DNA-protein interactions on a cis-DNA element essential for ethylene regulation

The PRB-1b gene encodes for a basic-type component of the pathogenesis-related PR-1 protein family. In leaves of tobacco plants, PRB-1b mRNA accumulation is rapidly induced by the application of exogenous ethylene. Promoter deletion analysis was performed in transgenic tobacco plants to delineate cis-acting elements necessary for ethylene responsiveness of the PRB-1b gene. The promoter sequence from position -213 was sufficient to enhance a 20 fold increase of β-glucuronidase reporter gene expression in transgenic tobacco leaves exposed to 20 μl/l of ethylene, however -141 bp were not. The functional study was correlated with in vitro analysis of the nuclear protein-DNA complexes formed on the promoter element identified as necessary for ethylene induction. Gel-shift analysis using restriction fragments spanning the sequence between position -237 and -143 revealed two distinct nuclear protein-DNA interactions. The protein-binding sequences were mapped to the contiguous regions G (-200 to -178) and Y (-179 to -154) by gel-shift analysis using oligonucleotides. Fractionation of crude nuclear extract by heparin-agarose chromatography resulted in the differential elution of the two binding activities. The DNA-nuclear protein interactions characterized in vitro can be part of the molecular events which mediate the transcriptional regulation of the PRB-1b gene by ethylene.