A Study On The Roles Of CuAO And Its Catalysate H₂O₂ In Light/dark-regulated Stomatal Movement

Now,people show increasingly interest in the sources and the physiological roles of hydrogen peroxide(H₂O₂),which is an important plant signalling molecule.Previous studies proved that H₂O₂ generated by the plasma membrane NADPH oxidase was involved in abscisic acid(ABA)- and light/dark-regulated stomatal movement,and a lot of evidence indicated that H₂O₂,a catalysate of copper amine oxidase(CuAO) and Polyamine Oxidase(FAD-PAO),participated in many key plant physiological processes,such as plant defense reaction,development of vascular tissues, programmed cell death(PCD),occurrence of lateral root.Recent research reported that H₂O₂ generated by CuAO was involved in ABA-induced stomatal closure.However,the physiological roles of CuAO and its catalysate H₂O₂ in light/dark - regulated stomatal movement was still unclear. In the present study,using the abaxial epidermis of Vicia faba as materials,by means of the epidermal strip bioassay and the laser scanning confocal microscopy(LSCM),the roles of CuAO and its catalysate H₂O₂ in light/dark-regulated stomatal movement were investigated,and the relationship between H₂O₂ generated by CuAO and nitric oxide(NO),another important plant signalling molecule,was explored.The present results had an important theoretical significance for the further understanding of the guard cells signal transduction pathways and the mechanisms of responding to light/dark stimulus.The results were as follows:1.Aminoguanidine(AG) and 2-Bromoethylamine(BEA),which are the inhibitors of the CuAO, had no significant impacts on the stomatal aperture in light,but both could significantly prevent dark-induced stomata closure.The results indicated that CuAO involved in light/dark-regulated stomatal movement and the activity of CuAO seemed higher in dark than in light.2.The products from putrescine(Put) oxidation by CuAO include H₂O₂,ammonia(NH₃) and 4-aminobutanal.4-aminobutanal can be easily catalyzed toγ-aminobutyric acid(GABA),which is subsequently transaminated and oxidized to succinic acid(Succ).The present results suggested that only H₂O₂ had significant effects on stomatal aperture in light,and could reverse AG- and BEA-promoted stomatal opening in darkness.These results indicated that H₂O₂ generated by CuAO was involved in light/dark-regulated stomatal movement. 3.Put is a substrate of CuAO,so CuAO inhibitors-induced stomatal opening might relate to the accumulation of Put.The present results showed that exogenous Put did not induce stomatal opening in darkness,showing that the effect of AG and BEA on stomatal opening is not related to the accumulation of Put.4.By means of LSCM based on H₂DCF-DA,a specific molecular probe of H₂O₂,we provided evidence that dark indeed induced the generation of endogenous H₂O₂ and the fluorescence derived by darkness was very striking compared with that in light condition.The striking fluorescence was prevented by AG and BEA,the inhibitors of CuAO,indicating that dark induced H₂O₂ generation could through CuAO pathway.5.By means of LSCM based on DCF-2DA,a specific molecular probe of NO,we provided evidence that both darkness and exogenous H₂O₂ induced NO increasing,and the effect of darkness were also suppressed by the inhibitors of CuAO,AG and BEA,suggesting that NO generated by darkness was dependent on H₂O₂ synthesis through CuAO pathway.In summary,the present results shown that light/dark adjusted the production of H₂O₂ by governing the activity of CuAO,and then regulated NO synthesis,finally regulating stomatal movement.