Peroxidases are involved in biosynthesis and biodegradation of β-thujaplicin in fungal elicitor-treated Cupressus lusitanica cell cultures

Here, collaboration between peroxidases and H2O2 in biosynthesis and biodegredation of beta-thujaplicin in elicited Cupressus lusitanica was investigated. The accumulation of a phytoalexin, beta-thujaplicin, in C. lusitanica cell cultures can be stimulated by a yeast elicitor. A transient low peak was followed by a high level lasting for 2 d, and then a decrease, while peroxidases were activated to a high level just when amounts of beta-thujaplicin decreased. In vitro tests revealed that horseradish peroxidase can transform c. 80% of beta-thujaplicin in the presence of H2O2, and the culture medium was also able to transform beta-thujaplicin. A transient production of H-2 O-2 occured in the cell cultures immediately after elicitation, following a increase in NAD(P)H-oxidase activity. This H2O2 production may mediate the elicitor-induced accumulation of beta-thujaplicin, because inhibiting H2O2 production or removing H2O2 from the cell cultures suppressed elicitor-induced beta-thujaplicin accumulation, while exogenously applied H2O2 or H2O2 generation system can stimulate beta-thujaplicin accumulation. Both NAD(P)H oxidase inhibitors and peroxidase inhibitors partially inhibited NAD(P)H-dependent O-2(-) and H2O2 production. In-gel assay of peroxidase and superoxide anion synthase activity demonstrated that peroxidase isoforms have NAD(P)H-dependent superoxide anion synthase activity. These results suggest that peroxidases can act as superoxide anion synthases to contribute to genecation of H2O2 that promotes beta-thujaplicin production in elicited C. lusitanica cell cultures.