New insights into tobacco leaf browning during flue-curing: antioxidant capacity–ROS imbalance is closely associated with browning development

Browning in tobacco leaves during the flue-curing process severely reduces the quality and industrial usability, and the reasons and mechanisms are still unclear. Here, the reactive oxygen species (ROS) levels, antioxidant capacity, and membrane lipid oxidation in upper, middle, and lower leaves during the early flue-curing process were comprehensively compared with the severe-browning cultivar Dabaijin and the low-browning cultivar Yunyan87. During the later yellowing stage and early color-fixing stage, higher H₂O₂ content and greater membrane lipid oxidation, but lower O₂•− content were exhibited in the leaves of Dabaijin, while significantly lower SOD activity and higher POD and CAT activities were shown in Yunyan87. The antioxidant-related pathways (glutathione metabolism, carotenoid biosynthesis) were enriched in the shared differentially expressed genes (DEGs) and differentially accumulated metabolites (DAMs) from integrated transcriptomic and metabolomic analyses. We identified 62 ROS and antioxidant-related DEGs. Among these, 9 genes associated with ROS generation were upregulated in Dabaijin, while most antioxidant enzyme genes were downregulated. Meanwhile, 100 non-enzymatic antioxidant-related DAMs were screened, and most of the DAMs were significantly accumulated in Yunyan87. Additionally, exogenous application of H₂O₂ to Yunyan87 significantly promoted browning, while the antioxidants (200 µM glutathione and 0.1% calcium ascorbate) to Dabaijin efficiently mitigated browning. The investigation revealed that the imbalance between ROS generation and antioxidant capacity led to membrane lipid oxidation and then destruction, and may be closely associated with and likely promotes tobacco leaf browning. This study firstly simultaneous analysis of dynamic multi-omics and physiology across cultivars with different browning characteristics, provides a theoretical reference for further elucidating the molecular mechanisms of browning in post-harvest crops.