Contrary operations of Box-I element of pea phenylalanine ammonia-lyase gene 1 promoter for organ-specific expression

Expression of genes (PSPAL) encoding phenylalanine ammonia-lyase from pea (Pisum sativum L.) is regulated in response to various environmental stimuli and during plant development. We examined the cis-regulatory elements in PSPAL1 promoter for organ-specific expression by determining the sequences specifically associated with nuclear proteins on its promoter in each pea organ. In vivo dimethyl sulfate (DMS) footprinting analysis showed putative protein bindings on AC-rich sequences including Box-I in particular in roots and stems in which PSPAL mRNA were highly accumulated. The potential role of the AC-rich element was investigated by fusing PSPAL1 promoter with Box-I deleted to the reporter gene β-glucuronidase (GUS) and transforming the construct into tobacco plants (Nicotiana tabacum). GUS activity controlled under this Box-I deletion promoter was significantly reduced in roots and leaves, whereas drastically increased in stems as compared with the activity of wild-type PSPAL1 promoter. Histochemical GUS staining indicated the activity was elevated in vascular tissues of stem by deleting Box-I. These results suggest that Box-I in PSPAL1 contributes to a positive regulation in root and leaf, but might also function as a negative regulator in stem, especially in xylem tissue.