| Programmed cell death (PCD) is a active and orderly death process of cells coded with their own genes, which appears in the biological growth of organisms. It’s a normal physiological reaction of the metabolism and the development of organisms. The early studies of our laboratory showed that the differentiation of phloem SEs is a programmed cell semi-death process; at the same time, PCD was also proved to be in cells of root cap, wheat aleurone and many other tissues. All the PCDs of these cells occured in the stage of tissue development, and were closely related to the promotion of their developments. Therefore, the question of whether related regulation mechanisms and similar factors exist in PCDs of these different tissues is the one this study try to solute.This study was carried on wheat (Triticum aestivum L.cv.Huamai8). It aimed to study the regulation mechanism of PCD and the role of cathepsin B-like protease in this PCD happening in the development of root tip and caryopsis by multidisciplinary technical, including fluorescence microscope techniques, immunohistochemical techniques, in situ fluorescence staining techniques, molecular biological techniques, immune electron microscopy techniques, Autometallography (AMG) and TSQ fluorescence probe techniques. The main results were as follows:1. It was found that with the development of SEs cell wall began to thicken and accordingly, the intensity of the autofluorescence also changed. The results provided a time reference for our study of cathepsin B in SEs.2. Immunohistochemical techniques showed that the tissue from the top of root tip at a distance of112μm is root cap whose peripheral1-3layer cells have strong positive signal of cathepsin B-like protease. In the tissue from the top of root tip at a distance of490μm to700μm, it was found that the positive signal decreased and the signal in the middle of cortex declined faster than the signal in exodermis and stele. Along with the development of SEs and TEs, intracellular cathepsin B-ike protein positive signal also gradually disappeared. Cathepsin B-like protein may maintain the cell activity in this process.3. The techniques of in situ fluorescence staining showed that the cathepsin B-like protease could degrade the substrate which is special to cathepsin B protease, and present red fluorescence as the positive signal. RT-PCR showed that there were two types cathepsin B-like proteins’ mRNA accumulated in wheat root tip, which were named AI16and2529.4. The results of immune electron microscopy showed that cathepsin B-like protein distributed in cytoplasm and nuclei of the cells which in PCD, vacuole also had a few proteins scattered. The existence of cathepsin B-like proteins were not found in Autophagosome.5. Autometallography (AMG) suggested that zinc exits in the nucleolus of root tip cells. With the development of tissues, nucleolus and Zn2+in SEs and TEs disappeared gradually.6. TSQ fluorescence probe showed that the exogenous zinc always accumulated in the stele of the root tip by blue fluorescence. The fluorescence declines with the increase of the distance from the top of root tip.7. Immunohistochemical techniques showed that with the increase of the concentration of ZnCl2, the number of the cells which expressed cathepsin B-like protease decreases and the signal strength also declined.8. Immunohistochemical techniques showed that cathepsin B-like proteins distribute mainly in cells of both sides’ vascular bundle of caryopsis and endocarp cells at1DAF to9DAF. The content of cathepsin B-like protein in caryopsis aleurone layer increased, while the protein in cells of both sides’ vascular bundles disappearing at12DAF to24DAF, the cathepsin B-like protein concentrated in periphery of cell and reduced in endocarp cells of caryopsis back also can be observed.Results showed that in some PCDs during wheat growth there exist common acting factor and it is cathepsin B-like proteins; in this process cathepsin B-like proteins maintain the activity of cells; Zn2+can inhibit the expression of cathepsin B-like protein. |
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