ABSTRACT: Background: Tobacco leaves are the core raw materials for cigarette production, and their quality has direct and significant impacts on the sensory qualities of cigarette products. Cembrane diterpenoid alcohols are the main precursors of the aroma and fragrance components of tobacco leaves, accounting for approximately 60% of their chemical composition and considerably contributing to their aroma. Initially, we used ⁶⁰Co-γ radiation to induce mutagenesis in the tobacco wildtype variety, GDH64, to obtain the aphid-resistant mutant, MuGHD64. Cembrane diterpenoid alcohol levels were significantly increased in MuGHD64, indicating altered aromatic quality of its tobacco leaves. However, characteristic traits of and molecular mechanisms underlying the increased cembrane diterpenoid alcohol levels in MuGHD64 remain unknown. Therefore, MuGHD64 and GDH64 were investigated in this study. Results: Physiological and biochemical indicator levels were measured in MuGHD64 and GDH64, and transcriptome sequencing was performed to determine the mechanisms responsible for the increased cembrane diterpenoid alcohol levels in MuGHD64. Notably, cembratriene-ol and -diol levels were significantly higher in MuGHD64 than in GDH64. Additionally, MuGHD64 exhibited optimal chemical component levels and improved coordination. Its sensory quality indicators, such as aroma quality and intensity, sweetness, smoothness, and fineness, were superior to those of GDH64. Leaf surface secretory trichome density was significantly higher in MuGHD64 than in GDH64 at all stages. Levels of the cembrane diterpenoid alcohol metabolic precursors, isopentenyl and geranylgeranyl diphosphates, were also significantly higher in MuGHD64 than in GDH64. Transcriptome sequencing revealed that differentially expressed genes were mainly enriched in the metabolic processes related to terpenoid biosynthesis, sesquiterpenoid biosynthesis, isoprenoid biosynthesis, diterpenoid metabolism, diterpenoid biosynthesis, terpenoid skeleton biosynthesis, and plant hormone signal transduction. Cloning and sequencing of the cembratriene-ol synthase gene, NtCYC, revealed that the coding sequence of MuGHD64 harbored nucleotide substitutions at eight sites, resulting in six amino acid alterations. This significantly increased the gene expression levels, enhancing cembratriene-ol synthesis. Consequently, cembratriene-ol and -diol levels were significantly elevated, markedly improving the aroma quality of MuGHD64. Conclusions: Overall, this study revealed new genetic resources for the enhanced synthesis of cembratriene-ol, providing a foundation for breeding aroma-rich high-quality tobacco plants.
