Name: Baer, Stephanie
Date: Spring 2012
Major: Biology
Thesis Committee: Dr. Catharina Coenen, Dr. Scott Wissinger
Title: Effects of the Plant Growth Promoting Rhizobacteria Azospirillum brasilense and Nitrogen on Raphanus sativus
Nitrogen and phosphorus are two of the most limiting nutrients in plant growth. Both nitrogen and phosphorus require a chemical change from their most abundant form to become available for plant uptake; these changes are effected by a wide range of nitrogen-fixing and phosphate-solubilizing bacteria. Plant growth promoting rhizobacteria (PGPR) are a category of bacteria that colonize the rhizosphere of many different species of plants where they transform nutrients, suppress plant disease, or produce phytohormones that stimulate plant growth. Greenhouse experiments were conducted to evaluate the effects of the PGPR, Azospirillum brasilense, on Raphanus sativus (radish) grown under three different nitrogen conditions (low, intermediate and high). Various growth parameters and plant shoot chlorophyll contents were analyzed. The results indicate that A. brasilense enhanced lateral root formation at both low and high doses but did not significantly affect primary root length, root dry weight, or shoot dry weight. A. brasilense did have a significant effect on shoot chlorophyll content, which may be caused by its nitrogen-fixing ability. As is true for many plant species, nitrogen alone had a significant effect on radish primary root length, dry root weight, dry shoot weight, and shoot chlorophyll content. Nitrogen deficiency induced smaller roots and shoots. Interactions between nitrogen and A. brasilense effects on plant growth were complex, indicating that the nitrogen-fixing ability of A. brasilense is not the only major effector of plant growth. Beyond nitrogen-fixation, A. brasilense also produces plant hormones and other biologically active compounds, which may determine its effects on plant development.
Name: Briggs, Hannah
Date: Spring 2012
Major: Biology
Thesis Committee: Dr. Catharina Coenen, Dr. Tricia Humphreys
Title: Auxin activity and root development in phosphate-starved and phosphate-sufficient Nicotiana plumbaginifolia seedlings
Phosphate is an essential nutrient for plants. The model plant Arabidopsis thaliana demonstrates a phosphate starvation response characterized by a shortened primary root and increased lateral root growth. This response is dependent on the plant hormone auxin. Auxin-responsive gene activity in Arabidopsis lateral root primordia oscillates with a six hour period, but phosphate effects on this oscillation have not been investigated. This study assessed phosphate-dependent auxin activity in roots of Nicotiana plumbaginifolia seedlings transformed with a GH3::Luciferase promoter::reporter gene construct. This construct uses the gene that codes for luciferase as a reporter for auxin-inducible GH3 promoter activity: when auxin is active, the corresponding region of the plant will emit photons, or glow. This glow can be captured by luminescent photography. Changes in auxin activity were recorded in seedlings growing in phosphate-starved and phosphate-sufficient conditions. N. plumbaginifolia seedlings failed to exhibit a consistent phosphate starvation response in vivo. Auxin activity of a limited number of N. plumbaginifolia roots oscillated similar to A. thaliana, but with a longer period; however, this pattern was not repeatable. The role of auxin oscillations in determining species-specific differences in root architecture requires further investigation in plants with disparate root systems and phosphate starvation responses.
Name: Frick, Elizabeth
Date: Spring 2012
Major: Biology
Thesis Committee: Dr. Catharina Coenen, Dr. Margaret Nelson
Title: The Effects of Auxin Influx Carriers on Auxin-inducible Gene Expression in Nicotiana plumbaginifolia
The plant growth hormone auxin, indole-3 acetic acid (IAA) is crucial in a number of vital plant responses such as growth, organ initiation, and tropisms. Because it is produced in the apical meristem, polar transport of auxin through the plant is vital for these responses. Both influx and efflux carriers are important for speed and directionality of auxin transport, but how these carriers recognize and transport auxins is insufficiently understood. This project examines the effect of two synthetic auxins, indole-3-methanesulfonic acid (IMS) and 1-methylnaphtyl sodium sulfonate (NSS), on auxin-inducible gene expression. Because both IMS and NSS are fully dissociated at any pH, they cannot diffuse into plant cells to activate intracellular auxin receptors, unless their uptake is facilitated by influx carriers. I used transgenic Nicotiana plumbaginifolia seedlings containing the GH3::luciferase gene construct to quantify auxin-inducible gene expression by measuring luminescence produced by excised hypocotyls.In this system, IMS activated theGH3 promoter; however, it did so much more slowly than IAA. Consistent with the hypothesis that naphthalenic auxins are poorly transported by uptake carriers, NSS did not activate the GH3 promoter, whereas naphthalene acetic acid elicited IAA-like responses. Inability of NSS to elicit a full response indicates that auxin-induced gene expression requires activation intracellular auxin receptors and confirms that tobacco contains no auxin influx carriers capable of binding and transporting the naphthalenic backbone.
