Engineering glandular trichomes in Nicotiana tabacum for enhanced specialized metabolite production

Glandular trichomes are specialized epidermal structures with a well-documented role in plant defense, serving as biosynthetic sites for an array of high-value specialized metabolites, including terpenoids, alkaloids, and flavonoids. The ability to engineer these structures for enhanced metabolite production represents a promising strategy in plant molecular farming, offering potential for pharmaceutical, cosmetic, and industrial applications. In this study, we explored the molecular genetic network underpinning glandular trichome development and function in Nicotiana tabacum, leveraging transcription factor-driven induction and pathway optimization strategies to improve their development in plants as well as alter their metabolic output. While investigating the genetic regulation of glandular trichome formation, we identified an agamous-like gene that emerged as a crucial player in the formation of the glandular head of trichomes. Functional validation through reverse genetics, confocal live cell imaging, and DAP-seq assays confirmed its regulatory role in trichome development in N. tabacum. In parallel, we aimed to enhance the biosynthetic capacity of N. tabacum trichomes by implementing a synthetic pathway for triterpenic acid production1. While endogenous genes encoding key biosynthetic enzymes are present in N. tabacum, their expression levels in glandular trichomes are insufficient for efficient metabolite accumulation. To address this limitation, we introduced five transgenes encoding a farnesyl-diphosphate synthase, a squalene synthase, a squalene epoxidase, a beta-amyrin synthase, and a beta-amyrin 28-monooxygenase, all driven by a trichome-specific promoter (Figure 1). The primary objective was to assess whether rerouting isoprenoid precursors from plastids into the cytosolic terpenoid biosynthetic pathway could enhance metabolic flux and lead to increased accumulation of triterpenic acids. Metabolite analysis of whole-leaf extracts from transgenic plants detected low but measurable amounts of ursolic acid, demonstrating that the introduced pathway was at least partially functional in glandular trichomes. However, the relatively low yields highlight the need for further pathway optimization. Our findings underscore the potential of engineering glandular trichomes for the production of specialized metabolite. By integrating transcriptional regulation insights with metabolic pathway reconstruction, this study lays the foundation for future efforts in optimizing trichome-based biofactories for sustainable production of high-value plant-derived compounds.