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The genomes of DNA-containing cell organelles (mitochondria, chloroplasts) can be laterally transmitted between organisms, a process known as organelle capture. Organelle capture often occurs in the absence of detectable nuclear introgression, and the capture mechanism is unknown. Here, we have considered horizontal genome transfer across natural grafts as a mechanism underlying chloroplast capture in plants. By grafting sexually incompatible species, we show that complete chloroplast genomes can travel across the graft junction from one species into another. We demonstrate that, consistent with reported phylogenetic evidence, replacement of the resident plastid genome by the alien genome occurs in the absence of intergenomic recombination. Our results provide a plausible mechanism for organelle capture in plants and suggest natural grafting as a path for horizontal gene and genome transfer between sexually incompatible species.

The plant genus Oenothera has played an important role in the study of plant evolution of genomes and plant defense and reproduction. Here, we build on the 1kp transcriptomic dataset by creating 44 new transcriptomes and analyzing a total of 63 transcriptomes to present a large-scale comparative study across 29 Oenothera species. Our dataset included 30.4 million reads per individual and 2.3 million transcripts on average. We used this transcriptome resource to examine genome-wide evolutionary patterns and functional diversification by searching for orthologous genes and performed gene family evolution analysis. We found wide heterogeneity in gene family evolution across the genus, with section Oenothera exhibiting the most pronounced evolutionary changes. Overall, more significant gene family expansions occurred than contractions. We also analyzed the molecular evolution of phenolic metabolism by retrieving proteins annotated for phenolic enzymatic complexes. We identified 1,568 phenolic genes arranged into 83 multigene families that varied widely across the genus. All taxa experienced rapid phenolic evolution (fast rate of genomic turnover) involving 33 gene families, which exhibited large expansions, gaining about 2-fold more genes than they lost. Upstream enzymes phenylalanine ammonia-lyase (PAL) and 4-coumaroyl: CoA ligase (4CL) accounted for most of the significant expansions and contractions. Our results suggest that adaptive and neutral evolutionary processes have contributed to Oenothera diversification and rapid gene family evolution.

Abstract Plant mitochondrial genomes display an enormous structural complexity, as recombining repeat-pairs lead to the generation of various sub-genomic molecules, rendering these genomes extremely challenging to assemble. We present a novel bioinformatic data-processing pipeline called SAGBAC (Semi-Automated Graph-Based Assembly Curator) that identifies recombinogenic repeat-pairs and reconstructs plant mitochondrial genomes. SAGBAC processes assembly outputs and applies our novel ISEIS (Iterative Sequence Ends Identity Search) algorithm to obtain a graph-based visualization. We applied this approach to three mitochondrial genomes of evening primrose (Oenothera), a plant genus used for cytoplasmic genetics studies. All identified repeat pairs were found to be flanked by two alternative and unique sequence-contigs defining so-called ‘double forks’, resulting in four possible contig-repeat‐contig combinations for each repeat pair. Based on the inferred structural models, the stoichiometry of the different contig-repeat-contig combinations was analyzed using Illumina mate-pair and PacBio RSII data. This uncovered a remarkable structural diversity of the three closely related mitochondrial genomes, as well as substantial phylogenetic variation of the underlying repeats. Our model allows predicting all recombination events and, thus, all possible sub-genomes. In future work, the proposed methodology may prove useful for the investigation of the sub-genome organization and dynamics in different tissues and at various developmental stages.

Due to reciprocal chromosomal translocations, many species of Oenothera (evening primrose) form permanent multichromosomal meiotic rings. However, regular bivalent pairing is also observed. Chiasmata are restricted to chromosomal ends, which makes homologous recombination virtually undetectable. Genetic diversity is achieved by changing linkage relations of chromosomes in rings and bivalents via hybridization and reciprocal translocations. Although the structural prerequisite for this system is enigmatic, whole-arm translocations are widely assumed to be the mechanistic driving force. We demonstrate that this prerequisite is genome compartmentation into two epigenetically defined chromatin fractions. The first one facultatively condenses in cycling cells into chromocenters negative both for histone H3 dimethylated at lysine 4 and for C-banding, and forms huge condensed middle chromosome regions on prophase chromosomes. Remarkably, it decondenses in differentiating cells. The second fraction is euchromatin confined to distal chromosome segments, positive for histone H3 lysine 4 dimethylation and for histone H3 lysine 27 trimethylation. The end-segments are deprived of canonical telomeres but capped with constitutive heterochromatin. This genomic organization promotes translocation breakpoints between the two chromatin fractions, thus facilitating exchanges of end-segments. We challenge the whole-arm translocation hypothesis by demonstrating why reciprocal translocations of chromosomal end-segments should strongly promote meiotic rings and evolution toward permanent translocation heterozygosity. Reshuffled endsegments, each possessing a major crossover hot spot, can furthermore explain meiotic compatibility between genomes with different translocation histories.

