Figure 6. Chronic exposure to paraquat causes irreversible mitochondrial impairment by sustained mtDNA editing.

(A) Paraquat adapting cell populations (G=generations of exposure to paraquat) ultimately lose their capacity to recover respiratory (glycerol) growth (right y-axis, purple line, log₂ doubling time relative to founder) and the loss coincides with the genetic fixation of the paraquat adaptation (left y-axis, green line). Shade: S.E.M. of 5 populations, each measured at n=5. (B) All but one (ρ⁺) sequenced cell population adapted to long-term paraquat stress (t₅₀) retain only small (6–30 kb; ρ^-) or very small (<2 kb, ρ^--) mtDNA segments. Panels: Representative populations. y-axis: mtDNA copy number (median coverage in 1 kb windows relative to haploid nuclear genome). Gene positions are indicated. (C) Number of ρ^- populations after long-term paraquat exposure (t₅₀) in which the specified mtDNA gene was lost. (D) The ρ^-- populations became less fit than the ρ^- populations during a long-term exposure to paraquat. See also Figure 6—figure supplements 1–5.

Figure 6—figure supplement 1. Long-term exposure to paraquat causes genetic fixation of adaptation.

We released 96 populations from paraquat exposure after 6, 10, 19, 24, 33, and 242 generations (means) of adaptation (panels), and by re-exposing the populations to paraquat after a given number of generations of growth on a paraquat-free medium we could monitor the fraction of populations where the paraquat adaptation had become genetically fixed. Lines: 96 populations (each measured at n=5). Note that after six generations of adaptation, all populations rapidly lose their acquired paraquat adaptation, implying no genetic fixation, while after 242 generations the adaptation has become genetically fixed in all populations.

Figure 6—figure supplement 2. mtDNA loss during long-term exposure to paraquat.

(A) Sequenced populations (n=44) adapted (t₅₀) to chronic paraquat exposure were classified (color) as ρ⁺ (mtDNA intact, green, n=1), ρ^- (6–30 kb mtDNA segments retained, blue, n=18) and or near ρ⁰ (here called ρ^-- ,<2 kb mtDNA segments retained, red, n=25). y-axis: mtDNA copy number (median coverage in 0.5 kb windows relative to the euploid nuclear genome). x-axis: mtDNA position. Below: gene positions. Read map: a zoom-in on a 300 bp mtDNA stretch which is mapped to by the ρ^-- population B8 mtDNA sequence reads. (B) ρ^-- cells with low mtDNA sequence coverage retain very short (<300 bp) mtDNA segments. Micrographs: Light (top; DIC) and fluorescence (bottom) microscopy of DAPI stained DNA in ρ^-- (B8 at t₅₀), ρ⁺ (founder), ρ^- (A7 at t₅₀), and true ρ⁰ (mip1Δ) cells. Insets: Zoom-in on indicated ρ^-- and true ρ⁰ cells. Note that ρ^-- cells contain mitochondrial DNA (red arrow) while true ρ⁰ cells do not. (C) Schematic view of a PCR of the retained mtDNA segment in cells from paraquat-adapted population D1 (t₅₀), with primers directing the reaction outwards across the segment ends. Note that the presence of Sanger sequenced PCR product shows that at least some of the mtDNA molecules are circularized or linear tandem amplifications of the retained segment.

Figure 6—figure supplement 3. Dot-plot mapping of long mtDNA reads from Nanopore sequencing of clones from the D1 and A7 paraquat adapted populations.

Dot-plot mapping of 5 random long mtDNA reads (panels) from Nanopore sequencing of 1 clone from each the D1 (left panels) and A7 (right panels) populations (t₅₀), exposed to chronic paraquat stress to the founder reference mitochondrial genome. Nucleotide position in the reference (x-axis) and sequenced clone (y-axis) mtDNA genomes are shown. Note that reads tend to cover most or all of the retained segment (grey field), which therefore corresponds to a continuous stretch of DNA.

Figure 6—figure supplement 4. Nuclear genome evolution during long-term exposure to paraquat (PQ).

(A) Doubling time (h) of mip1Δ cell populations (n=432) growing in the presence of 400 μg/mL of paraquat, compared to that of founder cell populations (n=768) growing on ordinary medium. p-values Welch two-sided t-test. (B) mtDNA deletion and chromosome II, III and V duplications recur across populations adapted to long-term paraquat stress, but nuclear genes with point mutations rarely do. Upper x-axis: Number of populations in which a gene carries de novo point mutations, a chromosome is duplicated or a mtDNA segment is deleted. Dotted line: number of sequenced populations. Lower x-axis (grey line): For genes containing SNPs, the line shows the mean allele frequencies of SNPs in the gene. For chromosome or mtDNA copy number variations the line shows the mean sequence coverage across the chromosome or mtDNA relative to that of the haploid nuclear genome. y-axis: Genes with point mutations, chromosomes with duplications and mtDNA. Bar color: Type of variation. (C) The early, swift adaptation to paraquat (right y-axis, green line, A, shade: S.E.M., n=6) precedes point mutations (left y-axis, non-green lines, allele frequency). x-axis: Generations of exposure to paraquat. Panels: Sequenced populations (A7, A8, B5, B8, and B12; same as in Figure 3A). Line color: variant type. Variants pre-dating adaptation, supported by few (<10) reads or (<2) time points or shared across environments (>2) were filtered out.

Figure 6—figure supplement 5. Chromosome duplications explain the second phase of adaptation to paraquat.

(A) Chromosome II, III, and V duplications are common after 50 cycles (mean of G=242 generations) of paraquat exposure. Color: Chromosome copy number log₂ median coverage relative to haploid nuclear genome. (B) Chromosome II, III, and V duplications appear in the second phase of paraquat adaptation. Panels: five populations (A7, A8, B5, B8, and B12). Left y-axis (non-green lines): Chromosome copy number (log₂ of median sequence coverage across the chromosome relative to the median of the nuclear genome). Color: chromosome (II=blue, III=red and V = yellow). Right y-axis (green line): paraquat adaptation. Shade: S.E.M. (n=6). Broken lines: no data. (C–D) Chromosome II and III duplications reduce the cell doubling time under paraquat stress (C) but cannot explain the complete loss of respiratory growth in the parent populations (D). We backcrossed (x3) cells adapted to 242 generations (t₅₀) of paraquat exposure to founder cells over consecutive meioses and compared the growth on paraquat of 2–3 segregants with and without duplicated chromosome. x-axis: cells w. (+) and w/o (-) individual chromosome duplications. Error bars: S.E.M. (n=6). p-values: Welch two-sided t-test. Broken line: No growth, corresponding to doubling time >24 hr (the measurement limit).