Plant Molecular Biology

, Volume 40, Issue 6, pp 921–933 | Cite as

Elicitor- and A23187-induced expression of WCK-1, a gene encoding mitogen-activated protein kinase in wheat

  • Daisuke Takezawa


Wheat cultured cells were used to study the role of Ca2+ in regulating protein kinases during the induction of defense-related genes by fungal elicitor treatments. Manipulation of intracellular Ca2+ concentrations by treatment with calcium ionophore A23187 in the presence of high extracellular Ca2+ resulted in the induction of mRNA expression of WCK-1, a gene encoding mitogen-activated protein (MAP) kinase. The induction of WCK-1 mRNA by A23187 did not occur when extracellular Ca2+ was chelated by 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA). The WCK-1 mRNA was also induced by Typhula ishikariensis-derived elicitors, suggesting a possible involvement of WCK-1 in the plant defense response against pathogens. BAPTA and a calcium channel blocker, La3+, inhibited the elicitor-induced expression of the WCK-1 mRNA. A recombinant fusion protein of WCK-1 (GST-WCK-1) autophosphorylated at the Tyr residue and exhibited an autophosphorylation-dependent protein kinase activity towards myelin basic protein. Alteration of Tyr-196 in the conserved ‘TEY’ motif in GST-WCK-1 to Phe by site-directed mutagenesis abolished the autophosphorylation. The GST-WCK-1 protein was activated by elicitor-treated wheat cell extracts but not by the control extract. These results suggest that fungal elicitors activate WCK-1, a specific MAP kinase in wheat. Furthermore, the results suggest a possible involvement of Ca2+ in enhancing the MAP kinase signaling cascade in plants by controlling the levels of the MAP kinase transcripts.

