Extended Data Fig. 4: Reprogramming induced by transient zeocin treatment occurs independently of dead cells.

a Representative leaves without (top panels) or with visible dead cells (bottom panels) after transient zeocin or BLM treatment for 6 hours, followed by 7 days of cultivation without DNA damage reagents. Red arrows indicate dead cells in brown. b Percentage of gametophores with at least one visible dead leaf cell. Dark red dots and bars represent the mean and SD from three independent experiments (30 ≤ n ≤ 42, gametophores), respectively. c Bright-field (BF) and PI fluorescence images (PI) of a representative gametophore leaf without dead cells during and after 50 µg/ml zeocin treatment for 6 hours. Images taken at 0, 6 (just before washing out zeocin), 12, 24, 36, 48, 60, 72, and 84 h from the time-lapse images (Supplementary Video 1) are shown. Yellow arrows indicate reprogrammed cells with protrusion. Although PI does not penetrate into apoplastic space of intact gametophore tissue, staining of cell wall with PI became visible after 64 h (Supplementary Video 1). After protrusion started in reprogrammed cells, PI fluorescence in cell walls of surrounding cells became visible, likely because of higher penetration in reprogrammed cells with tip growth. Similar results have been observed in 6 different gametophores. Since only the intercellular space of surrounding cells were stained by PI and none of the cells show nucleus staining, no imperceptible dead cells existed around the protruding reprogrammed cells to trigger the reprogramming. d Bright-field (BF) and PI fluorescence images (PI) of a representative gametophore leaf with dead cells during and after 50 µg/ml zeocin treatment for 6 hours. Images were taken from the time-lapse images (Supplementary Video 2) at the same timepoints as those in (c). Yellow and pink arrows indicate reprogrammed cells with protrusion and dead cells, respectively. After protrusion started in reprogrammed cells, PI fluorescence in cell walls of surrounding cells became visible, likely because of higher penetration in reprogrammed cells with tip growth. Also, PI fluorescence was detected in cell walls surrounding dead cells. Similar results have been observed in 2 different gametophores. Scale bars: 100 µm in (a); 100 µm in (c, d).