methyl-jasmonate and sclareolide

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.

Topics: Acetates; ATP-Binding Cassette Transporters; Base Sequence; Cell Line; Cyclopentanes; Deoxyribonuclease I; Diterpenes; DNA Footprinting; DNA Mutational Analysis; Electrophoretic Mobility Shift Assay; Ethylenes; Gene Expression Regulation, Plant; Glucuronidase; Molecular Sequence Data; Nicotiana; Oxylipins; Plant Proteins; Promoter Regions, Genetic; Recombinant Fusion Proteins; Regulatory Sequences, Nucleic Acid; Salicylic Acid; Sequence Deletion; Sequence Homology, Nucleic Acid