【作者】 桑建荣；

【导师】 蒋明义；

【作者基本信息】 南京农业大学 ， 植物学， 2007， 博士

【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。一氧化氮（nitric oxide,NO）是一种多功能生物活性分子,作为信号参与基因活化、蛋白质表达和酶活性的调节使植物对逆境胁迫做出应答。最近的研究表明ABA和水分胁迫都可以引起NO和过氧化氢（hydrogenperoxide,H₂O₂）的产生,以及细胞质Ca²⁺([Ca²⁺]i)和钙调素（calmodulin,CaM）增加,诱导抗氧化基因的表达,提高植物抗氧化防护能力。NO在ABA诱导的抗氧化防护中起重要作用。然而,有关NO作为信号分子介导ABA诱导的抗氧化防护信号转导机制仍有待阐明。本研究以玉米叶片为材料,研究了在ABA和水分胁迫诱导的抗氧化防护中NO的作用以及H₂O₂,Ca²⁺/CaM与NO之间的关系。主要的研究结果如下:用NO特异的荧光染料4’5’—二氨基荧光素二乙酸酯（4,5-diaminofluoresceindiacetate,DAF-2DA）对小块叶片进行染色,并用激光共聚焦显微镜（confocal laserscanning microscopy,CLSM）观察荧光变化。10 mM CaCl₂处理迅速诱导NO产生和NOS活性增加。CaCl₂处理1h时NO达到最大值并一直持续到2h,2h后开始减弱。在CaCl₂处理1h时,细胞溶质和微粒体部分一氧化氮合成酶（nitric oxide synthase,NOS）活性达到最大值,分别比对照增加了92.7%和148%。NO清除剂c-PTIO（2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide）,NOS抑制剂_L-NAME（N^G-nitro-_L-Arg methylester）、PBITU（S,S’-1,3-phenylene-bis（1,2-ethanediyl）-bis-isothiourea）和CaM拮抗剂预处理都抑制了CaCl₂诱导的NO产生和细胞溶质及微粒体部分NOS活性增加。另一方面,Ca²⁺抑制剂和CaM拮抗剂预处理几乎完全抑制了ABA和H₂O₂诱导的NO产生和细胞溶质及微粒体部分NOS活性的增加,表明玉米叶片中ABA和H₂O₂是通过Ca²⁺-CaM依赖型的NOS途径诱导NO产生的。研究还发现外源NO处理显著提高了玉米叶片叶肉细胞[Ca²⁺]_i、玉米叶片CaM1基因表达和CaM含量。NO清除剂c-PTIO和NOS抑制剂_L-NAME预处理部分抑制了ABA处理后期[Ca²⁺]_i和CaM含量的提高,表明NO参与ABA诱导的Ca²⁺/CaM水平增加。此外,Ca²⁺鳌合剂、Ca²⁺通道阻塞剂和CaM拮抗剂预处理还显著抑制了NO诱导的抗氧化防护基因SOD4、cAPX、GRl表达和叶绿体及细胞溶质抗氧化酶SOD、APX、GR活性的增加,说明玉米叶片中NO诱导的Ca²⁺/CaM增加参与了NO诱导的亚细胞抗氧化防护系统。这些结果暗示了Ca²⁺/CaM作为NO的上游和下游信号分子参与ABA和H₂O₂诱导玉米叶片抗氧化防护。水分胁迫处理能够诱导玉米叶片叶肉细胞NO的产生。水分胁迫处理1h时NO开始产生,一直持续到4h达到最大,4h后减弱。水分胁迫诱导了NOS活性增加,水分胁迫处理4h时细胞溶质部分NOS活性是对照的4.3倍,微粒体部分的NOS活性是对照的7.2倍。这些结果表明微粒体部分NOS活性比细胞溶质部分高且更容易受到水分胁迫的影响。NOS抑制剂_L-NAME和PBITU预处理显著抑制了水分胁迫诱导的细胞溶质和微粒体部分NOS活性的增加,而NO清除剂c-PTIO和硝酸还原酶_{nitrate reductase,NR}抑制剂KCN、NaN₃抑制了水分胁迫诱导的NO产生,但并不影响NOS活性。结果表明NOS是水分胁迫诱导NO产生的主要来源,NR也部分参与了水分胁迫诱导的玉米叶片NO的产生。分别用DAB（3,3-diaminobenzidine）染色的组织化学、分光光度计法和CeCl₃染色的细胞化学方法检测了NO对水分胁迫诱导的H₂O₂累积的影响。结果显示SNP提高了水分胁迫诱导的叶绿体和细胞溶质谷胱甘肽还原酶（glutathione reductase,GR）、超氧化物歧化酶（superoxide dismutase,SOD）和抗坏血酸过氧化物酶（ascorbate peroxidase,APX）的活性,减少了水分胁迫诱导的H₂O₂的积累,而[Fe（Ⅲ）CN]在一定程度上降低了亚细胞抗氧化防护酶的活性,不影响H₂O₂的积累。施用NO清除剂、NOS抑制剂和NR抑制剂的研究结果表明NO参与外源H₂O₂和水分胁迫诱导的SOD4、cAPX、GRl的表达和叶绿体及细胞溶质抗氧化防护酶SOD、APX、GR活性的提高。这些结果表明NO能够清除水分胁迫诱导产生的H₂O₂至少部分归因于NO诱导的不同亚细胞分隔抗氧化防护的协同作用。对水分胁迫下ABA积累、Ca²⁺/CAM增加和NO产生之间的关系的研究结果显示,水分胁迫积累的内源ABA是水分胁迫诱导NO产生和激活NOS活性的关键诱导因子;NO参与了水分胁迫诱导的ABA积累、[Ca²⁺]_i提高、CaM1基因表达和CaM含量的增加。另一方面,Ca²⁺抑制剂和CaM拮抗剂预处理几乎完全抑制了水分胁迫诱导的NO的产生和细胞溶质及微粒体部分NOS活性的增加,表明Ca²⁺/CaM介导了水分胁迫诱导的玉米叶片NO的产生。此外,研究还发现水分胁迫诱导叶绿体和线粒体NO产生,Ca²⁺鳌合剂和CaM拮抗剂预处理只抑制了水分胁迫诱导的线粒体NO产生而对叶绿体NO的产生没有影响,表明在玉米叶片叶绿体和线粒体中合成NO的NOS种类可能有所不同。上述研究表明,ABA和水分胁迫诱导的[Ca²⁺]_i和CaM含量的增加激活了NOS活性合成NO,NO又反过来影响Ca²⁺/CaM水平,进而提高不同亚细胞分隔抗氧化酶活性及相关基因的表达。NO和Ca²⁺/CaM之间的交叉谈话在外源ABA或水分胁迫积累的ABA信号转导过程中起重要作用。更多还原

【Abstract】 Abscisic acid（ABA） can regulate a variety of physiological and molecular responses for plant against stresses,including drought,chilling,salinity,and so forth.Nitric oxide（NO） is a multifunctional bioactive molecule and as a signal is involved in gene activation,protein expression and activity regulation of enzymes response to stresses in plants.Recent work showed that ABA can cause the generation of NO and hydrogen peroxide（H₂O₂）,and the increases in the levels of calcium([Ca²⁺]_i) and calmodulin（CAM） in maize leaves,which induce the expression of antioxidant genes,and enhance the capacity of antioxidant defense systems.NO is an important intermediate component in the ABA signaling.However,the detailed mechanism about how NO mediates