| 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. |
