Browsing by Subject "Plants -- Metabolism"
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Item Localization of glutathione-s-transferase and its effect on seedling development(Texas Tech University, 1998-12) Lodhi, Sundus AminaPlants have evolved various defense mechanisms that include antioxidants (easily oxidizable compounds of small molecular weight) and antioxidant enzymes to combat the effects of reactive oxygen species produced by abiotic and biotic stress. One such enzyme family is glutathione-S-transferase (GST). These enzymes detoxify various electrophilic and hydrophobic compounds via conjugation to the thiol group of glutathione (GSH), an antioxidant molecule that is abundantly present in plant cells. Tobacco plants transformed with NtI07, a cDNA clone that encodes a GST with glutathione peroxidase activity, have been shown to have increased tolerance to salt and temperature stress, as well as to have increased levels of oxidized glutathione (Roxas et al., 1997). Although GST has been established as an effective enzyme to protect seedlings under stressful conditions, exactly how the plant system is affected remains to be completely understood. We have attempted to examine the effects of glutathione on seedling growth by using the specific glutathione synthesis inhibitor, buthionine sulfoximine (BSO). Results indicated that glutathione is required for normal seedling growth, and seedlings treated with BSO had higher lipid peroxidation levels than controls. Although we observed that exogenous glutathione also had an inhibitory effect on growth, seedlings treated with glutathione did not show higher levels of lipid peroxidation. Seed storage protein mobilization patterns in GST-overexpressing and wildtype seedlings have also been investigated under normal and stress conditions. We found that GST-overexpressing seedlings mobilized their storage proteins more slowly than did wildtype under normal conditions, however the mobilization in the transgenic seedlings was less affected under salt stress than it was in wildtype. Knowledge about the localized activity of GST could also help elucidate the mechanism of stress resistance. We used polyclonal antibodies raised against the protein to localize GST in two-week old tobacco seedlings and found the primary localization to be in the nucleus of both transgenic and wildtype seedlings, indicating that GST may be involved in protection against DNA damage.Item Molecular analysis of monodehydroascorbate reductase and dehydroascorbate reductase in plants(Texas Tech University, 2000-12) Youn, HyewonOxidative stress is an inevitable phenomenon in green plants because they generate toxic reactive oxygen species. Through normal metabolism plants produce copious amount of molecular oxygen in cellular organelles that carry out highly energetic electron transfer reactions, resulting in the reduction of oxygen to form reactive oxygen intermediates (ROI's). Therefore, scavenging systems to remove reactive oxygen species are necessary. Ascorbate is the primary soluble antioxidant compound that can scavenge ROI's in plant cells. Monodehydroascorbate reductase (MR, EC 1.6.5.4) and dehydroascorbate reductase (DR, EC 1.8.5.1) are the enzymes responsible for the regeneration of ascorbate. As little is known about the role of MR and DR in oxidative stress in plants, this study was focused on determining the protective roles of these enzymes against oxidative stress using molecular genetic approaches. In order to fulfill this goal, transgenic tobacco plants that overexpress cucumber MR were developed. Analyses of the stress tolerance of the MR overexpressing plants were also investigated. Some plants showed increased tolerance against photoinhibition and methyl viologen exposure. However, they showed no increased stress tolerance differences during germination under low temperature and salt stress. Identification of a tobacco MR using PCR was also performed. The nucleotide sequence and amino acid comparison of tobacco MR with other plant MRs showed significant similarity. It was shown that FAD"" and NAD* binding sites were highly conserved between species. In addition, the ascorbate regeneration systems in honey locust leaves were investigated in this research. The Sunburst variety of honey locust showed higher sensitivity to light. Leaves of these plants were green in the shade but yellow in the light. This photosensitivity could be caused by the loss of chlorophyll due to oxidative stress. Surprisingly, Sunburst honey locust leaves had less DR activity and more MR activity than wild type leaves. In this system, DR seems to be required ascorbate regeneration. Although Sunburst leaves had elevated MR leaves, this was not support to overcome the photo bleaching phenotype. From this research, it was possible to better understand the function of MR and DR in oxidative stress. Understanding the role of these enzymes could lead to development of stress resistant transgenic crops capable of increased production.