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

Abstract

The biosynthesis of a phytoalexin, β-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 β-thujaplicin. The elicitor signalling pathway involved in β-thujaplicin induction was further investigated using pharmacological and biochemical approaches. Treatment of the cells with calcium ionophore A23187 alone stimulated the production of β-thujaplicin. A23187 also enhanced the elicitor-induced production of β-thujaplicin. EGTA, LaCl3, and verapamil pretreatments partially blocked A23187- or yeast elicitor-induced accumulation of β-thujaplicin. These results suggest that Ca2+ influx is required for elicitor-induced production of β-thujaplicin. Treatment of cell cultures with mastoparan, melittin or cholera toxin alone or in combination with the elicitor stimulated the production of β-thujaplicin or enhanced the elicitor-induced production of β-thujaplicin. The G-protein inhibitor suramin inhibited the elicitor-induced production of β-thujaplicin, suggesting that receptor-coupled G-proteins are likely to be involved in the elicitor-induced biosynthesis of β-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 β-thujaplicin.