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Synthetic biology is a rapidly growing field that aims to treat cellular biological networks in an analogous way to electrical circuits. However, the field of plant synthetic biology has not grown at the same pace as bacterial and yeast synthetic biology, leaving a dearth of characterized tools for the community. Due to the need for tools for the synthetic plant biologist, I have endeavored to create a library of well-characterized TATA box variants in the cauliflower mosaic virus (CaMV) 35S promoter using the standardized assembly method Golden Braid 2.0. I introduced single nucleotide changes in the TATA box of the CaMV 35S promoter, a genetic part widely used in plant gene expression studies and agricultural biotechnology. Using a dual-luciferase reporter system, I quantified the transcriptional strength of the altered TATA box sequences and compared to the wild-type sequence, both in transient protoplast assays and stable transgenic Arabidopsis thaliana plants. The library of TATA-box modified CaMV 35S promoters with varying transcriptional strengths created here can provide the plant synthetic biology community with a series of modular Golden Braid-adapted genetic parts that can be used dependably and reproducibly by researchers to fine-tune gene expression levels in complex, yet predictable, synthetic genetic circuits.

Master of Science / Department of Biology / Jesse B. Nippert / The Konza Prairie in northern Kansas, USA contains over 550 vascular plant species; of which, few have been closely studied. These species are adapted to environmental stress as imposed by variable temperature, precipitation, fire, and grazing. Understanding which plant traits relate to drought responses will allow us to both predict drought tolerance and potential future shifts in plant community composition from changes in local climate. Morphological and physiological measurements were taken on 121 species of herbaceous tallgrass prairie plants grown from seed in a growth chamber. Gas exchange measurements including maximum photosynthetic rate, stomatal conductance to water vapor, and intercellular CO[subscript]2 concentration were measured. All plants were exposed to a drought treatment and were monitored daily until stomatal conductance was zero. At this point, critical leaf water potential (Ψ[subscript]crit), an indicator of physiological drought tolerance was assessed. Other measurements include root length, diameter, volume, and mass, leaf area, leaf tissue density, root tissue density, and root to shoot ratio. Traits were compared using pair-wise bivariate analysis and principal component analysis (PCA). A dichotomy was found between dry-adapted plants with thin, dense leaves and roots, high leaf angle, and highly negative Ψ[subscript]crit and hydrophiles which have the opposite profile. A second axis offers more separation based on high photosynthetic rate, high conductance rate, and leaf angle, but fails to provide a distinction between C[subscript]3 and C[subscript]4 species. When tested independently, grasses and forbs both showed drought tolerance strategies similar to the primary analysis. Matching up these axes with long term abundance data suggests that species with drought tolerance traits have increased abundance on Konza, especially in upland habitats. However, traits that relate to drought tolerance mirror relationships with nutrient stress, confounding separation of low water versus low nutrient strategies. My results not only illustrate the utility of morphological and physiological plant traits in classifying drought responses across a range of species, but as functional traits in predicting both drought tolerance in individual species and relative abundance across environmental gradients of water availability.

Doctor of Philosophy / Department of Horticulture, Forestry, and Recreation Resources / Dale J. Bremer / Jack D. Fry / Researchers and practitioners can use numerous techniques to measure or estimate evapotranspiration (ET) from turfgrass but little is known about how they compare to ET using standard lysimeters. An investigation was conducted to compare measurements of ET from lysimeters (LYS[subscript]E[subscript]T) with ET estimates from the FAO56 Penman-Monteith (PM[subscript]E[subscript]T) and Priestley-Taylor (PT[subscript]E[subscript]T) empirical models, atmometers (AT[subscript]E[subscript]T), eddy covariance (EC[subscript]E[subscript]T), and a canopy stomatal conductance model that estimates transpiration (COND[subscript]T). Methods were compared at the same site during the 2010, 2011, and 2012 growing seasons. Overall, PT[subscript]E[subscript]T and EC[subscript]E[subscript]T were not different from LYS[subscript]E[subscript]T, whereas PM[subscript]E[subscript]T, AT[subscript]E[subscript]T, and COND[subscript]T, increasingly underestimated LYS[subscript]E[subscript]T. Differences exist among ET measurement techniques and one should employ the technique that best fits their situation. An atmometer is an inexpensive tool that can be used to measure turfgrass ET within microclimates, such as those typically found in an urban home lawn. An investigation was conducted to compare AT[subscript]E[subscript]T estimates with PM[subscript]E[subscript]T estimates within a number of lawn microclimates. Home lawns in Manhattan and Wichita, KS, were selected for study during the growing seasons of 2010 and 2011. Open sward AT[subscript]E[subscript]T was 4.73 mm d[superscript]-[superscript]1, whereas PM[subscript]E[subscript]T was 5.48 mm d[superscript]-[superscript]1. Within microclimates, AT[subscript]E[subscript]T was 3.94 mm d[superscript]-[superscript]1 and PM[subscript]E[subscript]T 3.23 mm d[superscript]-[superscript]1. Atmometers can provide practitioners with reliable estimates of PM[subscript]E[subscript]T within microclimates. Zoysiagrass (Zoysia spp.) is a common turfgrass used on home lawns and golf courses. However, poor shade tolerance and cold hardiness have limited its use in the transition zone. A study was conducted to determine changes and differences in growth and physiology among selected Zoysia over a three-year period (2010-2012) in the transition zone. The genotypes were 'Emerald' [Z. japonica × Z. pacifica], 'Zorro' [Z. matrella], 'Meyer' and Chinese Common [Z. japonica], and experimental progeny Exp1 [Z. matrella × Z. japonica], and Exp2 and Exp3 [(Z. japonica × Z. pacifica) × Z. japonica]. 'Zorro' and 'Emerald' experienced winter injury. 'Meyer', Chinese Common, and Exp1 showed poor performance over the three-years. The Exp2 and Exp3 progeny, maintained high percent cover, visual quality, and tiller density, and may provide practitioners more shade-tolerant cultivar choices in the transition zone.

