Synergistic interaction of AaMYC2 and AaMYC2-LIKE enhances artemisinin production in Artemisia annua L.

Abstract

Artemisinin-based combination therapies recommended by WHO marks Artemisia annua as the only natural source of artemisinin fighting deadly disease, Malaria. In this study, we isolated two transcription factors, AaMYC2 and AaMYC2-LIKE, from A. annua and investigated their role in regulating artemisinin biosynthetic pathway. Our findings depict that both AaMYC2 and AaMYC2-LIKE are transcriptionally active and, when co-transformed in yeast cells, significantly enhance β-galactosidase activity in transactivation assays as compared to their individual transformations. Furthermore, Yeast two-hybrid (Y2H) and Biomolecular fluorescence complementation assays revealed AaMYC2 physically interacts with AaMYC2-LIKE in yeast cells and in the nucleus of onion epidermal cells respectively. Generation of transient transgenic over expression and co-expression lines of AaMYC2 and AaMYC2-LIKE resulted in elevated expression of artemisinin biosynthetic genes and trichome development genes in co-expression lines as compared to individual transgenic lines and wildtype. Importantly, the glandular trichome density and artemisinin content was also significantly higher in co-transformed transgenic lines compared to individual AaMYC2 and AMYC2-LIKE transgenic lines. Conversely, artemisinin content was markedly reduced in AaMYC2-RNAi lines, underscoring the critical role of functional AaMYC2 in synergistic regulation with AaMYC2-LIKE. Altogether the above studies provide valuable insights into the regulatory networks of MYC type bHLH transcription factors in controlling economically and medically important pathway in A. annua.