Cytoplasmic male sterility (CMS), encoded by the interacting mitochondrial and nuclear genes, causes pollen abortion or non-viability. CMS is widely used in agriculture and extensively studied in crops. Much less is known about CMS in wild species. We performed a comparative transcriptomic analysis of male sterile and fertile individuals of Silene vulgaris, a model plant for the study of gynodioecy, to reveal the genes responsible for pollen abortion in this species. We used RNA-seq datasets previously employed for the analysis of mitochondrial and plastid transcriptomes of female and hermaphrodite flower buds, making it possible to compare the transcriptomes derived from three genomes in the same RNA specimen. We assembled de novo transcriptomes for two haplotypes of S. vulgaris and identified differentially expressed genes between the females and hermaphrodites, associated with stress response or pollen development. The gene for alternative oxidase was downregulated in females. The genetic pathways controlling CMS in S. vulgaris are similar to those in crops. The high number of the differentially expressed nuclear genes contrasts with the uniformity of organellar transcriptomes across genders, which suggests these pathways are evolutionarily conserved and that selective mechanisms may shield organellar transcription against changes in the cytoplasmic transcriptome.

• Keywords
• Silene vulgaris, cytoplasmic male sterility, differential gene expression,
• MeSH
• Molecular Sequence Annotation MeSH
• Cell Nucleus genetics   MeSH
• Down-Regulation genetics   MeSH
• Gene Ontology MeSH
• Haplotypes genetics   MeSH
• RNA, Messenger genetics   metabolism   MeSH
• Mitochondrial Proteins genetics   metabolism   MeSH
• Plant Infertility genetics   MeSH
• Oxidative Stress genetics   MeSH
• Oxidoreductases genetics   metabolism   MeSH
• Pollen genetics   MeSH
• Gene Expression Regulation, Plant * MeSH
• Plant Proteins genetics   metabolism   MeSH
• Silene genetics   physiology   MeSH
• Gene Expression Profiling * MeSH
• Transcriptome genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• alternative oxidase MeSH Browser
• RNA, Messenger MeSH
• Mitochondrial Proteins MeSH
• Oxidoreductases MeSH
• Plant Proteins MeSH

BACKGROUND: Silene vulgaris (bladder campion) is a gynodioecious species existing as two genders - male-sterile females and hermaphrodites. Cytoplasmic male sterility (CMS) is generally encoded by mitochondrial genes, which interact with nuclear fertility restorer genes. Mitochondrial genomes of this species vary in DNA sequence, gene order and gene content. Multiple CMS genes are expected to exist in S. vulgaris, but little is known about their molecular identity. RESULTS: We assembled the complete mitochondrial genome from the haplotype KRA of S. vulgaris. It consists of five chromosomes, two of which recombine with each other. Two small non-recombining chromosomes exist in linear, supercoiled and relaxed circle forms. We compared the mitochondrial transcriptomes from females and hermaphrodites and confirmed the differentially expressed chimeric gene bobt as the strongest CMS candidate gene in S. vulgaris KRA. The chimeric gene bobt is co-transcribed with the Cytochrome b (cob) gene in some genomic configurations. The co-transcription of a CMS factor with an essential gene may constrain transcription inhibition as a mechanism for fertility restoration because of the need to maintain appropriate production of the necessary protein. Homologous recombination places the gene cob outside the control of bobt, which allows for the suppression of the CMS gene by the fertility restorer genes. We found the loss of three editing sites in the KRA mitochondrial genome and identified four sites with highly distinct editing rates between KRA and another S. vulgaris haplotypes (KOV). Three of these highly differentially edited sites were located in the transport membrane protein B (mttB) gene. They resulted in differences in MttB protein sequences between haplotypes. CONCLUSIONS: Frequent homologous recombination events that are widespread in plant mitochondrial genomes may change chromosomal configurations and also the control of gene transcription including CMS gene expression. Posttranscriptional processes, e.g. RNA editing shall be evaluated in evolutionary and co-evolutionary studies of mitochondrial genes, because they may change protein composition despite the sequence identity of the respective genes. The investigation of natural populations of wild species such as S. vulgaris are necessary to reveal important aspects of CMS missed in domesticated crops, the traditional focus of the CMS studies.