Master of Science / Agronomy / Mithila Jugulam / Common waterhemp (Amaranthus rudis) is a problematic weed species of cropping systems throughout the Midwestern states, including Kansas. Recently, waterhemp populations from Kansas were found to have evolved resistance to the widely used herbicide glyphosate as a result of amplification of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), the enzyme target of glyphosate. The objectives of this research were to 1) perform glyphosate dose-response study and determine the relationship between relative EPSPS genomic copies and EPSPS gene expression in glyphosate-resistant waterhemp, and 2) characterize the genomic configuration and distribution of EPSPS copies using florescence in situ hybridization (FISH) in three glyphosate-resistant waterhemp populations. Waterhemp populations from eastern Kansas were screened with 868 g ae haˉ¹ (field used rate) of glyphosate, and genomic DNA and total RNA was isolated from the survivors to determine the EPSPS genomic copies and EPSPS gene expression relative to the acetolactate synthase (ALS) gene using qPCR. Furthermore, waterhemp specific EPSPS probes were synthesized to perform florescence in situ hybridization (FISH) on these glyphosate-resistant plants. Results of these experiments indicate a positive correlation between level of glyphosate resistance, EPSPS copies, and their expression. As expected, a negative correlation was found between shikimate accumulation and EPSPS copies. Sequencing of the EPSPS gene showed no presence of the proline 106 mutation, which is known to be associated with glyphosate resistance suggesting that an insensitive EPSPS enzyme was not involved in the mechanism of glyphosate resistance. FISH analysis of resistant plants illustrated presence of amplified EPSPS copies on two homologous chromosomes, likely near the centromeric region. . This is the first report demonstrating a positive relationship between EPSPS copies and expressions, as well as chromosome configuration of EPSPS copies in glyphosate- resistant waterhemp from Kansas.

Doctor of Philosophy / Department of Horticulture, Forestry, and Recreation Resources / Sunghun Park / Plants are continuously exposed to numerous abiotic stresses, which adversely affect plant growth, development, and yield. Plants have developed different signaling pathways to cope with abiotic stresses, and some of the pathways converge to help plants tolerate simultaneous stresses. Here, we report ectopic expression of an Arabidopsis glutaredoxin AtGRXS17 that confers tolerance to multiple abiotic stresses in tomato plants. In yeast assays, AtGRXS17 co-localized with yeast ScGrx3 in the nucleus and suppressed the sensitivity of yeast grx3grx4 double mutants to oxidative stress and heat shock. In plants, GFP-AtGRXS17 fusion proteins initially localized in the cytoplasm but migrated to the nucleus during heat stress. Ectopic expression of AtGRXS17 in tomato plants minimized photo-oxidation of chlorophyll and reduced oxidative damage of cell membrane systems under heat stress. Furthermore, expression of the heat shock transcription factor (HSF) and heat shock protein (HSP) genes was up-regulated in AtGRXS17-expressing tomato plants during heat stress when compared to wild-type controls. Under cold, drought, and oxidative stress conditions, AtGRXS17-expressing tomato plants also displayed more vigorous growth and less physiological damage than those of the wild-type control plants. Quantitative real-time PCR (qRT-PCR) analysis indicated that expression of AtGRXS17 alters multiple stress defense signaling pathways, including the Abscisic Acid (ABA) and C-Repeat Binding Factors (CBF) pathways. The results revealed a conserved function for a glutaredoxin protein in abiotic stress adaptation, and manipulation of AtGRXS17 may be a useful approach to improve crop stress tolerance and understand plant signaling under abiotic stress conditions. Deregulated expression of an Arabidopsis H[superscript]+/Ca[superscript]2[superscript]+ antiporter (sCAX1) in agricultural crops increases total calcium (Ca[superscript]2[superscript]+) but may result in yield loses due to calcium-deficiency like symptoms. Here we demonstrate that co-expression of a maize calreticulin (CRT, a Ca[superscript]2[superscript]+ binding protein located at endoplasmic reticulum) in sCAX1-expressing plants mitigated these adverse effects while maintaining enhanced Ca[superscript]2[superscript]+ content. Co-expression of CRT and sCAX1 could alleviate the hypersensitivity to ion imbalance in tobacco plants. Furthermore, blossom-end rot (BER) in tomato may be linked to changes in CAX activity and enhanced CRT expression mitigated BER in sCAX1 expressing lines. These findings suggest that co-expressing Ca[superscript]2[superscript]+ transporters and binding protein at different intracellular compartments can alter the content and distribution of calcium within the plant matrix.