A23187 calcium elicitor MAP kinase Typhula ishikariensis wheat 


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  1. Alessandrini, A., Crews, C.M. and Erikson, R.L. 1992. Phorbol ester stimulates a protein tyrosine/threonine kinase that phosphorylates and activates the Erk-1 gene product. Proc. Natl. Acad. Sci. USA 89: 8200–8204.PubMedGoogle Scholar
  2. Berridge, M.J. 1993. Inositol triphosphate and calcium signaling. Nature 361: 315–325.CrossRefPubMedGoogle Scholar
  3. Boller, T. 1995. Chemoperception of microbial signals in plant cells. Annu. Rev. Plant Physiol. Plant Mol. Biol. 46: 189–214.Google Scholar
  4. Bögre, L., Ligterink, W., Meskiene, I., Barker, P.J., Heberie-Bors, E., Huskisson, N.S. and Hirt, H. 1997. Wounding induces the rapid and transient activation of a specific MAP kinase pathway. Plant Cell 9: 75–83.CrossRefPubMedGoogle Scholar
  5. Bohland, C., Balkenhohl, T., Loers, G., Feussner, I. and Grambow, H. J. 1997. Differential induction of lipoxygenase isoforms in wheat upon treatment with rust fungus elicitor, chitin oligosaccharides, chitosan, and methyl jasmonate. Plant Physiol. 114: 679–685.PubMedGoogle Scholar
  6. Braam, J. 1992. Regulated expression of the calmodulin-related TCH genes in cultured Arabidopsiscells: Induction by calcium and heat shock. Proc. Natl. Acad. Sci. USA 89: 3213–3216.PubMedGoogle Scholar
  7. Bush, D.S. 1995. Calcium regulation in plant cells and its role in signaling. Annu. Rev. Plant Physiol. PlantMol. Biol. 46: 95–122.Google Scholar
  8. Clapham, D.E. 1995. Calcium signaling. Cell 80: 259–268.PubMedGoogle Scholar
  9. Cobb, M.H., Hepler, J.E., Cheng, M. and Robbins, D. 1994. The mitogen-activated protein kinases, ERK1 and ERK2. Semin. Cancer Biol. 5: 261–268.PubMedGoogle Scholar
  10. Cohen, P. 1992. Signal integration at the level of protein kinases, protein phosphatases and their substrates. Trends Biochem. Sci. 17: 408–413.PubMedGoogle Scholar
  11. Conrath, U., Jeblick, W. and Kauss, H. 1991. The protein kinase inhibitor, K-252a, decreases elicitor-induced Ca2C uptake and KC release, and increases coumarin synthesis in parsley cells. FEBS Lett. 279: 141–144.PubMedGoogle Scholar
  12. Dietrich, A., Mayer, J.E. and Hahlbrock, K. 1990. Fungal elicitor triggers rapid, transient, and specific protein phosphorylation in parsley cell suspension cultures. J. Biol. Chem. 265: 6360–6368.Google Scholar
  13. Dische, Z. 1955. New color reactions for determination of sugars in polysaccharides. In: D. Glick (Ed.), Methods of Biochemical Analysis Vol. 2, Interscience Publishers, New York, pp. 313–358.Google Scholar
  14. Duclos, B., Marcandier, S. and Cozzone, A.J. 1991. Chemical properties and separation of phosphoamino acids by thin-layer chromatography. Meth. Enzymol. 201: 10–21.PubMedGoogle Scholar
  15. Duerr, B., Gawienowski, M., Ropp, T. and Jacobs, T. 1993. MsERK1: a mitogen-activated protein kinase from a flowering plant. Plant Cell 5: 87–96.CrossRefPubMedGoogle Scholar
  16. Ebel, J. and Cosio, E.G. 1994. Elicitors of plant defense responses. Int. Rev. Cytol. 148: 1–36.Google Scholar
  17. Elion, E.A., Grisafi, P.L. and Fink, G.R. 1990. FUS3 encodes a cdc2C/CDC28-related kinase required for the transition from mitosis into conjugation. Cell 60: 649–664.PubMedGoogle Scholar
  18. Felix, G., Grosskopf, D.G., Regenass, M. and Boller, T. 1991. Rapid changes of protein phosphorylation are involved in transduction of the elicitor signal in plant cells. Proc. Natl. Acad. Sci. USA 88: 8831–8834.PubMedGoogle Scholar
  19. Fransworth, C.L., Freshney, N.W., Rosen, L.B., Ghosh, A., Greenberg, M.E. and Feig, L.A. 1995. Calcium activation of Ras mediated by neuronal exchange factor Ras-GRF. Nature 376: 524–527.PubMedGoogle Scholar
  20. Ghosh, A. and Greenberg, M.E. 1995. Calcium signaling in neurons: molecular mechanisms and cellular consequences. Science 268: 239–247.Google Scholar
  21. Gilroy, S. 1996. Signal transduction in barley aleurone protoplasts is calcium dependent and independent. Plant Cell 8: 2193–2209.CrossRefPubMedGoogle Scholar
  22. Görlach, J., Volrath, S., Knauf-Beiter, G., Hengy, G., Beckhove, U., Kogel, K.H., Oostendorp, M., Staub, T., Ward, E., Kessmann, H. and Ryals, J. 1996. Benzothiadiazole, a novel class of inducers of systemic acquired resistance, activates gene expression and disease resistance in wheat. Plant Cell 8: 629–643.CrossRefPubMedGoogle Scholar
  23. Gotoh, Y., Nishida, E., Yamashita, T., Hoshi, M., Kawakami, M. and Sakai, H. 1990. Microtubule-associated-protein (MAP) kinase activated by nerve growth factor and epidermal growth factor in PC12 cells. Identity with the mitogen-activated MAP kinase of fibroblastic cells. Eur. J. Biochem. 193: 661–669.PubMedGoogle Scholar
  24. Han, J., Lee, J.-D., Bibbs, L. and Ulevitch, R.J. 1994. AMAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells. Science 265: 808–811.PubMedGoogle Scholar
  25. Hanks, S.K. and Lawton, M.A. 1993. Homology-based approaches for identifying cDNAs that encode eukaryotic protein (serine/ threonine) and protein (tyrosine) kinases. In: D.G. Hardie (Ed.), Protein Phosphorylation: A Practical Approach, IRL Press, New York, pp. 173–196.Google Scholar
  26. Hanks, S.K. and Hunter, T. 1995. Eukaryotic protein kinase family: kinase (catalytic) domain structure and classification. FASEB J. 9: 576–596.PubMedGoogle Scholar
  27. Hunter, T. 1987. A thousand and one protein kinases. Cell 50: 823–829.CrossRefPubMedGoogle Scholar
  28. Jonak, C., Kiegerl, S., Ligterink, W., Barker, P.J., Huskisson, N.S. and Hirt, H. 1996. Stress signaling in plants: a mitogen-activated protein kinase pathway is activated by cold and drought. Proc. Natl. Acad. Sci. USA 93: 11274–11279.PubMedGoogle Scholar
  29. Kim, C.Y., Gal, S.W., Choe, M.S., Jeong, S.Y., Lee, S.I., Cheong, Y.H., Lee, S.H., Choi, Y.J., Han, C.-d., Kang, K.Y. and Cho, M.J. 1998. A new class II rice chitinase, Rcht2, whose induction by fungal elicitor is abolished by protein phosphatase 1 and 2A inhibitor. Plant Mol. Biol. 37: 523–534.PubMedGoogle Scholar
  30. Knetsch, M.L.W., Wang, M., Snaar-Jagalska, B.E. and Heimovaara-Dijkstra, S. 1996. Abscisic acid induces mitogen-activated protein kinase activation in barley aleurone protoplasts. Plant Cell 8: 1061–1067.CrossRefPubMedGoogle Scholar
  31. Knight, M.R., Campbell, A.K., Smith, S.M. and Trewavas, A.J. 1991. Transgenic plant aequorin reports the effects of touch and cold-shock and elicitors on cytoplasmic calcium. Nature 352: 524–526.CrossRefPubMedGoogle Scholar
  32. Knight, M.R., Smith, S.M. and Trewavas, A.J. 1992. Wind-induced plant motion immediately increases cytosolic calcium. Proc. Natl. Acad. Sci. USA 89: 4967–4971.PubMedGoogle Scholar
  33. Koike, M., Takezawa, D., Arakawa, K. and Yoshida, S. 1997. Accumulation of 19-kDa plasma membrane polypeptide during induction of freezing tolerance in wheat suspension-cultured cells by abscisic acid. Plant Cell Physiol. 38: 707–716.PubMedGoogle Scholar
  34. Kuwabara, C., Takezawa, D., Arakawa, K. and Yoshida, S. 1998. Molecular cloning of ABA-induced secretory protein (WAS-2) in winter wheat cultured cells. Plant Cell Physiol. Suppl 39: 92.Google Scholar
  35. Kyriakis, J.M., Banerjee, P., Nikolakak, E., Dai, T., Rubie, E.A., Ahmad, M.F., Avruch, J. and Woodgett, J.R. 1994. The stressactivated protein kinase subfamily of c-Jun kinases. Nature 369: 156–160.CrossRefPubMedGoogle Scholar
  36. Lamb, C.J., Lawton, M.A., Dron, M. and Dixon, R.A. 1989. Plant disease resistance genes in signal perception and transduction. Cell 56: 215–224.CrossRefPubMedGoogle Scholar
  37. Ligterink, W., Kroj, T., Nieden, U., Hirt, H. and Scheel, D. 1997. Receptor-mediated activation of a MAP kinase in pathogen defense response. Science 276: 2054–2057.Google Scholar
  38. Linsmaier, E.M. and Skoog, F. 1965. Organic growth factor requirements of tobacco tissue cultures. Physiol. Plant. 18: 100–127.Google Scholar
  39. McAinsh, M.R., Brownlee, C. and Hetherington, A.M. 1997. Calcium ions as second messengers in guard cell signal transduction. Physiol. Plant. 100: 16–29.Google Scholar
  40. Mizoguchi, T., Hayashida, N., Yamaguchi-Shinozaki, K., Kamada, H. and Shinozaki K. 1993. AtMPKs: a gene family of plant MAP kinases in Arabidopsis thaliana. FEBS Lett. 336: 440–444.PubMedGoogle Scholar
  41. Mizoguchi, T., Irie, K., Hirayama, T., Hayashida, N., Yamaguchi-Shinozaki, K., Matsumoto, K. and Shinozaki, K. 1996. A gene encoding a mitogen-activated protein kinase kinase kinase is induced simultaneously with genes for a mitogen-activated protein kinase and an S6 ribosomal protein kinase by touch, cold, and water stress in Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 93: 765–769.PubMedGoogle Scholar
