Identification of an ethylene-responsive region in the promoter of a tobacco class I chitinase gene

The Chn48 gene is a representative of a family of tobacco class I basic chitinase genes, and the expression is induced by the stress hormone ethylene. To investigate the molecular basis for transcriptional regulation by ethylene we have examined the Chn48 promoter to identify cis-elements and trans-acting factors that are involved in the chitinase gene expression. In transgenic tobacco plants, a chimeric gene construct containing a 2 kb Chn48 promoter fused to a β-glucuronidase reporter gene was induced by ethylene in leaf tissues. Deletion analysis indicated that a positive ethylene-responsive region is located between nucleotides -503 and -358 relative to the transcription initiation site. This 146 bp sequence was found to confer ethylene-responsive reporter gene expression when inserted in either orientation upstream of the heterologous promoter, indicating that the sequence functions as a regulatory enhancer. The ethylene-responsive region contains two copies of a GCC-box (TAAGAGCCGCC), which is conserved in a number of ethylene-responsive defense genes. The sequences within this ethylene-responsive region that are necessary for ethylene-responsive transcription were further localized to the 71 bp sequence between positions -480 and -410 containing two copies of the GCC-box by loss-of-function analysis. Gel mobility-shift experiments showed the presence of leaf nuclear factors that interact with the DNA sequences included in the ethylene-responsive region.