Nitrogen allocation to biochemical fractions shapes functional traits and N use efficiency in tobacco cultivars

Nitrogen is essential for plant growth, however, excessive application leads to environmental pollution and increased production costs. Improving nitrogen use efficiency (NUE) is therefore critical for sustainable agriculture. This study aimed to elucidate how N fractions and functional allocations jointly regulate N accumulation and biomass production in flue-cured tobacco, with a focus on understanding physiological mechanisms underlying cultivar differences. Two contrasting tobacco cultivars, Hongda (higher NUE) and K326 (lower NUE), were grown under three N application levels: 0 g N per plant (CK), 4 g N per plant (T1) and 8 g N per plant (T2). Biomass and N accumulation were monitored across multiple growth stages. N was divided into four solubility fractions: water-soluble (Nw), sodium dodecyl sulfate (SDS)-soluble (Ns), SDS-insoluble (Nin-SDS) and non-protein N. These were then further divided into four functional categories: photosynthetic (Npsn), respiratory (Nresp), structural and storage N. Stepwise regression analysis was used to identify key N components influencing growth. Under N-deficient conditions (CK), greater allocation to Npsn and Nresp supported continued growth. Under moderate N input (T1), increased stem Nin-SDS contributed to leaf biomass accumulation. At high N input (T2), elevated root Nw enhanced total biomass, reducing genotypic differences. N partitioning into distinct functional forms played a central role in regulating biomass production and overall NUE. The superior NUE of tobacco cv. Hongda was associated with its effective N allocation toward physiologically active pools and organs. These findings provide a physiological basis for optimizing N management and breeding tobacco cultivars with reduced fertilizer requirements.