Harvest maturity is associated with carbon reallocation and fresh-to-flue-cured metabolic changes in Nicotiana tabacum leaves.

Harvest maturity is associated with the biochemical status of leaf-harvested crops before postharvest processing, but the extent to which maturity-established metabolic differences are retained or transformed during flue-curing remains unclear in Nicotiana tabacum. Here, middle leaves of Yunyan 87 grown at a single experimental site were harvested at five harvest-maturity treatments (16, 18, 20, 22, and 24 days after topping). T1 and T5 were selected as contrasting harvest-maturity endpoints for paired metabolomic analyses of fresh leaves (F_T5 vs. F_T1), flue-cured leaves (FC_T5 vs. FC_T1), and direct fresh-to-flue-cured metabolite abundance changes at the same harvest-maturity treatment (FC_T1 vs. F_T1 and FC_T5 vs. F_T5). Phenotype, SPAD values, color traits, enzyme activities, conventional chemical constituents, auxiliary flue-cured leaf traits, and untargeted UPLC-HRMS metabolomics were integrated. Increasing maturity was accompanied by progressive yellowing, decreased SPAD and H° values, and increased L*, a*, b*, and C*. Fresh T5 leaves showed metabolic changes linked to carbon allocation, secondary metabolism, hormone/terpenoid-related pathways, phenylpropanoid-related metabolism, and redox adjustment. After flue-curing, maturity-associated differences were mainly reflected in aromatic amino acid metabolism, especially tryptophan and phenylalanine metabolism, together with secondary metabolism, metabolites assigned to transporter-associated KEGG categories, carbohydrate-related substrates, amino acid-related compounds, and lipid-associated remodeling. Direct fresh-to-flue-cured comparisons identified 84 and 85 differential metabolites at T1 and T5, respectively, including 70 shared metabolites and harvest-maturity-associated, comparison-specific components. These results identify a continuous physiological and conventional chemical maturity gradient across T1-T5 and endpoint metabolomic differences between the contrasting T1 and T5 treatments. Together, the findings suggest that harvest maturity is associated with both the fresh-leaf biochemical starting point and the metabolite abundance profile observed after flue-curing, although intermediate metabolomic states and causal mechanisms underlying individual metabolite changes remain to be determined.