Abstract
The rapid production of reactive oxygen species (ROS) in response to biotic and abiotic cues is a conserved hallmark of plant responses. The detection and quantification of ROS generation during immune responses is an excellent readout to analyze signaling triggered by the perception of pathogens. The assay described here is easy to employ and versatile, allowing its use in a multitude of variations. For example, ROS production can be analyzed using different tissues including whole seedlings, roots, leaves, protoplasts, and cultured cells, which can originate from different ecotypes or mutants. Samples can be tested in combination with any ROS-inducing elicitors, such as the FLS2-activating peptide flg22, but also lipids or even abiotic stresses. Furthermore, early (PAMP-triggered) and late (effector-triggered) ROS production induced by virulent and avirulent bacteria, respectively, can also be assayed.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Doke N (1983) Generation of superoxide anion by potato tuber protoplasts during the hypersensitive response to hyphal cell wall components of Phytophthora infestans and specific inhibition of the reaction by suppressors of hypersensitivity. Physiol Plant Pathol 23:359–367
Doke N (1983) Involvement of superoxide anion generation in the hypersensitive response of potato tuber tissues to infection with an incompatible race of Phytophthora infestans and to the hyphal wall components. Physiol Plant Pathol 23:345–357. https://doi.org/10.1016/0048-4059(83)90019-X
Wu F, Chi Y, Jiang Z, Xu Y, Xie L, Huang F, Wan D, Ni J, Yuan F, Wu X, Zhang Y, Wang L, Ye R, Byeon B, Wang W, Zhang S, Sima M, Chen S, Zhu M, Pei J, Johnson DM, Zhu S, Cao X, Pei C, Zai Z, Liu Y, Liu T, Swift GB, Zhang W, Yu M, Hu Z, Siedow JN, Chen X, Pei ZM (2020) Hydrogen peroxide sensor HPCA1 is an LRR receptor kinase in Arabidopsis. Nature 578(7796):577–581. https://doi.org/10.1038/s41586-020-2032-3
Wrzaczek M, Brosche M, Kangasjarvi J (2013) ROS signaling loops - production, perception, regulation. Curr Opin Plant Biol 16(5):575–582. https://doi.org/10.1016/j.pbi.2013.07.002
Lamb C, Dixon RA (1997) The oxidative burst in plant disease resistance. Annu Rev Plant Physiol Plant Mol Biol 48:251–275. https://doi.org/10.1146/annurev.arplant.48.1.251
Kadota Y, Sklenar J, Derbyshire P, Stransfeld L, Asai S, Ntoukakis V, Jones JD, Shirasu K, Menke F, Jones A, Zipfel C (2014) Direct regulation of the NADPH oxidase RBOHD by the PRR-associated kinase BIK1 during plant immunity. Mol Cell 54(1):43–55. https://doi.org/10.1016/j.molcel.2014.02.021
Gomez-Gomez L, Boller T (2000) FLS2: an LRR receptor-like kinase involved in the perception of the bacterial elicitor flagellin in Arabidopsis. Mol Cell 5(6):1003–1011. https://doi.org/10.1016/s1097-2765(00)80265-8
Chinchilla D, Bauer Z, Regenass M, Boller T, Felix G (2006) The Arabidopsis receptor kinase FLS2 binds flg22 and determines the specificity of flagellin perception. Plant Cell 18(2):465–476. https://doi.org/10.1105/tpc.105.036574
Bent AF, Mackey D (2007) Elicitors, effectors, and R genes: the new paradigm and a lifetime supply of questions. Annu Rev Phytopathol 45:399–436. https://doi.org/10.1146/annurev.phyto.45.062806.094427
Mersmann S, Bourdais G, Rietz S, Robatzek S (2010) Ethylene signaling regulates accumulation of the FLS2 receptor and is required for the oxidative burst contributing to plant immunity. Plant Physiol 154(1):391–400. https://doi.org/10.1104/pp.110.154567
Daudi A, Cheng Z, O’Brien JA, Mammarella N, Khan S, Ausubel FM, Bolwell GP (2012) The apoplastic oxidative burst peroxidase in Arabidopsis is a major component of pattern-triggered immunity. Plant Cell 24(1):275–287. https://doi.org/10.1105/tpc.111.093039
Chen Z, Kloek AP, Boch J, Katagiri F, Kunkel BN (2000) The pseudomonas syringae avrRpt2 gene product promotes pathogen virulence from inside plant cells. Mol Plant-Microbe Interact MPMI 13(12):1312–1321. https://doi.org/10.1094/MPMI.2000.13.12.1312
Nishinaka Y, Aramaki Y, Yoshida H, Masuya H, Sugawara T, Ichimori Y (1993) A new sensitive chemiluminescence probe, L-012, for measuring the production of superoxide anion by cells. Biochem Biophys Res Commun 193(2):554–559. https://doi.org/10.1006/bbrc.1993.1659
Melcher RL, Moerschbacher BM (2016) An improved microtiter plate assay to monitor the oxidative burst in monocot and dicot plant cell suspension cultures. Plant Methods 12:5. https://doi.org/10.1186/s13007-016-0110-1
Trujillo M, Ichimura K, Casais C, Shirasu K (2008) Negative regulation of PAMP-triggered immunity by an E3 ubiquitin ligase triplet in Arabidopsis. Curr Biol 18(18):1396–1401
Smith JM, Heese A (2014) Rapid bioassay to measure early reactive oxygen species production in Arabidopsis leave tissue in response to living pseudomonas syringae. Plant Methods 10(1):6. https://doi.org/10.1186/1746-4811-10-6
Acknowledgments
The authors would like to acknowledge the support by the University of Freiburg, the Deutsche Forschungsgemeinschaft (DFG), and the SERB Postdoctoral Fellowship (India).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Saeed, B., Trujillo, M. (2022). Analysis of Immunity-Related Oxidative Bursts by a Luminol-Based Assay. In: Duque, P., Szakonyi, D. (eds) Environmental Responses in Plants. Methods in Molecular Biology, vol 2494. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2297-1_24
Download citation
DOI: https://doi.org/10.1007/978-1-0716-2297-1_24
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2296-4
Online ISBN: 978-1-0716-2297-1
eBook Packages: Springer Protocols