An atypical bHLH transcription factor NtPRE4.1 negatively regulates tobacco glandular trichome density

As the world's largest producer and consumer of tobacco, it holds significant socio-economic value in China. Trichomes, specialized hair-like structures on the epidermal cells of tobacco, play a crucial role in the synthesis and secretion of defense-related secondary metabolites, such as terpenoids and alkaloids, which directly influence leaf quality. However, the molecular mechanisms underlying trichome development remain poorly understood. In this study, we identified and characterized the previously unknown TF NtPRE4.1 from tobacco (Nicotiana tabacum). Phylogenetic tree analysis revealed that the protein encoded by NtPRE4.1 belongs to the atypical bHLH TF family. NtPRE4.1 was found to localize in both the nucleus and cytoplasm. In Nicotiana tabacum, overexpression of NtPRE4.1 resulted in a 40-48 % reduction in trichome density, while VIGS-treated lines exhibited a 44-69 % increase in trichome density compared to non-transgenic controls. Additionally, Y2H and Co-IP experiments confirmed that NtPRE4.1 interacts with the R2R3-MYB TF NtMYB330, which is highly expressed in trichomes, suggesting that NtMYB330 may play a role in regulating glandular trichome development. Finally, our results demonstrated that NtPRE4.1 significantly modulated the expression of marker genes (NtGA20ox1 and NtGA3ox1) involved in GA biosynthesis, as well as two trichome development-related genes (NtGIS and NtCycB2). Overexpression of NtPRE4.1 significantly decreased the GA₃ content in tobacco leaves, while the expression of NtPRE4.1 was significantly suppressed in tobacco plants treated with GA₃.These findings provide new insights into the role of atypical bHLH TFs in regulating tobacco trichome formation through GA signaling.