Reactive oxygen species, nitric oxide, and their interactions play different roles in Cupressus lusitanica cell death and phytoalexin biosynthesis

beta-Thujaplicin is a natural troponoid with strong antifungal, antiviral, and anticancer activities. beta-Thujaplicin production in yeast elicitor-treated Cupressus lusitanica cell culture and its relationships with reactive oxygen species (ROS) and nitric oxide (NO) production and hypersensitive cell death were investigated. Superoxide anion radical (O-2(center dot-)) induced cell death and inhibited beta-thujaplicin accumulation, whereas hydrogen peroxide (H2O2) induced beta-thujaplicin accumulation but did not significantly affect cell death. Both elicitor and O-2(center dot-) induced programmed cell death, which can be blocked by protease inhibitors, protein kinase inhibitors, and Ca2+ chelators. Elicitor-induced NO generation was nitric oxide synthase (NOS)-dependent. Inhibition of NO generation by NOS inhibitors and NO scavenger partly blocked the elicitor-induced beta-thujaplicin accumulation and cell death, and NO donors strongly induced cell death. Interaction among NO, H2O2, and O-2(center dot-) shows that NO production and H2O2 production are interdependent, but NO and O-2(center dot-) accumulation were negatively related because of coconsumption of NO and O-2(center dot-). NO- and O-2(center dot-)-induced cell death required each other, and both were required for elicitor-induced cell death. A direct interaction between NO and O-2(center dot-) was implicated in the production of a potent oxidant peroxynitrite, which might mediate the elicitor-induced cell death.