A subset of synthetic chemicals present in the environment, such as organochlorine pesticides and plastics by-products, are able to mimic endogenous estrogen-induced signaling in representatives of each vertebrate class; as such, they are termed endocrine disrupting chemicals (EDCs). Similarly, estrogen-like signaling has also been observed when vertebrates are exposed to plant products called phytoestrogens. Previous research has focused on what effects synthetic and plant estrogens may have on estrogen receptor (ER) function and estrogenic signaling in humans and wildlife, while the evolutionary signaling targets of phytoestrogens, namely soil bacteria capable of nitrogen-fixing symbiosis with leguminous plants, had not been studied to determine if EDCs may interfere with crucial phytoestrogen-bacterial signaling Here I show that, in much the same way estrogen signaling with ERs is disrupted by the presence of EDCs, phytoestrogen signaling with its natural target, soil bacterial nodulation D (NodD) transcriptional activator proteins, is also disrupted by a similar profile of endocrine disrupting pesticides and pollutants. The similarities between ERs and NodD proteins may be explained by convergent evolution in which both NodD and ER separately evolved to adapt to the presence of natural estrogenic ligands, both vertebrate estrogens and phytoestrogens, as well as synthetic chemicals found in the environment that are able to mimic the natural ligands of both ER and NodD. I hypothesize that synthetic environmental compounds with similar structural properties to phytoestrogens, such as EDCs, may disrupt phytoestrogen-NodD signaling My studies herein show that the presence of a subset of EDCs: (1) inhibit phytoestrogen-NodD signaling and reduce the activation of key nod genes as measured in vitro, (2) the same EDCs tested in an in vivo plant-bacterial ecosystem inhibit symbiosis, nitrogen fixation, and adversely affect crop yield in treated plants. I demonstrate that both estrogenic and NodD signaling systems are adversely affected by the presence of a similar profile of EDCs at environmentally relevant concentrations. Therefore, I conclude that signaling components used by plants and pathways present in bacteria may have previously unrecognized similarities to those present in animals, and both of these systems are vulnerable to endocrine disruptors present in the environment / acase@tulane.edu

<p> The Brownsberg Nature Park (BNP) in Suriname is home to eight monkey species: <i>Saguinus midas, Saimiri sciureus, Cebus apella, Alouatta seniculus. Pithecia pithecia, Cebus olivaceus, Chiropotes satanas (sagulatus), </i>and <i>Ateles paniscus.</i> Several studies have undertaken the task to better study the feeding and behavioral ecology of these species within the park. However, studies on the black spider monkey (<i>Ateles paniscus</i>) have been absent. As part of my thesis, I decided to conduct a baseline feeding and behavioral ecology study of this species during the period May 2008 &ndash; July 2008. In addition, I developed a field method for determining mercury levels (in parts per million = ppm) in fecal and urine samples of wild monkeys. Since the park is under enormous pressure from illegal gold mining activities, I decided to collect baseline data on potential exposure of wild monkeys to mercury in the environment. I also collected samples from monkeys at the zoo in Paramaribo and monkeys born in captivity at Hiram College in Ohio. I collected data on the frequency of feeding, resting, and traveling by black spider monkey subgroups every 10 minutes during all day follows. Feeding ecology data consisted of identifying fruits eaten by these subgroups. For the mercury analysis I used the OSUMEX LTD. home testing kit. Results from the behavioral data show the following frequencies of activities for the entire study period: 32% feeding, 43% resting, and 25% traveling. The feeding data further justifies spider monkeys as ripe fruit frugivores: 76% of food items consisted of ripe fruit, while 22% consisted of leaves, and 2% was comprised of flowers. The mercury testing results from the Brownsberg and zoo populations ranged between 0.025 ppm to 0.1 ppm (toxic level = 0.8 ppm). The Hiram College monkeys all displayed levels at 0.000 ppm. The results from the mercury analyses indicate that 1) wild monkeys in the vicinity of gold mining activities may not be under the same threat as humans, with regards to mercury exposure through food, and 2) that wild monkeys are still relative exposed to mercury in the environment whether it be natural or anthropogenic.</p>

