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Artemisinin, a sesquiterpene lactone obtained from Artemisia 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, have not yet been elucidated. AaWRKY9 protein is a positive regulator of artemisinin biosynthesis that activates AaGSW1 (glandular trichome–specific WRKY1) and AaDBR2 (double-bond reductase 2). 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. Toward 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 overexpression plants, the expression of GSW1 was found to be significantly increased when compared to that of AaYABBY5 antisense or control plants. In addition, AaGSW1 was identified as an upstream activator of AaYABBY5. Further, it was found that AaJAZ8, a transcriptional repressor of jasmonate signaling, interacted with AaYABBY5 and attenuated its activity. Co-expression of AaYABBY5 and anti- AaJAZ8 in A. annua increased the activity of AaYABBY5 toward artemisinin biosynthesis. This current study provides the first indication of the molecular basis of regulation of artemisinin biosynthesis through YABBY–WRKY interactions, which are regulated through AaJAZ8. This knowledge presents AaYABBY5 overexpression plants as a powerful genetic resource for artemisinin biosynthesis. [ABSTRACT FROM AUTHOR]

Artemisinin, the frontline drug against malaria, is a sesquiterpenoid extracted from Artemisia annua. Light has been proposed to play an important role in the activation of artemisinin biosynthesis. Here, we report the basic leucine zipper transcription factor (TF) AaHY5 as a key regulator of light-induced biosynthesis of artemisinin. We show that AaHY5 transcription overlaps with that of artemisinin biosynthesis genes in response to light and in A. annua tissues. Analysis of AaHY5 overexpression and RNAi-suppression lines suggests that AaHY5 is a positive regulator of the expression of artemisinin biosynthesis genes and accumulation of artemisinin. We show that AaHY5 complements the hy5 mutant in Arabidopsis thaliana. Our data further suggest that AaHY5 interacts with AaCOP1, the ubiquitin E3 ligase CONSTITUTIVE PHOTOMORPHOGENIC1 in A. annua. In yeast one-hybrid and transient expression assays, we demonstrate that AaHY5 acts via the TF GLANDULAR TRICHOME-SPECIFIC WRKY 1 (AaGSW1) in artemisinin regulation. In summary, we present a novel regulator of artemisinin gene expression and propose a model in which AaHY5 indirectly controls artemisinin production in response to changing light conditions. [ABSTRACT FROM AUTHOR]

Artemisinin is a sesquiterpene lactone produced by the Chinese traditional herb Artemisia annua and is used for the treatment of malaria. It is known that salicylic acid (SA) can enhance artemisinin content but the mechanism by which it does so is not known. In this study, we systematically investigated a basic leucine zipper family transcription factor, AaTGA6, involved in SA signaling to regulate artemisinin biosynthesis. We found specific in vivo and in vitro binding of the AaTGA6 protein to a 'TGACG' element in the AaERF1 promoter. Moreover, we demonstrated that AaNPR1 can interact with AaTGA6 and enhance its DNA-binding activity to its cognate promoter element 'TGACG' in the promoter of AaERF1 , thus enhancing artemisinin biosynthesis. The artemisinin contents in AaTGA6-overexpressing and RNAi transgenic plants were increased by 90–120% and decreased by 20–60%, respectively, indicating that AaTGA6 plays a positive role in artemisinin biosynthesis. Importantly, heterodimerization with AaTGA3 significantly inhibits the DNA-binding activity of AaTGA6 and plays a negative role in target gene activation. In conclusion, we demonstrate that binding of AaTGA6 to the promoter of the artemisinin-regulatory gene AaERF1 is enhanced by AaNPR1 and inhibited by AaTGA3. Based on these findings, AaTGA6 has potential value in the genetic engineering of artemisinin production. [ABSTRACT FROM AUTHOR]