RNA Sequencing Reveals Transcriptomic Changes in Tobacco (Nicotiana Tabacum) Following NtCPS2 Knockout

Abstract 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-knockout model, using CRISPR/Cas9 genome-editing technology to inhibit NtCPS2 function in vitro. In mutant plants, cis-abienol and labdene-diol contents decreased, whereas gibberellin and abscisic acid (ABA) contents increased compared with those in wild-type tobacco plants. RNA sequencing analysis revealed the presence of 9,514 differentially expressed genes (DEGs; 4,279 upregulated, 5,235 downregulated) when the leaves of wild-type and NtCPS2-knockout 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, plant-pathogen interactions, etc. Conclusions: Our findings provide clues to the molecular regulatory mechanism underlying NtCPS2 activity, allowing for a better understanding of the interactions among related genes in tobacco.