Trichomes are hair-like structures found on the surface of virtually all terrestrial plants (Yang et al., 2015). They are epidermal outgrowths that can occur on all of the aerial parts of a plant, varying markedly in size, shape, distribution, and in their ability to produce secondary metabolites. About 30% of all vascular plants carry the glandular trichomes capable of producing secondary metabolites (Glas et al., 2012). Trichomes are vitally important to plants as a defence mechanism, they are highly significant commercially, and they are of interest to plant biologists in that they serve as an excellent model system to study all aspects of plant differentiation at the single-cell level (Hulskamp, 2004). The simple, non-glandular trichomes found in Arabidopsis have been studied extensively. However the glandular trichomes of the kind found on the surface of Antirrhinum are much less well understood. The primary aim of the research reported here is to identify the gene(s) responsible for variation in epidermal hair (trichome) distribution between Antirrhinum species. Following an introduction which provides essential background on trichomes and on Antirrhinum, the thesis is presented in four parts. The first part describes a RAD-seq experiment used to produce linkage maps for the eight chromosomes making up the Antirrhinum genome and estimates the position of the Hairy gene on linkage group 8. The results are cross-validated against maps produced independently by the Xue group. It also describes novel methods developed to address a number of problems that arose during the course of the analysis, and explores the value of imputation methods in helping to overcome gaps and inconsistencies in the data. The second part presents the findings from a fine-mapping Pool-seq experiment designed to estimate the position of Hairy more precisely. The findings suggest that Hairy lies on one of a small number of scaffolds, with Scaffold 1097 being the most likely candidate. Also covered are the findings of another experiment to estimate the position of the gene that determines whether flowers are pale or dark. In this case the results indicate that the gene lies on one of a small number of scaffolds on linkage group 5. The third part presents the results of an RNA-seq experiment which, when combined with the results of the Pool-seq experiment provides evidence that Hairy might be a glutaredoxin gene on Scaffold 1097. Finally the interim results of three experiments designed to confirm that the gene identified as Hairy controls the distribution of trichomes in Antirrhinum are presented.

Doctor of Philosophy / Department of Horticulture and Natural Resources / Sunghun Park / Drought and heat stress are two of the biggest constraints to global food production. Abiotic stress response pathways are complex and consist of osmotic adjustors, macromolecule stabilizers, and antioxidants to counteract the damaging nature of abiotic stress induced reactive oxygen species (ROS) accumulation. In this work, we studied the effect of overexpression of an Arabidopsis glutaredoxin, AtGRXS17, on heat tolerance in maize (Zea mays L.) and drought tolerance in rice (Orzya sativa L.). Glutaredoxins (GRXs) are proteins cable of reducing disulfide bonds, therefore regulating the cellular redox status, and require glutathione for regeneration. Ectopic expression of AtGRXS17 in maize resulted in increased heat stress tolerance during flowering. AtGRXS17 enhanced heat tolerance by increasing kernel set and total grain yield during heat treatments, compared to wild type controls. Our results indicated that AtGRXS17-expressing maize plants produce heat tolerant pollen with higher germination rates than wild type when challenged during heat treatments. Furthermore, AtGRXS17-expressing plants were less susceptible to post pollination heat induced kernel abortion. Rice plants expressing AtGRXS17 were also tolerant to abiotic stress. AtGRXS17-expressing rice was more tolerant to drought stress challenges and consistently survived drought treatments. A nontargeted metabolomics study revealed distinct changes in profiles of key metabolite groups in response to drought stress. Soluble sugars and amino acids accumulate as osmotic adjustors while antioxidants, such as glutathione, accumulate to mediate ROS accumulation and regulate redox activity. All genotypes accumulated amino acids, soluble sugars, and raffinose family oligosaccharides in response to drought stress. Our results indicated AtGRXS17-expression affected several pathways known to increase drought tolerance. Altered sugar metabolites suggested a redox modulation of sucrose synthase activity and significant increases in the secondary sulfur assimilation pathway metabolites suggested altered sulfur metabolism. This research provides new insights into ability of GRXs to improve heat tolerance and crop yield in maize and functions of GRXs in affecting metabolite profiles contributing to increased drought tolerance in rice.

