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The essential oil of Pelargonium graveolens (rose-scented geranium), an important aromatic plant, comprising mainly mono- and sesqui-terpenes, has applications in food and cosmetic industries. This study reports the characterization of isoprenyl disphosphate synthases (IDSs) involved in P. graveolens terpene biosynthesis. The six identified PgIDSs belonged to different classes of IDSs, comprising homomeric geranyl diphosphate synthases (GPPSs; PgGPPS1 and PgGPPS2), the large subunit of heteromeric GPPS or geranylgeranyl diphosphate synthases (GGPPSs; PgGGPPS), the small subunit of heteromeric GPPS (PgGPPS.SSUI and PgGPPS.SSUII), and farnesyl diphosphate synthases (FPPS; PgFPPS).All IDSs exhibited maximal expression in glandular trichomes (GTs), the site of aroma formation, and their expression except PgGPPS.SSUII was induced upon treatment with MeJA. Functional characterization of recombinant proteins revealed that PgGPPS1, PgGGPPS and PgFPPS were active enzymes producing GPP, GGPP/GPP, and FPP respectively, whereas both PgGPPS.SSUs and PgGPPS2 were inactive. Co-expression of PgGGPPS (that exhibited bifunctional G(G)PPS activity) with PgGPPS.SSUs in bacterial expression system showed lack of interaction between the two proteins, however, PgGGPPS interacted with a phylogenetically distant Antirrhinum majus GPPS.SSU. Further, transient expression of AmGPPS.SSU in P. graveolens leaf led to a significant increase in monoterpene levels. These findings provide insight into the types of IDSs and their role in providing precursors for different terpenoid components of P. graveolens essential oil., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)