ETHYLENE CONTROL OF E4 TRANSCRIPTION DURING TOMATO FRUIT RIPENING INVOLVES 2 COOPERATIVE CIS-ELEMENTS

E4 gene transcription is controlled by ethylene during tomato fruit ri pening. To define the ethylene-responsive promoter elements, we have t ested the activity of mutations of the E4 promoter, and of chimeric ge nes in transient assay. Using a set of linker scan mutations of the re gion from -160 to -91, we determined that sequences located between -1 50 and -121 bp from the transcription start site are required for norm al levels of ethylene-regulated transcription. However, E4 sequences f rom -193 to -40 were not able to confer ethylene-responsiveness to the minimal (-46) 35S promoter. The E4/E8 binding protein (E4/E8 BP) inte racts with sequences in the 5'-flanking regions of both E4 and the coo rdinately regulated E8 gene, and its role in regulation of E4 transcri ption 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, suppor ting 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 leas t two cis elements are required for ethylene-responsive transcription of the E4 gene during fruit ripening, one between -150 and -121 and th e other between -40 and +65.