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.

• Klíčová slova
• Silene vulgaris, cytoplasmic male sterility, differential gene expression,
• MeSH
• anotace sekvence MeSH
• buněčné jádro genetika   MeSH
• down regulace genetika   MeSH
• genová ontologie MeSH
• haplotypy genetika   MeSH
• messenger RNA genetika   metabolismus   MeSH
• mitochondriální proteiny genetika   metabolismus   MeSH
• neplodnost rostlin genetika   MeSH
• oxidační stres genetika   MeSH
• oxidoreduktasy genetika   metabolismus   MeSH
• pyl genetika   MeSH
• regulace genové exprese u rostlin * MeSH
• rostlinné proteiny genetika   metabolismus   MeSH
• Silene genetika   fyziologie   MeSH
• stanovení celkové genové exprese * MeSH
• transkriptom genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• alternative oxidase MeSH Prohlížeč
• messenger RNA MeSH
• mitochondriální proteiny MeSH
• oxidoreduktasy MeSH
• rostlinné proteiny MeSH

BACKGROUND: Gynodioecious species exist in two sexes - male-sterile females and hermaphrodites. Male sterility in higher plants often results from mitonuclear interaction between the CMS (cytoplasmic male sterility) gene(s) encoded by mitochondrial genome and by nuclear-encoded restorer genes. Mitochondrial and nuclear-encoded transcriptomes in females and hermaphrodites are intensively studied, but little is known about sex-specific gene expression in plastids. We have compared plastid transcriptomes between females and hermaphrodites in two haplotypes of a gynodioecious species Silene vulgaris with known CMS candidate genes. RESULTS: We generated complete plastid genome sequences from five haplotypes S. vulgaris including the haplotypes KRA and KOV, for which complete mitochondrial genome sequences were already published. We constructed a phylogenetic tree based on plastid sequences of S. vulgaris. Whereas lowland S. vulgaris haplotypes including KRA and KOV clustered together, the accessions from high European mountains diverged early in the phylogram. S. vulgaris belongs among Silene species with slowly evolving plastid genomes, but we still detected 212 substitutions and 112 indels between two accessions of this species. We estimated elevated Ka/Ks in the ndhF gene, which may reflect the adaptation of S. vulgaris to high altitudes, or relaxed selection. We compared depth of coverage and editing rates between female and hermaphrodite plastid transcriptomes and found no significant differences between the two sexes. We identified 51 unique C to U editing sites in the plastid genomes of S. vulgaris, 38 of them in protein coding regions, 2 in introns, and 11 in intergenic regions. The editing site in the psbZ gene was edited only in one of two plastid genomes under study. CONCLUSIONS: We revealed no significant differences between the sexes in plastid transcriptomes of two haplotypes of S. vulgaris. It suggests that gene expression of plastid genes is not affected by CMS in flower buds of S. vulgaris, although both sexes may still differ in plastid gene expression in specific tissues. We revealed the difference between the plastid transcriptomes of two S. vulgaris haplotypes in editing rate and in the coverage of several antisense transcripts. Our results document the variation in plastid genomes and transcriptomes in S. vulgaris.

• MeSH
• genom plastidový genetika   MeSH
• Silene genetika   metabolismus   MeSH
• transkriptom genetika   MeSH
• Publikační typ
• časopisecké články 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
• cytochromy b genetika   metabolismus   MeSH
• editace RNA MeSH
• genom mitochondriální * MeSH
• haplotypy MeSH
• homologní rekombinace * MeSH
• membránové glykoproteiny genetika   MeSH
• mitochondriální protonové ATPasy genetika   MeSH
• mitochondrie genetika   MeSH
• neplodnost rostlin genetika   MeSH
• otevřené čtecí rámce genetika   MeSH
• rostlinné proteiny genetika   MeSH
• Silene genetika   MeSH
• transkriptom MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cytochromy b MeSH
• membránové glykoproteiny MeSH
• mitochondriální protonové ATPasy MeSH
• rostlinné proteiny MeSH