  42. Monroy, A.F. and Dhindsa, R.S. 1995. Low-temperature signal transduction: induction of cold acclimation-specific genes of alfalfa by calcium at 25 °C. Plant Cell 7: 321–331.CrossRefPubMedGoogle Scholar
  43. Morris, P.C., Guerrier, D., Leung, J. and Giraudat, J. 1997. Cloning and characterization of MEK1, an Arabidopsisgene encoding a homologue of MAP kinase kinase. Plant Mol. Biol. 35: 1057–1064.Google Scholar
  44. Poovaiah, B.W. and Reddy, A.S.N. 1993. Calcium and signal transduction in plants. CRC Crit. Rev. Plant Sci. 12: 185–211.PubMedGoogle Scholar
  45. Posada, J., Sanghera, J., Pelech, S., Aebersold, R. and Cooper, J.A. 1991. Tyrosine phosphorylation and activation of homologous protein kinases during oocyte maturation and mitogenic activation of fibroblasts. Mol. Cell. Biol. 11: 2517–2528.PubMedGoogle Scholar
  46. Raz, V. and Fluhr, R. 1992. Calcium requirements for ethylene dependent responses. Plant Cell 4: 1123–1130.CrossRefPubMedGoogle Scholar
  47. Roberts, D.M. and Harmon, A.C. 1992. Calcium-modulated proteins: targets of intracellular calcium signals in higher plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 43: 375–414.Google Scholar
  48. Rosen, L.B. and Greenberg, M.E. 1996. Stimulation of growth factor receptor signal transduction by activation of voltage-sensitive calcium channels. Proc. Natl. Acad. Sci. USA 93: 1113–1118.PubMedGoogle Scholar
  49. Rossomando, A.J., Wu, J., Michel, H., Shabanowitz, J., Hunt, D.F., Weber, M.J., and Sturgill, T.W. 1992. Identification of Tyr-185 as the site of tyrosine autophosphorylation of recombinant mitogenactivated protein kinase p42-mapk. Proc. Natl. Acad. Sci. USA 89: 5779–5783.PubMedGoogle Scholar
  50. Sambrook, J., Fritsch, E.F. and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.Google Scholar
  51. Sanchez, I., Hughes, R.T., Mayer, B.J., Yee, K., Woodgett, J.R., Avruch, J., Kyriakis, J.M. and Zon, L.I. 1994. Role of SAPK/ERK kinase-1 in the stress-activated pathway regulating transcription factor c-Jun. Nature 372: 794–798.PubMedGoogle Scholar
  52. Seger, R. and Krebs, E.G. 1995. The MAPK signaling cascade. FASEB J. 9: 726–735.PubMedGoogle Scholar
  53. Seo, S., Okamoto, M., Seto, H., Ishizuka, K., Sano, H. and Ohashi, Y. 1995. Tobacco MAP kinase: a possible mediator in wound signal transduction pathways. Science 270: 1988–1992.PubMedGoogle Scholar
  54. Sheen, J. 1996. Ca2C-dependent protein kinases and stress signal transduction in plants. Science 274: 1900–1902.Google Scholar
  55. Subramaniam, R., Després, C. and Brisson, N. 1997. A functional homolog of mammalian protein kinase C participation in the elicitor-induced defense response in potato. Plant Cell 9: 653–664.PubMedGoogle Scholar
  56. Suzuki, K. and Shinshi, H. 1995. Transient activation and tyrosine phosphorylation of a protein kinase in tobacco cells treated with a fungal elicitor. Plant Cell 7: 639–647.CrossRefPubMedGoogle Scholar
  57. Takezawa, D., Ramachandiran, S., Paranjape, V. and Poovaiah, B. W. 1996. Dual regulation of a chimeric plant serine/threonine kinase by calcium and calcium/calmodulin. J. Biol. Chem. 271: 8126–8132.CrossRefPubMedGoogle Scholar
  58. Xu, H. and Heath, M.C. 1998. Role of calcium in signal transduction during the hypersensitive response caused by basidiosporederived infection of the cowpea rust fungus. Plant Cell 10: 585–597.PubMedGoogle Scholar
  59. Zhang, S. and Klessig, D.F. 1997. Salicylic acid activates a 48-kDa MAP kinase in tobacco. Plant Cell 9: 809–824.CrossRefPubMedGoogle Scholar
  60. Zwick, E., Daub, H., Aoki, N., Yamaguchi-Aoki, Y., Tonhofer, I., Maly, K. and Ullrich, A. 1997. Critical role of calciumdependent epidermal growth factor receptor transactivation in PC12 cell membrane depolarization and bradykinin signaling. J. Biol. Chem. 272: 24767–24770.PubMedGoogle Scholar

Copyright information

© Kluwer Academic Publishers 1999

Authors and Affiliations

  • Daisuke Takezawa
    • 1
  1. 1.Institute of Low Temperature ScienceHokkaido UniversitySapporoJapan

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