Somatic hybrids between distant species offer a remarkable model to study genomic recombination events after mitochondrial fusion. Recently, our lab described highly chimeric mitogenomes in two somatic hybrids between the Solanaceae Nicotiana tabacum and Hyoscyamus niger resulting from interparental homologous recombination. To better examine the recombination map in somatic hybrid mitochondria, we developed a more sensitive bioinformatic strategy to detect recombination activity based on high-throughput sequencing without assembling the hybrid mitogenome. We generated a new intergeneric somatic hybrid between N. tabacum and Physochlaina orientalis and re-analyzed the somatic hybrids previously generated in our lab. We inferred 213 homologous recombination events across repeats of 2.1 kb on average. Most of them (~80%) were asymmetrical, consistent with the break-induced replication (BIR) pathway. Only rare (2.74%) non-homologous events were detected. Interestingly, independent events frequently occurred in the same regions within and across somatic hybrids, suggesting the existence of recombination hotspots in plant mitogenomes. BIR is the main pathway of interparental recombination in somatic hybrid mitochondria. Findings of this study are relevant to mitogenome editing assays and to mechanistic aspects of DNA integration following mitochondrial DNA horizontal transfer events.

Plant mitochondrial transcription is initiated from multiple promoters without an apparent motif, which precludes their identification in other species based on sequence comparisons. Even though coding regions take up only a small fraction of plant mitochondrial genomes, deep RNAseq studies uncovered that these genomes are fully or nearly fully transcribed with significantly different RNA read depth across the genome. Transcriptomic analysis can be a powerful tool to understand the transcription process in diverse angiosperms, including the identification of potential promoters and co-transcribed genes or to study the efficiency of intron splicing. In this work, we analyzed the transcriptional landscape of the Arabidopsis mitochondrial genome (mtDNA) based on large-scale RNA sequencing data to evaluate the use of RNAseq to study those aspects of the transcription process. We found that about 98% of the Arabidopsis mtDNA is transcribed with highly different RNA read depth, which was elevated in known genes. The location of a sharp increase in RNA read depth upstream of genes matched the experimentally identified promoters. The continuously high RNA read depth across two adjacent genes agreed with the known co-transcribed units in Arabidopsis mitochondria. Most intron-containing genes showed a high splicing efficiency with no differences between cis and trans-spliced introns or between genes with distinct splicing mechanisms. Deep RNAseq analyses of diverse plant species will be valuable to recognize general and lineage-specific characteristics related to the mitochondrial transcription process.

Mitochondria play a crucial role in eukaryotic organisms, housing their own genome with genes vital for oxidative phosphorylation. Coordination between nuclear and mitochondrial genomes is pivotal for organelle gene expression. Splicing, editing and processing of mitochondrial transcripts are regulated by nuclear-encoded factors. Splicing efficiency (SEf) of the many group II introns present in plant mitochondrial genes is critical for mitochondrial function since a splicing defect or splicing deficiency can severely impact plant growth and development. This study investigates SEf in free-living and holoparasitic plants, focusing on 25 group II introns from 15 angiosperm species. Our comparative analyses reveal distinctive splicing patterns with holoparasites exhibiting significantly lower SEf, potentially linked to their unique evolutionary trajectory. Given the preponderance of horizontal gene transfer (HGT) in parasitic plants, we investigated the effect of HGT on SEf, such as the presence of foreign introns or foreign nuclear-encoded splicing factors. Contrary to expectations, the SEf reductions do not correlate with HGT events, suggesting that other factors are at play, such as the loss of photosynthesis or the transition to a holoparasitic lifestyle. The findings of this study broaden our understanding of the molecular evolution in parasitic plants and shed light on the multifaceted factors influencing organelle gene expression.