Polyamines-induced Nitric Oxide Production And Their Requirement For Oxygen Or Hydrogen Peroxide In Suspended Cells Of Soybean Cotyledon Node Callus

Spermine （Spm）, spermidine （Spd） and their diamine obligate precursor putrescine（Put） are micro molecular aliphatic polycations that are ubiquitous in all plant cells.They have been proposed to be closely associated with plant development,morphogenesis and response to diverse stresses. Nitric oxide （NO） is a plant cellsignaling molecule which has been confirmed to play a pivotal role in the regulation ofseed germination, development of root system, stomatal closure, expression of defencegenes and programmed cell death. Several reports indicate that polyamines （PAs） inducenitric oxide （NO） production in plant tissues, and polyamine oxidases play importantroles in polyamine-induced NO generation, but not much information is available onbiochemical mechanisms underlying effects of PAs on NO induction.In the present study, suspended cells of soybean cotyledon-node callus wereapplied. Using a NO specific fluorescent dye DAF-FM DA and a laser confocalscanning microscopy, changes in NO generation induced by exogenous PAs werestudied after treatments with exogenous polyamines （Put, Spd and Spm）,L-aminoguanidine （L-AG, an inhibitor of polyamine oxidase）, L-NAME （a mammalinhibitor of nitric oxide synthase）, catalase and peroxidase （H₂O₂scavengers）,sodium dithionite （SDT, a chemical anoxia agent） and N’N-dimethylthiourea （DMTU,H₂O₂quencher）. The results showed that:（1） NO fluorescence was significantly induced above the endogenous levels whencallus cells were treated with0.05mM of PAs. The observed NO release by PAs wasvery rapid without apparent lag phase.（2） The response was quenched when suspended cells were treated with a NOspecific scavenger cPTIO, showing its NO fluorescent traits.（3）When0.01mML-aminoguanidine （L-AG） was applied prior to PA treatments the NO fluorescent intensity was diminished, and the inhibition of NO fluorescence correlated withdecreases in diamine oxidase （DAO） activity.1.0mM L-NAME also decreased NOfluorescent intensity induced by PAs but it could not inhibit CuAO activity.（4） When suspended cells were treated with0.1mM CAT and0.05mM DMTUbefore PA treatments, NO fluorescence was significantly reduced, but0.1mM POD hadno apparent effect on NO decrease.（5） When callus cells were incubated with1.0mM sodium dithionite （SDT） andcells were kept under an anoxia condition, NO fluorescent releases were significantlydiminished, and NO fluorescent intensity decreased with SDT concentration, while SDTdid not quench NO fluorescence generated by NO donor （SNP）.（6） When1.0mM SDT was applied prior to PA treatments, NO fluorescenceinduced by PAs was significantly reduced.Altogether, our data obtained evidence for PA-induced NO production insuspended cells of soybean cotyledon node callus，and tentatively demonstrated thatoxygen or peroxide might be involved in NO production induced by PAs, probably withDAO activity. This study provided a new insight into PA-mediated signalingtransduction and NO potential roles as PA mediator.