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Jan 2013

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Determination of Ferric Chelate Reductase Activity in the Arabidopsis thaliana Root    

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Abstract

Plants have developed two distinct mechanisms, i.e., strategy I (reduction strategy) and II (chelation strategy), to mobilize insoluble Fe(III) in the rhizosphere and transport it through the plasma membrane. Arabidopsis thaliana and other dicots rely on strategy I. In this strategy, the rhizosphere is first acidified by a PM-localized H+-ATPase, AHA2. Then, FERRIC CHELATE REDUCTASE 2 (FRO2) reduces Fe(III) to soluble Fe(II). Finally, the reduced Fe is taken up by a high-affinity transporter, IRON-REGULATED TRANSPORTER 1 (IRT1). Root ferric chelate reductase activity can be quantified spectrophotometrically by the formation of Purple-colored Fe(II)-ferrozine complex in darkness.

Keywords: Arabidopsis thaliana, Ferric chelate reductase, FRO, Enzyme activity, Root

Materials and Reagents

  1. Arabidopsis thaliana plants [wild-type Col-0 and T-DNA insertion line of FERRIC REDUCTASE DEFECTIVE 3 (frd3-1) are used as examples below]
  2. Murashige and Skoog (MS) salts
  3. Ethylenediaminetetraacetic acid ferric sodium salt [Fe(III)-EDTA] (Sigma-Aldrich, catalog number: E6760 )
  4. 3-(2-Pyridyl)-5,6-diphenyl-1,2,4-triazine-4’,4”-disulfonic acid sodium salt (Ferrozine) (Sigma-Aldrich, catalog number: P9762 )
  5. Assay solution (see Recipes)

Equipment

  1. 1.5 ml Eppendorf tubes
  2. Spectrophotometer (Shimadzu, model: UV-1700 )

Procedure

  1. Col-0 and frd3-1 seeds were placed on media containing 1/4 Murashige and Skoog (MS) salts, 50 μM Fe-EDTA, 0.5% sucrose, and 1.5% agar (basal medium).
  2. After stratification for 2 days at 4 °C, the plates were kept in a growth incubator under a long-day photoperiod (16 h light, 8 h darkness) at 25 °C.
  3. Fe deficiency was applied by transferring 7-day-old seedlings to basal medium without Fe-EDTA but containing 300 μM ferrozine [3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine sulfonate]. Then, the plants were grown for additional three days on this medium.
  4. 700 μl of assay solution is placed in a 1.5 ml eppendorf tube, tube is placed onto scale and the weight of the tube is tared (zeroed).
  5. Both primary and lateral roots of five plants are soaked totally in this assay solution in order to prevent their drying, the tube is weighed again and the fresh weight of the sample is recorded.
    Notes:
    1. The assay solution should be kept at dark during the experiment.
    2. Maximum fresh weight of the roots recommended for this assay is 200 mg.
    3. Roots are not cut into pieces.
  6. The tube is mixed by tapping several times for increasing the contact of roots with assay solution, and incubated for 30 min in darkness at room temperature.
  7. At the end of the incubation, purple-colored Fe(II)-ferrozine complex formation is observed around the roots in the solution (Figure 1a).
    Note: Much deeper purple color formation is observed around the roots of the plants treated with Fe deficiency (Figure 1b).


    Figure 1. Purple-colored Fe(II)-ferrozine complex formation of assay solution before and after Fe deficiency.

  8. The absorbance of the assay solution is determined in a spectrophotometer at 562 nm against an identical assay solution without any plants (blank).
  9. Purple-colored Fe(II)-ferrozine complex formation is quantified using a molar extinction coefficient of 28.6  mM-1 cm-1 as in the equation of



  10. The experimental results are presented in the unit of μM Fe(II)/g root FW/hr as the mean of three biological repeats with six technical replicates each (Figure 2).


    Figure 2. Ferric Chelate Reductase activity in roots of Col-0 and frd3-1 under Fe-sufficient or -deficient conditions.

Recipes

  1. Assay solution
    The assay solution is composed of 0.1 mM Fe(III)-EDTA and 0.3 mM ferrozine in distilled water. Prepare fresh before each experiment and kept at dark.

Acknowledgments

This protocol is adapted from Yi and Guerinot (1996).

References

  1. Yi, Y. and Guerinot, M. L. (1996). Genetic evidence that induction of root Fe(III) chelate reductase activity is necessary for iron uptake under iron deficiency. Plant J 10(5): 835-844.
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Aksoy, E. and Koiwa, H. (2013). Determination of Ferric Chelate Reductase Activity in the Arabidopsis thaliana Root. Bio-protocol 3(15): e843. DOI: 10.21769/BioProtoc.843.
Q&A
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If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.

If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.

Saradia Kar
Assam University,
I did the same but not in eppendorf but in petriplates with 10 ml of assay solution. In the formula therefore i have to put 10 ml instead of 700 microlitre right?
6/25/2016 1:16:04 AM Reply
Saradia Kar
Assam University,
What is '2' referred to in the formula?
6/25/2016 1:13:59 AM Reply
Lukas Häfner
University of Bayreuth
Great protocol, I am using it right now, but I have some problems with the formula:

I tried to reconstruct the mathematical steps that led to the formula (See the picture I added). Assuming the assay volume V ("700" in the formula) has the dimension [V] = 1 microL, and the fresh root weight m ("rootFW" in the formula) has the dimension [m] = 1 mg, the dimension of the FRO enzyme activity - [FRO] = 1 micromol/(g*h) - would be correct EVEN WITHOUT the factor 10^3 (which, I assume, was added to switch between micro and milli, for example?). So what does this factor stand for?

And what does the factor 180 represent? I assume it is the reaction time t, since it is the only necessary part left, but shouldn´t it be 0.5 (30 min incubation time = 0.5 h)?
11/12/2015 7:53:41 AM Reply
Santosh Satbhai
GMI-Vienna
Hi again..i have one more question related to formula to calculate the enzyme activity. You have presented the data in μM Fe(II)/g root FW/hr, however in assay you incubated roots for 30 min. Did you just double the O.D in that case or something else? Thanks in advance
4/14/2015 8:59:20 AM Reply
Santosh Satbhai
GMI-Vienna
Thanks for the detailed protocol. I would like to know whether do you cut out shoot parts before taking fresh weight or do you use whole seedlings for assay?
4/2/2015 4:02:57 AM Reply
EMRE EMRE AKSOY
Agricultural Genetic Engineering, Nigde Omer Halisdemir University

Shoots are separated from the roots by sharp scissors before taking the fresh weight. Make sure your assay solution is ready waiting in the tubes before you do all cutting and transfer of roots so that the roots do not dry out.

4/2/2015 4:57:03 AM Reply


Santosh Satbhai
GMI-Vienna

Thank you.

4/2/2015 5:28:45 AM Reply


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