• MeSH
• Cytochromes b genetics   metabolism   MeSH
• RNA Editing MeSH
• Genome, Mitochondrial * MeSH
• Haplotypes MeSH
• Homologous Recombination * MeSH
• Membrane Glycoproteins genetics   MeSH
• Mitochondrial Proton-Translocating ATPases genetics   MeSH
• Mitochondria genetics   MeSH
• Plant Infertility genetics   MeSH
• Open Reading Frames genetics   MeSH
• Plant Proteins genetics   MeSH
• Silene genetics   MeSH
• Transcriptome MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Cytochromes b MeSH
• Membrane Glycoproteins MeSH
• Mitochondrial Proton-Translocating ATPases MeSH
• Plant Proteins MeSH

Cytoplasmic male sterility (CMS) is a widespread phenomenon in flowering plants caused by mitochondrial (mt) genes. CMS genes typically encode novel proteins that interfere with mt functions and can be silenced by nuclear fertility-restorer genes. Although the molecular basis of CMS is well established in a number of crop systems, our understanding of it in natural populations is far more limited. To identify CMS genes in a gynodioecious plant, Silene vulgaris, we constructed mt transcriptomes and compared transcript levels and RNA editing patterns in floral bud tissue from female and hermaphrodite full siblings. The transcriptomes from female and hermaphrodite individuals were very similar overall with respect to variation in levels of transcript abundance across the genome, the extent of RNA editing, and the order in which RNA editing and intron splicing events occurred. We found only a single genomic region that was highly overexpressed and differentially edited in females relative to hermaphrodites. This region is not located near any other transcribed elements and lacks an open-reading frame (ORF) of even moderate size. To our knowledge, this transcript would represent the first non-coding mt RNA associated with CMS in plants and is, therefore, an important target for future functional validation studies.

• Keywords
• Cytoplasmic male sterility, Silene vulgaris, editing, mitochondrion, non-coding RNA, splicing, transcriptome.,
• MeSH
• RNA Editing MeSH
• Flowers genetics   growth & development   MeSH
• Genes, Mitochondrial * MeSH
• RNA, Untranslated * MeSH
• Plant Infertility * MeSH
• Plant Proteins genetics   metabolism   MeSH
• Silene genetics   physiology   MeSH
• Transcriptome * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Names of Substances
• RNA, Untranslated * MeSH
• Plant Proteins MeSH

An extraordinary variation in mitochondrial DNA sequence exists in angiosperm Silene vulgaris. The atp1 gene is flanked by very variable regions, as deduced from four completely sequenced mitochondrial genomes of this species. This diversity contributed to a highly variable transcript profile of this gene observed across S. vulgaris populations. We examined the atp1 transcript in the KOV mitochondrial genome and found three 5' ends, created most likely by the combination of transcription initiation and RNA processing. Most atp1 transcripts terminated about 70 bp upstream of the translation stop codon, which was present in only 10 % of them. Controlled crosses between a KOV mother and a geographically distant pollen donor (Krasnoyarsk, Russia) showed that nuclear background also affected atp1 transcription. The distant pollen donor introduced the factor(s) preventing the formation of a long 2,100 nt-transcript, because this long atp1 transcript reappeared in the progeny from self-crosses. The highly rearranged mitochondrial genomes with a variation in gene flanking regions make S. vulgaris an excellent model for the study of mitochondrial gene expression in plants.

• MeSH
• 5' Untranslated Regions genetics   MeSH
• Cell Nucleus genetics   MeSH
• Transcription, Genetic * MeSH
• Genome, Mitochondrial genetics   MeSH
• Genome, Plant genetics   MeSH
• Gene Rearrangement genetics   MeSH
• Crosses, Genetic MeSH
• RNA, Messenger genetics   metabolism   MeSH
• Molecular Sequence Data MeSH
• Blotting, Northern MeSH
• Open Reading Frames genetics   MeSH
• Pollen genetics   MeSH
• Gene Expression Regulation, Plant MeSH
• Plant Proteins genetics   metabolism   MeSH
• Base Sequence MeSH
• Sequence Alignment MeSH
• Silene genetics   MeSH
• Gene Expression Profiling MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 5' Untranslated Regions MeSH
• RNA, Messenger MeSH
• Plant Proteins MeSH

