A comparative study on quality traits of middle fresh tobacco leaves at different leaf positions under a topped condition based on transcriptomics, metabolomics, and plant hormone analysis

The biochemical composition of middle fresh tobacco leaves, which is closely related to quality traits, varies considerably among different leaf positions, yet the underlying for these positional differences remains largely unexplored. In this study, we systematically investigated agronomic traits, physiological parameters, phytohormone profiles, metabolomes, and transcriptomes of middle leaves at positions 6–10 (L6–L10) sampled at a Topped Condition. Leaf size, fresh weight, and leaf angle gradually decreased from L6 to L10, whereas total nitrogen and protein contents increased. Nicotine content remained relatively stable (0.52%–0.63%), whereas reducing sugars and total sugars peaked at L7 and then declined. Multi-omics analyses identified 604 differentially accumulated metabolites and differentially expressed genes enriched in plant hormone signal transduction, starch and sucrose metabolism, biosynthesis of various plant secondary metabolites, and the TCA cycle. Weighted gene co-expression network analysis revealed specific modules associated with nicotine and sugar contents, in which transcription factor families including AP2/ERF, bHLH, MYB, WRKY, bZIP, and B3-ARF formed hierarchical regulatory networks. Auxin, cytokinin, gibberellin, and jasmonic acid exhibited distinct accumulation across leaf positions and were significantly correlated with quality traits. In the comparative study of leaf positions, we observed that the leaf position is associated with hormone levels and the balance of nicotine and sugars in the mid-leaf. There may be complex interactions among these factors. These findings provide relevant insights for the quality variations associated with leaf positions in middle tobacco leaves.