This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Programmed cell death (PCD) is an essential cellular process characterized by cell shrinkage, nuclear condensation and fragmentation that leads to the selective elimination of cells and has been reported to play a major role in cystic fibrosis, oncogenesis and other disease occurrences. Interestingly, there appears to be some evolutionarily conserved signaling pathways for an apoptotic-like programmed cell death (AL-PCD) in plants. We propose to use a plant model, tobacco (Nicotiana tobacum), to characterize the role of glutathione in apoptosis. Glutathione (GSH) is considered to be the most important water-soluble antioxidant and is found in both plants and animals. Glutathione functions to reduced oxidative stress and levels in the cell via GSH reductase and decreases in GSH levels have been shown to directly activate pro-apoptotic pathways. Transgenic tobacco plants with the inducible expression of a glucose oxidase (GOX) gene will be employed. When expressed the GOX protein catalyzes the breakdown of glucose in the plant cell;a byproduct of this process is the generation of H2O2 and subsequently oxidative stress.
Our Specific Aims are (1) to measure changes in GSH expression, using western blot and LCMS analysis, as result of H2O2 production in transgenic GOX tobacco plants, (2) measure apoptosis using DNA laddering assays to determine the relationship of GSH levels and apoptosis in plants. It is our hypothesis that GSH levels will increase as a result of H2O2 production and result in AL-PCD. Alternately, (3) we plan to measure the effects of GOX expression on leaf physiology by measuring changes in photosynthetic parameters. If successful, our project will establish the viability of using plants as a model system to study PCD in animals. The use of plants as a model system is ideal for the undergraduate lab. Plants are easier and cheaper to maintain than animals systems and require less technical skill and experience.
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