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

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.