Diversity of the Arabidopsis mitochondrial genome occurs via nuclear-controlled recombination activity

Abstract

The plant mitochondrial genome is recombinogenic, with DNA exchange activity controlled to a large extent by nuclear gene products. One nuclear gene, MSH1, appears to participate in suppressing recombination in Arabidopsis at every repeated sequence ranging in size from 108 to 556 bp. Present in a wide range of plant species, these mitochondrial repeats display evidence of successful asymmetric DNA exchange in Arabidopsis when MSH1 is disrupted. Recombination frequency appears to be influenced by repeat sequence homology and size, with larger size repeats corresponding to increased DNA exchange activity. The extensive mitochondrial genomic reorganization of the msh1 mutant produced altered mitochondrial transcription patterns. Comparison of mitochondrial genomes from the Arabidopsis ecotypes C24, Col-0, and Ler suggests that MSH1 activity accounts for most or all of the polymorphisms distinguishing these genomes, producing ecotype-specific stoichiometric changes in each line. Our observations suggest that MSH1 participates in mitochondrial genome evolution by influencing the lineage-specific pattern of mitochondrial genetic variation in higher plants.

Figure 1.—

Sites of recombination in the Arabidopsis mitochondrial genome. (A) Computer-generated map of the 33 identified small mitochondrial repeats active in Arabidopsis msh1. Colored repeats represent those tested by gel blot hybridization, with corresponding colors designating interacting repeat pairs. Numbers in parentheses indicate lengths of the repeats (bp). (B) DNA gel blots (BamHI) displaying evidence of ectopic recombination at repeats D and F. A and B designate parental forms; R designates the recombinant product. Repeats D and F are proximal to genes CoxII and Atp8. Arrows indicate secondary recombination products. (C) Recombination is not evident at an imperfect repeat of 342 bp of 80% sequence homology. A and B designate the two repeats (BamHI), with arrows predicting the sizes of recombinant products. Probes used are the repeats.

Figure 2.—

Early (first-generation) and advanced-generation msh1 mutants differ in mitochondrial genome configuration. Evaluations were made with three different repeats—B, D, and F—to demonstrate recombination (A and B, parental molecules; C, recombinant molecules). Changes from early to advanced generation include altered stoichiometries, novel polymorphisms, and loss or reduction of parental forms. Of 20 repeats studied in more detail, 6 showed no further changes in the advanced mutant. For the remaining 12, later-occurring differences usually involved the loss of one of the parental forms. Data shown are from DNA gel blot hybridizations with repeats as probes.

Figure 3.—

Features of msh1-regulated recombination. (A) A gradient of recombination based on repeat size may exist. For example, molecules A and B recombine at repeat G (335 bp) to give product C, and molecules B and D (which do not hybridize with this probe) recombine at repeat L (249 bp) to give product E. The stoichiometry of recombinant product C is consistently higher than recombinant molecule E. Parental molecule A shifts to substoichiometric levels in the msh1 mutant. Two early generation (F₃) progeny show that there is initial plant-to-plant variability for the rate at which A is reduced in concentration. F₃ plants were derived from different F₂ progeny. (B) In the msh1 mutant, recombination products can serve as substrate for subsequent recombination events. Recombinant molecule C is substrate for recombination at repeat V, giving rise to recombinant molecule E. Molecule D does not hybridize to the probe used in this experiment. (C) Genetic variation is observed among F₃ progeny following recombination in Col-0 × msh1 populations. This variation is presumably the consequence of cytoplasmic sorting and often includes loss of one of the parental A or B forms (example indicated by arrow; also see panel A). All results shown are from DNA gel blot hybridization experiments.

Figure 4.—

Reciprocal recombinants can accumulate in the msh1 recA3 double mutant. Experiments using repeats D, K, and I as probes show that parental forms A and B are predominant in Col-0. In the msh1 mutant, only recombinant molecule C accumulates. In the double mutant, the reciprocal recombinant product D also accumulates.

Figure 5.—

Mitochondrial transcription is influenced by DNA rearrangement activity. RNA gel blot analysis of total RNA preparations probed with the mitochondrial coding sequence of orf452, Atp8, and CoxII to demonstrate examples of enhanced transcript levels (orf452) and novel transcripts emerging in response to mitochondrial rearrangement (Atp8 and CoxII). Ubiquitin is shown as a loading control.

Figure 6.—

Substoichiometric shifting accounts for DNA polymorphisms distinguishing Arabidopsis ecotypes. Recombination data are presented for two different repeats, B and L. In both panels, molecules A and B are predominant forms in Col-0. Molecule C is the product of A/B recombination and accumulates in the Col-0 msh1 mutant. Reciprocal recombinant D, while not visible in any ecotype in either panel, is present in very low stoichiometry and serves as substrate for recombination with molecule C in the Ler msh1 mutant to produce A and B in both panels (dashed line indicates the expected size of molecule D). Arrowheads indicate the “environments” not present in the parental ecotypes and emerging after disruption of the MSH1 locus.

Figure 7.—

Intermediate repeats within the Arabidopsis mitochondrial genome show evidence of clustering. (A) Position of the repeat clusters on the mitochondrial genome map. The y-axis represents the map position with a cluster represented by a symbol. Repeats present within each cluster are designated. (B) Four active repeats exist within an ∼1.8-kb segment that differentiates ecotype C24 from Col-0 (Forner et al. 2005). The schematic depicts dense organization of repeats E, H, L, and K. In this case, several partial (p) gene segments exist within the region that is located upstream to an intact copy of CoxIII.

Figure 8.—

Model representing the mitochondrial genomes of Arabidopsis ecotypes based on diagnostic gel blot hybridization experiments including intermediate repeat regions and an assembly script. The predominant form is represented as the larger forms, with smaller circles representing the more abundant of many substoichiometric forms. Different orientations for the same regions are represented by a different texture. The large repeat I is represented by red arrows and the large repeat II is represented by black arrows. A fragment absent in the C-24 published sequence, but present in the predominant form of Col-0 and as a subfragment in Ler, is denoted by a green triangle. The dominant form for Ler has lost one copy of large repeat I and four of the regions present in C-24. For simplicity of the depicted model, not all possible substoichiometric configurations are shown. Similarly, the various configurations arising from recombination between large repeats are omitted.