Functional effects of heterologous transcription factors driving trichome fate in Cannabis cell cultures and tobacco plants

Cannabis is an economically important species known for producing bioactive secondary metabolites with important metabolites pharmaceutical and industrial applications, particularly cannabinoids, which are mainly synthesized and secreted by glandular trichomes located on female inflorescences. Despite the increasing interest in cannabinoid production through synthetic biology and metabolic engineering, progress remains limited. To overcome these limitations, stably transformed Cannabis suspension cell lines were generated in our laboratory through biolistic transformation. The selected cell lines expressed either the tomato transcription factors (TFs) SlCD2, belonging to the HD-ZIP IV family or the peach PpMYB25, member of the R2R3-MYB MIXTA family, both selected for their regulatory roles in glandular trichome differentiation. Transgene expression was controlled by the dexamethasone-inducible pOp6/LhGR system. The transformed cell lines were studied on an in-solid culture system, promoting cell differentiation through a solid physical interface to support trichome-like development. Following the induction, transformed cells exhibited distinct morphological changes, as elongated shapes, resembling glandular trichome structures. To validate the effectiveness of the induction strategy, the expression of endogenous and exogenous genes involved in trichome development and cannabinoid biosynthesis was evaluated by qRT-PCR. Following the identification of differentially expressed metabolites in liquid cultures through HPLC-MS analysis, genes involved in their biosynthetic pathway were also investigated by qRT-PCR to determine whether the presence of these metabolites was supported by the activation of their biosynthetic pathway. To investigate the effect of these transcription factors at the organism level, the peach MIXTA gene was constitutively expressed in tobacco plants. Morphological analyses were carried out to evaluate its role as a positive regulator of glandular trichome development at leaf level and its effect on floral conical cells.