Non-coding RNA may be associated with cytoplasmic male sterility in Silene vulgaris

Abstract

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..

Fig. 1.

RNA-seq read coverages for genes and intergenic regions. Coverage was calculated based on a sliding window with a size of 75 bp and a step size of 25 bp for (A–C), and on a single base resolution for (D) and (E). Mean depth of coverage (DOC) values across individuals are given by the lines of the plots, while standard deviations in DOC are given by the blue bands. Values in (A–D) are for all six individuals examined, and in (E) they are split into female (n=3) and hermaphrodite (n=3). (A) The ccmB and rpl5 genes. (B) Exons and introns of the nad5 and nad1 genes. Antisense coverage is shown with negative y-values. (C) The matR gene with a ‘hole’ in the middle of the coding region. (D) The cob gene with transcripts lacking a stop codon. (E) FSNR, which is highly expressed in females and not expressed above background levels in hermaphrodites. The size of this region is defined according to the DOC threshold for ‘transcription islands’.

Fig. 2.

Relative expression and copy number of the selected genes and regions in mt DNA of S. vulgaris. (A) Gene expression relative to mt 18S rRNA. (B) Copy number relative to mt 18S rDNA. (C) Copy number relative to nuclear 18S rDNA. White bars are females (F), gray bars are hermaphrodites (H). B, flower buds; L, leaves; R, roots; P, pollen. Values are means ±SD, n=6.

Fig. 3.

RNA editing extent in Silene vulgaris mitochondria, Female vs. Hermaphrodite, organized by editing site location. Mean values (±SD) for females (n=3) and hermaphrodites (n=3) are plotted. A line representing equal editing extent for both genders is plotted as y=x, in red. A 95% confidence band for the observed data is given in gray within each plot. Within the intron panel, sites predicted to stabilize intron structure are indicated with filled circles, while sites not known to influence intron stability are depicted with open circles. In addition, within the intron panel, sites within cis-introns are colored red, while trans-intron sites are colored blue. Lastly, within the island panel, the editing site located in the FSNR is indicated with an open square.

Fig. 4.

Editing achieved prior to splicing, as a fraction of post-splice editing extent for all sites with sufficient pre- and post-splice reads available (n=106 sites). The x-axis shows the distance from the nearest splice junction. Downstream positions are positive, upstream positions are negative. Data are mean values (±SD) across all six individuals.

Fig. 5.

Editing extent before and after splicing for selected transcripts. Mean pre-splice edit extents are depicted on the y-axis with open circles and mean post-splice values are depicted with closed circles. Genomic coordinates are given on the x-axis. Exon boundaries are indicated above the x-axis with gray arrows and introns are depicted with open arrows. Cis-spliced introns are outlined in red and trans-spliced introns are outlined with a blue-to-gray fade, as distal trans-intron boundaries are not precisely known. In some cases, particularly within introns, only pre-splice editing extents could be calculated.