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 β‐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.