Polyamine-induced nitric oxide generation and its potential requirement for peroxide in suspension cells of soybean cotyledon node callus.

Polyamines (PAs) induce nitric oxide (NO) generation in plant tissues; however, their mechanism is still unclear. In the present study, suspension cells of soybean cotyledon-node callus were employed. Using a NO-specific fluorescent dye, DAF-FM-DA (3-amino, 4-aminomethyl-2', 7'-difluorescein, diacetate), and laser confocal scanning microscopy, changes in NO generation induced by exogenous PAs were examined. The results of this study showed that NO fluorescence was significantly induced above endogenous levels when callus cells were treated with 0.05 mM PAs. However, putrescine (Put) was the most active PA. The observed NO release by PAs was rapid and without an apparent lag phase. The response was quenched when the suspension cells were treated with the NO-specific scavenger cPTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-1-oxy-3-oxide). When 0.01 mM l-aminoguanidine (L-AG) was applied prior to the PA treatments, the NO fluorescence was diminished, and the inhibition of NO fluorescence was correlated with a decrease in diamine oxidase (DAO) activity. When callus cells were incubated with 0.1 mM catalase (CAT) and 1.0 mM N'N-dimethylthiourea (DMTU) prior to PA application, NO release was significantly reduced. In sum, our data provided evidence for PA-induced NO generation in suspension cells of soybean cotyledon node callus and demonstrated that peroxide, potentially derived from PA oxidative degradation, was involved in NO release induced by PAs.