Cytoplasmic Ca2+ influx mediates iron- and reactive oxygen species-dependent ferroptotic cell death in rice immunity

Iron- and reactive oxygen species (ROS)-dependent ferroptosis occurs in plant cells. Ca 2+ acts as a conserved key mediator to control plant immune responses. Here, we report a novel role of cytoplasmic Ca 2+ influx regulating ferroptotic cell death in rice immunity using pharmacological approaches. High Ca 2+ influx triggered iron-dependent ROS accumulation, lipid peroxidation, and subsequent hypersensitive response (HR) cell death in rice ( Oryza sativa ). During Magnaporthe oryzae infection, 14 different Ca 2+ influx regulators altered Ca 2+ , ROS and Fe 2+ accumulation, glutathione reductase ( GR ) expression, glutathione (GSH) depletion and lipid peroxidation, leading to ferroptotic cell death in rice. High Ca 2+ levels inhibited the reduction of glutathione isulphide (GSSG) to GSH in vitro . Ca 2+ chelation by ethylene glycol-bis (2-aminoethylether)- N, N, N’, N’ -tetra-acetic acid (EGTA) suppressed apoplastic Ca 2+ influx in rice leaf sheaths during infection. Blocking apoplastic Ca 2+ influx into the cytoplasm by Ca 2+ chelation effectively suppressed Ca 2+ -mediated iron-dependent ROS accumulation and ferroptotic cell death. By contrast, acibenzolar- S -methyl (ASM), a plant defense activator, significantly enhanced Ca 2+ influx, as well as ROS and iron accumulation to trigger ferroptotic cell death in rice. The cytoplasmic Ca 2+ influx through calcium-permeable cation channels, including the putative resistosomes, could mediate iron- and ROS-dependent ferroptotic cell death under reduced GR expression levels in rice immune responses.