Flavonoids are essential secondary metabolites that predominantly affect flower pigmentation in plants. Understanding the molecular mechanisms underlying flower color divergence is crucial for ornamental plant breeding. This study aimed to elucidate the factors responsible for the differences in color between white-petaled Cosmos bipinnatus and orange-petaled Cosmos sulphureus. We employed an integrated approach combining untargeted LC–MS/MS metabolomics and high-throughput transcriptome sequencing of fresh petals to analyze pigment composition and differential gene expression. Petal pigment extraction, total flavonoid quantification, and metabolomic profiling consistently revealed that differences in flavonoid abundance are responsible for flower color divergence between the two species. In contrast, carotenoids, previously considered potential contributors to flower coloration, were neither evident in the oil phase of the pigment extracts nor detected by metabolomic analysis. Flavonoid compounds accumulated at relatively high levels in the orange petals of C. sulphureus, reaching 11.36 times that of C. bipinnatus, contributing to its bright appearance. Transcriptomic analysis revealed differences in gene expression patterns between the two species, highlighting key candidate genes involved in the flavonoid biosynthesis pathway, such as chalcone synthase. These findings indicate that the orange coloration of C. sulphureus may be associated with CHS-regulated accumulation of naringenin chalcone and downstream compounds in the flavonoid metabolic pathway after CHS, providing valuable theoretical support for a deeper understanding of the causes underlying the differences in flower color between C. bipinnatus and the orange-petaled C. sulphureus.

Isopentenyl diphosphate isomerase (IPPI) is a key enzyme catalyzing the interconversion between isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), two isoprenyl C5 units that are precursors for terpenoid biosynthesis. Previous study has identified two IPPIs, namely AaIPPI1 and AaIPPI2 from Artemisia annua, and revealed the positive role of AaIPPI1 in regulating the biosynthesis of terpenoids. However, the role of AaIPPI2 in regulating terpenoid biosynthesis remains to be clarified. In this study, we explored the contribution of AaIPPI2 to terpenoid biosynthesis in Artemisia annua. Overexpression of AaIPPI2 in A. annua significantly enhanced the contents of diverse monoterpenes and sesquiterpenes. Suppression of AaIPPI2 by RNA interference (RNAi) in A. annua markedly reduced the biosynthesis of these sesquiterpenes, but only caused a modest decrease in several monoterpenes’ levels with no change in other monoterpenes’ levels, suggesting that AaIPPI2 plays a lesser part in the regulation of monoterpenes biosynthesis in plastids compared to AaIPPI1. Taken together, these results demonstrate that AaIPPI2 plays a positive role in regulating the biosynthesis of terpenoids. Our findings enhanced the understanding of the distinction between the roles of the two IPPIs in the regulation of terpenoid biosynthesis in A. annua and provided candidate genes for optimizing terpene production in plants.