Multiple signalling pathways mediate fungal elicitor-induced beta-thujaplicin biosynthesis in Cupressus lusitanica cell cultures

The biosynthesis of a phytoalexin, beta-thujaplicin, in Cupressus lusitanica cell cultures can be stimulated by a yeast elicitor, H(2)O(2), 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 beta-thujaplicin. The elicitor signalling pathway involved in beta-thujaplicin induction was further investigated using pharmacological and biochemical approaches. Treatment of the cells with calcium ionophore A23187 alone stimulated the production of beta-thujaplicin. A23187 also enhanced the elicitor-induced production of beta-thujaplicin. EGTA, LaCl(3), and verapamil pretreatments partially blocked A23187- or yeast elicitor-induced accumulation of beta-thujaplicin. These results suggest that Ca(2+) influx is required for elicitor-induced production of beta-thujaplicin. Treatment of cell cultures with mastoparan, melittin or cholera toxin alone or in combination with the elicitor stimulated the production of beta-thujaplicin or enhanced the elicitor-induced production of beta-thujaplicin. The G-protein inhibitor suramin inhibited the elicitor-induced production of beta-thujaplicin, suggesting that receptor-coupled G-proteins are likely to be involved in the elicitor-induced biosynthesis of beta-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 Ca(2+) flux suppressed elicitor-induced increases in lipoxygenase activity whereas activators of G-proteins and the Ca(2+) signalling pathway increased lipoxygenase activity. These observations suggest that Ca(2+) and G-proteins may mediate elicitor signals to the jasmonate pathway, and the jasmonate signalling pathway may then lead to the production of beta-thujaplicin.