In most eukaryotes, organellar genomes are transmitted preferentially by the mother, but molecular mechanisms and evolutionary forces underlying this fundamental biological principle are far from understood. It is believed that biparental inheritance promotes competition between the cytoplasmic organelles and allows the spread of so-called selfish cytoplasmic elements. Those can be, for example, fast-replicating or aggressive chloroplasts (plastids) that are incompatible with the hybrid nuclear genome and therefore maladaptive. Here we show that the ability of plastids to compete against each other is a metabolic phenotype determined by extremely rapidly evolving genes in the plastid genome of the evening primrose Oenothera. Repeats in the regulatory region of accD (the plastid-encoded subunit of the acetyl-CoA carboxylase, which catalyzes the first and rate-limiting step of lipid biosynthesis), as well as in ycf2 (a giant reading frame of still unknown function), are responsible for the differences in competitive behavior of plastid genotypes. Polymorphisms in these genes influence lipid synthesis and most likely profiles of the plastid envelope membrane. These in turn determine plastid division and/or turnover rates and hence competitiveness. This work uncovers cytoplasmic drive loci controlling the outcome of biparental chloroplast transmission. Here, they define the mode of chloroplast inheritance, as plastid competitiveness can result in uniparental inheritance (through elimination of the “weak” plastid) or biparental inheritance (when two similarly “strong” plastids are transmitted).

Spontaneous plastome mutants have been used as a research tool since the beginning of genetics. However, technical restrictions have severely limited their contributions to research in physiology and molecular biology. Here, we used full plastome sequencing to systematically characterize a collection of 51 spontaneous chloroplast mutants in Oenothera (evening primrose). Most mutants carry only a single mutation. Unexpectedly, the vast majority of mutations do not represent single nucleotide polymorphisms but are insertions/deletions originating from DNA replication slippage events. Only very few mutations appear to be caused by imprecise double-strand break repair, nucleotide misincorporation during replication, or incorrect nucleotide excision repair following oxidative damage. U-turn inversions were not detected. Replication slippage is induced at repetitive sequences that can be very small and tend to have high A/T content. Interestingly, the mutations are not distributed randomly in the genome. The underrepresentation of mutations caused by faulty double-strand break repair might explain the high structural conservation of seed plant plastomes throughout evolution. In addition to providing a fully characterized mutant collection for future research on plastid genetics, gene expression, and photosynthesis, our work identified the spectrum of spontaneous mutations in plastids and reveals that this spectrum is very different from that in the nucleus.

The fate of plastid DNA (ptDNA) during leaf development has become a matter of contention. Reports on little change in ptDNA copy number per cell contrast with claims of complete or nearly complete DNA loss already in mature leaves. We employed highresolution fluorescence microscopy, transmission electron microscopy, semithin sectioning of leaf tissue, and real-time quantitative PCR to study structural and quantitative aspects of ptDNA during leaf development in four higher plant species (Arabidopsis thaliana, sugar beet [Beta vulgaris], tobacco [Nicotiana tabacum], and maize [Zea mays]) for which controversial findings have been reported. Our data demonstrate the retention of substantial amounts of ptDNA in mesophyll cells until leaf necrosis. In ageing and senescent leaves of Arabidopsis, tobacco, and maize, ptDNA amounts remain largely unchanged and nucleoids visible, in spite of marked structural changes during chloroplast-to-gerontoplast transition. This excludes the possibility that ptDNA degradation triggers senescence. In senescent sugar beet leaves, reduction of ptDNA per cell to ~30% was observed reflecting primarily a decrease in plastid number per cell rather than a decline in DNA per organeile, as reported previously. Our findings are at variance with reports claiming loss of ptDNA at or after leaf maturation.

