Integrated Transcriptomic and Metabolomic Analyses Reveal the Synergistic Regulatory Mechanisms of Nitrogen Application Rate and Topping on Tobacco Leaf Quality

Nitrogen application rate and topping are two key agronomic factors affecting tobacco leaf quality, but the synergistic regulatory mechanisms remain unclear. This study aimed to elucidate the physiological and molecular mechanisms by which nitrogen application rate and topping synergistically regulate tobacco leaf quality, and to identify key candidate genes involved in nicotine biosynthesis. Using the tobacco cultivar K326, three nitrogen application levels were established: low (0 kg/ha), medium (120 kg/ha), and high (210 kg/ha). Agronomic traits and rhizosphere soil physicochemical properties were monitored, carbon and nitrogen metabolism enzyme activities were measured before and after topping, and mature leaf chemical composition was determined, integrated with transcriptomic and metabolomic analyses. The results showed that medium nitrogen achieved high yield while enhancing organic matter turnover efficiency, reducing rhizosphere water-soluble chloride content, and alleviating soil acidification and ammonium–potassium ion uptake competition caused by excessive fertilization. Combined with topping, it significantly enhanced carbon and nitrogen metabolism, promoted starch-to-soluble-sugar conversion, and increased nicotine accumulation, thereby optimizing leaf chemical composition. Integrated transcriptomic and metabolomic analyses revealed that medium nitrogen combined with topping exerted the most pronounced effects on gene expression and metabolite accumulation. Weighted gene co-expression network analysis (WGCNAs) identified a module highly correlated with nicotine biosynthesis, with hub genes mainly annotated as cytochrome P450 and terpene synthase family members. In conclusion, this study elucidated the molecular mechanisms by which the combination of medium nitrogen application and topping, as an integrated agronomic practice, improves tobacco leaf quality through coordinating carbon-nitrogen metabolism and optimizing chemical composition, and identified key candidate genes potentially regulating nicotine biosynthesis, providing a theoretical basis for nitrogen fertilizer management and high-quality cultivation of tobacco.