Detection of Reactive Oxygen Species in Oryza sativa L. (Rice)
水稻中活性氧的检测
发布: 2016年12月20日第6卷第24期 DOI: 10.21769/BioProtoc.2061 浏览次数: 24476
评审: Pooja SaxenaAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Jun 2016
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Abstract
Superoxide ions (O2-) and hydrogen peroxide (H2O2) are the reactive oxygen species (ROS) that play a significant role in regulation of many plant processes. The level of O2- ions is determined qualitatively using nitrobluetetrazolium (NBT) assay while the H2O2 is qualitatively estimated using 3,3-diaminobenzidine (DAB) and 2’,7’-dichlorodihydrofluorescein diacetate (H2DCFDA) assay. Further the aqueous content of H2O2 is estimated quantitatively using ferrous oxidation-xylenol orange (FOX) assay.
Keywords: Rice (水稻)
Reactive oxygen species (活性氧)
Superoxide (超氧化物)
Hydrogen peroxide (过氧化氢)
Nitrobluetetrazolium (四氮唑蓝)
3,3-diaminobenzidine (3,3-二氨基联苯胺)
Background
Superoxide ions (O2-) and hydrogen peroxide (H2O2) are the vital reactive oxygen molecules that play a central role in many processes involved in plant growth and development including abiotic stress tolerance. To get better insights into the ROS mediated regulation of these processes, qualitative and quantitative estimation of different types of ROS is of significant importance. O2- is produced by the transfer of electrons from NADPH to oxygen (O2) mediated by the NADPH oxidase enzyme system. These ions are estimated in rice seedlings using NBT assay which is based upon the principle of reduction of yellow coloured NBT into dark blue coloured insoluble formazan by O2- (Kaur et al., 2016).
H2O2 is another reactive oxygen molecule that acts as an important signaling molecule regulating different plant processes. The content of H2O2 is estimated qualitatively in rice seedlings using DAB and H2DCFDA assay (Kaur et al., 2016). DAB assay is based upon the principle of formation of deep brown polymerization product on the reaction of DAB with H2O2 while H2DCFDA assay is based upon the principle of fluorescent microscopy. When non fluorescent H2DCFDA binds to ROS (predominantly H2O2), it gets converted into highly fluorescent 2’,7’-dichlorofluorescein (DCF). DCF gives a green coloured fluorescence when excited with a laser beam of excitation 488 nm using confocal microscope. Further, the quantitative measurement of aqueous H2O2 is carried out using ferrous oxidation-xylenol orange (FOX) method (Kaur et al., 2016). FOX assay is based upon the principle of oxidation of ferrous ions by H2O2 to ferric ions. Ferric ions then bind with xylenol orange to give a coloured complex having absorption maxima at 560 nm.
Materials and Reagents
- Petri dish (35 mm) (Tarsons, catalog number: 460035 )
- Microscopic glass slides
- Glue or nail enamel
- Whatman filter paper No.1 (Thermo Fisher Scientific, Fisher Scientific, catalog number: 09-805 )
- Amber Eppendorf (Capacity: 2 ml) (Tarsons, catalog number: 500013 )
- Glass tubes
- Tubes (Capacity:15 ml and 50 ml) (Tarsons, catalog numbers: 546021 [15 ml]; 546041 [50 ml])
- Disposable cuvettes (Sigma-Aldrich, catalog number: Z330361 )
Note: This product has been discontinued. - Leaf of 14 days old fresh rice seedlings
- Tri-sodium citrate dihydrate (HiMedia Laboratories, catalog number: RM1415 )
- Glycerol (Sigma-Aldrich, catalog number: G5516 )
- Absolute ethanol
- Activated charcoal (HiMedia Laboratories, catalog number: PCT1001 )
- Trichloroacetic acid (Sigma-Aldrich, catalog number: T6399 )
- Liquid nitrogen
- 30 % hydrogen peroxide solution (H2O2) (Sigma-Aldrich, catalog number: H1009 )
- Hydrochloric acid (HCl) (Molychem, catalog number: 23540 )
- Diaminobenzidine (DAB) (Sigma-Aldrich, catalog number: D8001 )
- Nitrobluetetrazolium (NBT) (HiMedia Laboratories, catalog number: MB107 )
- Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: 1310-73-2 )
- 2’,7’-dichlorodihydrofluorescein diacetate (H2DCFDA) (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: D399 )
- Dimethyl sulphoxide (Minimum assay: 99.0%) (S D Fine-Chem, catalog number: 38216 )
- Ammonium ferrous sulfate (HiMedia Laboratories, catalog number: GRM1026 )
- Sulfuric acid (HiMedia Laboratories, catalog number: AS016 )
- Xylenol orange (LobaChemie, catalog number: 06507 )
- Methanol (HPLC grade) (Minimum Assay: 99.7%) (HiMedia Laboratories, catalog number: AS061 )
- Butylated hydroxytoluene (HiMedia Laboratories, catalog number: GRM797 )
- NBT solution (see Recipes)
- DAB solution (see Recipes)
- H2DCFDA solution (see Recipes)
- Ferrous oxidation-xylenol orange (FOX) reagent (see Recipes)
- Standard H2O2 solutions (see Recipes)
Equipment
- Pipette (Corning, model: Lambda Plus)
- Vacuum infiltration equipment (Dessicator connected to vacuum pump) (Vacuum pump: Rocker 300 , Rocker Scientific, model: Rocker 300]; Dessicator vacuum: tarsons 403010 [Tarsons, model: 403010 ] )
- Water bath (Polyscience, model: WB02S )
- Stereomicroscope (Olympus, model: SZ61 )
- Confocal microscope (Nikon A1R, Laser scanning confocal microscope system)
- Centrifuge (REMI, model: C-24 PLUS )
- Spectrophotometer (PerkinElmer, model: Lambda 25 )
Note: This product has been discontinued. - Weighing balance (Citizen Scale, model: CY220 )
- pH meter (Systronics, model: µ361 )
- Pestle mortar
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Kaur, N., Sharma, I., Kirat, K. and Pati, P. K. (2016). Detection of Reactive Oxygen Species in Oryza sativa L. (Rice). Bio-protocol 6(24): e2061. DOI: 10.21769/BioProtoc.2061.
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分类
植物科学 > 植物生物化学 > 其它化合物
生物化学 > 其它化合物 > 活性氧
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