title: Identification of regulatory sequence elements within the transcription promoter region of NpABC1, a gene encoding a plant ABC transporter induced by diterpenes.
abstract: Expression of NpABC1, a gene encoding a plasma membrane ATP binding cassette (ABC) transporter in Nicotiana plumbaginifolia, is induced by sclareol, an antifungal diterpene produced at the leaf surface, as well as by sclareolide, a close analog. A genomic fragment including the 1282-bp region upstream of the NpABC1 transcription start was fused to the reporter beta-glucuronidase (gus) gene and introduced into N. tabacum BY2 cells for stable transformation. A 25-fold increase in gus expression was observed when cells were treated with sclareolide and some other terpenes. The combined use of 5'-deletion promoter analysis, gel mobility shift assays, DNase I footprinting, and site-directed mutagenesis allowed us to identify three cis-elements (sclareol box 1 (SB1), SB2, and SB3) located, respectively, within nucleotides -827 to -802, -278 to -243, and -216 to -190 upstream of the NpABC1 transcription start. In vivo evaluation of these elements on sclareolide-induced expression showed that mutation of SB1 reduced expression by twofold, while that of SB2 had no effect. On the other hand, SB3 had a marked effect as it completely abolished sclareolide-mediated expression. NpABC1-gus expression was not induced by the stress signals, salicylic acid and ethylene, but was mediated, to some extent, by methyl jasmonate, which is known to promote diterpene synthesis.
authors.affiliation: Unité de Biochimie Physiologique, Institut des sciences de la vie, Université Catholique de Louvain, Croix du Sud 2-20, B-1348 Louvain la Neuve, Belgium.
authors.forename: Sébastien
authors.initials: S
authors.lastname: Grec
authors.forename: Delphine
authors.initials: D
authors.lastname: Vanham
authors.forename: Jeoffrey Christyn
authors.initials: JC
authors.lastname: de Ribaucourt
authors.forename: Bénédicte
authors.initials: B
authors.lastname: Purnelle
authors.forename: Marc
authors.initials: M
authors.lastname: Boutry
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics* / Q000378:metabolism; D000085:Acetates / Q000494:pharmacology; D001483:Base Sequence; D002460:Cell Line; D003517:Cyclopentanes / Q000494:pharmacology; D018983:DNA Footprinting / Q000379:methods; D004252:DNA Mutational Analysis / Q000379:methods; D003850:Deoxyribonuclease I / Q000378:metabolism; D004224:Diterpenes / Q000494:pharmacology*; D024202:Electrophoretic Mobility Shift Assay; D005030:Ethylenes / Q000494:pharmacology; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D054883:Oxylipins; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D020156:Salicylic Acid / Q000494:pharmacology; D017384:Sequence Deletion; D012689:Sequence Homology, Nucleic Acid; D014026:Nicotiana / Q000166:cytology / Q000235:genetics
chemical_list: D018528:ATP-Binding Cassette Transporters; D000085:Acetates; D003517:Cyclopentanes; D004224:Diterpenes; D005030:Ethylenes; D054883:Oxylipins; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; C109395:sclareolide; C072239:methyl jasmonate; C036216:ethylene; C070760:sclareol; D003850:Deoxyribonuclease I; D005966:Glucuronidase; D020156:Salicylic Acid