Master of Science / Department of Biology / Jyoti Shah / Plant defense responses are constitutively activated in the Arabidopsis thaliana ssi2 mutant plant. In addition, the ssi2 mutant allele confers a dwarf phenotype. The SSI2 gene encodes a stearoyl-ACP-desaturase, which converts stearic acid (18:0) to oleic acid (18:1), suggesting a role for lipids in plant defense. Microarray analysis identified several genes which encode putative acyl hydrolases/lipases that are expressed at elevated levels in the leaves of ssi2, in comparison to the wild type plant. One gene in particular, At5g14180, was expressed at 60-fold greater level in ssi2 than in the wild type plant. To study the involvement of At5g14180 in plant defense and lipid metabolism, two transgenic lines containing T-DNA insertions within the At5g14180 gene were identified. These two T-DNA insertional alleles of the At5g14180 gene attenuate the ssi2-conferred heightened resistance to a virulent strain of Pseudomonas syringae pv. maculicola in the ssi2 At5g14180 double mutant plant. Furthermore, pathogen growth was enhanced in the At5g14180 single mutant plants, as compared to the wild type plant. Profiling of lipid composition in leaf tissue identified changes in the lipid composition between the At5g14180 mutant and wild type plants, suggesting that the At5g14180 encoded protein may impact lipid metabolism in Arabidopsis leaves.

Gotukola (Centella asiatica L. Urban) (Apiaceae), its extracts and the pure compound asiaticoside were studied for anxiolytic activity in thirteen standardized rat trials. High performance liquid chromatography (HPLC) was used to conduct the phytochemical analysis. Among different models tested, the most promising positive response for anxiolytic activity was observed in the elevated plus maze test conducted with: (a) whole plant materials, (b) ethyl acetate and methanol fractions and (c) asiaticoside. The results show for the first time that asiaticoside and triterpene enriched fractions of gotukola have anxiolytic effects in animal models. Therefore, they are recommended for clinical trials. The findings of this study also support the ayurvedic use of gotukola for psychiatric disorders. Other supplementary investigations conducted show that methyl jasmonate and full sunlight enhance the expression of asiaticoside in gotukola plants. The stolon explants were more successful compared to the leaf explants in in vitro propagation of gotukola.

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Master of Science / Department of Agronomy / William T. Schapaugh Jr / Crops research moving forward faces many challenges to improve crop performance. In breeding programs, phenotyping has time and economic constraints requiring new phenotyping techniques to be developed to improve selection efficiency and increase germplasm entering the pipeline. The objectives of these studies were to examine the changes in spectral reflectance with soybean breeding from 1923 to 2010, evaluate band regions most significantly contributing to yield estimation, evaluate spectral reflectance data for yield estimation modeling across environments and growth stages and to evaluate the usefulness of spectral data as an optimized phenotyping technique in breeding programs. Twenty maturity group III (MGIII) and twenty maturity group IV (MGIV) soybeans, arranged in a randomized complete block design, were grown in Manhattan, KS in 2011 and 2012. Spectral reflectance data were collected over the growing season in a total of six irrigated and water- stressed environments. Partial least squares and multiple linear regression were used for spectral variable selection and yield estimation model building. Significant differences were found between genotypes for yield and spectral reflectance data, with the visible (VI) having greater differences between genotypes than the near-infrared (NIR). This study found significant correlations with year of release (YOR) in the VI and NIR portions of the spectra, with newer released cultivars tending to have lower reflectance in the VI and high reflectance in the NIR. Spectral reflectance data accounted for a large portion of variability for seed yield between genotypes using the red edge and NIR portions of the spectra. Irrigated environments tended to explain a larger portion of seed yield variability than water-stressed environments. Growth stages most useful for yield estimation was highly dependent upon the environment as well as maturity group. This study found that spectral reflectance data is a good candidate for exploration into optimized phenotyping techniques and with further research and validation datasets, may be a suitable indirect selection technique for breeding programs.

Doctor of Philosophy / Department of Agronomy / Scott A. Staggenborg / Grain sorghum yield has notably increased from the beginning of hybrid production and commercialization in the late 1950s. The yield increases were the result of improved agronomic practices and hybrid advancement. The objectives of my research were: (1) to determine the magnitude of yield change in the hybrid era in irrigated and rain fed sorghum production, (2) to determine the contribution of agronomic and hybrid changes for yield in the hybrid era, (3) to investigate changes in sorghum morphology, physiology, and water use that contributed to yield increases, (4) to investigate changes that accompanied yield increase with hybrid improvement, and (5) to understand sorghum water and nutrient use and variations between hybrids in these regards. Fifty-two years of grain sorghum hybrid performance trial data (1957-2008), were analyzed and greenhouse and field studies were conducted on five selected hybrids to meet our objectives. The greenhouse and field studies were conducted from the summer of 2007 to the fall of 2009 on five selected hybrids, each representing a decade from the past fifty years. Results indicated that there was an increase in hybrid yield of nearly 50 kg ha-1 yr-1 in dryland sites over the 52 yrs (1957-2008) analyzed. Irrigated grain sorghum yields, however, remained unchanged over the same period. Agronomic practices such as planting date, phosphorus fertilizer use, and planting density changed over these years but were not found to contribute to increased dryland sorghum yields. There was no difference found between old and new hybrids tolerance to different densities. Hybrid advancement and increased nitrogen fertilizer application were responsible for changes in dryland yields. Total water use changed with hybrid advancement. New hybrids used the greatest total water and also had greater root-to-total biomass ratio than the old hybrids. Leaf biomass was also greater for the newest hybrid. There was a difference in amount of total nutrients extracted by hybrids, and there were differences among hybrids in allocation of nutrients to different tissues. In general the yield focus of sorghum hybrid development was effective in dryland sorghum production, likely because of intentional or inadvertent selection of hybrids with better drought tolerance. Results indicated that breeding programs created hybrids with improved morphological characteristics that might have resulted in better resource use (water and nutrient) and ultimately increased yield.

