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Protein degradation poses a significant challenge for the efficient production of recombinant proteins in plants, affecting the stability and yield of the recombinant protein. In this study the E. coli-derived enzyme glutathione reductase (GR) was transiently expressed in transgenic tobacco plants constitutively expressing the cysteine protease inhibitor OC-I and non-transgenic plants. A protein resembling the GR was detected in infiltrated leaves. Transiently expressing GR in transgenic N tabacum plants resulted in almost two fold significant increases in GR activity. Transgenic tobacco plants expressing the rice cysteine protease inhibitor OC-I had significantly lower cysteine protease activity when compared to non-transgenic tobacco plants. Lower cysteine protease activity in transgenic plants was directly related to higher GR activity and also higher GR amounts in transgenic plants. The study has demonstrated that OC-I is an effective companion protease inhibitor candidate with the potential to protect other high value proteins such as GR, from cysteine protease degradation. / Dissertation (MSc)--University of Pretoria, 2012. / Plant Science / unrestricted

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Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

>Magister Scientiae - MSc / Sugargraze is a moderately drought tolerant sweet sorghum hybrid which is ideal for grazing, winter stand over and pit silage. A major advantage that Sugargraze has over other forages is its very high sugar content which improves feed quality thus increasing palatability and results in significantly reduced feed wastage. This study explored the influence of hyperosmotic stress on plant development, ROS accumulation, antioxidant capacity and the extent of cell death. Heat shock protein (Hsp70) expression immunoblotting assays were used to demonstrate whether the various treatment conditions induced stress within natural physiological parameters for the experimental material. This was coupled with the separation, visualization and identification of abundant proteins in Sugargraze leaves in response to hyperosmotic stress using two-dimensional gel electrophoresis (2-DE) in combination with mass spectrometry (MS). The results showed that hyperosmotic stress significantly influences plant development by reducing plant biomass and increasing the levels of ROS accumulation, proline content and subsequently reducing total chlorophyll content. An over accumulation of ROS in the form of hydrogen peroxide and lipid peroxidation was observed in the stressed plants which was supported by the extent of cell death. Although an increase in antioxidant enzyme activity (in the form of total enzymatic activity or individual isoform activity) in response to hyperosmotic stress was observed, this increase was not sufficient to counter the deleterious effects caused by the stress conditions hence the decrease in plant biomass and increase in cell death. Western blotting analysis of Sugargraze leaf tissues using Hsp70 antibodies showed that hyperosmotic stress induced Hsp70 expression to levels significantly higher than observed for the control plants. A total of thirteen CBB stained spots were selected for mass spectrometric identification, owing to their good resolution and abundance levels, and of these, nine were positively identified. Identified proteins were divided into functional categories including both known and novel/putative stress responsive proteins. Molecular and physiological functions of some of the proteins of interest identified will be subjected to further investigation via bioinformatic and molecular biology approaches.

Seed desiccation and germination have great potential for oxidative stress. Glutathione, one of the most abundant antioxidants in plant cells, is a crucial to the plant's defense mechanisms. To better understand glutathione's responses during these two stages, we examined its dynamics in wildtype Arabidopsis seeds and in a transgenic line containing an antisense glutathione reductase2 (anGR2) cDNA insert. Seeds from the two genotypes were compared morphologically. Glutathione levels in maturing and germinating seeds were measured by HPLC, and GR activity by native PAGE. Cytosolic glutathione was measured in situ by confocal laser scanning microscopy. Stress in the form of natural and accelerated ageing, and germination at high and low temperature and at low water potential was applied to both WT and anGR2 seeds to test vigor. Results show similar glutathione levels and GR activity (except during late imbibition) in WT and anGR2. In both genotypes, GSH/GSSG ratio increased and GR activity decreased during seed maturation. During imbibition, the glutathione pool becomes very reduced (<1% GSSG) and in WT seeds, GSH levels increase mostly by GSSG recycling. Cytosolic GSH in embryonic epidermal cells was estimated to be 1.1-1.6 mM. AnGR2 seeds aged faster, and were less tolerant of heat and drought stress than WT. Accumulation of glutathione during maturation indicated that glutathione is a major antioxidant in the seed during storage. Changes in GSH levels during imbibition coincided with ROS production during radicle protrusion. Under stress conditions, anGR2 seeds showed lower vigor, indicating perturbations in the ROS scavenging systems particularly GR2. / Master of Science

Thesis (Ph. D.)--University of Missouri-Columbia, 2005. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. "December 2005" Includes bibliographical references.

Macrophage death is likely to contribute to the transformation of fatty streaks into advanced atherosclerotic lesions. Previous work in the laboratory showed that OxLDL promotes cell death in human macrophages by a mechanism involving intracellular peroxide formation. Here we show that glutathione depletion induced by OxLDL occurs independent of peroxyl radical formation. Our data suggest that the depletion of glutathione is the fundamental defect that renders macrophages susceptible to OxLDL-induced cell injury, but alone is not sufficient to kill macrophages. We indicate that increased protein-Sglutathionylation is involved in OxLDL-induced macrophage death. A potentiation of OxLDL toxicity was observed in macrophages transfected with siRNA directed against either glutathione reductase or glutaredoxin. Our data suggests that OxLDL-induced cell injury in human macrophage is mediated by the depletion of GSH, a decreased in the GSH/GSSG ratio and peroxyl radical formation. All three signals are required for OxLDL-induced macrophage death. Our results also show that the glutathione reductase/glutaredoxin system protects macrophages from OxLDL-induced cell death.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

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