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. 2022 Jul 8;11:e76095. doi: 10.7554/eLife.76095

Figure 3. mtDNA editing causes the early adaptation to paraquat.

(A) mtDNA copy number change (left y-axis, red line, median coverage relative to the haploid nuclear genome) during paraquat adaptation (right y-axis, green line, n=6) for 5 populations (panels). Shade: S.E.M. (B) mtDNA deletions associate with the paraquat adaptation. Circle: mtDNA (77 kb) before exposure to paraquat. Genes, origins of replication and position (kb) are indicated. Coloured fields: mtDNA deletions with concerted copy number change. Diagrams: mtDNA copy number change (left y-axis, purple line) of individual mtDNA genes during adaptation (right y-axis, green line) in population A7. Shade: S.E.M. (n=3). (C) The early-phase paraquat adaptation coincides with the loss of respiratory (glycerol) growth. Shade: S.E.M. 96 populations, each measured at n=5. Broken line indicates no growth (cell doubling time >24 h). (D) Recovery of the copy number of deleted mtDNA (right y-axis, red line) after release from 6 generations of paraquat exposure coincides with loss of the early-phase paraquat adaptation (left y-axis, green line, shade = S.E.M (n=15)) in populations A7, A8, and B12. (E) Doubling time (h) of wild type and mip1Δ cell populations in paraquat. Error bars: S.E.M. (n=191). p-values: Welch two-sided t-test. See also Figure 3—figure supplements 1–3.

Figure 3—source data 1. Doubling time data of 96 populations adapted to paraquat for G generations exposed; doubling times are in paraquat and respiratory media (glycerol).
Data are shown in Figure 3C.
Figure 3—source data 2. Mean log2 coverage of 1 kb windows spanning the mitochondrial genome of five sequenced paraquat adapting populations over generations G of selection.
Data are shown in Figure 3A.
elife-76095-fig3-data2.xlsx (218.6KB, xlsx)
Figure 3—source data 3. Mean log2 coverage of 1 kb windows spanning the mitochondrial genome of sequenced populations adapting to paraquat and then released from this selection; data is given as a function of generations G of relaxation of selection.
Data are shown in Figure 3D and in Figure 3—figure supplement 2C.
elife-76095-fig3-data3.xlsx (179.1KB, xlsx)
Figure 3—source data 4. qPCR data for mitochondrial DNA genes and nuclear DNA controls over generations of paraquat adaptation.
Data are shown in Figure 3B and Figure 3—figure supplement 1.
elife-76095-fig3-data4.xlsx (104.9KB, xlsx)

Figure 3.

Figure 3—figure supplement 1. Editing of mtDNA during the early adaptation to paraquat.

Figure 3—figure supplement 1.

Circle: mtDNA (77 kB) before exposure to paraquat stress, with genes, known origins of replication and position (numbers) indicated. Diagrams: mtDNA copy number (left y-axis, qPCR, purple line) of each protein and rRNA encoding gene, and the associated temporal adaptation profile (right y-axis, adjusted log2(D) ratio relative to founder, green line), in populations A4, A9, A8, B8, B5, B12, and D1. Error bars: S.E.M. (n=2). Colored fields describe concomitant copy number.
Figure 3—figure supplement 2. Homeostatic restoration of mtDNA copy numbers and ability for respiration after release from paraquat (PQ) stress.

Figure 3—figure supplement 2.

(A) mtDNA copy numbers at pre-adaptation levels. y-axis, median mtDNA coverage, in 0.5 kb windows, relative to that of the haploid nuclear genome. x-axis: nucleotide position. Data is from long read sequencing. (B) Respiratory (glycerol) growth (right y-axis, purple line, log2 doubling time relative to founder), and loss of adaptation (left y-axis, green line), in cell populations not exposed to paraquat (G=0) (left panel, founder) and cell populations exposed to 6 generations of paraquat exposure (right panel) and then released from this selection over G generations (x-axis) of growth in absence of paraquat. The mean of five cell populations (A7, A8, B5, B8, and B12; same as in Figure 3A), each measured at n=5, is shown. Shade: S.E.M. (C) Recovery of mtDNA copy numbers (y-axis, median coverage in 1 kb windows relative to the euploid nuclear genome) in cell populations released from six generations of paraquat exposure (dot color: growth cycles after release from paraquat). Red bars: positions of mtDNA deletions.
Figure 3—figure supplement 2—source data 1. Doubling time data of populations adapted to paraquat for Gs generations and then released from selection for Gr generations; doubling times are in paraquat and respiratory media (glycerol).
Data are shown in Figure 3—figure supplement 2B and Figure 6A.
Figure 3—figure supplement 3. Control of paraquat resistance through mtDNA deletions.

Figure 3—figure supplement 3.

(A) Comparisons of the growth of wild type (mean of n=64 populations; left panel) and mip1Δ (mean of n=24 populations; right panel) cells on paraquat (400 μg/mL), before and after 10 growth cycles (mean=82 generations) of adaptation to paraquat. Shade: S.E.M across cell populations (each measured at n=3). (B) Cell doubling time (log2 doubling time relative to wildtype) in presence of menadione (0.25 mM) as a function of generations of adaptation to paraquat (400 μg/mL). Boxplot shows median (horizontal line), interquartile range (box), 95% confidence interval (whiskers) and outliers (dots) of n=96 cell populations, each measured at n=1.
Figure 3—figure supplement 3—source data 1. Doubling time data of 96 populations adapted over G generations to paraquat; doubling times are in 0 and 0.25 mM of menadione.
Data are shown in Figure 3—figure supplement 3B.