6°Co-? radiation-induced GDH64 NtCYC mutation enhances the glandular trichome density and aroma quality of tobacco via physiological, biochemical, and molecular mechanisms

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 6°Co-? 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. Overall design: MuGDH64 and GDH64 were the experimental materials used in this study. MuGHD64 was obtained via radiation mutagenesis breeding through 6°Co-? irradiation of GDH64. Among the stable mutant strains, MuGHD64 was identified as an aphid-resistant mutant. Field experiments were conducted using a randomized block design with three replicates, each consisting of four rows, with 15 plants per row. Trials were conducted at the Tobacco Research Base of Guizhou University (106°16'E, 26°06'N) in 2022 and 2023. The tobacco field had yellow soil with loose texture and moderate fertility. Seedlings were propagated using the floating seedling method, and field cultivation and management were performed according to the local high-quality flue-cured tobacco cultivation techniques. MuGHD64 and GDH64 samples were collected at 40, 55, 70, and 85 d post-transplantation. In each plot, three healthy tobacco plants with uniform growth were randomly selected, and 9th to 11th leaves from the bottom were collected from each plant. Using a punch sampler, leaf portions were collected from each plant, mixed, placed in a centrifuge tube, immediately treated with liquid nitrogen, and stored at –80 °C for physiological indicator and transcriptome sequencing analyses. After middle leaf maturation, 9th to 11th leaves of MuGHD64 and GDH64 were harvested, dried, ground, and stored for cembrane diterpenoid alcohol measurement, with some portions used for sensory quality evaluation.