An Assay to Detect Oxidative Burst in Arabidopsis Leaf Discs upon Immune Elicitation

Obtain Arabidopsis thaliana leaf discs. Immerse them in microplate wells containing double-distilled water to prevent desiccation.

Incubate the discs to allow recovery from wounding.

Post-incubation, remove the water. Add a reaction mixture containing peptides derived from bacteria, horseradish peroxidase enzyme, and luminol.

Using a microplate reader, measure the luminescence over time.

Pattern recognition receptors, or PRRs, on the plant cells, bind the bacterial peptides, which are microbe-associated molecular patterns.

Further, the PRR associates with its co-receptor, undergoing mutual phosphorylation, activating receptor-like protein kinases, and causing intracellular calcium influx.

This, in turn, activates the transmembrane respiratory burst oxidase homolog, or RBOH proteins.

Activated RBOH catalyzes the production of extracellular reactive oxygen species, or ROS, causing an oxidative burst. Further, ROS gets converted into hydrogen peroxide.

Horseradish peroxidase utilizes hydrogen peroxide to oxidize luminol into an excited state, emitting luminescence upon returning to the ground state.

Measure the luminescence to monitor the rapid oxidative burst following microbial pattern recognition.