Cloning and DNA-binding properties of a tobacco Ethylene-Insensitive3 (EIN3) homolog.
Ethylene-Insensitive3 (EIN3) is a transcription factor that works in the ethylene signaling pathway in Arabidopsis. We isolated a tobacco cDNA encoding an EIN3 homolog as a sequence-specific DNA-binding protein. The encoded protein TEIL (tobaccoEIN3-like) shares 60% identity in amino acid sequence with EIN3. The DNA-binding domain was localized in the N-terminal half, which shows 92% identity in amino acid sequence with the corresponding region of EIN3, suggesting a conserved function in DNA-binding specificity. TEIL was indeed functionally similar to EIN3 because, like EIN3-overexpressing plants, transgenic Arabidopsis seedlings overexpressing TEIL cDNA exhibited constitutive triple response phenotypes. Random binding site selection analysis revealed that the consensus binding sequence for TEIL is AYGWAYCT, where Y and W represent A or C and A or T, respectively. A reporter plasmid containing the TEIL binding sites showed a 7- to 10-fold higher activation relative to that containing a mutated TEIL-binding sequence in tobacco protoplasts. A further 2- to 3-fold increase in activation was observed when a plasmid 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-binding 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 regulated at least in part by modulation of the DNA-binding activity through ethylene signaling.
Kosugi
S
S
National Institute of Agrobiological Resources, Tsukuba, Ibaraki 305-8602, Japan.
Ohashi
Y
Y
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics; D029681:Arabidopsis Proteins; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D018744:DNA, Plant / Q000378:metabolism*; D004268:DNA-Binding Proteins; D017930:Genes, Reporter; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D010641:Phenotype; D030821:Plants, Genetically Modified; D010947:Plants, Toxic; D017386:Sequence Homology, Amino Acid; D014026:Nicotiana / Q000235:genetics*; D014157:Transcription Factors
D029681:Arabidopsis Proteins; D018744:DNA, Plant; D004268:DNA-Binding Proteins; C106987:EIN3 protein, Arabidopsis; D009687:Nuclear Proteins; D014157:Transcription Factors