title: Studies on the expression of sesquiterpene synthases using promoter-β-glucuronidase fusions in transgenic Artemisia annua L.
abstract: In order to better understand the influence of sesquiterpene synthases on artemisinin yield in Artemisia annua, the expression of some sesquiterpene synthases has been studied using transgenic plants expressing promoter-GUS fusions. The cloned promoter sequences were 923, 1182 and 1510 bp for β-caryophyllene (CPS), epi-cedrol (ECS) and β-farnesene (FS) synthase, respectively. Prediction of cis-acting regulatory elements showed that the promoters are involved in complex regulation of expression. Transgenic A. annua plants carrying promoter-GUS fusions were studied to elucidate the expression pattern of the three sesquiterpene synthases and compared to the previously studied promoter of amorpha-4,11-diene synthase (ADS), a key enzyme of artemisinin biosynthesis. The CPS and ECS promoters were active in T-shaped trichomes of leaves and stems, basal bracts of flower buds and also in some florets cells but not in glandular secretory trichome while FS promoter activity was only observed in leaf cells and trichomes of transgenic shoots. ADS, CPS, ECS and FS transcripts were induced by wounding in a time depended manner. The four sesquiterpene synthases may be involved in responsiveness of A. annua to herbivory. Methyl jasmonate treatment triggered activation of the promoters of all four sesquiterpene synthases in a time depended manner. Southern blot result showed that the GUS gene was inserted into genomic DNA of transgenic lines as a single copy or two copies. The relative amounts of CPS and ECS as well as germacrene A synthase (GAS) transcripts are much lower than that of ADS transcript. Consequently, down-regulation of the expression of the CPS, ECS or GAS gene may not improve artemsinin yield. However, blocking the expression of FS may have effects on artemisinin production.
authors.affiliation: Department of Chemistry and Biomedicine, Linnaeus University, Kalmar, Sweden.
authors.forename: Hongzhen
authors.initials: H
authors.lastname: Wang
authors.forename: Junli
authors.initials: J
authors.lastname: Han
authors.forename: Selvaraju
authors.initials: S
authors.lastname: Kanagarajan
authors.forename: Anneli
authors.initials: A
authors.lastname: Lundgren
authors.forename: Peter E
authors.initials: PE
authors.lastname: Brodelius
mesh_terms: D000085:Acetates / Q000494:pharmacology; D019883:Alkyl and Aryl Transferases / Q000235:genetics* / Q000378:metabolism; D031172:Artemisia annua / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D015139:Blotting, Southern; D003517:Cyclopentanes / Q000494:pharmacology; D035264:Flowers / Q000187:drug effects / Q000235:genetics; D020869:Gene Expression Profiling; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D005966:Glucuronidase / Q000235:genetics* / Q000378:metabolism; D054883:Oxylipins / Q000494:pharmacology; D018515:Plant Leaves / Q000187:drug effects / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified; D011106:Polyisoprenyl Phosphates / Q000737:chemistry / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012717:Sesquiterpenes / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity / Q000187:drug effects
chemical_list: D000085:Acetates; D003517:Cyclopentanes; D054883:Oxylipins; D010940:Plant Proteins; D011106:Polyisoprenyl Phosphates; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D012717:Sesquiterpenes; C004808:farnesyl pyrophosphate; C072239:methyl jasmonate; D019883:Alkyl and Aryl Transferases; C108877:terpene synthase; D005966:Glucuronidase