American Journal of Plant Sciences, 2011, 2, 619-628 doi:10.4236/ajps.2011.24073 Published Online October 2011 (http://www.SciRP.org/journal/ajps) Copyright © 2011 SciRes. AJPS 619 Trichome-Specific Expression of Amorpha-4,11-Diene Synthase, a Key Enzyme of Artemisinin Biosynthesis in Artemisia annua L., as Reported by a Promoter-GUS Fusion Hongzhen Wang, Linda Olofsson, Anneli Lundgren, Peter E. Brodelius School of Natural Sciences, Linnaeus University, Kalmar, Sweden. Email: peter.brodelius@lnu.se Received August 16 th , 2011; revised September 7 th , 2011; accepted September 20 th , 2011. ABSTRACT Artemisia annua L. produces small amounts of the sesquiterpenoid artemisinin, which is used for treatment of malaria. A worldwide shortage of the drug has led to intense research to increase the yield of artemisinin in the plant. In order to study the regulation of expression of a key enzyme of artemisinin biosynthesis, the promoter region of the key enzyme amorpha-4,11-diene synthase (ADS) was cloned and fused with the β-glucuronidase (GUS) reporter gene. Transgenic plants of A. annua expressing this fusion were generated and studied. Transgenic plants expressing the GUS gene were used to establish the activity of the cloned promoter by a GUS activity staining procedure. GUS under the control of the ADS promoter showed specific expression in glandular trichomes. The activity of the ADS promoter varies temporally and in old tissues essentially no GUS staining could be observed. The expression pattern of GUS and ADS in aerial parts of the transgenic plant was essentially the same indicating that the cis-elements controlling glandular trichome specific expression are included in the cloned promoter. However, some cis-element(s) that control expression in root and old leaf appears to be missing in the cloned promoter. Furthermore, qPCR was used to compare the activity of the wild-type ADS promoter with that of the cloned ADS promoter. The latter promoter showed a considerably lower activ- ity than the wild-type promoter as judged from the levels of GUS and ADS transcripts, respectively, which may be due to the removal of an enhancing cis-element from the ADS promoter. The ADS gene is specifically expressed in stalk and secretory cells of glandular trichomes of A. annua. Keywords: Agrobacterium Tumefaciens, Amorpha-4,11-Diene Synthase, Artemisia annua, Artemisinin Biosynthesis, β-Glucuronidase, Gene Regulation, Promoter Activity, Stable Transformation 1. Introduction Artemisinin is an effective anti-malarial drug, which has become an important component of artemisinin-based combination therapies (ACTs) [1]. The content of ar- temisinin in Artemisia annua is, however, very low and ranges between 0.1% and 0.8% of dry weight [2]. Selec- tion of high-producing varieties of A. annua has met lim- ited success but hybrids producing increased amounts of artemisinin have been produced [3]. However, there is still a shortage in the supply of artemisinin [4]. Different methods have been tried to improve the artemisinin pro- duction such as treatment with abscisic acid [5], gibbere- lic acid [6-8] and elicitors [9-11]. Various transgenic A. annua plants have been produced to increase the yield of artemisinin. These attempts include down-regulation of squalene synthase [12,13] or up-regulation of farnesyl diphosphate synthase (FDS) [14]. Cyclization of farnesyl diphosphate (FDP) to amor- pha-4,11-diene by amorpha-4,11-diene synthase (ADS) is the initial step of the artemisinin biosynthetic pathway [15] and amorpha-4,11-diene is the committed precursor [16]. Tissue specificity of ADS expression has been shown by GUS expression in Arabidopsis thaliana using a fusion of the ADS promoter and the reporter gene [17]. In the following step of the artemisinin biosynthesis, amorpha-4,11-diene is hydroxylated to yield artemisinic alcohol. This reaction is catalyzed by a cytochrome P450 dependent amorpha-4,11-diene 12-hydroxylase (CYP71AV1)