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Plant and Cell Physiology Advance Access published online on October 30, 2009
Plant and Cell Physiology, doi:10.1093/pcp/pcp149
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© The Author 2009. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Isolation and Characterization of AaWRKY1, an Artemisia annua Transcription Factor that Regulates the Amorpha-4,11-diene Synthase Gene, a Key Gene of Artemisinin Biosynthesis

Dongming Ma, Gaobin Pu, Caiyan Lei, Lanqing Ma, Huahong Wang, Yanwu Guo, Jianlin Chen, Zhigao Du, Hong Wang, Guofeng Li, Hechun Ye and Benye Liu*

Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, the Chinese Academy of Sciences, Nanxincun 20, Haidian District, 100093 Beijing, China

* To whom correspondence should be addressed. Corresponding author Porf. Dr. Benye Liu Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, the Chinese Academy of Sciences, Nanxincun 20, Haidian District, 100093 Beijing, China Phone: +86-10-62836249 Fax: +86-10-82591016 E-mail: benyel{at}ibcas.ac.cn


  Abstract

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 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. Coexpression of the effector construct 35S::AaWRKY1 with a reporter construct ADSpro1::GUS highly activated expression of the GUS gene in stably transformed tobacco. Furthermore, transient expression experiments in agroinfiltrated N. 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 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.

Keywords: Amorpha-4,11-diene synthase - Artemisia annua - Artemisinin - W-box - WRKY transcription factor

The nucleotide sequence reported in this paper has been submitted to the GenBank under accession number FJ390842 [GenBank]

(Received September 22, 2009; Accepted October 16, 2009)
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