Organellar-specific ROS dynamics drive differential cross-compartmental responses between chloroplast and mitochondria in C. reinhardtii

Reactive oxygen species (ROS) act as key signaling intermediates in plant metabolism, defense, and stress adaptation. In photosynthetic organisms, chloroplast and mitochondria serve as the major hubs of ROS production, thereby coordinating stress responses across cellular compartments. However, the extent of cross-organellar communication following compartmentalized oxidative stress remains poorly understood. Using C. reinhardtii as a model system, we induced compartmentalized oxidative stress in chloroplast and mitochondria to investigate whether the ROS generation in one organelle triggers any functional response in the other organelle. Methyl viologen (MV) was used to induce ROS production in the chloroplast, while menadione (MD) was used to trigger mitochondrial ROS production. Real-time monitoring of compartment-specific roGFP strains showed specific, localized, and reversible ROS production following MV and MD treatment as a function of time within the respective target organelles. Comprehensive functional analyses following compartmentalized ROS perturbations revealed that the mitochondrial ROS remained localized to their site of origin and did not detectably affect chloroplast function, as evidenced by the unchanged chlorophyll a fluorescence parameters and photosystem stoichiometry. In contrast, chloroplast-derived ROS diffused from its site of origin and transiently suppressed the mitochondrial oxygen consumption rates. Together, these findings underscore a dynamically regulated system, where chloroplast ROS leaks out and affects mitochondrial function while the mitochondrial ROS remains localized and does not impact chloroplast function. These compartment-specific ROS responses likely represent an adaptive mechanism for coordinating cellular energy metabolism in response to fluctuating environmental conditions.