Whirly Transcription Factor <scp>NtWHY1</scp> Positively Regulates the Biosynthesis of Cembranoid Diterpenoids by Directly Targeting <scp><i>NtCBTS</i></scp> in Tobacco

Cembranoid diterpenoids, as crucial secondary metabolites in tobacco, play significant physiological roles and exhibit notable biological activities, while the transcriptional regulators governing their biosynthesis remain largely unexplored. A whirly transcription factor NtWHY1 is screened out by DNA pull down using the promoter of NtCBTS (cembratrien-ol synthase), a known key gene in the pathway of cembranoid diterpenoid biosynthesis. Further experiments revealed that NtWHY1 encodes a protein with dual localization in chloroplasts and the nucleus, and it is highly transcriptionally active in tobacco's glandular trichomes and leaves. The expression level of NtWHY1 is positively correlated with the expression level of NtCBTS, as well as the products of α-cembrenediol (α-CBD) and β-cembrenediol (β-CBD), two main cembranoid diterpenoids in tobacco. We also proved that NtWHY1 can directly bind to the promoter region of NtCBTS, with evidence from chromatin immunoprecipitation (ChIP), dual-Luciferase (Dual-LUC) and electrophoretic mobility shift (EMSA) assays. Furthermore, ChIP assays have revealed that NtWHY1 silencing is correlated with reduced H3K9 acetylation and increased H3K27 methylation levels within the promoter region of NtCBTS. Collectively, our results elucidate a novel regulatory role of NtWHY1 in the biosynthesis of cembranoid diterpenoids, thereby advancing our understanding of plant secondary metabolism.