The Role And Mechanism Of Copper Amine Oxidase In ABA-Mediated H₂O₂ Production And Stomatal Movement In Vicia Faba

In stress conditions,abscisic acid(ABA) modulates stomatal apertures through an intricate signalling network to reduce water loss and this is crucial to improve plant tolerance.H₂O₂ is an important signalling molecule that involved in ABA-induced stomatal movement and it comes from NADPH oxidase,amine oxidase and other sources in plants. It has been reported that the H₂O₂ generated by NADPH oxidase is involved in ABA-induced stomatal closure.The amine oxidase in plants catalyzes the oxidation of diamine or polyamine and produce H₂O₂.But whether the H₂O₂ generated by amine oxidase is involved in ABA-induced stomatal closure has not been reported.It has been reported that the phospholipase D(PLD) in Arabidopsis hydrolyzes phospholipids, producing phosphatidic acid(PA).PA activates NADPH oxidase producing H₂O₂ and then induces stomatal closure.But whether this route is exist in Vicia faba and its relationship with the route that the H₂O₂ generated by CuAO have not been reported.In this study,we investigated the role and the relationship of CuAO,PLD and NADPH oxidase in ABA-induced stomatal closure in Vicia faba using pharmacological, physiological,cytobiological and biochemical approaches.Our results showed that the CuAO inhibitor inhibited the ABA-induced stomatal closure significantly and in the metabolized produces of CuAO,only H₂O₂ could induce stomatal closure.The CuAO in Vicia faba mainly existed in apoplast.ABA elevated the CuAO activity and the H₂O₂ level in guard cells rapidly,and the CuAO inhibitor inhibited the process.CuAO in Vicia faba oxidized putrescine(put) but not spermidine(spd) or spermine(spm) to produce H₂O₂.As the substrate of CuAO,put also activated the CuAO activity,and induced the elevation of H₂O₂ content in guard cells and stomatal closure.The CuAO inhibitor inhibited the process significently.These results indicate that CuAO is involved in ABA-induced stomatal closure via its degradation of put to produce H₂O₂.ABA and put elevated the Ca²⁺ content in Vicia faba guard cells and the CuAO inhibitor inhibited the process.This indicates that the H₂O₂ generated by CuAO is involved in ABA-induced stomatal closure via its elevation the Ca²⁺ content in guard cells.ABA elevated the activity of NADPH oxidase in Vicia faba plasma membrane which is another source of H₂O₂.The inhibitor of NADPH oxidase inhibited the ABA-induced elevation of H₂O₂ content in guard cells and stomatal closure.The elevation of the H₂O₂ content was more inhibited when the CuAO inhibitor was used together.These results indicated that NADPH oxidase and CuAO are involved in ABA-induced H₂O₂ production in guard cells independently.The CuAO inhibitor had no effect on the elevation of NADPH oxidase activity induced by ABA.The inhibitor of NADPH oxidase had no effect on the elevation of CuAO activity induced by ABA and put.These results further prove that the two enzymes are involved in ABA-induced H₂O₂ production in guard cells and stomatal closure independently.Inhibition of stomatal opening is another role of ABA in modulation stomatal movement.As the downstream signal of ABA,H₂O₂ inhibited stomatal opening in vicia faba.The H₂O₂ scavengers partly relieved the H₂O₂-or ABA-suppressed stomatal opening. This indicates that H₂O₂ is involed in ABA-suppressed stomatal opening.CuAO inhibitor partly relieved the ABA-suppressed stomatal opening and in the metabolized produces of CuAO only H₂O₂ inhibited stomatal opening.This indicates that the H₂O₂ generated by CuAO is involved in ABA-inhibited stomatal opening.As the substrate of CuAO,put also inhibited stomatal opening via elevation CuAO activity to produce H₂O₂.CuAO inhibitor partly relieved the put-suppressed stomatal opening.These results indicate that CuAO is involved in ABA-suppressed stomatal opening via its degradation of put to produce H₂O₂.PLD inhibitor partly inhibited the elevation of H₂O₂ content in vicia faba guard cells and stomatal closure induced by ABA,and a more inhibition was found when CuAO inhibitor was used together.This indicates that the PLD in Vicia faba guard cells is involved in ABA-induced H₂O₂ production and stomatal closure and PLD and CuAO may involved in this process independently.As the product of PLD,PA also induced the elevation of H₂O₂ content in vicia faba guard cells and stomatal closure and the CuAO inhibitor had no effect on the process,but the inhibitor of NADPH oxidase partly inhibited the process.This indicates that PA induces stomatal closure via activation NADPH oxidase to produce H₂O₂. The elevation of CuAO activity in vicia faba leaves induced by ABA was not mediated by PLD.This further proves that ABA induces H₂O₂ production and stomatal closure via its activation of PLD,NADPH oxidase and CuAO activity,and the two routes are independent.Based on these results,we propose a new signal route in vicia faba guard cells: ABA→CuAO→H₂O₂→Ca²⁺→stomatal closure.The signal route:ABA→PLD→NADPH oxidase→H₂O₂→stomatal closure is also exist in Vicia faba guard cells which is similar to that in Arabidopsis and the two routes are independently.These discoveries have important significance for thorough understanding and consummation of the ABA signalling pathway network in guard cells.