Skip to main content
SpringerLink
Log in

Using Foldscope to Monitor Superoxide Production and Cell Death During Pathogen Infection in Arabidopsis Under Different Nitrogen Regimes

  • Protocol
  • First Online:
Nitrogen Metabolism in Plants

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2057))

Abstract

Nitrogen nutrition plays a role in plant growth development and resistance against biotic and abiotic stress. During pathogen infection various signal molecules such as reactive oxygen species, calcium, reactive nitrogen species, salicylic acid, and ethylene plays an important role. The form of nitrogen nutrition such as nitrate or ammonium plays a role in production of these molecules. Under nitrate nutrition NO is predominant. The produced NO plays a role in reacting with superoxide to generate peroxynitrite to induce cell death during hypersensitive response elicited by avirulent pathogens. Excess of ROS is also detrimental to plants and NO plays a role in regulating ROS. Hence it is important to observe superoxide production during infection. By using an avirulent Pseudomonas syringae and Arabidopsis differential N nutrition we show superoxide production in leaves using a paper microscope called Foldscope, which can be applied as a simple microscope to observe objects. The data also compared with root system infected with pathogenic Fusarium oxysporum. Taken together here we show that Foldscope is a cost-effective and powerful technique to visualize superoxide and cell death in plants during infection.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 99.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 129.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 199.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Mittler R (2017) ROS are good. Trends Plant Sci 22:11–19

    Article  CAS  Google Scholar 

  2. Schieber M, Chandel NS (2014) ROS function in redox signaling and oxidative stress. Curr Biol 24:R453–R462

    Article  CAS  Google Scholar 

  3. Reczek CR, Chandel NS (2015) ROS-dependent signal transduction. Curr Biol 33:8–13

    Article  CAS  Google Scholar 

  4. Delledonne M, Xia Y, Dixon RA, Lamb C (1998) Nitric oxide functions as a signal in plant disease resistance. Nature 394:585

    Article  CAS  Google Scholar 

  5. Wany A, Gupta AK, Kumari MS et al (2018) Nitrate nutrition influences multiple factors in order to increase energy efficiency under hypoxia in Arabidopsis. Ann Bot 123(4):691–705

    Article  Google Scholar 

  6. De Gara L, de Pinto MC, Tommasi F (2003) The antioxidant systems vis-à-vis reactive oxygen species during plant–pathogen interaction. Plant Physiol Biochem 41:863–870

    Article  Google Scholar 

  7. Tommasi F, Paciolla C, de Pinto, MC, Gara LD (2001) A comparative study of glutathione and ascorbate metabolism during germination of Pinus pinea L seeds. J Exp Bot 52:1647–1654

    Google Scholar 

  8. Halliwell B (1978) Lignin synthesis: the generation of hydrogen peroxide and superoxide by horseradish peroxidase and its stimulation by manganese (II) and phenols. Planta 140:81–88

    Article  CAS  Google Scholar 

  9. Adam A, Farkas T, Somlyai G, Hevesi M, Kiraly Z (1989) Consequence of O2·− generation during a bacterially induced hypersensitive reaction in tobacco: deterioration of membrane lipids. Physiol Mol Plant Pathol 34:13–26

    Article  CAS  Google Scholar 

  10. Levine A, Tenhaken R, Dixon R, Lamb C (1994) H2O2 from the oxidative burst orchestrates the plant hypersensitive disease resistance response. Cell 79:583–593

    Article  CAS  Google Scholar 

  11. Mittler R, Hallak Herr E, Orvar BL, Van Camp W, Willekens H, Inzé D, Ellis BE (1999) Transgenic tobacco plants with reduced capability to detoxify reactive oxygen intermediates are hyperresponsive to pathogen infection. Proc Natl Acad Sci U S A 96:14165–14170

    Google Scholar 

  12. Van Breusegem F, Dat JF (2006) Reactive oxygen species in plant cell death. Plant Physiol 141:384–390

    Google Scholar 

  13. Vanacker H, Foyer CH, Carver TL (1999) Changes in apoplastic antioxidants induced by powdery mildew attack in oat genotypes with race non-specific resistance. Planta 208:444–452

    Article  CAS  Google Scholar 

  14. Mittler R, Feng X, Cohen M (1998) Post-transcriptional suppression of cytosolic ascorbate peroxidase expression during pathogen-induced programmed cell death in tobacco. Plant Cell 10:461–473

    Article  CAS  Google Scholar 

  15. Vishwakarma A, Kumari A, Mur LA, Gupta KJ (2018) A discrete role for alternative oxidase under hypoxia to increase nitric oxide and drive energy production. Free Radic Biol Med 122:40–51

    Article  CAS  Google Scholar 

  16. Samsatly J, Copley TR, Jabaji SH (2018) Antioxidant genes of plants and fungal pathogens are distinctly regulated during disease development in different Rhizoctonia solani pathosystems. PLoS One 13:e0192682

