Trichome-specific expression of the amorpha-4,11-diene 12-hydroxylase (cyp71av1) gene, encoding a key enzyme of artemisinin biosynthesis in Artemisia annua, as reported by a promoter-GUS fusion

Abstract

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.

OSC2 and CYP716A14v2 Catalyze the Biosynthesis of Triterpenoids for the Cuticle of Aerial Organs of Artemisia annua

“…In addition, the glandular trichomes have been proposed to be the primary site for artemisinin biosynthesis and storage in the plant (Duke et al, 1994;Van Nieuwerburgh et al, 2006;Olsson et al, 2009;Wang et al, 2013). In our comparative transcriptome analysis, transcripts corresponding to putative or known monoterpenoid, diterpenoid, and (most) sesquiterpenoid biosynthesis genes were overrepresented in the glandular trichomes, as opposed to the triterpenoid transcripts, which were abundant in the filamentous trichomes (Soetaert et al, 2013).…”

Reference gene selection in Artemisia annua L., a plant species producing anti-malarial artemisinin

“…ADS is the first committed enzyme involved in artemisinin biosynthesis, which catalyzed the cyclization of farnesyl diphosphate to form amorpha-4,11-diene, the unique cyclic precursor for artemisinin (Picaud et al 2005). The ADS gene was specifically expressed in glandular trichomes which were the location of artemisinin biosynthesis in A. annua (Wang et al 2013b). A lot of publications reported that the expression level of ADS was much higher in leaf than that in root, because root did not have glandular trichomes (Olofsson et al 2011).…”

“…Methyl jasmonate (MeJA) can greatly up-regulate the expression levels of ADS (Wang et al 2013a) and CYP71AV1 (Wang et al 2013b) and as a result, the content of artemisinin is consequently increased (Wang et al 2010). DBR2 expression level was not up-regulated by MeJA treatment when the UBQ gene was been used as the reference gene (Caretto et al 2011).…”

Plant Glandular Trichomes: Natural Cell Factories of High Biotechnological Interest

“…In addition to trichome-specific EST resources, numerous glandular trichome-specific gene promoters have been reported in the literature for a variety of plants, including (but not restricted to) Antirrhinum majus, A. annua, C. sativus, H. lupulus, Mentha spp., N. tabacum, S. lycopersicum, and S. habrochaites (Okada and Ito, 2001;Wang et al, 2002Wang et al, , 2011Wang et al, , 2013Gutiérrez-Alcalá et al, 2005;Liu et al, 2006;Jaffé et al, 2007;Kim et al, 2008;Shangguan et al, 2008;Ennajdaoui et al, 2010;Choi et al, 2012;Sallaud et al, 2012;Spyropoulou et al, 2014;Kortbeek et al, 2016;Laterre et al, 2017;Vining et al, 2017;for review, see Tissier, 2012b). It is worth noting that most of these trichome-specific promoters are active in mature glandular trichomes (mostly in glandular cells at the tip of the trichome).…”