Skip to main content
SpringerLink
Log in
Book cover

Plant Kinases pp 165–183Cite as

Modulating and Monitoring MAPK Activity During Programmed Cell Death in Pollen

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 779))

Abstract

Signal transduction through mitogen-activated protein kinase (MAPK) cascades regulates many cellular responses. One example of a stimulus-mediated MAPK signaling network in plants is the self-incompatibility (SI) response in Papaver rhoeas, which represents an important mechanism to prevent self-fertilization. This involves interaction of pistil S-locus determinants with a pollen receptor in an incompatible interaction, resulting in a Ca2+-dependent signaling network involving activation of a MAPK, p56, and stimulation of several caspase-like activities, resulting in programmed cell death (PCD). MAPK inhibitors provide a useful tool to dissect these mechanisms and distinguish their regulation by different signaling pathways. U0126 is a potent, noncompetitive, and specific inhibitor of MAPK signaling pathways that result in the inhibition of MAPK activation. Here, we describe the use of this drug in combination with a TEY (threonine–glutamic acid–tyrosine) antibody to alter and monitor MAPK activation, together with a range of markers for PCD to implicate a role for MAPK activation in signaling to PCD in pollen tubes. These techniques may be potentially adapted for use in other plant tissues to investigate MAPK activation in other physiologically relevant systems.

This is a preview of subscription content, 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   84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   139.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   109.99
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

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Samaj, J., Ovecka, M., Hlavacka, A., Lecourieux, F., Meskiene, I., Lichtscheidl, I., Lenart, P., Salaj, J., Volkmann, D., Bögre, L., Baluška, F., and Hirt, Heribert. (2002) Involvement of the mitogen-activated protein kinase SIMK in regulation of root hair tip growth. EMBO J. 2, 3296–3306.

    Article  Google Scholar 

  2. Chang, L., and Karin, M. (2001) Mammalian MAP kinase signalling cascades. Nature410, 37–40.

    Article  CAS  Google Scholar 

  3. Ligterink, W., Kroj, T., Nieden, U., Hirt, H., and Scheel, D. (1997) Receptor-mediated activation of a MAP kinase in pathogen defense of plants. Science276, 2054–2057.

    Article  CAS  Google Scholar 

  4. Yang, K. Y., Liu, Y. D., and Zhang, S. Q. (2002) Activation of a mitogen-activated protein kinase pathway is involved in disease resistance in tobacco. Proc. Natl. Acad. Sci. USA98, 741–746.

    Article  Google Scholar 

  5. Zhang, S. Q., and Klessig, D. F. (2001) MAPK cascades in plant defense signaling. Trends Plant Sci.6, 520–527.

    Article  CAS  Google Scholar 

  6. Asai, T., Tena, G., Plotnikova, J., Willmann, M. R., Chiu, W. L., Gomez-Gomez, L., Boller, T., Ausubel, F. M., and Sheen, J. (2002) MAP kinase signalling cascade in Arabidopsis innate immunity. Nature415, 977–983.

    Article  CAS  Google Scholar 

  7. Wheeler, M. J., de Graaf, B. H. J., Hadjiosif, N., Perry, R. M., Poulter, N. S., Osman, K., Vatovec, S., Harper, A., Franklin, F. C. H., and Franklin-Tong, V. E. (2009) Identification of the pollen self-incompatibility determinant in Papaver rhoeas. Nature459, 992–995.

    Article  CAS  Google Scholar 

  8. Franklin-Tong, V. E., Ride, J. P., Read, N. D., Trewavas, A. J., and Franklin, F. C. H. (1993) The self-incompatibility response in Papaver rhoeas is mediated by cytosolic-free calcium. Plant J.4, 63–77.

    Article  Google Scholar 

  9. Snowman, B. N., Kovar, D. R., Shevchenko, G., Franklin-Tong, V. E., and Staiger, C. J. (2002) Signal-mediated depolymerization of actin in pollen during the self-incompatibility response. Plant Cell4, 2613–2626.

    Article  CAS  Google Scholar 

  10. Thomas, S. G., Huang, S., Li, S. T., Staiger, C. J., and Franklin-Tong, V. E. ( 2006) Actin depolymerization is sufficient to induce programmed cell death in self-incompatible pollen. J. Cell Biol.74, 22–29.

    Google Scholar 

  11. Poulter, N. S., Vatovec, S., and Franklin-Tong, V. E. (2008) Microtubules are a target for self-incompatibility signaling in Papaver pollen. Plant Physiol.146, 1358–1367.

    Article  CAS  Google Scholar 

  12. de Graaf, B. H. J., Rudd, J. J., Wheeler, M. J., Perry, R. M., Bell, E. M., Osman, K., Franklin, F. C. H., and Franklin-Tong, V. E. (2006) Self-incompatibility in Papaver targets soluble inorganic pyrophosphatases in pollen. Nature444, 490–493.

    Article  CAS  Google Scholar 

  13. Rudd, J. J., Osman, K., Franklin, F. C. H., and Franklin-Tong, V. E. (2003) Activation of a putative MAP kinase in pollen is stimulated by the self-incompatibility (SI) response. FEBS Letters547, 223–227.