Thioredoxins (TRXs) and glutaredoxins (GRXs) are ubiquitous small redox proteins belonging to the thioredoxin (TRX) superfamily. They regulate several cellular functions via mediating a dithiol/disulphide exchange in target proteins. Thioredoxins have been classified into several subgroups based on their structural homologs. In an attempt to identify thioredoxin proteins which have not been characterized, an EST database survey of Lycopersicon esculentum, Glycine max, Helianthus annus, Secale cereale, Solanum tuberosum, Apis mellifera ligustica, Oncorhynchus mykiss, Salmo salar, and whole genome survey for Drosophila melanogaster, Rattus norvegicus and Caenorhabditis briggsae was performed. Several glutaredoxin and glutaredoxin-like proteins from Ricinus communis, Vercinia fordii, Lycopersicon esculentum, Tilia platyphyllos, Populus tremuloides, Triticum aestivum and Oryza sativa were also characterized. The deduced amino acid sequences were aligned and phylogenetic trees were constructed to determine the consensus sequences and for establishing interrelationships amongst the new and established thioredoxin and glutaredoxins. Based on the alignments, proteins were designated to their respective classes and subcellular localization predictions were used to predict their possible site of actions. In silico analysis has identified several new thioredoxins, glutaredoxins and related proteins and provided insight into their evolutionary relationships.

Oxidative stress is commonly associated with diet-induced metabolic syndrome (MetS) and left ventricular cardiac remodeling, but much remains unknown about the role of redox signaling, sensors, and switches in mediating the effects of high fat and sugar intake. In this work, I describe and apply an optimized method for quantifying changes in reversible protein-cysteine oxidation in the heart. This method uses isobaric tagging of cysteine thiols and mass spectrometry in a modified biotin switch on whole tissue lysate. Analyzing the resulting data with systems biology approaches helped delineate redox pathways playing a role in disease development, while cysteine-specificity provided exact targets for mutation-based mechanistic studies. Initial findings in a mouse model for MetS, wherein C57Bl6J mice were fed a high fat/high sucrose diet, identified energy pathways as the primary target of changing reversible cysteine oxidation. In follow-up studies, our collaborators helped validate the pathophysiological role of two particular cysteines in complex II; their early reversible oxidation and later irreversible oxidation contributed to decreased ATP output from cardiac mitochondria. A subsequent, more robust study revealed a weakness in our original method. While investigating the role of hydrogen peroxide-induced oxidative post-translational modifications (OPTMs) in the development of MetS sequelae, analysis of four mouse groups, each with an n=5, revealed that measurements of reversibly oxidized cysteine thiols were highly variable compared to those of all available thiols. Thus, I developed a strategy to address the source of variability and, in the process, improved many additional steps in the switch protocol. Finally, in an effort to clarify the role of the most stable reversible OPTM, glutathionylation (RSSG), we characterized the HFHS diet response in mice engineered to have more or less RSSG via genetic manipulation of glutaredoxin-1 expression. Those with more RSSG suffered worsened cardiac function, making them an ideal model for future studies with the methods optimized in this work. Studying the progression from poor diet to cardiac involvement in these and other mouse models using the methods described herein will aid in the design of diagnostics and targeted therapies against the growing burden of metabolic CVD.