Chimeric genes are significant sources of evolutionary innovation that are normally created when portions of two or more protein coding regions fuse to form a new open reading frame. In plant mitochondria astonishingly high numbers of different novel chimeric genes have been reported, where they are generated through processes of rearrangement and recombination. Nonetheless, because most studies do not find or report nucleotide variation within the same chimeric gene, evolution after the origination of these chimeric genes remains unstudied. Here we identify two alleles of a complex chimera in Silene vulgaris that are divergent in nucleotide sequence, genomic position relative to other mitochondrial genes, and expression patterns. Structural patterns suggest a history partially influenced by gene conversion between the chimeric gene and functional copies of subunit 1 of the mitochondrial ATP synthase gene (atp1). We identified small repeat structures within the chimeras that are likely recombination sites allowing generation of the chimera. These results establish the potential for chimeric gene divergence in different plant mitochondrial lineages within the same species. This result contrasts with the absence of diversity within mitochondrial chimeras found in crop species.

• MeSH
• Alleles MeSH
• DNA Primers genetics   MeSH
• Species Specificity MeSH
• Phylogeny MeSH
• Transcription, Genetic MeSH
• Genetic Variation MeSH
• Genome, Plant MeSH
• Codon MeSH
• Crosses, Genetic MeSH
• RNA, Messenger metabolism   MeSH
• Genes, Mitochondrial * MeSH
• Models, Genetic MeSH
• Evolution, Molecular MeSH
• Mosaicism MeSH
• Polymerase Chain Reaction MeSH
• Likelihood Functions MeSH
• Arabidopsis Proteins genetics   MeSH
• Proton-Translocating ATPases genetics   MeSH
• Gene Expression Regulation MeSH
• Recombination, Genetic MeSH
• Genes, Plant MeSH
• Silene genetics   MeSH
• Blotting, Southern MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Names of Substances
• ATP1 protein, Arabidopsis MeSH Browser
• DNA Primers MeSH
• Codon MeSH
• RNA, Messenger MeSH
• Arabidopsis Proteins MeSH
• Proton-Translocating ATPases MeSH

BACKGROUND: Although rapid changes in copy number and gene order are common within plant mitochondrial genomes, associated patterns of gene transcription are underinvestigated. Previous studies have shown that the gynodioecious plant species Silene vulgaris exhibits high mitochondrial diversity and occasional paternal inheritance of mitochondrial markers. Here we address whether variation in DNA molecular markers is correlated with variation in transcription of mitochondrial genes in S. vulgaris collected from natural populations. RESULTS: We analyzed RFLP variation in two mitochondrial genes, cox1 and atp1, in offspring of ten plants from a natural population of S. vulgaris in Central Europe. We also investigated transcription profiles of the atp1 and cox1 genes. Most DNA haplotypes and transcription profiles were maternally inherited; for these, transcription profiles were associated with specific mitochondrial DNA haplotypes. One individual exhibited a pattern consistent with paternal inheritance of mitochondrial DNA; this individual exhibited a transcription profile suggestive of paternal but inconsistent with maternal inheritance. We found no associations between gender and transcript profiles. CONCLUSIONS: Specific transcription profiles of mitochondrial genes were associated with specific mitochondrial DNA haplotypes in a natural population of a gynodioecious species S. vulgaris.Our findings suggest the potential for a causal association between rearrangements in the plant mt genome and transcription product variation.

• MeSH
• DNA, Plant genetics   MeSH
• Haplotypes * MeSH
• DNA, Mitochondrial genetics   MeSH
• Genes, Mitochondrial * MeSH
• Polymorphism, Restriction Fragment Length MeSH
• Genetics, Population * MeSH
• Sequence Analysis, DNA MeSH
• Silene genetics   MeSH
• Gene Expression Profiling * MeSH
• Inheritance Patterns MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Plant MeSH
• DNA, Mitochondrial MeSH