SEPARATION OF CIS-ELEMENTS RESPONSIVE TO ETHYLENE, FRUIT-DEVELOPMENT,AND RIPENING IN THE 5'-FLANKING REGION OF THE RIPENING-RELATED E8 GENE

The E8 gene is expressed at a high level during fruit ripening, and is transcriptionally activated by ethylene. We have identified a 428 bp fragment of the E8 5'-flanking region, from -1528 to -1100, that makes a minimal 35S promoter responsive to ethylene, This fragment confers ethylene-responsiveness only in the 5'-to-3' orientation; in the rever se orientation it results in increased expression in unripe fruit. Int erestingly, this ethylene-responsive construct does not have high leve ls of expression during fruit ripening, indicating that sequences requ ired for high level expression during fruit ripening are separate from sequences required for ethylene response. The ethylene-responsive seq uences of the E8 5'-flanking region interact with the same DNA-binding protein that interacts with sequences required for ethylene responsiv eness of the coordinately regulated E4 gene. We also conducted experim ents to test the function of a second DNA-binding protein that interac ts with both E4 and E8 5'-flanking sequences, the E4/E8-binding protei n (E4/E8BP). We examined the effect of an internal deletion from -1088 to -863, which includes the binding site for E4/E8BP, on gene express ion This deletion did not affect expression in ripening fruit, and did not impair ethylene responsiveness. The deletion had a negative effec t on expression in unripe fruit, but resulted in increased expression in leaves. These results suggest that the E4/E8BP is not critical for high levels of expression during fruit ripening or for ethylene respon se, but may play a role in organ-specific gene transcription.