Figure 7. The bimodal chl-roGFP oxidation in response to H₂O₂ is light-dependent.

The effects of a short exposure to darkness during daytime on chl-roGFP oxidation patterns were examined. (A–C) Flow cytometry measurements of chl-roGFP OxD distribution in the population over time. Cells were treated with 0 µM (control, A), 50 µM (B), and 80 µM H₂O₂ (C), and were then transitioned to the dark at time 0 (within 5 min post H₂O₂ treatment). Cells were kept in the dark for 90 min (green) and were then transferred back to the light (cyan). The same H₂O₂ treatment without transition to the dark (light ctrl, black) and maximum oxidation (200 µM H₂O₂, red) and reduction (2 mM DTT, blue) are shown for reference. The experiment was done in triplicates that were highly similar, for visualization the first replica is shown. Each histogram consists of >8000 cells. (D) Mean ± SEM basal (control) chl-roGFP OxD over time of cells transitioned to the dark for 90 min (gray box) at time 0 (‘dark’) and cells kept in light conditions (‘light’), n = 3 biological repeats. Pre-treatment baseline under light conditions is shown. SEM lower than 0.5% are not shown. (E) The fraction of dead cells 24 hr post H₂O₂ treatment, with or without transition to the dark (‘dark’ and ‘light’ respectively), as measured by positive Sytox staining. Data is shown as mean ± SEM, n = 3 biological repeats. P values: *=0.0064, **=0.0026, ***=2·10⁻⁵, t-test.

Figure 7—figure supplement 1. Redox response of chl-roGFP to transition to the dark.

Flow cytometry measurements of chl-roGFP oxidation in the population over time. Cells were treated with 0 µM (basal, A, same as in Figure 7A), 10 µM (B), and 30 µM H₂O₂ (C), and were then transitioned to the dark at time 0 (within 10 min post treatment). Cells were kept in the dark for 90 min (green) and were then transferred back to the light (cyan). For reference, chl-roGFP OxD following the same H₂O₂ treatment but without transition to the dark (black) and following maximum oxidation (200 µM H₂O₂, red) and maximum reduction (2 mM DTT, blue) are shown. The experiment was done in triplicates that were highly similar, for visualization purposes the first replica is shown. Each histogram consists of >8000 cells.

Figure 7—figure supplement 2. Responses of chloroplast and nucleus targeted roGFP to transition to the dark.

Measurements of roGFP oxidation response in the chloroplast and nucleus to a temporal transition to the dark. (A–D) Flow cytometry measurements of roGFP oxidation dynamics in the population over time in the chloroplast (A–B) and nucleus (C–D). Cells were treated with 80 µM H₂O₂ (B, D) or untreated control (A, C), and were then transitioned to the dark at time 0 (within 4 min post treatment). Cells were kept in the dark for 120 min (green) and were then transferred back to the light (dark cyan). For reference, roGFP OxD following the same H₂O₂ treatment but without transition to the dark (black, ~130 min post treatment) and following maximum oxidation (200 µM H₂O₂, red) and maximum reduction (2 mM DTT, blue) are shown. (E) The fraction of dead cells 24 hr post H₂O₂ treatment with or without transition to the dark (‘dark’ and ‘light’ respectively) as measured by positive Sytox staining in chl-roGFP strain. Data is shown as mean ± SEM, n = 3 biological repeats.