    Article  Google Scholar 

  17. Gupta KJ, Brotman Y, Segu S, Zeier T, Zeier J, Persijn ST, Cristescu SM, Harren FJM, Bauwe H, Fernie AR (2013) The form of nitrogen nutrition affects resistance against Pseudomonas syringae pv. phaseolicola in tobacco. J Exp Bot 64:553–568

    Article  CAS  Google Scholar 

  18. Modolo LV, Augusto O, Almeida IM, Magalhaes JR, Salgado I (2005) Nitrite as the major source of nitric oxide production by Arabidopsis thaliana in response to Pseudomonas syringae. FEBS Lett 579:3814–3820

    Article  CAS  Google Scholar 

  19. Liszkay A, van der Zalm E, Schopfer P (2004) Production of reactive oxygen intermediates (O2−, H2O2, and OH) by maize roots and their role in wall loosening and elongation growth. Plant Physiol 136:3114–3123

    Google Scholar 

  20. Warwar N, Mor A, Fluhr R, Pandian RP, Kuppusamy P, Blank A (2011) Detection and imaging of superoxide in roots by an electron spin resonance spin-probe method. Biophys J 101:1529–1538

    Article  CAS  Google Scholar 

  21. Grellet Bournonville CF, Díaz-Ricci JC (2011) Quantitative determination of superoxide in plant leaves using a modified NBT staining method. Phytochem Anal 22:268–271

    Article  CAS  Google Scholar 

  22. Wohlgemuth H, Mittelstrass K, Kschieschan S, Bender J, Weigel HJ et al (2002) Activation of an oxidative burst is a general feature of sensitive plants exposed to the air pollutant ozone. Plant Cell Environ 25:717–726

    Article  CAS  Google Scholar 

  23. Vleeshouwers VG, van Dooijeweert W, Govers F, Kamoun S, Colon LT (2000) The hypersensitive response is associated with host and nonhost resistance to Phytophthora infestans. Planta 210(6):853–864

    Article  CAS  Google Scholar 

  24. Johansson ON, Nilsson AK, Gustavsson MB, Backhaus T, Andersson MX, Ellerström M (2015) A quick and robust method for quantification of the hypersensitive response in plants. PeerJ e1469:3

    Google Scholar 

  25. Planchet E, Sonoda M, Zeier J, Kaiser WM (2006) Nitric oxide (NO) as an intermediate in the cryptogein-induced hypersensitive response–a critical re-evaluation. Plant Cell Environ 29:59–69

    Article  CAS  Google Scholar 

  26. Keogh RC, Deverall BJ, McLeod S (1980) Comparison of histological and physiological responses to Phakopsora pachyrhizi in resistant and susceptible soybean. Trans Br Mycol Soc 74(2):329–333

    Article  Google Scholar 

  27. Bouarab K, Melton R, Peart J, Baulcombe D, Osbourn A (2002) A saponin-detoxifying enzyme mediates suppression of plant defences. Nature 418(6900):889

    Article  CAS  Google Scholar 

  28. Zhou L, Cheung MY, Li MW, Fu Y, Sun Z, Sun SM, Lam HM (2010) Rice hypersensitive induced reaction protein 1 (OsHIR1) associates with plasma membrane and triggers hypersensitive cell death. BMC Plant Biol 10(1):290

    Article  CAS  Google Scholar 

  29. Pogany M, von Rad U, Grün S, Dongo A, Pintye A, Simoneau P et al (2009) Dual roles of reactive oxygen species and NADPH oxidase RBOHD in an Arabidopsis-Alternaria pathosystem. Plant Physiol 151(3):1459–1475

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work is supported by Foldscope project funded by DBT-Prakash lab initiative. PS and PKP are funded by UGC SRF and SRF.

Author information

Author notes

  1. Reena Arora and Pooja Singh contributed equally to this work.

Authors and Affiliations

  1. National Institute of Plant Genome Research, Aurna Asaf Ali Marg, New Delhi, India

    Reena Arora, Pooja Singh, Aprajita Kumari, Pradeep Kumar Pathak & Kapuganti Jagadis Gupta

Authors
  1. Reena Arora
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Pooja Singh
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Aprajita Kumari
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Pradeep Kumar Pathak
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Kapuganti Jagadis Gupta
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Kapuganti Jagadis Gupta .

Editor information

Editors and Affiliations

  1. National Institute of Plant Genome Research, New Delhi, India

    Kapuganti Jagadis Gupta

Rights and permissions

Reprints and permissions

Copyright information

© 2020 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Arora, R., Singh, P., Kumari, A., Pathak, P.K., Gupta, K.J. (2020). Using Foldscope to Monitor Superoxide Production and Cell Death During Pathogen Infection in Arabidopsis Under Different Nitrogen Regimes. In: Gupta, K. (eds) Nitrogen Metabolism in Plants. Methods in Molecular Biology, vol 2057. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9790-9_9

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9790-9_9

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9789-3

  • Online ISBN: 978-1-4939-9790-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation