strand-displacement activities during short or long-patch BER sub-pathways, respectively. The plant model Arabidopsis thaliana encodes two family-A DNAPs paralogs, AtPolIA and AtPolIB, which are the sole DNAPs in plant organelles identified to date. Herein we demonstrate that both AtPolIs present 5′-dRP lyase activities. AtPolIB performs efficient strand-displacement on a BER-associated 1-nt gap DNA substrate, whereas AtPolIA exhibits only moderate strand-displacement activity. Both lyase and strand-displacement activities are dependent on an amino acid insertion that is exclusively present in plant organellar DNAPs. Within this insertion, we identified that residue AtPollB-Lys593 acts as nucleophile for lyase activity. Our results demonstrate that AtPolIs are functionally equipped to play a role in short-patch BER and suggest a major role of AtPolIB in a predicted long-patch BER sub-pathway. We propose that the acquisition of insertion 1 in the polymerization domain of AtPolIs was a key component in their evolution as BER associated and replicative DNAPs.

Complete mitochondrial genome sequence of Anthoceros angustus: conservative evolution of the mitogenomes in hornworts

The architecture and composition of the mitochondrial genome of bryophytes is currently considered much more stable and invariant than that of vascular plants. For example, liverworts and mosses appear to have nearly static mitochondrial genomes, whereas neither of two vascular plant mitogenomes sequenced to date share the same gene order. By contrast, the two available mitogenomes of hornworts, Nothoceros aenigmaticus and Phaeoceros laevis, show high consistency in gene content but differ in their gene order. To explore the patterns of hornwort mitogenome structure at a broader phylogenetic breadth and provide insights into plant mitogenome evolution, we assembled the complete mitochondrial genome from a third hornwort species, Anthoceros angustus Steph. Its mitogenome comprises 242,410 base pairs, and encodes 21 protein coding genes, three rRNA genes and 20 tRNA genes, with 38 introns disrupting 16 protein-coding genes, including three newly described introns, making it the most intron-rich plant mitochondrial genome determined to date. A total of ten putatively pseudogenized protein coding genes were found in Anthoceros; eight of them are shared with Nothoceros and Phaeoceros. The genic composition of the Anthoceros mitogenome differs from that of the other two hornworts by only one or two gene insertions or losses. The gene order in the Anthoceros mtDNA differs from that in Phaeoceros and Nothoceros by only one and three events of inversion or translocations, respectively. Overall, hornworts appear to have kept a stable mt genome structure, which could have characterized early land plant mt genome evolution.

A RAPID AND EFFICIENT METHOD FOR THE ISOLATION OF MITOCHONDRIAL DNA FROM SMALL AMOUNTS OF SOME FRUIT TREES

the plant mitochondrial genome is a source of potentially useful markers for studies of phylogeny and population genetics. The isolation of high quality mtDNA from plants containing a high content of polyphenolics has been a difficult problem because the presence of metabolites that interfere with DNA isolation procedures and downstream applications such as PCR and DNA restriction. The present study describes a rapid and easy protocol that provides mtDNA of sufficient purity from small amounts (milligrams to 1gram) leaves tissue from different species of fruit trees such as pear (Pyrus betulaefolia), apple (Anna), olive (Kalamata), mango (Sukkary), and banana (Grand Nain), containing a high content of polyphenolics. The purity of mtDNA was confirmed by PCR amplification of genomic, mitochondrial gene and RAPD profiles. The mitochondrial COXIII gene of 400 bp was amplified while the genomic β-actin gene was not amplified. The technique is fast, inexpensive, reproducible, and suitable for PCR-based markers.

Mitochondrial genome evolution in Alismatales: Size reduction and extensive loss of ribosomal protein genes.

The order Alismatales is a hotspot for evolution of plant mitochondrial genomes characterized by remarkable differences in genome size, substitution rates, RNA editing, retrotranscription, gene loss and intron loss. Here we have sequenced the complete mitogenomes of Zostera marina and Stratiotes aloides, which together with previously sequenced mitogenomes from Butomus and Spirodela, provide new evolutionary evidence of genome size reduction, gene loss and transfer to the nucleus. The Zostera mitogenome includes a large portion of DNA transferred from the plastome, yet it is the smallest known mitogenome from a non-parasitic plant. Using a broad sample of the Alismatales, the evolutionary history of ribosomal protein gene loss is analyzed. In Zostera almost all ribosomal protein genes are lost from the mitogenome, but only some can be found in the nucleus.

