Characterization of Nuclear Factors for Elicitor-Mediated Activation of the Promoter of the Pea Phenylalanine Ammonia-Lyase Gene 1

Abstract

The nuclear factors presumably associated with the activation of the gene encoding phenylalanine ammonia-lyase by a fungal elicitor were characterized in pea (Pisum sativum L.) epicotyls. The TATA-proximal region was dissected and putative cis-regulatory elements in the promoter of pea phenylalanine ammonia-lyase gene 1 were examined by gel-mobility shift and in vitro footprinting analyses. Specific binding of the nuclear factors to the promoter-proximal regions of pea phenylalanine ammonia-lyase gene 1 associated with elicitor-mediated activation was detected at a region containing consensus sequence motifs of boxes 2 and 4 and other AT-rich sequences. The analyses of DNA fragments containing the deleted promoter regions suggested that a residue from -183 to -173 (ATTAGTAAGTGAT) was essential for a maximal activity of forming low-mobility complex (LMC) in the gel-mobility shift assay, and synthetic oligonucleotides confirmed the presence of at least one nuclear component associated with the formation of an active LMC. Competition experiments and treatment with Hoechst 33258 provided direct evidence that the formation of LMC with the promoter fragments from genes encoding phenylalanine ammonia-lyase and chalcone synthase in pea contained one or more of the same proteins that recognize AT-rich sequence motifs for binding. It also suggests that common high-mobility group-like proteins might be involved in the regulation of elicitor-inducible genes in pea.