Ethylene control of E4 transcription during tomato fruit ripening involves two cooperative cis elements.

E4 gene transcription is controlled by ethylene during tomato fruit ripening. To define the ethylene-responsive promoter elements, we have tested the activity of mutations of the E4 promoter, and of chimeric genes in transient assay. Using a set of linker scan mutations of the region from -160 to -91, we determined that sequences located between -150 and -121 bp from the transcription start site are required for normal levels of ethylene-regulated transcription. However, E4 sequences from -193 to -40 were not able to confer ethylene-responsiveness to the minimal (-46) 35S promoter. The E4/E8 binding protein (E4/E8 BP) interacts with sequences in the 5'-flanking regions of both E4 and the coordinately regulated E8 gene, and its role in regulation of E4 transcription was investigated. The E4 binding site spans the E4 TATA box, and so mutations of this site were limited to those that did not disrupt the E4 TATA box. Mutations of this site which reduced affinity for the E4/E8 BP also resulted in reduced activity in transient assay, supporting a role for this element in normal regulation of the gene. Fusion of the 35S enhancer to E4 sequences from -85 to +65 did not result in an ethylene-responsive promoter, indicating that the E4/E8 BP-binding site is not sufficient for ethylene response. We conclude that at least two cis elements are required for ethylene-responsive transcription of the E4 gene during fruit ripening, one between -150 and -121 and the other between -40 and +65.