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.

Reconstructing the plant mitochondrial genome for marker discovery: a case study using Pinus.

Whole-genome-shotgun (WGS) sequencing of total genomic DNA was used to recover ~1Mbp of novel mitochondrial (mtDNA) sequence from Pinus sylvestris (L.) and three members of the closely related Pinus mugo species complex. DNA was extracted from megagametophyte tissue from six mother trees from locations across Europe, and 100-bp paired-end sequencing was performed on the Illumina HiSeq platform. Candidate mtDNA sequences were identified by their size and coverage characteristics, and by comparison with published plant mitochondrial genomes. Novel variants were identified, and primers targeting these loci were trialled on a set of 28 individuals from across Europe. In total, 31 SNP loci were successfully resequenced, characterizing 15 unique haplotypes. This approach offers a cost-effective means of developing marker resources for mitochondrial genomes in other plant species where reference sequences are unavailable.

Plant Mitochondrial Genome Evolution and Cytoplasmic Male Sterility

ABSTRACTMitochondria are responsible for providing energy currency to life processes in the molecular form of ATP and are therefore typically referred to as the power factories of cells. Plant mitochondria are also relevant to the common phenomenon of cytoplasmic male sterility, which is agronomically important in various crop species. Cytoplasmic male sterility (CMS) is a complex trait that may be influenced by patterns of mitochondrial genome evolution, and by intergenomic gene transfer among the organellar and nuclear compartments of plant cells. Here, we review patterns and processes that shape plant mitochondrial genomes, some relevant interactions between organelles, and the general features shared by the majority of cytoplasmic male-sterile genes in plants to further the goal of understanding CMS.

Plant mitochondrial DNA.

Plants possess mitochondrial genomes that are large and complex compared to animals. Nearly all animal mitochondrial genomes are about 16.5. kbp in length, whereas plant mitochondrial genomes range between 200-2,000 kbp. This is curious if we assume modern mitochondria originated from a common alpha-proteobacterial ancestor. Despite their size, plant mitochondrial genomes do not contain significantly more genes than their animal counterparts. Most of the additional DNA found in plant mitochondrial genomes consists of large introns, repeats and non-coding regions. Furthermore, plant mtDNA does not exist as large circular DNA molecules but mostly as a collection of linear DNA with combinations of smaller circular and branched molecules. Studies into these highly fragmented genomes heavily imply that recombination is the main mechanism driving replication of plant mtDNA.

Plant mitochondrial genomes and attempts to transform them

Plant mitochondrial genomes and attempts to transform them

Novel CMS lines in pigeonpea [Cajanus cajan (L.) Millspaugh] derived from cytoplasmic substitutions, and their effective restoration and deployment in hybrid breeding

The availability of stable cytoplasmic male sterile (CMS or A) lines coupled with a robust restoration system (R lines) is an essential prerequisite for efficient hybrid breeding. CMS-enabled hybrid technology holds immense potential to enhance the long-stagnant productivity of pigeonpea. In the present investigation, cytoplasmic substitutions were made in the nuclear backgrounds of early-maturing pigeonpea varieties or lines. Three new CMS lines (ICPL 88039A, Pusa 992A, and DPP 3-2A) resulted from genetic crosses involving cytoplasmic donors from A2 (GT 288A) and A4 (ICPA 2089) categories. In addition to visual inspection of anthers, pollen-staining techniques and scanning electron microscopy (SEM) analysis were used to confirm pollen sterility. Further, given the relevance of the plant mitochondrial genome to CMS manifestation, 25 mitochondrion-specific DNA markers were assayed on these newly developed A lines and isogenic maintainer (B) lines. DNA polymorphism between Pusa 992A and Pusa 992B as revealed by the nad7a_del marker confirmed the successful combination of sterilizing cytoplasm (A4) and nonrestoring nuclear background (Pusa 992). Such cytoplasm-specific DNA markers are required for A2-CMS as well. Further, to assess restoration ability, potential restorers were crossed with these CMS lines, and as a consequence, promising A×R combinations exhibiting 100% pollen fertility could be identified. In parallel, we also analyzed the inheritance patterns underlying fertility restoration using ICPL 88039A-derived F2 and BC1F1 populations, and established a monogenic dominant model to explain the phenomenon of A2-CMS restoration. In summary, we report the successful development of new CMS lines and describe their effective deployment in hybrid breeding of pigeonpea.

