AN ETHYLENE-RESPONSIVE ENHANCER ELEMENT IS INVOLVED IN THE SENESCENCE-RELATED EXPRESSION OF THE CARNATION GLUTATHIONE-S-TRANSFERASE (GSTI) GENE

The increased production of ethylene during carnation petal senescence regulates the transcription of the GST1 gene encoding a subunit of gl utathione-S-transferase. We have investigated the molecular basis for this ethylene-responsive transcription by examining the cis elements a nd trans-acting factors involved in the expression of the GST1 gene. T ransient expression assays following delivery of GST1 5' flanking DNA fused to a beta-glucuronidase reporter gene were used to functionally define sequences responsible for ethylene-responsive expression. Delet ion analysis of the 5' flanking sequences of GST1 identified a single positive regulatory element of 197 bp between -667 and -470 necessary for ethylene-responsive expression. The sequences within this ethylene -responsive region were further localized to 126 bp between -596 and - 470. The ethylene-responsive element (ERE) within this region conferre d ethylene-regulated expression upon a minimal cauliflower mosaic viru s-35S TATA-box promoter in an orientation-independent manner. Gel elec trophoresis mobility-shift assays and DNase I footprinting were used t o identify proteins that bind to sequences within the ERE. Nuclear pro teins from carnation petals were shown to specifically interact with t he 126-bp ERE and the presence and binding of these proteins were inde pendent of ethylene or petal senescence. DNase I footprinting defined DNA sequences between -510 and -488 within the ERE specifically protec ted by bound protein. An 8-bp sequence (ATTTCAAA) within the protected region shares significant homology with promoter sequences required f or ethylene responsiveness from the tomato fruit-ripening E4 gene.