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Determination of Ferric Chelate Reductase Activity in the Arabidopsis thaliana Root
拟南芥根中铁还原酶活性的测定   

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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(拟南芥), Ferric chelate reductase(铁螯合物还原酶), FRO(FRO), Enzyme activity(酶活), Root(根)

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.

简介

植物已经开发了两种不同的机制,即,策略I(还原策略)和II(螯合策略),以在根际中移动不溶性Fe(III)并将其运输通过质膜。 拟南芥和其它双子叶植物依赖策略I.在该策略中,根际首先通过PM局部化H + -ATP酶AHA2酸化。 然后,FERRIC CHELATE REDUCTASE 2(FRO2)将Fe(III)还原为可溶性Fe(II)。 最后,还原的Fe被高亲和性转运蛋白IRON-REGULATED TRANSPORTER 1(IRT1)摄取。 根铁螯合物还原酶活性可以通过分光光度法通过在黑暗中形成紫色Fe(II) - ferrozine复合物来定量。

关键字:拟南芥, 铁螯合物还原酶, FRO, 酶活, 根

材料和试剂

  1. 使用拟南芥植物[以下作为实例使用了FERRIC REDUCTEASE DEFECTIVE 3(frd3-1)的野生型Col-0和T-DNA插入线]]
  2. Murashige和Skoog(MS)盐
  3. 乙二胺四乙酸铁钠盐[Fe(III)-EDTA](Sigma-Aldrich,目录号:E6760)
  4. 3-(2-吡啶基)-5,6-二苯基-1,2,4-三嗪-4',4" - 二磺酸钠盐(Ferrozine)(Sigma-Aldrich,目录号:P9762)
  5. 测定解决方案(参见配方)

设备

  1. 1.5 ml Eppendorf管
  2. 分光光度计(Shimadzu,型号:UV-1700)

程序

  1. 将Col-0和emd3-1种子置于含有1/4 Murashige和Skoog(MS)盐,50μMFe-EDTA,0.5%蔗糖和1.5%琼脂(基础培养基)的培养基上。
  2. 在4℃下分层2天后,将板在25℃下在长日光周期(16小时光照,8小时黑暗)下保持在生长培养箱中。
  3. 通过将7天龄的幼苗转移到不含Fe-EDTA但含有300μM的[3-(2-吡啶基)-5,6-二苯基-1,2,4-三嗪磺酸盐]的基础培养基中来施加Fe缺乏。然后,使植物在该培养基上再生长三天
  4. 将700μl测定溶液置于1.5ml eppendorf管中,将管放置在天平上并称重管的重量(归零)。
  5. 将五种植物的初级和侧根全部浸泡在该测定溶液中以防止它们干燥,再次称量管并记录样品的鲜重。
    注意:
    1. 在实验过程中,测定溶液应保持黑暗。
    2. 此测定推荐的根的最大鲜重为200mg。
    3. 根不会被切成碎片。
  6. 通过轻敲几次混合管以增加根与测定溶液的接触,并在室温下在黑暗中孵育30分钟。
  7. 在孵育结束时,在溶液中的根周围观察到紫色的Fe(II) - ferrozine复合物形成(图1a)。
    注意:在用Fe缺乏处理的植物的根周围观察到更深的紫色形成(图1b)。


    图1. Fe缺乏前后测定溶液的紫色Fe(II) - 铁复合物形成。

  8. 在分光光度计中在562nm下针对没有任何植物的相同测定溶液(空白)测定测定溶液的吸光度。
  9. 紫色的Fe(II) - ferrozine复合物形成使用摩尔消光系数为28.6 mM -1 cm -1 ,如公式



  10. 实验结果以μMFe(II)/g root FW/hr为单位表示为具有六个技术重复的三个生物重复的平均值(图2)。


    图2.在足够或不足的条件下在Col-0和frd3-1的根中的铁螯合物还原酶活性。

食谱

  1. 测定溶液
    测定溶液由在蒸馏水中的0.1mM Fe(III)-EDTA和0.3mM ferrozine组成。 在每次实验前准备新鲜,并保持黑暗

致谢

该协议改编自Yi和Guerinot(1996)。

参考文献

  1. Yi,Y。和Guerinot,M.L。(1996)。 基因证据表明,根系Fe(III)螯合物还原酶活性的诱导对于铁缺乏时的铁吸收是必要的 。 Plant J  10(5):835-844。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用: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.
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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
Horticultural Sciences Department, Texas A&M University, USA

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


Santosh Satbhai
GMI-Vienna

Thank you.

4/2/2015 5:28:45 AM