Assembly and analysis of the complete Salix purpurea L. (Salicaceae) mitochondrial genome sequence.

Plant mitochondrial (mt) genomes possess several complex features, including a variable size, a dynamic genome structure, and complicated patterns of gene loss and gain throughout evolutionary history. Studies of plant mt genomes can, therefore, provide unique insights into organelle evolution. We assembled the complete Salix purpurea L. mt genome by screening genomic sequence reads generated by a Roche-454 pyrosequencing platform. The pseudo-molecule obtained has a typical circular structure 598,970 bp long, with an overall GC content of 55.06%. The S. purpurea mt genome contains 52 genes: 31 protein-coding, 18 tRNAs, and three rRNAs. Eighteen tandem repeats and 404 microsatellites are distributed unevenly throughout the S. purpurea mt genome. A phylogenetic tree of 23 representative terrestrial plants strongly supports S. purpurea inclusion in the Malpighiales clade. Our analysis contributes toward understanding the organization and evolution of organelle genomes in Salicaceae species.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-016-3521-6) contains supplementary material, which is available to authorized users.

Multi-step formation, evolution, and functionalization of new cytoplasmic male sterility genes in the plant mitochondrial genomes.

New gene origination is a major source of genomic innovations that confer phenotypic changes and biological diversity. Generation of new mitochondrial genes in plants may cause cytoplasmic male sterility (CMS), which can promote outcrossing and increase fitness. However, how mitochondrial genes originate and evolve in structure and function remains unclear. The rice Wild Abortive type of CMS is conferred by the mitochondrial gene WA352c (previously named WA352) and has been widely exploited in hybrid rice breeding. Here, we reconstruct the evolutionary trajectory of WA352c by the identification and analyses of 11 mitochondrial genomic recombinant structures related to WA352c in wild and cultivated rice. We deduce that these structures arose through multiple rearrangements among conserved mitochondrial sequences in the mitochondrial genome of the wild rice Oryza rufipogon, coupled with substoichiometric shifting and sequence variation. We identify two expressed but nonfunctional protogenes among these structures, and show that they could evolve into functional CMS genes via sequence variations that could relieve the self-inhibitory potential of the proteins. These sequence changes would endow the proteins the ability to interact with the nucleus-encoded mitochondrial protein COX11, resulting in premature programmed cell death in the anther tapetum and male sterility. Furthermore, we show that the sequences that encode the COX11-interaction domains in these WA352c-related genes have experienced purifying selection during evolution. We propose a model for the formation and evolution of new CMS genes via a "multi-recombination/protogene formation/functionalization" mechanism involving gradual variations in the structure, sequence, copy number, and function.

Localized Retroprocessing as a Model of Intron Loss in the Plant Mitochondrial Genome.

Loss of introns in plant mitochondrial genes is commonly explained by retroprocessing. Under this model, an mRNA is reverse transcribed and integrated back into the genome, simultaneously affecting the contents of introns and edited sites. To evaluate the extent to which retroprocessing explains intron loss, we analyzed patterns of intron content and predicted RNA editing for whole mitochondrial genomes of 30 species in the monocot order Alismatales. In this group, we found an unusually high degree of variation in the intron content, even expanding the hitherto known variation among angiosperms. Some species have lost some two-third of the cis-spliced introns. We found a strong correlation between intron content and editing frequency, and detected 27 events in which intron loss is consistent with the presence of nucleotides in an edited state, supporting retroprocessing. However, we also detected seven cases of intron loss not readily being explained by retroprocession. Our analyses are also not consistent with the entire length of a fully processed cDNA copy being integrated into the genome, but instead indicate that retroprocessing usually occurs for only part of the gene. In some cases, several rounds of retroprocessing may explain intron loss in genes completely devoid of introns. A number of taxa retroprocessing seem to be very common and a possibly ongoing process. It affects the entire mitochondrial genome.

Monilophyte mitochondrial rps1 genes carry a unique group II intron that likely originated from an ancient paralog in rpl2.