abstract: This study was designed to produce a comprehensive flora of Usery Mountain Regional Park and Pass Mountain of the Tonto National Forest. A total of 168 vascular plant species representing 46 families and 127 genera were collected or documented at this study area. Sixteen species were not native to the flora of Arizona and represent 9.5% of the flora. Nevertheless, the study area does not appear to be significantly damaged or degraded in spite of its historical and current land use. The location and types of invasive species recorded in this study will assist with implementing preventative measures to prevent further spreading of certain species. The complete list of all vascular species recorded in this study will provide a valuable tool for land management decisions and future restoration projects that may occur at this area or similar sites and invasive species control. The distribution of the saguaro (Carnegiea gigantea) population on Pass Mountain was documented through the measurement of saguaros by random sampling. ArcGIS was used to generate 50 random points for sampling the saguaro population. Analysis to determine saguaro habitat preferences based on the parameters of aspect, slope and elevation was conducted through ArcGIS. The saguaro population of Pass Mountain significantly favored the southern aspects with the highest concentration occurring in the southwest aspects at an average density of 42.66 saguaros per hectare. The large numbers of saguaros recorded in the younger size classes suggests a growing populations. / Dissertation/Thesis / M.S. Applied Biological Sciences 2011

Mon projet de maîtrise avait pour but d’évaluer la faisabilité d’une solution de phytoremédiation par phyto-extraction d’un sol modérément contaminé en zinc et en arsenic. Cette évaluation s’est basée sur les résultats d’une expérience en pots réalisée dans un tunnel de croissance de l’Institut de recherche en biologie végétale (IRBV) durant l’été 2015. Le sol utilisé provenait directement du site contaminé. Les plantes choisies pour cette expérience étaient deux cultivars de saule, Salix purpurea ‘Fish Creek’ et Salix miyabeana ‘SX67’ ainsi que trois herbacées Medicago sativa, Festuca arundinacea et un mélange de plantes utilisé pour la stabilisation de sol. L’impact d’un agent chélateur (acide nitrilotriacétique, NTA) sur la phytoremédiation et le développement des espèces a également été étudié. Les saules ont extrait deux à trois fois plus de zinc que les herbacées. La tendance était inversée pour l’arsenic avec une extraction deux à trois fois plus grande chez les herbacées. Le chélateur NTA a eu un effet positif sur la biomasse aérienne des plantes et sur la phyto-extraction du zinc, mais pas sur la phyto-extraction de l’arsenic. Une diminution importante de la quantité de zinc et d’arsenic a été observée dans les sols. Cette diminution provient des effets combinés de la phyto-extraction et d’autres facteurs, comme la variabilité intrinsèque du sol ainsi que son lessivage. La phyto-extraction de l’arsenic a donné des résultats médiocres, quels que soient les traitements et les plantes utilisés. Les résultats obtenus démontrent l’efficacité des saules et le potentiel du chélateur NTA pour la phyto-extraction du zinc. / My master project aimed to evaluate if phytoremediation by phytoextraction was a viable solution in cleaning up a moderately contaminated soil in zinc and arsenic. This evaluation was based on the results of a potted experiment done within Montreal Botanical Garden, in a growth tunnel set up by Institut de recherche en biologie végétale (IRBV). The soil was collected from a contaminated site. We chose the following plants species: two willow cultivars, Salix purpurea ‘Fish Creek’ and Salix miyabeana ‘SX67’, two grasses Medicago sativa L., Festuca arundinacea and a mix of grasses used for soil stabilization. The impact of a chelating agent (nitrilotriacetic acid, NTA) on phytoextraction and plant growth was also assessed. Salix extracted two to three time more zinc than the grasses. For arsenic, the opposite trend was observed, with two to three times more arsenic extracted by grasses. Chelator NTA had a positive effect on above ground biomass and overall phytoextraction of zinc, but not for arsenic phytoextraction. A significant diminution of zinc and arsenic was observed in soil after the experiment. This diminution comes from the combined effects of phytoextraction and other factors, such as intrinsic soil variability and its leaching. Arsenic phytoextraction was overall mediocre regardless of the species and treatments used. These results show the effectiveness of willows and potential from chelator NTA for zinc phytoextraction.