Journal of Experimental Botany, Vol. 54, No. 383, pp. 647-656,
February 1, 2003
© 2003 Oxford University Press
Multiple signalling pathways mediate fungal elicitor-induced ß-thujaplicin biosynthesis in Cupressus lusitanica cell cultures
Received 4 April 2002; Accepted 7 October 2002
1 Laboratory of Forest Biochemistry, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan
2 Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
3 Present address and to whom correspondence should be sent: Department of Biochemistry, 104 Willard Hall, Kansas State University, Manhattan, KS 66506 USA. Fax: +1 785 532 7572. E-mail: jzhao{at}ksu.edu
Abbreviations: EGTA, ethylene glycol-bis-ß-aminoethylether-N,N,N’,N'-tetraacetic acid; G-proteins, GTP-binding proteins; DPI, diphenylene iodonium; MeJA, methyl jasmonate.
The biosynthesis of a phytoalexin, ß-thujaplicin, in Cupressus lusitanica cell cultures can be stimulated by a yeast elicitor, H2O2, or methyl jasmonate. Lipoxygenase activity was also stimulated by these treatments, suggesting that the oxidative burst and jasmonate pathway may mediate the elicitor-induced accumulation of ß-thujaplicin. The elicitor signalling pathway involved in ß-thujaplicin induction was further investigated using pharmacological and biochemical approaches. Treatment of the cells with calcium ionophore A23187 alone stimulated the production of ß-thujaplicin. A23187 also enhanced the elicitor-induced production of ß-thujaplicin. EGTA, LaCl3, and verapamil pretreatments partially blocked A23187- or yeast elicitor-induced accumulation of ß-thujaplicin. These results suggest that Ca2+ influx is required for elicitor-induced production of ß-thujaplicin. Treatment of cell cultures with mastoparan, melittin or cholera toxin alone or in combination with the elicitor stimulated the production of ß-thujaplicin or enhanced the elicitor-induced production of ß-thujaplicin. The G-protein inhibitor suramin inhibited the elicitor-induced production of ß-thujaplicin, suggesting that receptor-coupled G-proteins are likely to be involved in the elicitor-induced biosynthesis of ß-thujaplicin. Indeed, both GTP-binding activity and GTPase activity of the plasma membrane were stimulated by elicitor, and suramin and cholera toxin affected G-protein activities. In addition, all inhibitors of G-proteins and Ca2+ flux suppressed elicitor-induced increases in lipoxygenase activity whereas activators of G-proteins and the Ca2+ signalling pathway increased lipoxygenase activity. These observations suggest that Ca2+ and G-proteins may mediate elicitor signals to the jasmonate pathway, and the jasmonate signalling pathway may then lead to the production of ß-thujaplicin.
Key words: Calcium influx, Cupressus lusitanica, G-proteins, jasmonate signalling, signal transduction, ß-thujaplicin.
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