Cloning and DNA-binding properties of a tobacco Ethylene-Insensitive3 (EIN3) homolog

Ethylene-Insensitive3 (EIN3) is a transcription factor that works in the et hylene signaling pathway in Arabidopsis, We isolated a tobacco cDNA encodin g an EIN3 homolog as a sequence-specific DNA-binding protein, The encoded p rotein TEIL (tobacco EIN3-like) shares 60% identity in amino acid sequence with EIN3, The DNA-binding domain was localized in the N-terminal half, whi ch shows 92% identity in amino acid sequence with the corresponding region of EIN3, suggesting a conserved function in DNA-binding specificity, TEIL w as indeed functionally similar to EIN3 because, like EIN3-overexpressing pl ants, transgenic Arabidopsis seedlings overexpressing TEIL cDNA exhibited c onstitutive triple response phenotypes. Random binding site selection analy sis revealed that the consensus binding sequence for TEIL is AYGWAYCT, wher e Y and W represent A or C and A or T, respectively, A reporter plasmid con taining the TEIL binding sites showed a 7- to 10-fold higher activation rel ative to that containing a mutated TEIL-binding sequence in tobacco protopl asts. A further 2- to 3-fold increase in activation was observed when a pla smid for TEIL overproduction was co-transfected, indicating that TEIL is a transcriptional activator, Moreover, nuclear extracts from ethylene-treated leaves showed an increase in DNA-binding activity specific to the TEIL-bin ding sequence, despite the level of the transcripts being unchanged. These observations suggest that TEIL functions as a transcription activator with a relatively redundant DNA-binding specificity, and its function may be reg ulated at least in part by modulation of the DNA-binding activity through e thylene signaling.