Synergistic interaction of two bHLH transcription factors positively regulates artemisinin biosynthetic pathway in Artemisia annua L.

The wonder drug artemisinin, a sesquiterpene lactone endoperoxide from Artemisia annua is the million-dollar molecule required to curb the deadliest disease, Malaria. One of the major challenges even today is to increase the concentration of artemisinin within plants. The transcription factors are important regulators of plant secondary metabolites and have the potential to regulate key steps or the whole biosynthetic pathway. In this study, we have identified and characterised two bHLH transcription factors (Aa6119 and Aa7162) from A. annua. Both the transcription factors turned out to be transcriptionally active and nuclear-localised typical bHLH proteins. In our study, we found that Aa6119 specifically binds to the E-box element present on the promoter of artemisinin biosynthetic gene, AMORPHA-4,11-DIENE SYNTHASE (ADS). The protein-DNA interaction confirmed by Yeast one-hybrid assay was specific as Aa6119 was unable to bind to the mutated E-boxes of ADS. Further, Aa6119 interacted physically with Aa7162, which was confirmed in vitro by Yeast two-hybrid assay and in vivo by Bimolecular Fluorescent complementation assay. Our quantitative expression studies have confirmed that Aa6119 and Aa7162 act synergistically in the regulation of artemisinin biosynthetic and trichome developmental genes. The higher accumulation of artemisinin content in the transient co-transformed transgenic plants than in the individual over-expression transgenic plants has further validated that Aa6119 and Aa7162 act positively and synergistically to regulate artemisinin accumulation.