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 reverse orientation it results in increased expression in unripe fruit. Interestingly, this ethylene-responsive construct does not have high levels of expression during fruit ripening, indicating that sequences required for high level expression during fruit ripening are separate from sequences required for ethylene response. The ethylene-responsive sequences of the E8 5'-flanking region interact with the same DNA-binding protein that interacts with sequences required for ethylene responsiveness of the coordinately regulated E4 gene. We also conducted experiments to test the function of a second DNA-binding protein that interacts with both E4 and E8 5'-flanking sequences, the E4/E8-binding protein (E4/E8BP). We examined the effect of an internal deletion from -1088 to -863, which includes the binding site for E4/E8BP, on gene expression This deletion did not affect expression in ripening fruit, and did not impair ethylene responsiveness. The deletion had a negative effect 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 response, but may play a role in organ-specific gene transcription.