【作者】 薛贝贝；

【导师】 蒋明义；

【作者基本信息】 南京农业大学 ， 细胞生物学， 2008， 硕士

【摘要】 植物激素脱落酸（abscisic acid,ABA）可以调节植物抗逆（包括干旱、冷害、盐害等）的多种生理反应和分子生物学效应。过去的研究结果已经表明ABA能够诱导过氧化氢（hydrogen peroxide,H₂O₂）产生以及抗氧化基因的表达,但有关ABA诱导植物抗氧化防护能力提高的详细机制仍有许多问题尚不清楚。多胺氧化酶（polyamine oxidases,PAO）是一种主要分布在禾谷类等单子叶植物中的胺氧化酶。它分子中含有黄素蛋白（flavoprotein,FAD）,能将细胞产生的胺类物质（如亚精胺和精胺）氧化成醛,并释放NH₃和H₂O₂。目前关于PAO的研究多集中于其在细胞壁硬化以及程序性细胞死亡中的作用,有关ABA诱导的H₂O₂产生中,PAO的贡献及功能研究还非常少,特别是PAO对亚细胞区室细胞溶质抗氧化防护系统的影响,目前尚未见报道。为此,本研究以玉米（Zea mays L.）叶片为材料,应用组织化学和细胞化学、酶活性分析以及半定量RT-PCR相结合的手段,进一步研究玉米多胺氧化酶（maize polyamine oxidases,MPAO）是否参与ABA诱导的玉米叶片中H₂O₂产生及其在细胞溶质抗氧化防护的作用。主要的研究结果如下:采用半定量RT-PCR的方法,100μM ABA处理后,玉米叶片中MPAO基因转录水平与对照相比,在10 min至20 min时出现一个快速、瞬时的上调;另一方面,相对于对照组,质外体MPAO活性在ABA处理后迅速上升,在0.5 h达到最大后开始下降,即外源ABA处理能诱导其基因表达和酶活性的提高。DAB在过氧化物酶催化下能与H₂O₂反应生成深棕色的多聚化合物。本研究用DAB染色检测H₂O₂量的变化来揭示MPAO在外源ABA诱导H₂O₂产生中的作用。从组织化学图片中可以看出,在外源ABA的处理下则迅速地引起了玉米叶片H₂O₂的大量积累。分别采取MPAO的两种抑制剂,并结合ABA处理来观察它们对外源ABA诱导玉米叶片H₂O₂积累的影响。结果表明不同浓度的两种抑制剂（0.1,0.5和1 mM）均对ABA诱导的玉米叶片H₂O₂的积累有一定抑制作用。用细胞化学方法来进一步揭示MPAO在外源ABA诱导H₂O₂积累中的作用。电镜照片表明,在外源ABA处理条件下,玉米叶肉细胞位于朝向细胞间隙的细胞壁上有大量的黑色沉积物,表明外源ABA诱导质外体产生大量的H₂O₂。抑制剂实验中,guazatine和DADD处理后叶肉细胞壁上的沉积物明显减少,这与组织化学的结论是一致的。而抑制剂预处理8 h后结合纯水处理,未发现在玉米叶肉细胞质外体有H₂O₂的产生,表明抑制剂本身对于实验结果没有影响。MPAO酶活性结合半定量RT-PCR分析以及组织化学结合细胞化学方法的研究进一步提示,MPAO可能参与了ABA诱导的玉米叶片H₂O₂的产生,MPAO是玉米叶片中活性氧的来源之一。研究表明,ABA上调了玉米叶片细胞溶质抗氧化防护酶SOD,APX的活性。为了明确MPAO与玉米叶片细胞溶质抗氧化防护的关系,本研究用MPAO抑制剂guazatine和DADD预处理8 h,再用100μM ABA处理8 h或12 h来进行研究。结果表明这两种抑制剂均部分抑制ABA诱导的抗氧化防护酶cAPX和SOD4基因的表达以及细胞溶质APX和SOD活性的提高,并且抑制剂浓度越高抑制程度越明显。这提示MPAO可能参与了ABA诱导玉米叶片细胞溶质抗氧化防护系统的调节。为了进一步明确MPAO对玉米叶片细胞溶质抗氧化防护系统的影响,用MPAO的最适底物（Spd,Spm）处理玉米离体植株。1 mM Spd处理引起了细胞溶质抗氧化防护能力的提高。外源多胺处理提高了离体玉米植株抗氧化防护基因cAPX和SOD4的表达,也提高了细胞溶质抗氧化防护酶APX,SOD的活性。另一方面,MPAO抑制剂（guazatine、DADD）和H₂O₂清除剂（CAT、DMTU）的实验结果表明抑制剂或清除剂均明显抑制了多胺诱导的抗氧化防护酶cAPX和SOD4基因表达以及细胞溶质APX和SOD活性的提高,并且抑制剂浓度越高抑制程度越明显。这说明MPAO是通过分解多胺产生的代谢产物H₂O₂诱导玉米叶片细胞溶质抗氧化防护,H₂O₂是一个重要的中间信使。更多还原

【Abstract】 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.更多还原