Due to translocation heterozygosity for all chromosomes in the cell complement, the oyster plant (Tradescantia spathacea) forms a complete meiotic ring. It also shows Rabl-arrangement at interphase, featured by polar centromere clustering. We demonstrate that the pericentromeric regions of the oyster plant are homogenized in concert by three subtelomeric sequences: 45S rDNA, (TTTAGGG)n motif, and TSrepI repeat. The Rabl-based clustering of pericentromeric regions may have been an excellent device to combine the subtelomere-pericentromere sequence migration (via inversions) with the pericentromere-pericentromere DNA movement (via whole arm translocations) that altogether led to the concerted homogenization of all the pericentromeric domains by the subtelomeric sequences. We also show that the repetitive sequence landscape of interstitial chromosome regions contains many loci consisting of Arabidopsis-type telomeric sequence or of TSrepI repeat, and it is extensively heterozygous. However, the sequence arrangement on some chromosomal arms suggest segmental inversions that are fully or partially homozygous, a fact that could be explained if the inversions started to create linkages already in a bivalent-forming ancestor. Remarkably, the subterminal TSrepI loci reside exclusively on the longer arms that could be due to sharing sequences between similarly-sized chromosomal arms in the interphase nucleus. Altogether, our study spotlights the supergene system of the oyster plant as an excellent model to link complex chromosome rearrangements, evolution of repetitive sequences, and nuclear architecture.

Plastid genomes (plastomes) are part of the integrated compartmentalised genetic system of photoautotrophic eukaryotes. They are highly redundant and generally dispersed in several regions (nucleoids) within organelles. DNA quantities and number of DNA-containing regions per plastid vary and are developmentally regulated in a way not yet understood. Reliable quantitative data describing these patterns are scarce. We present a protocol to isolate fractions of pure plastids with varying average sizes from leaflets (≤1 mm) and leaves of different developmental stages continuously up to maturity (25 cm) from Beta vulgaris L. (sugar beet) to determine DNA amounts per organelle. The approach is based on plastid purification from homogenates of moderately fixed tissue by differential and isopycnic gradient centrifugations and on application of two different DNA specific colorimetric reactions after removing potentially interfering compounds. The sensitive fluorochrome DAPI (4′,6-diamidino-2-phenylindole) was used to estimate numbers and emission intensity of nucleoids per plastid. The amounts determined ranged from 0.15 to 4.9 × 10⁻² pg DNA for plastids of 1→8 μm average diameter, corresponding from approximately a dozen to 330 genome equivalents per organelle and on average four to seven copies per nucleoid. The ratio of plastid/nuclear DNA changed continuously during leaf development from as little as 0.4% to about 20% in fully developed leaves. On the other hand, mesophyll cells of mature leaves differing in ploidy (di-, tri- and tetraploid) appeared to maintain a relatively constant nuclear genome/plastome ratio, equivalent to about 1,700 copies per C-value.

The plant genus Oenothera has played an important role in the study of plant evolution of genomes and plant defense and reproduction. Here, we build on the 1kp transcriptomic dataset by creating 44 new transcriptomes and analyzing a total of 63 transcriptomes to present a large-scale comparative study across 29 Oenothera species. Our dataset included 30.4 million reads per individual and 2.3 million transcripts on average. We used this transcriptome resource to examine genome-wide evolutionary patterns and functional diversification by searching for orthologous genes and performed gene family evolution analysis. We found wide heterogeneity in gene family evolution across the genus, with section Oenothera exhibiting the most pronounced evolutionary changes. Overall, more significant gene family expansions occurred than contractions. We also analyzed the molecular evolution of phenolic metabolism by retrieving proteins annotated for phenolic enzymatic complexes. We identified 1,568 phenolic genes arranged into 83 multigene families that varied widely across the genus. All taxa experienced rapid phenolic evolution (fast rate of genomic turnover) involving 33 gene families, which exhibited large expansions, gaining about 2-fold more genes than they lost. Upstream enzymes phenylalanine ammonia-lyase (PAL) and 4-coumaroyl: CoA ligase (4CL) accounted for most of the significant expansions and contractions. Our results suggest that adaptive and neutral evolutionary processes have contributed to Oenothera diversification and rapid gene family evolution.