Widely targeted metabolomics reveals the physiological and metabolic mechanisms of browning in tobacco leaves induced by starvation stress

Browning widely occurs during the processing of crops and their products, reducing both quality and economic value. However, the mechanisms underlying postharvest leaf browning under starvation-dominated conditions remain unclear. Here, compared with the standard curing practice (CK), lower tobacco leaves were subjected to a one-day dark treatment prior to curing to induce starvation-induced browning (SB), and were analyzed through integrated phenotypic characterization, enzyme activity assays, carbohydrate profiling, and widely targeted metabolomics during curing. The results showed that SB significantly intensified leaf browning and tissue shrinkage, accompanied by increased oxidative enzyme activities and malondialdehyde (MDA) content compared with CK during curing, as well as depletion of carbohydrate reserves, reflecting a pronounced metabolic imbalance. LC-MS/MS analysis identified a total of 812 metabolites in flue-cured tobacco leaves. Comparative analysis between CK and SB revealed 117 differentially accumulated metabolites (DAMs), including 73 up-regulated and 44 down-regulated metabolites. Notably, KEGG pathway analysis revealed extensive metabolic reprogramming, particularly involving in phenylpropanoid biosynthesis, flavone and flavonol biosynthesis pathways and phenylalanine, tyrosine and tryptophan biosynthesis pathways. It is worth emphasizing that SB treatment led to the accumulation of antioxidant-related metabolites (e.g., liquiritin and mangiferin) and the decrease in amino acids (e.g., phenylalanine, tryptophan and proline), highlighting the metabolic reprogramming underlying starvation-induced browning. Correlation analysis revealed significant correlations among metabolic changes, enzyme activities, browning severity, and water structure traits. The results indicated that coordinated changes in enzymatic oxidation, substrate availability, and tissue water–structure properties are closely associated with postharvest leaf browning under starvation-dominated conditions, providing potential physiological and metabolic indicators for monitoring and mitigating browning in tobacco and other crops.