Intron patterns in plant mitochondrial genomes differ significantly between the major land plant clades. We here report on a new, clade-specific group II intron in the rps1 gene of monilophytes (ferns). This intron, rps1i25g2, is strikingly similar to rpl2i846g2 previously identified in the mitochondrial rpl2 gene of seed plants, ferns, and the lycophyte Phlegmariurus squarrosus. Although mitochondrial ribosomal protein genes are frequently subject to endosymbiotic gene transfer among plants, we could retrieve the mitochondrial rps1 gene in a taxonomically wide sampling of 44 monilophyte taxa including basal lineages such as the Ophioglossales, Psilotales, and Marattiales with the only exception being the Equisetales (horsetails). Introns rps1i25g2 and rpl2i846g2 were likewise consistently present with only two exceptions: Intron rps1i25g2 is lost in the genus Ophioglossum and intron rpl2i846g2 is lost in Equisetum bogotense. Both intron sequences are moderately affected by RNA editing. The unprecedented primary and secondary structure similarity of rps1i25g2 and rpl2i846g2 suggests an ancient retrotransposition event copying rpl2i846g2 into rps1, for which we suggest a model. Our phylogenetic analysis adding the new rps1 locus to a previous data set is fully congruent with recent insights on monilophyte phylogeny and further supports a sister relationship of Gleicheniales and Hymenophyllales.

Mitochondrial genome structure of photosynthetic eukaryotes

Current ideas of plant mitochondrial genome organization are presented. Data on the size and structural organization of mtDNA, gene content, and peculiarities are summarized. Special emphasis is given to characteristic features of the mitochondrial genomes of land plants and photosynthetic algae that distinguish them from the mitochondrial genomes of other eukaryotes. The data published before the end of 2014 are reviewed.

The Whole Genome Assembly and Comparative Genomic Research of Thellungiella parvula (Extremophile Crucifer) Mitochondrion.

The complete nucleotide sequences of the mitochondrial (mt) genome of an extremophile species Thellungiella parvula (T. parvula) have been determined with the lengths of 255,773 bp. T. parvula mt genome is a circular sequence and contains 32 protein-coding genes, 19 tRNA genes, and three ribosomal RNA genes with a 11.5% coding sequence. The base composition of 27.5% A, 27.5% T, 22.7% C, and 22.3% G in descending order shows a slight bias of 55% AT. Fifty-three repeats were identified in the mitochondrial genome of T. parvula, including 24 direct repeats, 28 tandem repeats (TRs), and one palindromic repeat. Furthermore, a total of 199 perfect microsatellites have been mined with a high A/T content (83.1%) through simple sequence repeat (SSR) analysis and they were distributed unevenly within this mitochondrial genome. We also analyzed other plant mitochondrial genomes' evolution in general, providing clues for the understanding of the evolution of organelles genomes in plants. Comparing with other Brassicaceae species, T. parvula is related to Arabidopsis thaliana whose characters of low temperature resistance have been well documented. This study will provide important genetic tools for other Brassicaceae species research and improve yields of economically important plants.

The complete mitochondrial genome of Tibetan hulless barley

Hulless barley (Hordeum vulgar L. var. nudum) is one of the staple foods for Tibetans and an important livestock feed in the Tibetan Plateau. We report the complete mitochondrial genome of Tibetan hulless barely. The complete mitochondrial genome size is 416,675 bp. Hulless barely mitochondrial genome encode 34 protein-coding genes, 19 tRNA genes and three rRNA genes. The mitochondrial genome has 50 forward and palindrome repeats totally. Nucleotide sequence of coding region takes over 13.60%, GC content is 44.33%. The maximum-likelihood (ML) phylogenetic tree based on 11 protein-coding genes common to seven plant mitochondrial genomes using Arabidopsis thaliana as out-group support that hulless barely is close to Triticum species.

Plant mitochondria contain the protein translocase subunits TatB and TatC.

Twin-arginine translocation (Tat) pathways have been well-characterized in bacteria and chloroplasts. Genes encoding a TatC protein are found in almost all plant mitochondrial genomes but to date these have not been extensively investigated. For the first time it could be demonstrated that this mitochondrial-encoded TatC is a functional gene that is translated into a protein in the model plant Arabidopsis thaliana A TatB--like subunit localized to the inner membrane was also identified that is nuclear-encoded and is essential for plant growth and development, indicating that plants potentially require a Tat pathway for mitochondrial biogenesis.