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Asthma is a multi-faceted chronic inflammatory disease accompanied by loss of airway epithelial integrity leading to remodeling of the airways. Perturbations to the lung redox environment, including alterations in glutathione (GSH) content, have been reported in asthma. GSH can be conjugated to protein cysteines, controlling protein function in an oxidant-dependent process known as protein S-glutathionylation (PSSG). The thioltransferase, glutaredoxin-1 (Glrx1), deglutathionylates proteins under physiological conditions, restoring sulfhydryl groups of target proteins. Glrx1 is emerging as a critical player in settings of allergic airway disease, but its function in regulating epithelial cell responses to asthma-relevant cytokines has not been examined. Furthermore, the role of Glrx1 in controlling the extent of airway remodeling in response to house dust mite (HDM) in vivo is still not well understood. Interleukin-17A (IL-17A) is a potent cytokine that stimulates epithelial cells to produce pro-inflammatory mediators, in part by activating the nuclear factor kappaB (NF-κB) pathway, a key regulator of inflammation. We demonstrate that interleukin-17A (IL-17A) induces rapid activation of both classical and alternative NF-κB, while simultaneously resulting in protein oxidation and PSSG. In particular, we show IL 17A induces S-glutathionylation of RelA (RelA-SSG) and IKKα (IKKα-SSG), which is enhanced following siRNA-mediated knockdown of Glrx1. We also demonstrate that absence of Glrx1 leads to increased nuclear content of RelA and RelB and enhanced production of NF-κB-driven pro-inflammatory genes, KC and CCL20 while decreasing IL-6 expression. Finally, we show that siRNA-mediated knockdown of IKKα attenuates nuclear RelA and RelB and dampens pro-inflammatory gene production. Together, these data indicate a crucial role for the Glrx1/PSSG axis in controlling RelA-SSG, IKKα-SSG and epithelial cell responsiveness to IL-17A. Mice lacking Glrx1 were previously shown to display enhanced resolution of allergic airway disease induced by ovalbumin (Ova) challenge. In this study, we determined the role of Glrx1 in a HDM model of allergic airway disease. Wild type (WT) mice and Glrx1 deficient (Glrx1-/-) mice demonstrated similar total lung cell counts, but Glrx1-/- mice displayed fewer neutrophils than WT mice. Conversely, mice overexpressing Glrx1 specifically in CCSP positive cells in the lung (Epi-Glrx1) showed attenuated total lung cell counts and lung eosinophils compared to control mice. Immunohistological analysis of remodeling markers revealed that Glrx1-/- mice displayed increased HDM-induced mucus metaplasia, α smooth muscle actin (αSMA) positivity and collagen staining compared to WT mice. Evaluation of total lung collagen showed that Glrx1-/- mice had significantly higher collagen content compared to WT mice. In Epi-Glrx1 mice, attenuation of mucus metaplasia, αSMA content and collagen staining was observed compared to control mice. Furthermore, Epi-Glrx1 mice also demonstrated significantly impaired collagen production compared to control mice. We also demonstrate that Glrx1 absence results in decreased expression of the epithelial cell marker, E-cadherin, and increased expression of αSMA, a mesenchymal marker. Together, these studies demonstrate a critical role for Glrx1 in controlling epithelial cell responses to IL-17A and in mediating in vivo collagen production in response to chronic allergen exposure.

OBJECTIVES: Recent studies have elucidated that diabetes mellitus (DM) patients exhibit an accelerated tumor progression, but the mechanism of its regulation is not yet fully understood. The following study seeks to examine the role of angiogenic factors in the growth of subcutaneously injected melanoma cancer using a diet-induced type II diabetic mouse model. METHODS: C57BL/6 mice were fed either a regular or high-fat, high-sucrose (HFHS) diet for 2 months (T2DM model; confirmed through a GTT) and subcutaneously injected with B16F0 melanoma cells. After a 1-week or 2-week incubation period, the tumor was extracted to examine its size, weight, vascularity, and gene/protein expression. In vitro studies were performed using endothelial cells to assess the effects of high-glucose on endothelial cell proliferation, migration, and tube formation. GLRX expression was examined in both tumor samples and endothelial cells. RESULTS: The results of the study showed that T2DM induced by a HFHS diet is able to promote tumor growth in both weight (2-week, p = 0.0070) and volume (1-week, p = 0.0351; 2-week, p = 0.0002). Tumors extracted from the HFHS diet group showed reduced expressions of angiogenic markers (ACTA2 (1-week, p = 0.0239; 2-week, p = 0.0123), KDR (1-week, p = 0.0091)) by western blot and a slightly reduced trend of angiogenesis (PECAM1) in histological analyses. GLRX expression was reduced in HFHS tumor samples (1-week, p = 0.0090) and, interestingly, lower amounts of GSH adducts (2-week, p = 0.0317) could be seen in 2-week tumors as well. In vitro studies of endothelial cells showed reduced trends of endothelial cell function (proliferation, migration, and tube formation) in high glucose medium. Also, it has been observed that high glucose may be able to stimulate GLRX expression in endothelial cells. CONCLUSION: The results of the following study have confirmed that B16F0 melanoma growth is, in fact, augmented in diet-induced diabetic mice; however, the vascularity and levels of angiogenic markers from the tumor tissues did not parallel the growth in its size. In vitro studies suggested that high glucose can impair EC function (i.e. proliferation, migration, and tube formation capabilities) as well as promote GLRX expression, which may be related to this discrepancy. Glutaredoxin-1 (GLRX), an enzyme which controls redox signaling, is upregulated in DM. Endothelial cell-specific GLRX overexpression in transgenic mice was found to stimulate subcutaneously injected melanoma (B16F0) growth, despite hindering limb revascularization after hind limb ischemia. The augmented tumor progression in DM may be associated with GLRX upregulation, alongside impaired ischemic limb revascularization and tumor angiogenesis; however, the mechanism of tumor growth in diabetes still lies inconclusive and further studies need to be examined to elucidate this phenomenon.

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