ROS-mediated membrane damage and antioxidant imbalance drive apple flesh browning during cold storage.

Introduction: Flesh browning is a major postharvest disorder in apples during cold storage, yet its physiological basis remains unclear. This study examined reactive oxygen species (ROS) metabolism and antioxidant systems in flesh-browning (FB) and normal (FN) tissues of browning apples, compared with healthy fruit (Control). Methods: 'Fuji' apples were stored at -1-0°C for 120 days. Flesh color, membrane damage indicators, phenolic metabolism, antioxidant contents (AsA, GSH), ROS levels (H2O2, O2 •-), and antioxidant enzyme activities (SOD, CAT, APX) were analyzed, together with relevant gene expression. Results: FB tissues showed the highest PPO and POD activities, enhanced membrane damage (higher EC, MDA, LOX), and markedly elevated ROS. Total antioxidant capacity decreased by ~25% in FB, accompanied by significant reductions in AsA and GSH and lower CAT/APX activities. Although phenylpropanoid-related enzymes and genes were upregulated, the resulting phenolics and flavonoids were insufficient for ROS mitigation. Strong correlations were found between browning intensity (a*), ROS accumulation, membrane damage, and antioxidant depletion. Discussion: Prolonged cold storage induces severe ROS accumulation and membrane disruption in FB tissues. Simultaneous reductions in non-enzymatic antioxidants (AsA/GSH) and antioxidant enzymes (CAT/APX) create a self-reinforcing imbalance in ROS metabolism, ultimately triggering enzymatic browning.