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 ethyl ene. Promoter deletion analysis was performed in transgenic tobacco pl ants to delineate cis-acting elements necessary for ethylene responsiv eness of the PRB-1b gene. The promoter sequence from position -213 was sufficient to enhance a 20 fold increase of beta-glucuronidase report er gene expression in transgenic tobacco leaves exposed to 20 mul/l of ethylene, however -141 bp were not. The functional study was correlat ed 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 con tiguous regions G (-200 to -178) and Y (-179 to -154) by gel-shift ana lysis using oligonucleotides. Fractionation of crude nuclear extract b y heparin-agarose chromatography resulted in the differential elution of the two binding activities. The DNA-nuclear protein interactions ch aracterized in vitro can be part of the molecular events which mediate the transcriptional regulation of the PRB-1b gene by ethylene.