LAUSR

Background Amber-like compounds form in tobacco ( Nicotiana tabacum ) during leaf curing and impact aromatic quality. In particular, cis -abienol, a polycyclic labdane-related diterpenoid, is of research interest as a precursor of these compounds. Glandular trichome cells specifically express copalyl diphosphate synthase ( NtCPS2 ) at high levels in tobacco, which, together with NtABS , are major regulators of cis -abienol biosynthesis in tobacco. Results To identify the genes involved in the biosynthesis of cis -abienol in tobacco, we constructed transgenic tobacco lines based on an NtCPS2 gene-knockdown model using CRISPR/Cas9 genome-editing technology to inhibit NtCPS2 function in vitro. In mutant plants, cis -abienol and labdene diol contents decreased, whereas the gibberellin and abscisic acid (ABA) contents increased compared with those in wild-type tobacco plants. RNA sequencing analysis revealed the presence of 9514 differentially expressed genes (DEGs; 4279 upregulated, 5235 downregulated) when the leaves of wild-type and NtCPS2 -knockdown tobacco plants were screened. Among these DEGs, the genes encoding cis -abienol synthase, ent-kaurene oxidase, auxin/ABA-related proteins, and transcription factors were found to be involved in various biological and physiochemical processes, including diterpenoid biosynthesis, plant hormone signal transduction, and plant-pathogen interactions. Conclusions The present study provides insight into the unique transcriptome profile of NtCPS2 knockdown tobacco, allowing for a better understanding of the biosynthesis of cis -abienol in tobacco.