Updated project metadata. Physical damage to cells leads to the release of immunomodulatory peptides in order to elicit wound defense responses in the surrounding tissue. In Arabidopsis thaliana, the intracellular PROPEP1 protein precursor is processed into the mature 2.5 kDa PEP1 before release. However, the maturation mechanism remained unknown. Here, we demonstrate that both at tissue level and upon highly precise single-cell damage by multiphoton laser wounding, the cysteine protease METACASPASE4 is instantly activated in a spatiotemporal Ca2+ dependent manner, and is necessary and sufficient for PEP1 maturation. Our results reveal a robust and most likely conserved mechanism that links the intracellular and calcium dependent activation of a specific cysteine protease with the maturation of damage induced wound defense signals. Peptide coverage was obtained for the PROPEP1-YFP-HA fusion protein used in this study and its lower molecular weight processed forms (derived from in vivo proteolysis). In gel digest of immunoprecipitated bands was subjected to mass spectrometry analysis, repeated twice on different mass spectrometers (Velos and Q-Exactive HF).