Quantification of Three Key Enzymes Involved in Artemisinin Biogenesis in Artemisia annua by Polyclonal Antisera-Based ELISA

To elucidate the fine-tuned temporal and spatial modulation of artemisinin production in annual wormwood (Artemisia annua), we conducted enzyme-linked immunosorbent assay-based immunoquantification of three key enzymes involved in artemisinin biosynthesis, amorpha-4,11-diene synthase (ADS), cytochrome P450 monooxygenase (CYP71AV1), and cytochrome P450 reductase (CPR), in various tissues and under different growth conditions. The field-grown plants accumulate abundant ADS and CYP71AV1 but a trace amount of CPR in all tested tissues. Furthermore, ADS and CYP71AV1 accumulations in leaves are 16- and eightfold higher than in roots, and ten- and fourfold higher than in stems, respectively, demonstrating a tissue-specific expression pattern. Interestingly, the flowering field plants and cold-acclimated cultural plants produce higher levels of ADS and CYP71AV1 than non-flowering field plants or untreated cultural plants, indicating the environmental and developmental induction on ADS and CYP71AV1 genes and providing possible explanation for the observation that elevation of artemisinin level occurs after flowering.