Amorpha-4,11-diene synthase (ADS) of Artemisia annua catalyzes the conversion of farnesyl diphosphate into amorpha-4,11-diene, the first committed step in the biosynthesis of the antimalarial drug artemisinin. The promoters of ADS contain two reverse-oriented TTGACC W-box cis-acting elements, which are the proposed binding sites of WRKY transcription factors. A full-length cDNA (AaWRKY1) was isolated from a cDNA library of the glandular secretory trichomes (GSTs) in which artemisinin is synthesized and sequestered. AaWRKY1 encodes a 311 amino acid protein containing a single WRKY domain. AaWRKY1 and ADS genes were highly expressed in GSTs and both were strongly induced by methyl jasmonate and chitosan. Transient expression analysis of the AaWRKY1-GFP (green fluorescent protein) reporter revealed that AaWRKY1 was targeted to nuclei. Biochemical analysis demonstrated that the AaWRKY1 protein was capable of binding to the W-box cis-acting elements of the ADS promoters, and it demonstrated transactivation activity in yeast. Co-expression of the effector construct 35S::AaWRKY1 with a reporter construct ADSpro1::GUS greatly activated expression of the GUS (beta-glucuronidase) gene in stably transformed tobacco. Furthermore, transient expression experiments in agroinfiltrated Nicotiana benthamiana and A. annua leaves showed that AaWRKY1 protein transactivated the ADSpro2 promoter activity by binding to the W-box of the promoter; disruption of the W-box abolished the activation. Transient expression of AaWRKY1 cDNA in A. annua leaves clearly activated the expression of the majority of artemisinin biosynthetic genes. These results strongly suggest the involvement of the AaWRKY1 transcription factor in the regulation of artemisinin biosynthesis, and indicate that ADS is a target gene of AaWRKY1 in A. annua.

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