    Article  CAS  Google Scholar 

  14. Li. S. T., Šamaj, J., and Franklin-Tong, V. E. (2007) A mitogen-activated protein kinase signals to programmed cell death induced by self-incompatibility in Papaver pollen. Plant Physiol.45, 236–245.

    Article  CAS  Google Scholar 

  15. Bosch, M., and Franklin-Tong, V. E. (2007) Temporal and spatial activation of caspase-like enzymes induced by self-incompatibility in Papaver pollen. Proc. Natl. Acad. Sci. USA104, 18327–18332.

    Article  Google Scholar 

  16. Thomas, S. G., and Franklin-Tong, V. E. (2004) Self-incompatibility triggers programmed cell death in Papaver pollen. Nature429, 305–309.

    Article  CAS  Google Scholar 

  17. Kroj, T., Rudd, J.J., Nurnberger, T., Gabler, Y., Lee, J., and Scheel, D. (2003) Mitogen-activated protein kinases play an essential role in oxidative burst-independent expression of pathogenesis-related genes in parsley. J. Biol. Chem.278, 2256–2264.

    Article  CAS  Google Scholar 

  18. Samuel, M. A., Miles, G. P., and Ellis, B. E. (2000) Ozone treatment rapidly activates MAP kinase signalling in plants. Plant J.22, 367–376.

    Article  CAS  Google Scholar 

  19. Lee, J., Klessig, D. F., and Nurnberger, T. (2001) A harpin binding site in tobacco plasma membranes mediates activation of the pathogenesis-related gene HIN1 independent of extracellular calcium but dependent on mitogen-activated protein kinase activity. Plant Cell13, 1079–1093.

    Article  CAS  Google Scholar 

  20. Piater, L. A., Nurnberger, T., and Dubery, I. A. (2004) Identification of a lipopolysaccharide responsive ERK-like MAP kinase in tobacco leaf tissue. Mol. Plant Pathol.5, 331–341.

    Article  CAS  Google Scholar 

  21. Favata, M. F., Horiuchi, K. Y., Manos, E. J., Daulerio, A. J., Stradley, D. A., Feeser, W. S., van Dyk, D. E., Pitts, W. J., Earl, R. A., Hobbs, F., Copeland, R. A., Magolda, R. .L, Scherle, P. A., and Trzaskos, J. M. (1998) Identification of a novel inhibitor of mitogen-activated protein kinase kinase. J. Biol. Chem.273, 18623–18632.

    Article  CAS  Google Scholar 

  22. Schmid, R. S., Pruitt, W. M., and Maness, P. F. (2000) A MAP kinase-signaling pathway mediates neurite outgrowth on L1 and requires Src-dependent endocytosis. J. Neurosci.20, 4177–4188.

    Article  CAS  Google Scholar 

  23. Foote, H. C. C., Ride, J. P., Franklin-Tong, V. E., Walker, E. A., Lawrence, M. J., Franklin, F. C. H. (1994) Cloning and expression of a distinctive class of self-incompatibility (S) gene from Papaver rhoeas. Proc. Natl. Acad. Sci. USA91, 2265–2269.

    Article  CAS  Google Scholar 

  24. Kakeda, K., Jordan, N. D., Conner, A., Ride, J. P., Franklin-Tong, V. E., and Franklin, F. C. H. (1998) Identification of residues in a hydrophilic loop of the Papaver rhoeas S protein that play a crucial role in recognition of incompatible pollen. Plant Cell10, 1723–1731.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The authors would like to thank Kim Osman, Chris Franklin, Barend de Graaf, Steve Thomas, and Maurice Bosch for helpful comments and technical assistance. This work was supported by the Royal Society and the Biotechnology and Biological Sciences Research Council (BBSRC).

Author information

Authors and Affiliations

  1. Department of Botany, University of Osnabrück, Osnabrück, Germany

    Shutian Li

  2. School of Biosciences, University of Birmingham, Edgbaston, Birmingham, UK

    Shutian Li

  3. School of Biosciences, University of Birmingham, Birmingham, UK

    Vernonica E. Franklin-Tong

Authors
  1. Shutian Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Vernonica E. Franklin-Tong
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Shutian Li .

Editor information

Editors and Affiliations

  1. Leibniz Institute of Plant Biochemistry, Weinberg 3, Halle (Saale), 06120, Sachsen-Anhalt, Germany

    Nico Dissmeyer

  2. rue du Général Zimmer 12, Strasbourg, F-67000, France

    Arp Schnittger

Rights and permissions

Reprints and permissions

Copyright information

© 2011 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Li, S., Franklin-Tong, V.E. (2011). Modulating and Monitoring MAPK Activity During Programmed Cell Death in Pollen. In: Dissmeyer, N., Schnittger, A. (eds) Plant Kinases. Methods in Molecular Biology, vol 779. Humana, Totowa, NJ. https://doi.org/10.1007/978-1-61779-264-9_9

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-264-9_9

  • Published:

  • Publisher Name: Humana, Totowa, NJ

  • Print ISBN: 978-1-61779-263-2

  • Online ISBN: 978-1-61779-264-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation