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BackgroundRecently, Artemisia annua L. (annual or sweet wormwood) has received increasing attention due to the fact that the plant produces the sesquiterpenoid endoperoxide artemisinin, which today is widely used for treatment of malaria. The plant produces relatively small amounts of artemisinin and a worldwide shortage of the drug has led to intense research in order to increase the yield of artemisinin. In order to improve our understanding of terpene metabolism in the plant and to evaluate the competition for precursors, which may influence the yield of artemisinin, we have used qPCR to estimate the expression of 14 genes of terpene metabolism in different tissues.ResultsThe four genes of the artemisinin biosynthetic pathway (amorpha-4,11-diene synthase, amorphadiene-12-hydroxylase, artemisinic aldehyde ∆11(13) reductase and aldehyde dehydrogenase 1) showed remarkably higher expression (between ~40- to ~500-fold) in flower buds and young leaves compared to other tissues (old leaves, stems, roots, hairy root cultures). Further, dihydroartemisinic aldehyde reductase showed a very high expression only in hairy root cultures. Germacrene A and caryophyllene synthase were mostly expressed in young leaves and flower buds while epi-cedrol synthase was highly expressed in old leaves. 3-Hydroxy-3-methyl-glutaryl coenzyme A reductase exhibited lower expression in old leaves compared to other tissues. Farnesyldiphosphate synthase, squalene synthase, and 1-deoxy-D-xylulose-5-phosphate reductoisomerase showed only modest variation in expression in the different tissues, while expression of 1-deoxy-D-xylulose-5-phosphate synthase was 7-8-fold higher in flower buds and young leaves compared to old leaves.ConclusionsFour genes of artemisinin biosynthesis were highly expressed in flower buds and young leaves (tissues showing a high density of glandular trichomes). The expression of dihydroartemisinic aldehyde reductase has been suggested to have a negative effect on artemisinin production through reduction of dihydroartemisinic aldehyde to dihydroartemisinic alcohol. However, our results show that this enzyme is expressed only at low levels in tissues producing artemisinin and consequently its effect on artemisinin production may be limited. Finally, squalene synthase but not other sesquiterpene synthases appears to be a significant competitor for farnesyl diphosphate in artemisinin-producing tissues.

A clone encoding farnesyl diphosphate synthase (FPPS) was obtained by PCR from a cDNA library made from young leaves of Artemisia annua. A cDNA clone encoding the tobacco epi-aristolochene synthase (eAS) was kindly supplied by J. Chappell (University of Kentucky, Lexington, KY, USA). Two fusions were constructed, i.e. FPPS/ eAS and eAS/FPPS. The stop codon of the N-terminal enzyme was removed and replaced by a short peptide (Gly-SerGly) to introduce a linker between the two ORFs. These two fusions and the two single cDNA clones were separately introduced into a bacterial expression vector (pET32). Escherichia coli was transformed with the expression vectors and enzymatically active soluble proteins were obtained after induction with isopropyl thio-b-D-thiogalactoside. The recombinant enzymes were purified using immobilized metal affinity chromatography on Co 2+ columns. The fusion enzymes produced epi-aristolochene from isopentenyl diphosphate through a coupled reaction. The K m values of FPPS and eAS for isopentenyl diphosphate and farnesyl diphosphate, respectively, were essentially the same for the single and fused enzymes. The bifunctional enzymes showed a more efficient conversion of isopentenyl diphosphate to epi-aristolochene than the corresponding amount of single enzymes.

Artemisinin derivatives are effective anti-malarial drugs. In order to design transgenic plants of Artemisia annua with enhanced biosynthesis of artemisinin, we are studying the promoters of genes encoding enzymes involved in artemisinin biosynthesis. A 1,151 bp promoter region of the cyp71av1 gene, encoding amorpha-4,11-diene 12-hydroxylase, was cloned. Alignment of the cloned promoter and other cyp71av1 promoter sequences indicated that the cyp71av1 promoter may be different in different A. annua varieties. Comparison to the promoter of amorpha-4,11-diene synthase gene showed a number of putative cis-acting regulatory elements in common, suggesting a co-regulation of the two genes. The cyp71av1 promoter sequence was fused to the β-glucuronidase (GUS) reporter gene and two varieties of A. annua and Nicotiana tabacum were transformed. In A. annua, GUS expression was exclusively localized to glandular secretory trichomes (GSTs) of leaf primordia and top expanded leaves. In older leaves, there is a shift of expression to T-shaped trichomes (TSTs). Only TSTs showed GUS staining in lower leaves and there is no GUS staining in old leaves. GUS expression in flower buds was specifically localized to GSTs. The recombinant promoter carries the cis-acting regulatory elements required for GST-specific expression. The cyp71av1 promoter shows activity in young tissues. The recombinant promoter was up to 200 times more active than the wild type promoter. GUS expression in transgenic N. tabacum was localized to glandular heads. Transcript levels were up-regulated by MeJA. Wound responsiveness experiment showed that the cyp71av1 promoter does not appear to play any role in the response of A. annua to mechanical stress.

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