Papaver S-determinants trigger mitochondrially derived ROS production and disrupt energy metabolism in incompatible pollen tubes

Many plants use self-incompatibility (SI) mechanisms to prevent inbreeding. SI in Papaver rhoeas is triggered by allele-specific interaction between the pollen and pistil S-determinants, activating a Ca²⁺-dependent signaling network that leads to rapid reactive oxygen species (ROS) production and eventual programed cell death (PCD) in incompatible pollen. Expression of the Papaver pollen S-determinant (PrpS) in Arabidopsis thaliana recapitulates Papaver SI when challenged with the cognate pistil ligand (PrsS). Using roGFP2-Orp1, a genetically encoded hydrogen peroxide (H₂O₂) sensor, and measurements of mitochondrial metabolism, reveals a complex SI response. Within minutes, elevated cytosolic Ca²⁺ ([Ca²⁺]_cyt) and cytosolic acidification converge to trigger mitochondrial H₂O₂ production, mitochondrial membrane depolarization, decreased respiration rate, and ATP depletion. In parallel, oxidative inactivation of GAPDH inhibits glycolysis, resulting in decreased TCA cycle intermediates and providing a feedback loop to enhance mitochondrial disruption. Preceding mitochondrial ROS production, SI rapidly arrests pollen tube growth via inactivation of plasma membrane-localized NADPH oxidase (RBOH) mediated superoxide production. This provides insights into how ROS signatures from NADPH oxidase and mitochondria drive distinct processes. We demonstrate that early mitochondrial disruption, likely driven by interconnected Ca²⁺, pH, and redox signaling, is a central feature of this SI response, underpinning rapid disruption of energy metabolism in incompatible pollen tubes prior to PCD.