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Hydrogen Peroxide Measurement in Arabidopsis Root Tissue Using Amplex Red

Amplex Red荧光法测定拟南芥根组织中的过氧化氢酶

Tzvetina Brumbarova Tzvetina Brumbarova
Cham Thi Tuyet Le Cham Thi Tuyet Le
Petra Bauer Petra Bauer

发布: 2016年11月05日第6卷第21期 DOI: 10.21769/BioProtoc.1999 浏览次数: 17079

评审: Arsalan DaudiAnonymous reviewer(s)

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Cover of Plant Physiology, featuring study using the protocol.

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Abstract

This protocol describes the measurement of hydrogen peroxide (H2O2) content in Arabidopsis root tissue by using the Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit. When root tissue is disrupted and resuspended in phosphate buffer, H2O2 is released from the cells. The obtained root extracts containing H2O2 can be mixed with a solution containing Amplex® Red reagent (10-acetyl-3,7-dihydrophenoxazine). In the presence of horseradish peroxidase, the Amplex® Red reagent reacts with H2O2 in a 1:1 stoichiometry. The resulting product is the red-fluorescent compound resorufin which can be detected fluorometrically or spectrophotometrically. Our protocol is based on the manual of the Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit and describes a step-by-step procedure with a detailed description of the necessary controls and data analysis. We have also included modifications of the protocol, notes and examples that intend to aid the user in easily reproducing the assay with their own samples.

Background

Reactive oxygen species (ROS), such as H2O2, can be generated in the cell as a result of a developmental cue or a stress condition. In high amounts, ROS accumulation can be detrimental by causing cellular damage. However, increased ROS production can also have a signaling role and serve as a secondary messenger in controlling downstream cellular responses. In plants, a signaling role for ROS has been shown for many abiotic stresses, such as drought, salinity, temperature stress, and nutrient deprivation (Mittler, 2002; Mittler and Blumwald, 2015; Xia et al., 2015). In our recent publication Le et al. (2016), we have investigated the connection between ROS production and iron (Fe) deficiency response regulation by investigating the H2O2 content of roots from wild type and mutant Arabidopsis plant lines grown under sufficient and deficient Fe supply conditions.

Materials and Reagents

  1. Kimtech® science precision tissues (Carl Roth, catalog number: AA63.1 )
  2. 2 ml microcentrifuge tubes, safe-seal (SARSTEDT, catalog number: 72.695.500 )
  3. Pipet tips 
  4. Aluminium foil
  5. 96-well microtiter plates for absorbance measurement (e.g., UV-Star® microplate, 96 well, half area, µClear® [Greiner Bio-One, catalog number: 675801 ])
  6. 96-well microtiter plates for fluorescence measurement (e.g., 96 well, half area, black [Greiner Bio-One, catalog number: 675076 ])
  7. Arabidopsis seeds
  8. Sterile distilled water
  9. Plant agar (Duchefa Biochemie, catalog number: P1001.1000 )
  10. Liquid nitrogen
  11. Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: A22188 )
  12. H2O2 working solution
  13. HRP enzyme
  14. Horseradish peroxidase
  15. Sodium hypochlorite (NaOCl) (Carl Roth, catalog number: 9062.3 )
  16. Triton X-100 (SERVA Electrophoresis, catalog number: 37238 or Sigma-Aldrich, catalog number: X-100 )
  17. Magnesium sulfate heptahydrate (MgSO4·7H2O) (Sigma-Aldrich, catalog number: M9397 )
    Note: This product has been discontinued. Alternatively, MgSO4·7H2O from Carl Roth can be used (Carl Roth, catalog number: T888 )
  18. Potassium dihydrogen phosphate (KH2PO4) (Sigma-Aldrich, catalog number: P0662 ; or Carl Roth, catalog number: 3904 )
  19. Potassium nitrate (KNO3) (Carl Roth, catalog number: P021.2 )
  20. Calcium nitrate tetrahydrate [Ca(NO3)2·4H2O] (Carl Roth, catalog number: P740.2 )
  21. Potassium chloride (KCl) (Carl Roth, catalog number: 6781.1 )
  22. Boric acid (H3BO3) (Carl Roth, catalog number: 6943.1 )
  23. Manganese sulfate (MnSO4) (AppliChem, catalog number: A1038 ; or Carl Roth, catalog number: X890.1 )
  24. Zinc sulfate heptahydrate (ZnSO4·7H2O) (VWR, catalog number: VWRC29253.236 )
  25. Copper sulfate pentahydrate (CuSO4·5H2O) (Thermo Fisher Scientific, Fisher Scientific, catalog number: AC197720050 )
  26. Ammonium heptamolybdate tetrahydrate [(NH4)6Mo7O24·4H2O] (Sigma-Aldrich, catalog number: 431346 ; or Carl Roth, catalog number: 7311 )
  27. D(+)-sucrose (Carl Roth, catalog number: 4621.1 )
  28. Ferric sodium ethylenediaminetetraacetic acid (FeNaEDTA) (Carl Roth, catalog number: 8043.1 )
  29. 3-(2-Pyridyl)-5,6-diphenyl-1,2,4-triazine-4’,4’’-disulfonic acid sodium salt (Ferrozine) (Sigma-Aldrich, catalog number: 82950 )
  30. Sterilization solution (see Recipes)
  31. Hoagland medium (see Recipes)
  32. Phosphate buffer (see Recipes)

Equipment

  1. Tube rotator (e.g., VWR, catalog number: 10136-084 )
  2. Mortars and pestles (e.g., MTC Haldenwanger)
  3. Centrifuge
  4. Multi-channel pipet
  5. Analytical scales, e.g., ALJ160_4NM (Kern)
  6. Benchtop centrifuge with cooling (e.g., Thermo Fisher Scientific, Thermo ScientificTM, model: Heraeus Fresco 21 Centrifuge )
  7. Plate reader (e.g., Tecan Trading, model: Infinite® M200 Pro )
    Note: The plate reader should be able to excite in the range of 530-560 nm and detect fluorescence at approx. 590 nm, or detect absorbance at approx. 560 nm.
  8. Homogenizer (e.g., such as Precellys® 24 homogenizer) (VWR, catalog number: 432-3750 )

Software

  1. Microsoft Excel

Procedure

English
中文翻译

文章信息

版权信息

© 2016 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

  1. Brumbarova, T., Le, C. T. T. and Bauer, P. (2016). Hydrogen Peroxide Measurement in Arabidopsis Root Tissue Using Amplex Red. Bio-protocol 6(21): e1999. DOI: 10.21769/BioProtoc.1999.
  2. Le, C. T., Brumbarova, T., Ivanov, R., Stoof, C., Weber, E., Mohrbacher, J., Fink-Straube, C. and Bauer, P. (2016). ZINC FINGER OF ARABIDOPSIS THALIANA12 (ZAT12) interacts with FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT) linking iron deficiency and oxidative stress responses. Plant Physiol 170(1): 540-557.

    3. ris ris Download Citation in RIS Format

分类

植物科学 > 植物生物化学 > 其它化合物

植物科学 > 植物新陈代谢 > 其它化合物

生物化学 > 其它化合物 > 活性氧

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