Mechanosensing activates flashing Ca ²⁺ dynamics associated with cell regeneration in Physcomitrium patens

Mechanical wounding is the primary trigger for various forms of plant regeneration; however, the mechanism by which mechanical cues are rapidly translated into regenerative cell fate decisions remains elusive. Here, using live-cell imaging in Physcomitrium patens , we established a spatiotemporal framework of Ca ²⁺ signaling that bridges initial wound perception and cellular reprogramming. We demonstrate that wounding induces a primary Ca ²⁺ wave followed by two forms of secondary responses, including a unique stochastic flashing signature that persists for hours and restricts in wound-neighbor cells. Wound-induced membrane deformation was associated with the activation of Ca ²⁺ influx. In addition, osmotic stress-induced membrane tension also elicited a rapid Ca ²⁺ spike followed by broadly flashing signals without spatial specificity. Pharmacological blockade of plasma membrane Ca ²⁺ channels and chelation of extracellular Ca ²⁺ significantly reduced both primary and secondary Ca ²⁺ responses, indicating that these wound-induced Ca ²⁺ dynamics mainly rely on regulated influx rather than passive diffusion. Inhibition of Ca ²⁺ signaling also disrupted mechanosensitive F-actin reorganization and suppressed cell reprogramming, resulting in a severe reduction in regeneration capacity. Together, our results demonstrate a complex spatiotemporal Ca ²⁺ code that precedes cytoskeletal reorganization and cell reprogramming in wound-neighbor cells, providing new insights into plant tissue regeneration.