【作者】 王晨芳；

【导师】 黄丽丽； 康振生；

【作者基本信息】 西北农林科技大学 ， 植物病理学， 2008， 博士

【摘要】 在植物与病原菌互作过程中,植物会产生一系列的防卫反应来抵御病原菌的入侵与扩展。在这些防卫反应中,寄主最快的抗病反应就是在短时间内产生大量的活性氧物质,即氧化迸发(oxidative burst)。虽然过量的活性氧积累会对植物细胞造成很强的毒害,但其在植物的抗病反应中却起着重要作用。自1983年Doke首次发表了有关非亲和病原物可以引起植物组织内产生O2-的文章以来,有关活性氧生理功能、产生机制、化学变化和清除系统等方面已进行了大量的研究。然而,小麦与条锈菌互作中活性氧迸发的特征及在抗病性表达中的作用方面的研究,目前国内外尚无报道。因此,本研究以小麦品种水源11与条锈菌毒性和非毒性小种(CY31和CY23)组成的亲和与非亲和互作体系为研究对象,对不同互作体系中的组织学特征和细胞学特征进行了研究,并利用NBT、DAB组织化学染色法来明确不同组合间O2-和H2O2产生的时间变化特征,利用电镜细胞化学标记技术在亚细胞水平上对O2-、H2O2和过氧化物酶的空间分布进行了定位,并检测了不同组合间活性氧迸发与相关酶系的变化关系及活性氧清除剂处理对寄主细胞过敏性坏死发生的影响,以明确活性氧迸发在小麦抗条锈性中的作用,从而为进一步揭示小麦抗条锈性机制奠定基础。通过研究取得了以下主要结果:1.利用荧光显微技术、微分干涉技术和电镜技术揭示了小麦品种水源11抗锈性表达过程中的组织学和超微结构特征。结果表明,亲和与非亲和组合中,条锈菌扩展及寄主细胞对条锈菌的侵染,在组织学和超微结构上均表现出明显差异。组织学水平上,非亲和组合表现为菌丝生长受抑,菌落发育延迟或败育,吸器母细胞和吸器数目明显减少;同时,侵染点的寄主细胞表现出典型的过敏性坏死症状。在亚细胞水平上,非亲和组合中病菌胞间菌丝、吸器母细胞、吸器也发生了一系列异常变化。其中原生质凝集、液泡程度增加、原生质逐渐消解;胞间菌丝、吸器母细胞细胞壁不规则增厚;细胞器排列紊乱,菌丝逐渐解体;吸器母细胞细胞质最终空泡化后丧失其生理功能。吸器外质膜皱褶,吸器外间质加宽,吸器体最终畸形坏死。同时,寄主细胞产生一系列显著的结构防卫反应:形成胞壁沉积物、乳突、吸器鞘等结构,以及发生坏死,使病菌的发育及扩展显著受抑。2.利用氮蓝四唑(nitroblue tetrazolium, NBT)和二氨基联苯胺(3,3-diaminobenzidine, DAB)组织化学染色法研究了小麦-条锈菌互作过程中O2-和H2O2产生的时间变化特征和在组织间的定位。非亲和组合中,O2-在接种后12 h左右为产生的高发期,而H2O2产生的高发期大约在接种后12～24 h之间。亲和组合中,绝大多数侵染位点互作早期难以检测到明显的NBT、DAB染色。此外,在侵入前期,两种条锈菌生理小种(CY23和CY31)均可诱导保卫细胞中H2O2的积累。接种后48 h内,O2-和H2O2基本上是在与条锈菌初生吸器母细胞相接触的叶肉细胞中产生;随着时间点的推移,O2-也可以在侵染点处坏死寄主细胞周围的叶肉细胞中产生,而H2O2在坏死寄主叶肉细胞及周围叶肉细胞中均可以检测到。Evans Blue染色后发现,寄主细胞过敏性坏死反应发生的时间大约在接种后16～20 h,这一时间要晚于O2-和H2O2开始产生的时间。试验表明,O2-和H2O2的产生和积累与寄主细胞过敏性坏死有着密切关系,在过敏性坏死反应的发生中可能起着重要作用。3.采用生化测定的方法对活性氧迸发与相关酶活的变化关系进行了研究。非亲和组合中O2-和H2O2的含量要高于亲和组合的;SOD在亲和组合中的活性总体上要高于非亲和组合的;接种24 h后,CAT在两种组合中的活性均高于对照,在接种后36 h和48 h时,亲和组合中的CAT活性高于非亲和组合的,而在接种60 h后又开始低于非亲和组合的;POD活性在接种24 h后均明显升高,但亲和组合中POD活性增幅大;MDA在非亲和组合中于接种后72 h含量明显上升。结果表明,亲和与非亲和组合中活性氧含量及相关酶活变化都存在明显差异,这些差异与小麦抗锈性的表达可能有密切联系。4.采用细胞化学方法对O2-、H2O2和过氧化物酶的分布进行了亚细胞水平的定位,基本明确了它们的产生部位。O2-在与菌丝或坏死寄主细胞相邻的叶肉细胞的液泡膜、质膜和细胞壁上均有分布,并且以分布在液泡膜上的情况居多;而H2O2多分布在与菌丝或坏死寄主细胞相邻的叶肉细胞的细胞壁和质膜上,少数细胞的液泡膜上及寄主细胞间隙也可有H2O2的分布;部分病原菌细胞壁上也显示有活性氧的分布;过氧化物酶在寄主细胞中存在的主要部位为细胞壁和细胞质膜上。试验表明,O2-、H2O2和过氧化物酶在亲和与非亲和组合中的分布位置基本相似,但不同组合间活性氧及过氧化物酶含量有着明显差异。5.通过使用活性氧清除剂SOD和CAT处理小麦叶片后发现,SOD处理的叶片中,H2O2的含量要大于CAT处理的叶片,且出现过敏性坏死的侵染点的比例明显高于对照;而CAT可在一定程度上降低出现过敏性坏死的侵染点的比例,但不能完全阻止过敏性坏死的发生。说明在小麦与条锈菌互作的体系中,活性氧的产生在过敏性坏死反应的发生中起着重要作用。更多还原

【Abstract】 During the plant-pathogen interactions, host plants can induce some characteristic defense reactions to confine the infection and spread of pathogen. During the development of defense responses, pathogen recognition by plant cells leads to the rapid production of high levels of reactive oxygen species (ROS), which is oxidative burst. The superfluous accumulation of ROS is harmful for the growth of plant cells, but it plays an important role in plant defense reactions. Doke first reported generation of superoxide radical can be induced in plant tissue in response to inoculation by an avirulent isolate of pathogen. Since then, many investigations have been accomplished to elucidate the role of ROS about physiology function, production mechanism, chemical change and scavenging system. However, only few data are available on generation and function of ROS during disease resistance expression in the interaction between wheat and wheat stripe rust. In this work, histological and cytological characteristics of pathogen development were