Oxidative Burst in Suspension Culture of Taxus cuspidata Induced by a Laminar Shear Stress in Short-Term

Generation of active oxidative species induced by shear stress in suspension cultures of Taxus cuspidata was investigated in a Couette-type shear reactor. It was found that T. cuspidata cells respond to a shear rate of 95 s^-1 with oxidative bursts. Their triphasic characteristics in 6 h were similar in both intracellular H2O2 production and extracellular O2- (bullet) production. Additionally, inhibition studies with diphenylene iodonium and azide suggested that the key enzyme responsible for oxidative bursts under the shear rate of 95 s^-1 is primarily NADPH oxidase and the contribution of peroxidase for oxidative bursts was less. Investigation of the relationship between active oxidative species and defense responses induced by the shear stress indicated that the O2-(bullet) burst may account for the change of membrane permeability, and the H2O2 burst plays an important role in inducing secondary metabolites such as the activation of phenylalanine ammonia lyase enzyme and phenolic accumulation. Furthermore, oxidative bursts elicited by the shear rate of 95 s^-1 were suppressed by treatment with suramin, nifedipine, and neomycin prior to the shear stress treatment, suggesting that Gprotein, Ca2+ channel, and phospholipase C are involved in the signal pathway for oxidative bursts induced by the shear stress. A model is proposed to explain the oxidative burst in cultured T. cuspidata cells challenged with the shear stress.