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, Reactive oxygen species, Superoxide, Hydrogen peroxide, Nitrobluetetrazolium, 3,3-diaminobenzidine
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
Equipment
Procedure
Data analysis
Notes
Recipes
Acknowledgments
This protocol has been adopted and modified from DeLong et al. (2003), Kristiasen et al. (2009) and Wu et al. (2010). Authors acknowledge the Department of Biotechnology (DBT), Government of India for financial support (Project No. BT/PR13965/BRB/10/883/2010).
References
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