Plant MAP Kinases pp 107-115

Part of the Methods in Molecular Biology book series (MIMB, volume 1171) | Cite as

Immunofluorescent Localization of MAPKs and Colocalization with Microtubules in Arabidopsis Seedling Whole-Mount Probes

Protocol

Abstract

In all eukaryotes, signaling by mitogen-activated protein kinase (MAPK) pathways plays a crucial role in signal transduction during regulation of cell growth, differentiation, proliferation as well as death and stress responses. In this chapter we describe a reliable method to immunolocalize MAPKs in roots of Arabidopsis thaliana by using whole-mount seedling probes. This method relies on quick and efficient chemical fixation, partial cell wall digestion, plasma membrane permeabilization, subsequent antibody incubation, and visualization by high-end confocal laser scanning microscopy (CLSM) performed on whole Arabidopsis seedlings. Protocols are provided for immunofluorescent localization of MPK3, MPK4, and MPK6, representing three major developmentally and stress-regulated MAPKs of Arabidopsis. In addition, protocols for colocalization of these MAPKs with microtubules are also provided.

Key words

Arabidopsis thaliana Mitogen activated protein kinase Root Whole mount Immunolocalization 

References

  1. 1.
    Rodriguez MC, Petersen M, Mundy J (2010) Mitogen-activated protein kinase signaling in plants. Annu Rev Plant Biol 61:621–649PubMedCrossRefGoogle Scholar
  2. 2.
    Komis G, Illés P, Beck M, Šamaj J (2011) Microtubules and mitogen-activated protein kinase signalling. Curr Opin Plant Biol 14:650–657PubMedCrossRefGoogle Scholar
  3. 3.
    Šamajová O, Komis G, Šamaj J (2013) Emerging topics in the cell biology of mitogen-activated protein kinases. Trends Plant Sci 18:140–148PubMedCrossRefGoogle Scholar
  4. 4.
    Plotnikov A, Zehorai E, Procaccia S, Seger R (2011) The MAPK cascades: signaling components, nuclear roles and mechanisms of nuclear translocation. Biochim Biophys Acta 1813:1619–1633PubMedCrossRefGoogle Scholar
  5. 5.
    Pullikuth AK, Catling AD (2007) Scaffold mediated regulation of MAPK signaling and cytoskeletal dynamics: a perspective. Cell Signal 19:1621–1632PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Wortzel I, Seger R (2011) The ERK Cascade: distinct functions within various subcellular organelles. Genes Cancer 2:195–209PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Lazarides E, Weber K (1974) Actin antibody: the specific visualization of actin filaments in non-muscle cells. Proc Natl Acad Sci U S A 71:2268–2272PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Wick SM, Seagull RW, Osborn M, Weber K, Gunning BE (1981) Immunofluorescence microscopy of organized microtubule arrays in structurally stabilized meristematic plant cells. J Cell Biol 89:685–690PubMedCrossRefGoogle Scholar
  9. 9.
    Komis G, Apostolakos P, Galatis B (2001) Altered patterns of tubulin polymerization in dividing leaf cells of Chlorophyton comosum after a hyperosmotic treatment. New Phytol 149:193–207CrossRefGoogle Scholar
  10. 10.
    Beck M, Komis G, Ziemann A, Menzel D, Šamaj J (2011) Mitogen-activated protein kinase 4 is involved in the regulation of mitotic and cytokinetic microtubule transitions in Arabidopsis thaliana. New Phytol 189:1069–1083PubMedCrossRefGoogle Scholar
  11. 11.
    Müller J, Beck M, Mettbach U, Komis G, Hause G, Menzel D, Šamaj J (2010) Arabidopsis MPK6 is involved in cell division plane control during early root development, and localizes to the pre-prophase band, phragmoplast, trans-Golgi network and plasma membrane. Plant J 61:234–248PubMedCrossRefGoogle Scholar
  12. 12.
    Lampard GR, Lukowitz W, Ellis BE, Bergmann DC (2009) Novel and expanded roles for MAPK signaling in Arabidopsis stomatal cell fate revealed by cell type-specific manipulations. Plant Cell 21:3506–3517PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Brock AK, Willmann R, Kolb D, Grefen L, Lajunen HM, Bethke G, Lee J, Nürnberger T, Gust AA (2010) The Arabidopsis mitogen-activated protein kinase phosphatase PP2C5 affects seed germination, stomatal aperture, and abscisic acid-inducible gene expression. Plant Physiol 153:1098–1111PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Kilmartin JV, Wright B, Milstein C (1982) Rat monoclonal antitubulin antibodies derived by using a new nonsecreting rat cell line. J Cell Biol 93:576–582PubMedCrossRefGoogle Scholar
  15. 15.
    North AJ (2006) Seeing is believing? A beginners’ guide to practical pitfalls in image acquisition. J Cell Biol 172:9–18PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of SciencePalacký University OlomoucOlomoucCzech Republic

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