ABA signaling in plant cells remains to be determined.In the present study,the role of NO and the relationship between H₂O₂,Ca²⁺/CaM and NO in ABA-and water stress-induced antioxidant defense in leaves of maize（Zea mays L.） plants were investigated.The results are as follows:In order to investigate the production of NO in leaves of maize plants exposed to CaCl₂ treatment,the leaf segments were loaded with the NO-specific fluorescent dye 4,5-diaminofluorescein diacetate（DAF-2DA） and confocal laser scanning microscopy （CLSM） was used to monitor changes in NO-induced fluorescence,10 mM CaCl₂ led to a rapid increase of NO.The generation of NO maximized at 1 h and remained high for 2 h after CaCl₂ treatment,and then decreased after 2 h of CaCl₂ treatment.After the 1 h of CaCl₂ treatment,the activity of NOS reached the maximum values.Treatment with CaCl₂ for 1 h enhanced the activity of NOS in cytosolic and microsomal fractions by 92.7%and 148%, respectively,compared with the control values.To establish a link between NO,Ca²⁺/CaM and antioxidant defense in ABA signaling,the detached plants were pretreated with Ca²⁺ inhibitors or CaM antagonists,respectively,and then exposed to ABA,H₂O₂ or CaCl₂ treatment.Experimental results showed that pretreatments with Ca²⁺ inhibitors or CaM antagonists nearly fully arrested ABA-,H₂O₂- or CaCl₂-induced increases in the generation of NO and the activity of NOS,and also blocked the activities of the chloroplastic and cytosolic antioxidant enzymes SOD,APX,GR and the expression of the genes cAPX,GRl and SOD4 induced by SNP treatment in leaves of maize plants.These results indicate that the NOS, which mediates ABA- and H₂O₂-induced NO production,is Ca²⁺/CaM-dependent,and Ca²⁺/CaM is required for NO-induced subcellular antioxidant defense.On the other hand,our results showed also that exogenous NO led to significant increases in the levels of Ca²⁺/CaM. ABA-induced the increases in levels of Ca²⁺/CaM were partially blocked by pretreatments with the NO scavenger c-PTIO and the NOS inhibitor _L-NAME,suggesting that NO is involved in ABA-induced increases in the levels of Ca²⁺/CaM.These results suggest that Ca²⁺/CaM functions both upstream and downstream of NO production,which is mainly from NOS,in ABA- and H₂O₂-induced antioxidant defense in leaves of maize plants.Water stress induced the increases in the generation of NO in mesophyll of maize leaves. NO was detectable as early as 1 h after water stress treatment and maximized at 4 h,and then declined.Water stress induced the increases in the activity of NOS in maize leaves.The activity of NOS maintained at a continuously ascending trend during 4-h of water stress treatment.After 4 h of water stress treatment,the activity of NOS in cytosolic and microsomal fractions reached maximum values,which were 4.3-fold and 7.2-fold, respectively,higher than those in the controls.These results suggest that the activity of NOS in cytosolic and microsomal fractions is remarkably induced by water stress,and the activity of NOS in microsomal fraction was higher and more susceptible to water stress than that in cytosolic fraction.Pretreatments with _L-NAME and PBITU completely blocked the increases in the activity of NOS in cytosolic and microsomal fractions induced by water stress treatment. Pretreatments with c-PTIO,KCN