analyzed in the incompatible and compatible interactions between wheat and Puccinia striiformis f.sp. tritici; The temporal characteristics of O2- and H2O2 generation were studied histochemically using nitroblue tetrazolium (NBT) and 3,3-diaminobenzidine (DAB) in different interactions; The subcellular localization of O2- accumulation, H2O2 accumulation and peroxidase activity were studied using cytochemical technique; And to detect the relationship of the oxygen burst and the changes of protective enzymes and occurrence of hypersensitive response after using scavengers of ROS. The results were obtained as follows:1. The histology and cytology of resistant mechanism of wheat cultivar Suwon11 to Puccinia striiformis were examined by means of fluorescent microscopy,differential interference contrast microscopy and electron microscopy. The result indicated that there are striking differences in histology and ultrastructure were detected about pathogen development and host cells infected by stripe rust fungus in the incompatible and compatible interactions.The main histological features in the incompatible interaction included inhibition of hyphal growth, delay of hyphal branching and colony formation, decrease of formation of haustorial mother cells and haustoria, at the same time, occurrence of host cell necrosis. A series of abnormal cytological changes also occurred in intercellular hyphae, haustorial mother cells and haustoria in incompatible interaction. The cytoplasm became more electron-dense and vacuoles increased in number and in size which digested the protoplasm. The cell wall of intercellular hypha and haustorial mother cell were thichened irregularly. The organelles were distributed disorderly, then hypha disintegrated gradually. The cytoplasm were degraded into central vacuole gradually and haustorial mother cells lost their physiological function. The extrahaustorial membrane was wrinkled, the extrahaustorial matrix was widened,at the end, the haustorial body were malformed and necrosed. At the same time, the structural defense reactions such as formation of cell wall apposition, papilla, encasement of haustorium and necrosis of host cell were essentially more pronounced in th incompatible interaction than in compatible one. All constitute essential factors of resistance.2. During wheat and wheat stripe rust fungi interactions, the temporal characteristics and histological localization of O2- and H2O2 generation were studied histochemically using NBT and DAB, respectively. In the incompatible interaction, O2- generation was significant increased by 12h after inoculation (hai), About H2O2 accumulation, the rapid increase occurred from12 hai to 24 hai. Compared to the incompatible interaction, in the compatible interaction only a very small portion of infection sites showed O2- and H2O2 accumulation in the early infection stage. Moreover, at the pre-penetration stage, both stripe rust races (CY23 and CY31) induced H2O2 accumulation in guard cells. In addition, within 48 h after inoculation, the accumulation of O2- and H2O2 were detected in the mesophyll cells in contact with initial haustorial mother cells. At advanced incubation times, O2- accumulation also could be seen in the mesophyll cells around