Doctor of Philosophy / Department of Biochemistry and Molecular Biophysics / Lawrence C. Davis / Synthetic dyes are capable of producing the whole color spectrum on account of their structural diversity but this diversity poses challenges in the degradation of dyeing wastes. Laccases and peroxidases from bacterial or fungal sources and parts of plants in the presence of hydrogen peroxide (H₂O₂) plus a mediator have been exploited in the bioremediation of synthetic dyes. However, intact plants have not found much favor despite their phytoremediation potential. The goal of this research was to further clarify ways by which whole plants bring about decolorization of different types of synthetic dyes. Hydroponically cultivated plants from two dicot families namely Arabidopsis thaliana and sunflowers (Helianthus annuus) were exposed to representative dyes from several classes: monoazo (Methyl Red and Methyl Orange), disazo (Trypan Blue, Evans Blue and Chicago Blue 6B), and arylmethane (Brilliant Blue G, Bromocresol Green, Malachite Green and Phenol Red). Tests were done in presence or absence of externally added H₂O₂, with or without a free radical mediator, 1-hydroxybenzotriazole, using UV-Visible spectrophotometry. The initial rate of decolorization and the overall percentage decolorization was calculated for each dye in the different treatments. Decolorization of the dyes from different classes varied between plant species and depending on the treatment. Except for Methyl Red, all dyes required added H₂O₂ as well as mediator to achieve rapid decolorization. Added H₂O₂ was found to be the limiting factor since it was degraded by plants within a few hours. Both species were able to slowly decolorize dyes upon daily addition of fresh dye even in the absence of added H₂O₂ and mediator, provided that nutrients were supplied to the plants with the dye. A. thaliana was found to be more effective in dye decolorization per gram tissue than sunflower when treated under similar conditions. Analysis of the residual dye solution by ESI/MS did not reveal any potential by-products following the decolorization treatment with plants, suggesting that the plant roots might be trapping the by-products of dye decolorization and preventing their release into the solution. All these findings support the potential application of whole plants for larger scale remediation.

