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

• β-Thujaplicin is a natural troponoid with strong antifungal, antiviral, and anticancer activities. β-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 (O₂^•-) induced cell death and inhibited β-thujaplicin accumulation, whereas hydrogen peroxide (H₂O ₂) induced β-thujaplicin accumulation but did not significantly affect cell death. Both elicitor and O₂^•- 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 β-thujaplicin accumulation and cell death, and NO donors strongly induced cell death. • Interaction among NO, H₂O ₂, and O₂^•- shows that NO production and H₂O₂ production are interdependent, but NO and O ₂^•- accumulation were negatively related because of coconsumption of NO and O₂^•-. NO- and O ₂^•--induced cell death required each other, and both were required for elicitor-induced cell death. A direct interaction between NO and O₂^•- was implicated in the production of a potent oxidant peroxynitrite, which might mediate the elicitor-induced cell death.