necrotic host cells at infection sites, and that H2O2 accumulation could be observed both in necrotic mesophyll cells and the surrounding living mesophyll cells. Evane Blue staining showed that, in the incompatible interaction, the host mesophyll cells around penetrated sites took up hypersensitive response (HR) about 16 hai to 20 hai, which was later than the production of O2- and H2O2. The result suggested that, the generation and accumulation of O2- and H2O2 are closely associated with the HR of host cells, and which may play an important role in the occurrence of HR.3. The relationship of the oxygen burst and the changes of protective enzymes were analyzed use biochemical measuration. In the incompatible interaction, content of O2- and H2O2 were higher than in compatible interaction;The SOD activities in compatible interaction usually were higher than that in incompatible ones; At 24 hai, the CAT activities in both interactions were all higher than that in control, and CAT activities in compatible interaction were higher than that in incompatible ones at 36 h and 48 h, but after 60 hai, CAT activities in compatible interaction were lower than that in compatible ones again; The POD acivities increased obviously in both interactions at 24 hai, but POD activities increased more rapidly in compatible ones; The MDA content was increased significantly at 72 hai in incompatible interaction. It might be concluded that there are striking differences about ROS content and relative enzymes activities in the incompatible and compatible interactions, which may have close relationship with resistance expression of wheat against stripe rust fungus.4. The subcellular localization of O2- accumulation, H2O2 accumulation and peroxidase activity were studied and demonstrated by cytochemistry. O2- accumulation could be seen in the tonoplast, the plasma membrane and the cell wall of mesophyll cells adjacent to hyphae and necrotic host cells, and main distribution of O2- production was the tonoplast of host cells; The accunulation of H2O2 was observed mainly in the cell wall and the plasma membrane of mesophyll cells adjacent to hyphae and necrotic host cells, as well as in the tonoplast of some host cells and in the intercellular space; Accumulation of H2O2 was also observed in the cell wall of hyphae; peroxidase was mainly located in the cell wall and the plasma membrane of host cells. The result indicated that the subcellular localization of O2- accumulation, H2O2 accumulation and peroxidase activity in compatible interaction are similar to incompatible one, but there are striking differences about the content of ROS accumulation and peroxidase activity in different interactions.5. Treatment of wheat leaves with SOD and CAT which were scavengers of ROS showed that content of H2O2 in wheat leaves with SOD treatment was higher than in leaves with CAT treatment, and the percentage of infection sites of mesophyll cells with HR in wheat leaves with SOD treatment was higher than in control; CAT could decrease the percentage of infection sites of mesophyll cells with necrotic cells in certain degree, but could not avoid the occurrence of HR. The results suggest that the generation of ROS play an important role in the occurrence of HR.更多还原