The Role Of Polyamine Oxidase In Cytosolic Antioxidant Defense Induced By Abscisic Acid In Leaves Of Maize Plants

Abscisic acid(ABA) can regulate a variety of physiological and molecular reponses for plant against stresses,including drought,chilling,salinity,and so on.Previous studies showed that ABA can cause the generation of hydrogen peroxide(H₂O₂) and enhance the capacity of antioxidant defense systems.However,the detailed mechanism of ABA-induced antioxidant defense remains to be determined.PAO(polyamine oxidase) are highly expressed,particularly in monocots.PAO bears a non-covalently bound molecule of flavorprotein(FAD) as a cofactor and catalyses the oxidation of spermine(Spm), spermidine(Spd) and/or their acetylated derivatives at the secondary amino groups.In plants,the production of hydrogen peroxide(H₂O₂) deriving from polyamine oxidation has been correlated with cell wall maturation and lignification during development as well as wound-healing and cell wall reinforcement during pathogen invasion.As a signal molecule, H₂O₂ derived from polyamine oxidation mediates cell death,the hypersensitive response and the expression of defense genes.However,it is not clear that the specific contribution of PAO to ABA induced H₂O₂ accumulation and the effect of PAO to subcellular compartment cytosolic antioxidant defense.In this study,an effort was made to elucidate the contribution of MPAO(maize polyamine oxidase) in ABA induced H₂O₂ production and the effect of MPAO to subcellular compartment cytosolic antioxidant defense.The results are as follows:ABA treatment resulted in an enhancement in the transcript level of MPAO gene analyzed by RT-PCR.The time-course analysis of MPAO gene expression showed that the transcript level of MPAO reached the maximum value after 20 min of ABA treatment,and returned to the control level after 30 min.On the other hand,ABA treatment also resulted in the enhancement in the activity of apoplastic MPAO,which reached the maximum value after 0.5 h of ABA treatment.Previous studies showed that ABA only induced the apoplastic H₂O₂ accumulation in maize leaves.To test a possible effect of MPAO on H₂O₂ accumulation in leaves of maize plants exposed to ABA,a histochemical method for H₂O₂ detection was used.ABA treatment led to an obvious accumulation of H₂O₂.Pretreatments with the MPAO inhibitors guazatine(0.1 mM,0.5 mM,1 mM) and 1,12-diaminododecane(DADD)(0.1 mM,0.5 mM,1 mM),partly blocked the accumulation of H₂O₂ induced by ABA.The role of MPAO in the accumulation of H₂O₂ induced by ABA was further examined by a cytochemical technique with cerium chloride(CeCl₃),which reacts with H₂O₂ to produce electron-dense deposits of cerium perhydroxides.Treatment with ABA led to H₂O₂ accumulation in apoplast of the mesophyll cells,and the greatest accumulation of H₂O₂ was observed in the cell walls facing intercellular spaces.Pretreatments with the MPAO inhibitors guazatine(1 mM) and DADD(1 mM) significantly abolished the H₂O₂ accumulation detected with the CeCl₃ staining.Moreover,treatments with guazatine and DADD alone did not affect H₂O₂ production in control leaves.Previous study has shown that ABA can increase the activities of cytosolic antioxidant enzymes such as SOD and APX.In order to determine whether MPAO contributes to ABA-induced cytosolic antioxidant defense,the MPAO inhibitors guazatine and DADD were used.Pretreatments with two inhibitors partly blocked increases in the transcript levels of cytosolic ascorbate peroxidase(cAPX) and superoxide dismutase 4(SOD4) and the activities of cytosolic APX and SOD induced by ABA treatment in leaves of maize seedlings,and the inhibition on these parameters exhibited a dose-dependent manner,but guazatine and DADD alone had no effect,suggesting that MPAO is involved in ABA-induced cytosolic antioxidant defense in maize leaves.To further identify the effect of MPAO on the cytosolic antioxidant defense in maize leaves,the detached plants were treated with polyamine(Spd,Spm),the optimum substrate of MPAO,and then the leaves were analysed after 4,8,12 and 24 h of treatment.Treatment with polyamine induced the expression and the activities of the antioxidant enzymes.In order to determine whether polyamine-induced increases in the activities of cytosolic antioxidant enzymes result from MPAO-catalyzed H₂O₂ production,the detached plants were pretreated with the MPAO inhibitors guazatine and DADD and the H₂O₂ scavengers CAT and dimethylthiourea(DMTU) respectively,and then exposed to polyamine treatment. Pretreatment with guazatine,DADD,CAT and DMTU significantly blocked the increases in the transcript levels of cAPX and Sod4 and the activities of cytosolic APX and SOD induced by poiyamine treatment,and the inhibition exhibited a dose-dependent manner,but these inhibitors or scavengers alone had no effect on the transcript levels and the activities of antioxidant enzymes,suggesting that MPAO induces cytosolic antioxidant defense through H₂O₂,a polyamine catabolic product in maize leaves.