An Assay to Detect Oxidative Burst in Arabidopsis Leaf Discs upon Immune Elicitation
Published: January 31, 2024
Abstract
Source: Bredow, M., et al. Pattern-Triggered Oxidative Burst and Seedling Growth Inhibition Assays in Arabidopsis thaliana. J. Vis. Exp. (2019)
This video demonstrates an assay to measure oxidative burst in Arabidopsis leaves upon immune elicitation. Leaf discs, cut from plant leaves, are incubated with bacterial peptides functioning as microbe-associated molecular patterns or MAMPs. Upon detection of the MAMPs, plant cells induce the production of extracellular ROS, which is detected using a chemiluminescence assay.
Protocol
1. Detection of ROS burst in Arabidopsis leaf discs following immune elicitation Plant growth and maintenance. To synchronize germination, stratify Arabidopsis seeds by suspending approximately 50 seeds in 1 mL of sterile 0.1% agar [w/v] and store at 4 °C (no light) for 3-4 days. NOTE: Stratify a wild-type background control (for example, Col-0) and genotypes with high and low immune outputs (for example, cpk28-1 and bak…
Representative Results
Figure 1. Luminol-based oxidative burst assay following immune induction in Arabidopsis. (A) Grow plants on soil in short-day conditions for 4-5 weeks. (B) Use a 4 mm biopsy punch to collect leaf discs from each plant and recover overnight in ddH2O. (C) Add reaction solution (100 μM luminol, 10 μg/mL HRP, and the desired concentration of elicitor) and measure light emission ove…
Disclosures
The authors have nothing to disclose.
Materials
| 20-20-20 Fertilizer | Plant Prod | 10529 | Mix 1g/L in water and apply to plants every 2 weeks for optimal growth. |
| 4 mm Biopsy Punch | Medical Mart | 232-33-34-P | A cork borer set with a 0.125 cm^2 surface area can also be used. |
| BioHit mLine Mechanical 12 Multichannel Pipette (30-300 uL) | Sartorius | 725240 | Any multichannel pipette can be used, as can a single pipetter if necessary. |
| elf18 (Ac-SKEKFERTKPHVNVGTIG) | EZ Biolab | cp7211 | Store 10 mM stock peptide at -80C in low protein binding tubes. When thawed, store 100 uM working stock at -20C. |
| Forceps | Fisher Scientific | 22-327379 | |
| Horseradish Peroxidase | Sigma-Aldrich | P6782 | Dissolve in pure water. Store at -20C and away from light. |
| Luminol | Sigma-Aldrich | A8511 | Dissolve in DMSO. Store at -20C and away from light. |
| Murisage and Skoog Basal Salts | Cedarlane Labs | MSP09-100LT | Store at 4C. |
| Soil | SunGrow Horticulture | Sunshine Mix #1 | Other soil types can also be used to grow Arabidopsis. Mix with water when filling pots. |
| SpectraMax Paradigm Multi Mode Microplate Reader with LUM Module | Molecular Devices | Any plate reader capable of detecting luminescence can be used for these assays. | |
| White Polystyrene 96-Well Plates | Fisher Scientific | 07-200-589 |
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Cite This Article
An Assay to Detect Oxidative Burst in Arabidopsis Leaf Discs upon Immune Elicitation. J. Vis. Exp. (Pending Publication), e21909, doi: (2024).