Transcriptome-wide investigation and functional characterization reveal a terpene synthase involved in γ-terpinene biosynthesis in Monarda citriodora

Monarda citriodora Cerv. ex Lag. is a rich source of industrially important compounds like γ-terpinene, carvacrol, thymol and thymoquinone. Understanding the regulation of γ-terpinene biosynthesis, a precursor for other monoterpenes, could facilitate upscaling of these metabolites in M. citriodora. Therefore, the present study aimed to unravel and characterize the terpene synthase (TPS) involved in γ-terpinene biosynthesis. Homology searches revealed 33 TPS members in the transcriptome assembly of M. citriodora. Based on the correlation of expression patterns and phytochemical profile, McTPS22 emerged as the putative TPS for γ-terpinene biosynthesis. Molecular docking suggested geranyl diphosphate (GPP) as a potential substrate for McTPS22. Heterologous expression in Escherichia coli and Nicotiana benthamiana confirmed the role of McTPS22 in γ-terpinene biosynthesis. Both in-silico prediction and confocal microscopy indicated plastidial localization of the McTPS22. Gene co-expression network analysis revealed 507 genes interacting with McTPS22, including 80 transcription factors (TFs). Of these, 46 TFs had binding sites in the McTPS22 promoter, and 36 showed significant correlations with γ-terpinene accumulation, suggesting they may be potential regulators. Promoter analysis indicated regulation by phytohormones and abiotic factors, confirmed by phytohormone elicitation and QRT-PCR. The histochemical GUS staining suggested that McTPS22 is primarily active in the glandular trichomes of M. citriodora. The present work provides insights into the molecular regulation of biosynthesis of γ-terpinene in M. citriodora.