Figure 2. Organelle-specific oxidation of roGFP in response to H₂O₂ reveals heterogeneity at the single-cell level.

The distribution of roGFP OxD in the population over time was measured by flow cytometry in P. tricornutum cells expressing roGFP targeted to the chloroplast (chl-roGFP, (A–D, M)), nucleus (nuc-roGFP, (E–H, N)) and mitochondria (mit-roGFP, (I–L, O)). (A–L) Oxidation of roGFP in response to 0 µM (A,E,I), 50 µM (B,F,J), 80 µM (C,G,K), and 100 µM (D,H,L) H₂O₂. Maximum reduction (blue) and oxidation (red) of roGFP following additions of 2 mM Dithiothreitol (DTT) or 200 µM H₂O₂ respectively are shown as a reference. The ‘oxidized’ and ‘reduced’ subpopulations are marked by red and blue dashed boxes respectively (C, G, K). The experiment was done in triplicates, for visualization the first replica is shown except for the first 4 min in which all replicates are shown for higher temporal resolution. Each histogram consists of >8000 (A–D), >5900 (E–H) and >1400 (I–L) roGFP-positive cells. Measurements of >100% OxD may result from increased auto-fluorescence leakage after long exposure to stress (Figure 2—figure supplements 5–6). (M–O) The fraction of the ‘oxidized’ subpopulation over time upon exposure to 0–100 µM H₂O₂. Mean ± SEM, n = 3 biological repeats. SEM lower than 0.018 are not shown.

Figure 2—figure supplement 1. Organelle specific roGFP mean oxidation in response to H₂O₂ application in P. tricornutum.

Flow cytometry measurements of the mean ± SEM roGFP OxD in the population over time in response to oxidative stress in chl-roGFP (A), nuc-roGFP (B) and mit-roGFP (C) P. tricornutum strains, n = 3 biological repeats. Oxidation dynamics were measured following treatments with different H₂O₂ concentrations: 50 µM (green), 80 µM (blue), 100 µM (purple) and without treatment (control, gray). Maximum reduction (2 mM DTT, black) and maximum oxidation (200 µM H₂O₂, red) are shown for reference. Error bars below 1.5% are not shown.

Figure 2—figure supplement 2. Distribution of roGFP oxidation in different subcellular compartments of P. tricornutum cells at steady state.

Flow cytometry measurements of roGFP OxD and expression levels in P. tricornutum cells expressing roGFP targeted to the chloroplast (chl-roGFP, (A)), nucleus (nuc-roGFP, (B)) and mitochondria (mit-roGFP, (C)). (A–C) The distribution of roGFP OxD in the population at steady state conditions (without treatment, green) and following treatments of maximum oxidation (200 µM H₂O₂, red) and maximum reduction (2 mM DTT, blue). (D) Relative roGFP expression levels (AU) based on fluorescence intensity in chl-roGFP (red), nuc-roGFP (orange), mit-roGFP (green) and WT (no roGFP, cyan) strains. Relative roGFP expression was calculated by multiplication of the i405 and i488 (see Materials and methods). A threshold was set to exclude cells with low roGFP expression from roGFP oxidation calculations, shown as a gate with the label ‘roGFP+’. Each histogram consists of >12,000 cells (A, B, D) or >2,400 cells (C, due to lower roGFP expression). The experiment was done in triplicates, for visualization purposes one representative repeat is shown.

Figure 2—figure supplement 3. Oxidation of chl-roGFP reveals distinct subpopulations in response to H₂O₂ treatment.

(A–F) Density plots of flow cytometry fluorescence measurements of i405 over i488 (see Materials and methods) of chl-roGFP (A-D, roGFP positive cells) and WT cells that do not express roGFP (E-F, chlorophyll positive cells) 83–88 min post H₂O₂ treatments of 50 µM (A), 80 µM (B), 100 µM (C, F) and untreated control (D, E). The ratio i405/i488 increases upon roGFP oxidation and is used for calculating roGFP OxD (see Materials and methods). The ‘oxidized’ and ‘reduced’ chl-roGFP subpopulations are marked in red and blue respectively in B. (G) Flow cytometry measurements of mean roGFP OxD of the ‘oxidized’ (dashed, empty symbols) and ‘reduced’ (solid, full symbols) subpopulations over time post 50–200 µM H₂O₂ treatments and in untreated control (gray). Maximum reduction following 2 mM DTT (black) is shown for reference. Results are shown as mean ± SEM, n = 3 biological repeats.

Figure 2—figure supplement 4. Heterogeneity in mit-roGFP response to H₂O₂.

Flow cytometry measurements of mit-roGFP oxidation in response to 80 µM H₂O₂ in three independent experiments (in addition to the experiment shown in Figure 2): exp 1 (A), exp 2 (B, D, G) and exp 3 (C, E, F, H, I). (A–C) Distribution of mit-roGFP OxD at different times post 80 µM H₂O₂ (green). Measurements of untreated control (black) and following maximum oxidation (red, 200 µM H₂O₂) and maximum reduction (blue, 2 mM DTT) are shown for reference. (D–I) Density plots of roGFP fluorescence measurements of i405 vs. i488 of mit-roGFP (D, E, G, H) and WT (F, I) strains at different times post 80 µM H₂O₂ treatment (D–F) or untreated control (G–I). The ratio i405/i488 represents roGFP oxidation state (see Materials and methods). WT is shown for auto-fluorescence (AF) leakage reference, demonstrating the effect of lower expression level in mit-roGFP strain. A differential response was observed in exp 3 and in the exp shown in Figure 2K,L, but in exp 1 and 2 no distinct subpopulations were observed. This differential response was clearly observed only at later time points, which were not measured in exp 1 and 2, and may result from auto-fluorescecne leakage (see Figure 2—figure supplements 5–6). Measurements were done in triplicates, one repeat is shown for visualization.

Figure 2—figure supplement 5. Auto-fluorescence leakage into i405 channel in roGFP strains following H₂O₂ treatment.

Flow cytometry measurements of i405 auto-fluorescence leakage in P. tricornutum strains expressing roGFP targeted to the chloroplast (chl-roGFP, (A)), nucleus (nuc-roGFP, (B)) and mitochondria (mit-roGFP, (C)) over time following treatments of 0–200 µM H₂O₂ or 2 mM DTT. Leakage into i405 channel was calculated by the mean i405 of the WT strain (n = 3 biological repeats) divided by the mean i405 of the roGFP expressing strain (n = 3 biological repeats) at the same time-point following the same treatment.

Figure 2—figure supplement 6. Auto-fluorescence leakage into i488 channel in roGFP strains following H₂O₂ treatment.

Flow cytometry measurements of i488 auto-fluorescence leakage in P. tricornutum strains expressing roGFP targeted to the chloroplast (chl-roGFP, (A)), nucleus (nuc-roGFP, (B)) and mitochondria (mit-roGFP, (C)) over time following treatments of 0–200 µM H₂O₂ or 2 mM DTT. Leakage into i488 channel was calculated by the mean i488 of the WT strain (n = 3 biological repeats) divided by the mean i488 of the roGFP expressing strain (n = 3 biological repeats) at the same time-point following the same treatment.