This study investigates the oxidative burst in Arabidopsis thaliana leaf discs in response to microbial peptides. The methodology involves measuring luminescence to monitor reactive oxygen species production following pattern recognition receptor activation.
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.
At 4 to 5 weeks post-germination, use a sharp 4-millimeter biopsy punch to remove one leaf disk per Arabidopsis plant, avoiding the mid-vein and being careful to limit wounding. Collect the disks into individual wells of an unused 96-well luminometer plate containing 100 microliters of double-distilled water per well at axial side up to prevent desiccation.
If assessing multiple elicitors, remove a second leaf disk from the same leaf for each elicitor treatment, and cover the plate with the lid to allow the leaf disks to recover overnight at room temperature. The next morning, set the microplate reader parameters to a 1,000-millisecond integration time in 2-minute intervals over a 40 to 60-minute period to capture the dynamic oxidative burst.
Next, use a multichannel pipette to replace the water in each well with 100 microliters of freshly prepared reaction solution, including a control no-elicitor reaction for each genotype to assess the basal reaction oxygen species levels in the absence of elicitation. Then, immediately, measure the light emission for all of the wavelengths in the visible spectrum on the microplate reader.
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