Integrated proteomics and metabolomics analyses reveal the mechanism of calcium ascorbate inhibiting tobacco browning

Flue-cured tobacco often undergoes browning, which not only diminishes its commercial value but also increases potential health risks for smokers. Previous researches have demonstrated the potential of calcium ascorbate (CAAS) in preventing browning in fruits and vegetables. However, its efficacy in mitigating browning in tobacco leaf and the associated molecular mechanisms have not been extensively investigated. This study established a classification model for browning degree in tobacco leaves based on CIE color parameters and a multilayer perceptron neural network. In addition to evaluating the antioxidant effects of CAAS on tobacco leaves during the flue-curing process, we conducted integrated proteomic and metabolomic profiling to elucidate the molecular mechanisms by which CAAS prevents browning. The findings indicated that the overall accuracy of the classification model was above 94 %. According to the prediction results of the model, considering the physical characteristics, sensory attributes, economic significance, and chemical composition of the flue-cured leaves, 0.2 % CAAS was identified as the most effective inhibitor of browning in tobacco leaves. The mechanisms by which CAAS prevents browning indicated that it not only accelerated the yellowing process and enhanced antioxidant activity, but also inhibited enzymatic browning reactions by increasing POD activity and carotenoid content, while reducing the activity of PPO and the levels of MDA, chlorophyll, and H₂O₂. Furthermore, a combination of proteomic and metabolomic analysis showed that CAAS could upregulate proteins involved in photosynthesis while downregulating metabolites involved in purine metabolism, phenylpropanoid biosynthesis, and tyrosine metabolism, thereby further mitigating browning. The results of this study provide new insights into the mechanisms by which CAAS inhibits tobacco browning and suggest potential strategies for its application in the tobacco industry.