Cloning of a DNA-binding protein that interacts with the ethylene-responsive enhancer element of the carnation GST1 gene

Ethylene transcriptionally activates a glutathione S-transferase gene (GST1) at the onset of the senescence program in carnation (Dianthus caryophyllus L.) flower petals. A 126 bp region of the GST1 promoter sequence has been identified as an ethylene-responsive enhancer element (ERE). In this paper, we demonstrate the ability of nuclear proteins from senescing petals to recognize a 22 bp sequence within the ERE (ERE oligonucleotide). Mutation of the ERE oligonucleotide sequence significantly alters the strength of this nuclear protein-DNA association. The wild-type ERE oligonucleotide sequence was used to isolate a cDNA clone encoding a sequence-specific DNA binding protein. Nucleotide sequencing and deduced amino acid sequence analysis of this cDNA predicted a 32 kDa protein which we have designated carnation ethylene-responsive element-binding protein-1 (CEBP-1). The mRNA expression pattern of CEBP-1 suggests that it is not transcriptionally regulated by ethylene. The amino acid sequence homology of CEBP-1 with other plant nucleic acid binding proteins indicates a conserved nucleic acid binding domain. Within this domain are two highly conserved RNA-binding motifs, RNP-1 and RNP-2. An acidic region and a putative nuclear localization signal are also identified.