<p>Plants respond to changes in their local environment and, at the same time, influence the environment at a global scale. The molecular and physiological mechanisms regulating this interaction are not completely understood and this limits our capacity to predict the response of vegetation to future environmental changes. This dissertation combined tools from genomics, physiology, and ecology to examine the response of plants to environmental change. Specifically, it focused on processes affecting carbon and water exchange in forest trees because (1) trees are long-lived species that might face repeated environmental challenges; (2) relatively little information exists about the genes and the molecular mechanisms regulating structural and physiological traits in adult, long-lived woody plants; and (3) forest trees exchange a significant amount of carbon and water with the atmosphere and are therefore major players in the global carbon and water cycles. </p><p>Water flux through forests depends both on environmental conditions (e.g., soil moisture) and on the hydraulic architecture of individual trees. Resistance to xylem cavitation is an important hydraulic trait that is often associated with drought tolerance but potentially at the cost of reduced carbon uptake. The second chapter of this dissertation evaluated the variation in resistance to xylem cavitation, hydraulic conductivity, wood anatomy traits, and leaf gas exchange across 14 co-occurring temperate tree species including both angiosperms and gymnosperms. The relationship between vulnerability to cavitation (ψ₅₀) and hydraulic conductivity within specific organs (i.e. stems and roots) was not significant when considering the phylogenetic association between species. However, even after phylogenetic correction, photosynthetic carbon uptake (A) was positively correlated with both stem and root ψ₅₀, and stomatal conductance (g_s) was strongly correlated with root ψ₅₀ . These results suggest that there is a trade-off between vulnerability to cavitation and water transport capacity at the whole-plant level, and that this functional relationship reflects an adaptive response to the environment. </p><p>Forests are an important component of the global carbon cycle that can be directly impacted by a rise in atmospheric CO₂ concentration.. The third chapter of this dissertation investigated the effects of long-term exposure to elevated CO2 on the gene expression of mature, field-grown loblolly pine trees. Using cDNA microarrays, I compared the expression of 1784 pine transcripts in trees growing under ambient and those under elevated CO₂ at monthly intervals throughout a growing season. Overall, more genes were upregulated than downregulated by elevated CO₂, although the total number of genes differentially expressed varied throughout the season. The pattern of increasing number of differentially expressed genes until the peak of the growing season (July and August) followed by a decrease in that number, matched the seasonal trend of tree growth and photosynthetic response to elevated CO₂ in this species. The seasonal trend also reflected the interaction among multiple abiotic factors intrinsic to field conditions and emphasized the relevance of evaluating the role of genes in their natural environment. Genes consistently upregulated by elevated CO₂ were functionally associated with environmental sensing, cellular signaling, and carbon metabolism, in particular the degradation of carbohydrates through respiration. An increase in carbohydrates degradation is particularly relevant in the context of carbon balance of forest trees because of the potential for enhanced leaf and tree respiration leading to a reduced sink capacity for CO₂. </p><p>Loblolly pine produces several flushes of needles throughout the year each with an average lifespan of 19 months. Each year, two age classes of needles contribute to the annual carbon sequestration of the loblolly pine forest. To address the impact of leaf age on the effects of elevated CO₂ in carbon metabolism regulation, I compared the gene expression profiles from trees under ambient and elevated CO₂ conditions in two needle cohorts: one-year-old and current-year. Differential expression under elevated CO₂ was seven times more frequent in current-year than in one-year-old needles. Despite differences in magnitude, many of the patterns within specific groups of genes were similar across age classes. For instance, there was a trend for downregulation of genes involved in the light-reactions of photosynthesis and those in photorespiration in both age classes, while genes associated with dark respiration were largely upregulated by elevated CO₂ in both cases. The difference between the two cohorts was particularly evident in the group of genes related to energy production (ATP synthesis) and the group associated with carbon partitioning (sucrose and starch metabolism). Because sucrose and starch metabolism categories included many genes known to be important regulators of gene expression and plant physiological processes, this suggests that this stage of carbon metabolism might be an important control point in age-dependent foliar responses to elevated CO₂.</p><p>This dissertation examined both structural and physiological components of plant water and carbon relations (Chapter 2) across different biological scales of organization (whole-plant level in Chapter 2; gene-level response to ecosystem-level changes in Chapters 3 and 4) and reflecting adjustments at distinct temporal scales (life-span of the organism vs. evolutionary selection of traits). An integrative approach was used to advance our understanding of how plants acclimate and adapt to their environment, and to provide a mechanistic framework for predictive models of plant response to environmental change. </p> / Dissertation

<p>The problem is two fold: how does natural selection operate on systems of interacting genes and how does natural selection operate in natural populations. To address the first problem, I have conducted a theoretical investigation into the evolution of control and the distribution of mutations in a simple system of interacting genes, a linear metabolic pathway. I found that control is distributed unevenly between enzymes, with upstream enzymes possessing the greatest control and accumulating the most beneficial mutations during adaptive evolution. To address the second problem, I investigated the evolution of copper tolerance in the common yellow monkeyflower, Mimulus guttatus. I genetically mapped a major locus controlling copper tolerance, Tol1. A Dobzhansky-Muller incompatibility was hypothesized to also be controlled by Tol1, however, we have demonstrated that it maps to another, tightly linked locus, Nec1. Finally, we investigated the parallel evolution of copper tolerance in multiple new discovered mine populations. We found that copper tolerance has evolved in parallel multiple times via at least two distinct physiological mechanisms. In four mine populations, there was a strong signal of selection at markers linked to Tol1, implying that copper tolerance has evolved via the same genetic mechanisms in these populations.</p> / Dissertation

abstract: ATP synthase is a large multimeric protein complex responsible for generating the energy molecule adenosine triphosphate (ATP) in most organisms. The catalysis involves the rotation of a ring of c-subunits, which is driven by the transmembrane electrochemical gradient. This dissertation reports how the eukaryotic c-subunit from spinach chloroplast ATP synthase has successfully been expressed in Escherichia coli and purified in mg quantities by incorporating a unique combination of methods. Expression was accomplished using a codon optimized gene for the c-subunit, and it was expressed as an attachment to the larger, more soluble, native maltose binding protein (MBP-c1). The fusion protein MBP-c1 was purified on an affinity column, and the c1 subunit was subsequently severed by protease cleavage in the presence of detergent. Final purification of the monomeric c1 subunit was accomplished using reversed phase column chromatography with ethanol as an eluent. Circular dichroism spectroscopy data showed clear evidence that the purified c-subunit is folded with the native alpha-helical secondary structure. Recent experiments appear to indicate that this monomeric recombinant c-subunit forms an oligomeric ring that is similar to its native tetradecameric form when reconstituted in liposomes. The F-type ATP synthase c-subunit stoichiometry is currently known to vary from 8 to 15 subunits among different organisms. This has a direct influence on the metabolic requirements of the corresponding organism because each c-subunit binds and transports one H+ across the membrane as the ring makes a complete rotation. The c-ring rotation drives rotation of the gamma-subunit, which in turn drives the synthesis of 3 ATP for every complete rotation. The availability of a recombinantly produced c-ring will lead to new experiments which can be designed to investigate the possible factors that determine the variable c-ring stoichiometry and structure. / Dissertation/Thesis / Ph.D. Biochemistry 2011