Time-series analysis revealed the metabolic and transcriptomic dynamics regulated by nitrogen forms during the maturation process of flue-cured tobacco

This study investigated the regulatory mechanisms by which nitrogen forms influence the tobacco maturation process by establishing three nitrogen supply regimes: nitrate, ammonium, and a nitrate-ammonium mixture. Dynamic sampling was conducted at the under-ripe, ripe, and over-ripe stages, integrating physiological, biochemical, metabolomic, and transcriptomic data to reveal how different nitrogen sources affect leaf maturation. The findings demonstrated that nitrate nitrogen accelerated the maturation process, leading to a significant decline in photosynthetic pigment levels during ripening, accompanied by chloroplast degradation and starch granule accumulation, resulting in leaf yellowing and the onset of senescence. In contrast, ammonium nitrogen delayed yellowing, preserving higher photosynthetic pigment levels and intact chloroplast structures during ripening, which maintained greener leaves with reduced yellowing. Analysis of carbon and nitrogen metabolism revealed that under nitrate treatment, sugar, starch, and protein levels progressively declined at the ripe stage, whereas ammonium treatment sustained higher levels of these metabolites. The mixed nitrogen regime achieved a balanced coordination between photosynthesis and senescence, aligning maturation rhythms with quality development. MFUZZ time-series clustering identified the ripe stage as a critical period for metabolic transition and transcriptional activation during tobacco maturation, with different nitrogen sources modulating metabolic pathways and shaping distinct maturation trajectories. Nitrate nitrogen promoted cytokinin biosynthesis at early stages, triggered senescence signals during ripening, and subsequently upregulated pentose phosphate pathway activity to support metabolic reprogramming. Ammonium nitrogen, on the other hand, maintained growth signals and TCA cycle activity, inducing flavonoid accumulation during the ripe and over-ripe stages to alleviate ammonium toxicity, thereby delaying the maturation process. The mixed nitrogen treatment demonstrated superior coordination in regulating metabolic rhythms, C-N balance, and enzyme activities. This study provides a theoretical foundation and practical guidance for optimizing nitrogen fertilization strategies, precisely controlling maturation processes, and improving the quality of flue-cured tobacco. • Time-series multi-omics reveals nitrogen form regulation of leaf maturation. • Ripe stage is a window for coordinated transcriptional and metabolic shifts. • Nitrogen forms reshape metabolic pathway dynamics during tobacco leaf maturation.