The mitochondrial DNA of diplonemid and kinetoplastid protists is known for its suite of bizarre features, including the presence of concatenated circular molecules, extensive trans-splicing and various forms of RNA editing. Here we report on the existence of another remarkable characteristic: hyper-inflated DNA content. We estimated the total amount of mitochondrial DNA in four kinetoplastid species (Trypanosoma brucei, Trypanoplasma borreli, Cryptobia helicis, and Perkinsela sp.) and the diplonemid Diplonema papillatum. Staining with 4',6-diamidino-2-phenylindole and RedDot1 followed by color deconvolution and quantification revealed massive inflation in the total amount of DNA in their organelles. This was further confirmed by electron microscopy. The most extreme case is the ∼260 Mbp of DNA in the mitochondrion of Diplonema, which greatly exceeds that in its nucleus; this is, to our knowledge, the largest amount of DNA described in any organelle. Perkinsela sp. has a total mitochondrial DNA content ~6.6× greater than its nuclear genome. This mass of DNA occupies most of the volume of the Perkinsela cell, despite the fact that it contains only six protein-coding genes. Why so much DNA? We propose that these bloated mitochondrial DNAs accumulated by a ratchet-like process. Despite their excessive nature, the synthesis and maintenance of these mtDNAs must incur a relatively low cost, considering that diplonemids are one of the most ubiquitous and speciose protist groups in the ocean. © 2018 IUBMB Life, 70(12):1267-1274, 2018.

• Klíčová slova
• DNA content, kinetoplast DNA, mitochondrial DNA, protist,
• MeSH
• Euglenozoa genetika   MeSH
• fylogeneze MeSH
• Kinetoplastida genetika   MeSH
• mitochondriální DNA genetika   izolace a purifikace   ultrastruktura   MeSH
• mitochondrie genetika   MeSH
• trans-splicing genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mitochondriální DNA MeSH

Accurate gene expression measurements are essential in studies of both crop and wild plants. Reverse transcription quantitative real-time PCR (RT-qPCR) has become a preferred tool for gene expression estimation. A selection of suitable reference genes for the normalization of transcript levels is an essential prerequisite of accurate RT-qPCR results. We evaluated the expression stability of eight candidate reference genes across roots, leaves, flower buds and pollen of Silene vulgaris (bladder campion), a model plant for the study of gynodioecy. As random priming of cDNA is recommended for the study of organellar transcripts and poly(A) selection is indicated for nuclear transcripts, we estimated gene expression with both random-primed and oligo(dT)-primed cDNA. Accordingly, we determined reference genes that perform well with oligo(dT)- and random-primed cDNA, making it possible to estimate levels of nucleus-derived transcripts in the same cDNA samples as used for organellar transcripts, a key benefit in studies of cyto-nuclear interactions. Gene expression variance was estimated by RefFinder, which integrates four different analytical tools. The SvACT and SvGAPDH genes were the most stable candidates across various organs of S. vulgaris, regardless of whether pollen was included or not.

• MeSH
• komplementární DNA genetika   MeSH
• kvantitativní polymerázová řetězová reakce metody   MeSH
• polymerázová řetězová reakce s reverzní transkripcí metody   MeSH
• rostlinné geny * MeSH
• sekvenční analýza RNA MeSH
• Silene genetika   MeSH
• stanovení celkové genové exprese MeSH
• Publikační typ
• časopisecké články MeSH
• validační studie MeSH
• Názvy látek
• komplementární DNA 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.

• Klíčová slova
• Cytoplasmic male sterility, Silene vulgaris, editing, mitochondrion, non-coding RNA, splicing, transcriptome.,
• MeSH
• editace RNA MeSH
• květy genetika   růst a vývoj   MeSH
• mitochondriální geny * MeSH
• nekódující RNA * MeSH
• neplodnost rostlin * MeSH
• rostlinné proteiny genetika   metabolismus   MeSH
• Silene genetika   fyziologie   MeSH
• transkriptom * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• nekódující RNA * MeSH
• rostlinné proteiny 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' nepřekládaná oblast genetika   MeSH
• buněčné jádro genetika   MeSH
• genetická transkripce * MeSH
• genom mitochondriální genetika   MeSH
• genom rostlinný genetika   MeSH
• genová přestavba genetika   MeSH
• křížení genetické MeSH
• messenger RNA genetika   metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• northern blotting MeSH
• otevřené čtecí rámce genetika   MeSH
• pyl genetika   MeSH
• regulace genové exprese u rostlin MeSH
• rostlinné proteiny genetika   metabolismus   MeSH
• sekvence nukleotidů MeSH
• sekvenční seřazení MeSH
• Silene genetika   MeSH
• stanovení celkové genové exprese MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 5' nepřekládaná oblast MeSH
• messenger RNA MeSH
• rostlinné proteiny MeSH