and NaN₃ inhibited the generation of NO but hardly affected the activity of NOS in cytosolic and microsomal fractions induced by water stress treatment.These results suggest that NOS and NR are involved in water stress-induced NO production and NOS is the major source of NO.To investigate whether water stress-induced H₂O₂ production is regulated by NO,the effect of SNP on water stress-induced the activities of subcellular antioxidant enzymes and the accumulation of H₂O₂ were examined,by using the methods of histochemistry with 3,3-diamino benzidine（DAB） staining,spectrophotometry in leaf extracts,and cytochemistry with CeCl₃ staining and transmission electron microscopy,respectively.Experimental results showed that pretreatment with SNP resulted in remarkable increases in the activities of water stress-induced the chloroplastic and cytosolic antioxidant enzymes superoxide dismutase （SOD）,ascorbate peroxidase（APX）,and glutathione reductase（GR）,which decrease the accumulation of H₂O₂ induced by water stress treatment.However,pretreatment with Fe（Ⅲ）CN had a very little effect on the accumulation of H₂O₂,and reduced the activities of chloroplastic and cytosolic antioxidant enzymes GR,SOD and APX induced by water stress treatment.Pretreatments with NO scavenger,NOS inhibitors and NR inhibitors markedly blocked the increases in the activities of the chloroplastic and cytosolic antioxidant enzymes SOD,APX,GR and the expression of the genes cAPX,GRl and SOD4 induced by H₂O₂ or water stress,indicating that NO is involved in H₂O₂-and water stress-induced subcellular antioxidant defense.These results suggest that the potential ability of NO to scavenge H₂O₂ is, at least in part,due to the induction of subcellular antioxidant defense.The relationship between ABA,Ca²⁺/CaM and NO in water stress-induced antioxidant defense in leaves of maize plants were investigated.The application of ABA synthesis inhibitor blocked the generation of NO and the increase in the activity of NOS induced by water stress treatment,indicating that ABA is a key inducer of the induction of NO and the activation of NOS under water stress.NO is involved in water tress-induced accumulation of ABA and increases in the levels of Ca²⁺/CaM.On the other hand,pretreatments with Ca²⁺ inhibitors or CaM antagonists nearly fully arrested water stress-induced increases in the generation of NO and the activity of NOS,indicating that Ca²⁺/CaM mediates NO generation in leaves of maize plants exposed to water stress.In addition,our results showed also that water stress induced NO production in isolated chloroplasts and mitochondria of maize leaves. Pretreatments with Ca²⁺ chelator and CaM antagonist almost fully blocked water stress-induced NO production in mitochondria,but did not affect NO production in chloroplasts.These results suggest that there exists different species of NOS in chloroplasts and mitochondria of maize leaves.Taken together,our results suggest that the increases in the levels of Ca²⁺/CaM trigger NO production by activation of NOS,which then affects the levels of Ca²⁺/CaM,thus resulting in up-regulation in the activities of antioxidant enzymes and the expression of related genes in different subcellular compartments,and a crosstalk between NO and Ca²⁺/CaM plays a pivotal role in the ABA signaling transduction.更多还原