Citrus-specific truncation of CsGPX4 disrupts intracellular ROS homeostasis

Abstract Glutathione peroxidases (GPXs) are key antioxidant enzymes that contribute to cellular redox homeostasis through reactive oxygen species (ROS) detoxification and modulation of redox signaling pathways. Although GPX functions have been studied in many annual plants, their roles in woody perennial crops remain poorly understood. Here, we characterized the GPX gene family in Citrus sinensis and investigated the function of CsGPX4 , the closest homolog of Arabidopsis thaliana AtGPX8 because of its potential role in mediating oxidative stress signaling. Overexpression of CsGPX4 induced an oxidative stress phenotype that includes chlorosis, growth inhibition, cellular degeneration, and elevated intracellular ROS accumulation. Immunoblot and MALDI-TOF analyses showed that CsGPX4 overexpression resulted in a truncated proteoform, while the full length CsGPX4 was able to accumulate in Nicotiana benthamiana , indicating a species-specific post-translational truncation. The truncated CsGPX4 proteoform exhibited an altered subcellular localization shifting from cytoplasmic and nuclear localization to membrane-associated compartment in citrus cells. The truncated form of CsGPX4 had a reduced glutathione peroxidase activity compared to the full-length protein. Proteomic profiling suggested reprogramming of pathways associated with redox detoxification, cytoskeleton organization, hormone signaling, and stress responses. Our study identifies a citrus-specific post-translational regulatory mechanism that modulates CsGPX4 localization and enzymatic activity. This suggests that citrus may employ a species-specific proteolysis processing as a protective mechanism to maintain redox homeostasis during oxidative stress.