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

Area of Science:

• Plant Molecular Biology
• Plant Biochemistry
• Gene Regulation

Background:

• The PRB-1b gene encodes a pathogenesis-related protein (PR-1) family member.
• PRB-1b mRNA accumulation in tobacco leaves is rapidly induced by ethylene.
• Understanding ethylene's role in PRB-1b gene regulation is crucial for plant defense mechanisms.

Purpose of the Study:

• To delineate cis-acting elements responsible for ethylene responsiveness in the PRB-1b gene promoter.
• To identify and characterize nuclear proteins that interact with the ethylene-responsive promoter region.
• To elucidate the molecular mechanisms underlying ethylene-induced PRB-1b gene transcription.

Main Methods:

• Promoter deletion analysis in transgenic tobacco plants using a beta-glucuronidase reporter gene.
• In vitro gel-shift assays with nuclear protein extracts and promoter DNA fragments.
• Oligonucleotide-based gel-shift assays to map protein-binding sequences.
• Heparin-agarose chromatography for nuclear protein fractionation.

Main Results:

• A promoter fragment from -213 bp was sufficient for a 20-fold increase in reporter gene expression upon ethylene treatment.
• Gel-shift analysis identified two distinct nuclear protein-DNA interactions within the -237 to -143 bp region.
• Specific binding sequences (regions G and Y) were mapped, and the two binding activities showed differential elution during chromatography.

Conclusions:

• The region between -213 and -141 bp of the PRB-1b promoter contains essential elements for ethylene induction.
• Specific nuclear proteins bind to defined DNA sequences within this region, mediating ethylene responsiveness.
• These in vitro characterized DNA-nuclear protein interactions are likely involved in the transcriptional regulation of the PRB-1b gene by ethylene.