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

Overview

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

1. Plant growth and maintenance.
   1. 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 bak1-5, respectively) to serve as internal controls.
   2. Sow seeds on the soil and germinate under standard short-day conditions (22 °C, 10 h light, 150 μE/m²/s light intensity, and 65-70% relative humidity).
   3. After approximately 7 days, use forceps to transplant individual seedlings to new pots, separated by at least 4 cm to permit full rosette development. Transplant 6-12 seedlings per genotype and return to standard short-day conditions. Regularly water and fertilize plants (1 g/L of 20-20-20 fertilizer every 2 weeks).
2. Collect leaf discs in 96-well plates for overnight recovery.
   1. At 4-5 weeks post-germination (Figure 1A), use a sharp 4 mm biopsy punch to remove one leaf disc per plant, avoiding the mid-vein and being careful to limit wounding (Figure 1B). Place leaf discs in an unused 96-well luminometer plate containing 100 μL of double distilled water (ddH₂O) with the adaxial side facing upwards to prevent desiccation (Figure 1C). If assessing multiple elicitors, remove 1 leaf disc from the same leaf for each elicitor treatment.
      NOTE: Sample leaves that are fully expanded, third to fifth from the top of the rosette, and approximately the same size and age to limit variability. If possible, cut leaf discs in half using a razor blade prior to overnight recovery, as this increases the surface area exposed to elicitor solution.
   2. Recover overnight at room temperature to prevent the interference of ROS produced by wounding during leaf disc collection. Cover plates with a lid to prevent evaporation.
3. Perform the elicitor treatment and measure ROS production.
   1. Program the plate reader prior to adding the reaction solution, as this will reduce the time between ROS burst initiation and the first measurements recorded. Use a commercial software that is appropriate for the plate reader. In our case (see Table of Contents), click Settings, and select the LUM96 Cartridge and the Kinetic Read Type. Click the Read Area category and drag to select a subset of wells or the entire plate to be read. Under the PMT and Optics tab, set the integration time to 1,000 ms. Under the Timing tab, set the Total Run Time to 40-60 min and the Interval to 2 min.
   2. Remove water from each well using a multi-channel pipette.
      NOTE: Do not puncture leaf discs, as this may cause wounding stress and result in more variable ROS outputs.
   3. Prepare a reaction solution containing 100 μM luminol, 10 μg/mL horseradish peroxidase (HRP), and the desired concentration of elicitor (for example: elf18 at 1 nM, 10 nM, 100 nM, or 1,000 nM) in sterile ddH₂O using 10 mL of solution for one 96-well plate.
      NOTE: Dissolve lyophilized peptides in sterile H₂O in low-binding tubes to make a 10 mM stock, flash-freeze in liquid N₂, and store at -80 °C. When ready to use, dilute stocks in sterile H₂O to generate a 100 μM working stock and store at -20 °C.
   4. Use a multi-channel pipette to dispense 100 μL of the reaction mixture to each well, adding the solution to all leaf discs of the same treatment at the same time. Include a control reaction (no elicitor) for each genotype to assess basal ROS levels in the absence of elicitation. Immediately measure light emission for all wavelengths in the visible spectrum using a microplate reader.
      NOTE: Prepare and apply the reaction solution under low light, as luminol and HRP are light-sensitive reagents. Keep reagents on ice or at -20°C unless in use.
   5. Measure light emission with a 1,000 ms integration time in 2 min intervals over a 40-60 min period in a microplate reader in order to capture the dynamic oxidative burst (Figure 1D).
      NOTE: Use a longer integration time to improve assay sensitivity, while ensuring that all samples in a 96-well plate can be measured within one interval.

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 over 40-60 min, in 2 min intervals with an integration time of 1,000 ms. (D) Determine average photon counts for each genotype relative to Col-0 (shown in green), a high ROS control such as cpk28-1 (shown in purple), and a low ROS control such as bak1-5 (shown in orange).

Materials

Name	Company	Catalog Number	Comments
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