Aug 21, 2025
Protocol for Qualitative Root Ferric Reductase activity assay with Arabidopsis thaliana seedlings
- Petra Bauer1,
- Petra Bauer1
- 1Heinrich Heine University
Protocol Citation: Petra Bauer, Petra Bauer 2025. Protocol for Qualitative Root Ferric Reductase activity assay with Arabidopsis thaliana seedlings. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5k1bjv1b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 16, 2025
Last Modified: September 09, 2025
Protocol Integer ID: 222629
Keywords: Ferric reductase, FRO2, Arabidopsis thaliana, Root assay, Iron deficiency, protocol for qualitative root ferric reductase activity, qualitative root ferric reductase activity, activity of ferric reductase oxidase, ferric reductase activity, ferric reductase oxidase, spatial localization of the ferric reductase activity, arabidopsis thaliana seedlings aim of this experiment, arabidopsis thaliana seedlings aim, different root zone
Abstract
Aim of this experiment: examine the activity of Ferric reductase oxidases like FRO2. This is achieved by qualitative observation of colour changes. You might also see spatial localization of the ferric reductase activity in the different root zones.
Image Attribution
The photo was taken after 4h (photo taken by Jannik Hornbergs).
Exemplary picture: Comparison of +Fe Col-0 plants (left side) vs. -Fe Col-0 plants (right side). The photo was taken after 4h (photo taken by Jannik Hornbergs).
Materials
Needed materials:
- Intact Arabidopsis plants (Please note that the assay is performed with living and intact plants)
- Ferric reductase plates (see recipe)
- plastic forceps (iron-free, don't use metal/ steel!)
- A camera/ photo station
Compound table:
Troubleshooting
Before start
Preparations in advance:
Grow your plants in the desired system:
- 6 days vertical growth on Hoagland +Fe plates (50 µM final concentration of 10 mM FeNaEDTA stock solution) or Hoagland -Fe plates (no FeNaEDTA is added)
- 14+3 days system: Plants are grown for 14 days vertically on Hoagland +Fe plates, then all plants are transferred to fresh +Fe / -Fe Hoagland plates and grown vertically for three additional days.
Think of proper controls!
For analysis 5 plants are grouped in a bundle, at least 2 bundles are required = at least 10 plants are needed. Bundles are needed to have a stronger staining of the reduced Fe in the agar plates.
Protocol for the day of the assay:
Always prepare the plates freshly on the day of the assay.
Boil water with 0.5 mM CaSO₄ and 0.7 % (w/v) agar in the microwave until the agar is completely dissolved.
Let the medium cool down to approx. 50°C.
Add 0.25 mM FeNaEDTA and 0.25 mM ferrozine, fill up with millipore H₂O to the desired volume.
Pour your plates under the sterile hood and keep them in the dark before the assay (in the dark to avoid light-triggered Fe reduction in agar plates).
Transfer the plants to the assay plates with plastic forceps; we previously used at least 2 bundles of 5 seedlings per genotype and growth condition which worked well.
Wrap the plates with aluminium foil and incubate them in a drawer for approx. 2 hours (note down the time!).
Take photos with a camera stand, with low light from the bottom and the side. It might be useful to take photos after different time points, e.g. 2h, 4h, 24h.
Exemplary picture: Comparison of +Fe Col-0 plants (left side) vs. -Fe Col-0 plants (right side). The photo was taken after 4h (photo taken by Jannik Hornbergs).