IPPI, the core enzyme in C5-unit metabolism, regulates terpenoid biosynthesis in Artemisia annua

In plants, isopentenyl diphosphate isomerase (IPPI) plays a key role in regulating terpenoid biosynthesis by reversibly converting IPP and DMADP, two universal C 5 units derived from the plastidial MEP and cytoplasmic MVA pathways. However, the regulatory mechanisms of IPPIs across cellular compartments remain poorly understood. Artemisia annua , a medicinal plant renowned for its high yield of terpenoids, particularly the antimalarial drug artemisinin, serves as an excellent model for studying IPPIs function. In this study, we identified a novel dual-localized IPPI (AaIPPI2) in A. annua alongside the previously characterized plastid-localized AaIPPI1, and explored their contributions to terpenoid biosynthesis. Our results showed that AaIPPI2, primarily localized in cytoplasm, exhibited significantly lower catalytic activity toward IPP compared to AaIPPI1. Overexpression of AaIPPIs in their respective compartments enhanced the production of monoterpenes, particularly camphene and camphor. Interestingly, although artemisinin levels increased to 1.5 mg/g FW, the overall sesquiterpene synthesis did not significantly rise. This suggested that adequate levels of AaIPPI2 and its IPP substrates were manufactured in cytoplasm to compensate for its lower activity, thereby supporting sesquiterpene biosynthesis. This mechanism also facilitated the accumulation of cytoplasmic IPP and its flux into plastids for monoterpene biosynthesis. Conversely, monoterpene biosynthesis was limited by the availability of AaIPPI1 and IPP substrates. Additionally, interference with AaIPPI1 expression impaired terpenoid synthesis in both compartments, indicating that IPP formation and its normal flow between cytoplasm and plastids were disrupted. These findings enhance our understanding of terpenoid synthesis regulation in A. annua and suggest strategies for optimizing terpenoid production in plants. • A novel dual-localized IPPI member (AaIPPI2) was identified in A. annua . • AaIPPI1 and AaIPPI2 displayed different expression patterns in A. annua leaves. • AaIPPI1 and AaIPPI2 exhibited a clear preference in catalyzing IPP into DMAPP. • AaIPPI1 and AaIPPI2 synergistically regulated terpenoid biosynthesis in A. annua .