JA-regulated AaGSW1-AaYABBY5/AaWRKY9 complex regulates artemisinin biosynthesis in Artemisia annua.

Artemisinin, a sesquiterpene lactone from A. annua, is an essential therapeutic against malaria. YABBY family transcription factor; AaYABBY5 is an activator of AaCYP71AV1 (cytochrome P450-dependent hydroxylase) and AaDBR2 (double bond reductase 2); however, the protein-protein interactions of AaYABBY5, as well as the mechanism of its regulation, are not elucidated before. AaWRKY9 protein is a positive regulator of artemisinin biosynthesis that activates AaGSW1 (Glandular trichome specific WRKY1) and AaDBR2 (double bond reductase 2), respectively. In this study, YABBY-WRKY interactions are revealed to indirectly regulate artemisinin production. AaYABBY5 significantly increased the activity of the luciferase (LUC) gene fused to the promoter of AaGSW1. Towards the molecular basis of this regulation, AaYABBY5 interaction with AaWRKY9 protein was found. The combined effectors AaYABBY5 + AaWRKY9 showed synergistic effects toward the activities of AaGSW1, and AaDBR2 promoters, respectively. In AaYABBY5 over-expression plants, the expression of GSW1 was found significantly increase when compared to that of AaYABBY5 antisense or control plants. Secondly, AaGSW1 was seen as an upstream activator of AaYABBY5. Thirdly, it was found that AaJAZ8, a transcriptional repressor of jasmonates signaling, interacted with AaYABBY5 and attenuated its activity. Co-expression of AaYABBY5 and antiAaJAZ8 in A. annua increased the activity of AaYABBY5 towards artemisinin biosynthesis. For the first time, the current study provided the molecular basis of regulation of artemisinin biosynthesis through YABBY-WRKY interactions and its regulation through AaJAZ8. This knowledge provides AaYABBY5 overexpression plants as a powerful genetic resource for artemisinin biosynthesis.