Wound-induced Ca²⁺ wave and electrical signal propagation in M. polymorpha. (A) Bright field image and time-lapse imaging of cytosolic Ca²⁺ in Marchantia thali expressing fluorescent proteins GCaMP6f-mCherry after wounding with a needle. Binarized images were created with areas of significantly increased fluorescence intensity filled in black. The standard for significantly increasing the fluorescence intensity was F > F₀ + 2*SD (standard deviation). F₀ = average fluorescence intensity of 10 frames before wounding. The arrow indicates the wounded site. Scale bar = 3 mm. (B) Measurement of the GCaMP6f/mCherry ratio at positions 1–5 (ROI in image of (A)). Normalized by subtracting the average ratio value of the 10 frames before wounding from the ratio value at each time. The arrow indicates the time of wounding. (C) The time at which the Ca²⁺ wave arrived is shown. (D) Velocity of the Ca²⁺ wave between positions 1 to 2, 2 to 3 and 3 to 4 in Figure (C). n = 7–12. Mean ± SD. (E) Schematic diagram of the experiment. Surface potentials were measured at wounded branch 1 (positions 1), at the intermediate site between branches 1 and 2 (position 2), at the unwounded branch 2 (position 3) and branch 3 (position 4). (F) Representative recording of wound-induced surface potential changes at positions 1–4. The triangular arrow indicates the time of wounding.