Determination of ROS Generated by Arabidopsis Xanthine Dehydrogenase1 (AtXDH1) Using Nitroblue Tetrazolium (NBT) and 3,3'-Diaminobenzidine (DAP)

Overview

Journal Methods Mol Biol

Specialty Molecular Biology

Date 2024 Apr 8

PMID 38587736

Authors

Aigerim Soltabayeva

Moshe Sagi

Affiliations

Soon will be listed here.

Abstract

Plants generate reactive oxygen species (ROS) during different metabolic processes, which play an essential role in coordinating growth and response. ROS levels are sensitive to environmental stresses and are often used as a marker for stress in plants. While various methods can detect ROS changes, histochemical staining with nitroblue tetrazolium (NBT) and 3,3'-diaminobenzidine (DAB) is a popular method, though it has faced criticism. This staining method is advantageous as it enables both the quantification and localization of ROS and the identification of the enzymatic origin of ROS in plants, cellular compartments, or gels. In this protocol, we describe the use of NBT and DAP staining to detect ROS generation under different stresses such as nitrogen starvation, wounding, or UV-C. Additionally, we describe the use of NBT staining for detecting enzymatic generation of ROS in native and native SDS PAGE gels. Our protocol also outlines the separation and comparison of the origin of ROS generated by xanthine dehydrogenase1 (XDH1) using different substrates.

Citing Articles

Maize 4-coumarate coenzyme A ligase gene positively regulates drought stress response in .

Fan J, Luo Z, Wang Y, Jiao P, Wang Q, Dai Y GM Crops Food. 2025; 16(1):199-215.

PMID: 39988889 PMC: 11853610. DOI: 10.1080/21645698.2025.2469942.

References

Mittler R, Vanderauwera S, Suzuki N, Miller G, Tognetti V, Vandepoele K . ROS signaling: the new wave?. Trends Plant Sci. 2011; 16(6):300-9. DOI: 10.1016/j.tplants.2011.03.007. View

Baxter A, Mittler R, Suzuki N . ROS as key players in plant stress signalling. J Exp Bot. 2013; 65(5):1229-40. DOI: 10.1093/jxb/ert375. View

Van Breusegem F, Dat J . Reactive oxygen species in plant cell death. Plant Physiol. 2006; 141(2):384-90. PMC: 1475453. DOI: 10.1104/pp.106.078295. View

Gechev T, Van Breusegem F, Stone J, Denev I, Laloi C . Reactive oxygen species as signals that modulate plant stress responses and programmed cell death. Bioessays. 2006; 28(11):1091-101. DOI: 10.1002/bies.20493. View

Ortega-Villasante C, Buren S, Baron-Sola A, Martinez F, Hernandez L . In vivo ROS and redox potential fluorescent detection in plants: Present approaches and future perspectives. Methods. 2016; 109:92-104. DOI: 10.1016/j.ymeth.2016.07.009. View

Anjum N, Amreen , Tantray A, Khan N, Ahmad A . Reactive oxygen species detection-approaches in plants: Insights into genetically encoded FRET-based sensors. J Biotechnol. 2019; 308:108-117. DOI: 10.1016/j.jbiotec.2019.12.003. View

Yesbergenova Z, Yang G, Oron E, Soffer D, Fluhr R, Sagi M . The plant Mo-hydroxylases aldehyde oxidase and xanthine dehydrogenase have distinct reactive oxygen species signatures and are induced by drought and abscisic acid. Plant J. 2005; 42(6):862-76. DOI: 10.1111/j.1365-313X.2005.02422.x. View

Srivastava S, Brychkova G, Yarmolinsky D, Soltabayeva A, Samani T, Sagi M . Aldehyde Oxidase 4 Plays a Critical Role in Delaying Silique Senescence by Catalyzing Aldehyde Detoxification. Plant Physiol. 2017; 173(4):1977-1997. PMC: 5373044. DOI: 10.1104/pp.16.01939. View

Zarepour M, Kaspari K, Stagge S, Rethmeier R, Mendel R, Bittner F . Xanthine dehydrogenase AtXDH1 from Arabidopsis thaliana is a potent producer of superoxide anions via its NADH oxidase activity. Plant Mol Biol. 2009; 72(3):301-10. DOI: 10.1007/s11103-009-9570-2. View

10.

Soltabayeva A, Srivastava S, Kurmanbayeva A, Bekturova A, Fluhr R, Sagi M . Early Senescence in Older Leaves of Low Nitrate-Grown Uncovers a Role for Purine Catabolism in N Supply. Plant Physiol. 2018; 178(3):1027-1044. PMC: 6236613. DOI: 10.1104/pp.18.00795. View

11.

Soltabayeva A, Bekturova A, Kurmanbayeva A, Oshanova D, Nurbekova Z, Srivastava S . Ureides are accumulated similarly in response to UV-C irradiation and wounding in Arabidopsis leaves but are remobilized differently during recovery. J Exp Bot. 2021; 73(3):1016-1032. DOI: 10.1093/jxb/erab441. View

12.

Sagi M, Fluhr R . Superoxide production by plant homologues of the gp91(phox) NADPH oxidase. Modulation of activity by calcium and by tobacco mosaic virus infection. Plant Physiol. 2001; 126(3):1281-90. PMC: 116485. DOI: 10.1104/pp.126.3.1281. View