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 activity 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.
Highlights
Artemisinin is an effective anti-malarial drug, which has become an important component of artemisinin-based combination therapies (ACTs) [1]
For artemisinin biosynthesis the artemisinic aldehyde must be reduced to dihydroartemisinic aldehyde, which subsequently is oxidized to dihydroartemisinic acid and converted to artemisinin [20]
Transient expression of the A. annua transcription factor AaWRKY in agroinfiltrated leaves of A. annua resulted in increased levels of hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), ADS, CYP71AV1 and DBR2 indicating that several of the genes encoding enzymes involved in artemisinin biosynthesis are induced by binding of AaWRKY to the W-boxes [33]
Summary
Artemisinin is an effective anti-malarial drug, which has become an important component of artemisinin-based combination therapies (ACTs) [1]. For artemisinin biosynthesis the artemisinic aldehyde must be reduced to dihydroartemisinic aldehyde, which subsequently is oxidized to dihydroartemisinic acid and converted to artemisinin [20]. These two steps are catalyzed by artemisinic aldehyde 11 (13) reductase [21] and aldehyde dehydrogenase 1 [22], respectively. We have initiated studies on the regulation of terpene metabolism in different tissues of A. annua [25] and here we report on results from transgenic A. annua expressing a fusion of the reporter gene (GUS) and the promoter of the key enzyme ADS
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