Name: Gleeson, Garrett
Date: Spring 2012
Major: Biology
Thesis Committee: Dr. Catharina Coenen, Dr. Kristen Webb
Title: Auxin Inducible Gene Expression in Pseudomonas fluorescens str. Pf-5: The Role of Auxin Response Elements
The plant hormone auxin is produced by many rhizobacteria and serves as a signal in plant-microbe interactions. Auxin responsiveness has thus far been demonstrated in one plant-associated bacterium, Azospirillum brasilense, where it appears to be based on plant-like Auxin Response Elements (AuxRE) in the promoter region of auxin-responsive genes. However, no bacterial transcription factors binding these elements have been identified. This study investigates the mechanism of bacterial auxin signal transduction in the biocontrol bacterium Pseudomonas fluorescens str. Pf-5. To search for proteins that could bind to AuxRE in P. fluorescens, a Protein Basic Local Alignment Search Tool (pBLAST) of plant Auxin Response Factor (ARF) DNA Binding Domains (DBDs) was run against the Pf-5 genome, using a Position Specific Scoring Matrix (PSSM). The PSSM was built by four iterations of a PSI-BLAST of the consensus sequence of 23 Arabidopsis thaliana AR F DNA DBDs against all sequences in the NCBI database. Auxin inducibility of ten candidate genes was investigated by Quantitative Real Time PCR (qRT-PCR). Candidate genes were selected based on the number and position of elements in their promoter regions that match the consensus sequence, TGTCNC, of AuxREs. Understanding bacterial auxin signal transduction will provide insight into communication between plants and bacteria and shed light on the evolution of hormone signaling in plants.
Name: Lamoreaux, Gabrielle
Date: Spring 2012
Major: Biology
Thesis Committee: Dr. Catharina Coenen, Dr. Tricia Humphreys
Title: Effects of phosphate and auxin on the interactions between Pseudomonas fluorescens and tomato (Solanum lycopersicum L.)
Rhizobacteria can benefit plants through protection from disease and promoting growth. Many plant growth promoting bacteria are able to solubilize phosphate and produce the plant growth hormone auxin, although the roles of bacterial auxin production and phosphate solubilization in plant growth promotion are currently unclear. I examined the relationship between auxin and phosphate in interactions between biocontrol strains of Pseudomonas fluorescens and tomato seedlings through (1) surveying P. fluorescens strains for ability to solubilize phosphate while growing on different carbon sources, (2) assessing the influence of plant auxin in phosphate sensitivity on phosphate starvation response of tomato roots, and (3) analyzing the effects of several P. fluorescens strains on root morphology and growth of wild-type and auxin resistant tomato seedlings. The ability of P. fluorescens strains to solubilize phosphate was correlated to genotype as def ined by ADRA analysis of the phlD gene and was most pronounced in the presence of glucose, which is exuded by tomato roots. Auxin sensitivity effects on phosphate starvation responses could not be assessed, because tomato seedlings failed to alter root architecture in response to phosphate in our in vitro culture system. Bacterial effects on root morphology were inhibitory.
Name: Zinobile, Gabrielle
Date: Spring 2012
Major(s): Biology, Environmental Science
Thesis Committee: Dr. Catharina Coenen, Dr. Scott Wissinger
Title: Wild, Heirloom, and Hybrid (Solanum lycopersicum) Tomato Growth and Phosphorus Acquisition Response to Commercial Mycorrhizal Inoculation
Phosphorus is vital plant nutrient that is often heavily applied in readily available inorganic soluble forms to crops in industrial agriculture in the form of fertilizers. Excess soluble phosphorus not used by plants can lead to large of amounts of soluble phosphorus being converted into insoluble forms unavailable to plants. Nearly 80% of all terrestrial plants rely on arbuscular mycorrhizal fungi (AMF) root associations to solubilize unavailable phosphorus. This is especially true for plants that survive on naturally low soil concentrations of phosphorus. Through the modernization of plant genotypes for agriculture, it has been hypothesized that heirloom and hybrid plants under high available soluble phosphorus fertilizer conditions may not rely on AMF colonization for phosphorus acquisition as wild accessions in natural low phosphorus conditions. Wild, heirloom, and hybrid tomato (Solanum lycopersicum) plants were inoculated with a commerc ial AMF eight species mixture under low, intermediate, and high phosphorus and constant intermediate phosphorus conditions. The levels of total phosphorus in the shoot tissues and AMF root colonization examined the effects of inoculation on plant growth. I found that inoculation had no effect on fresh weight, dry weight, total P (μg/plant), and total P (mg/g dry tissue), except for Ailsa Craig under intermediate P conditions. Heirloom and hybrid tomatoes under high P soil conditions had higher fresh and dry weights than low and intermediate soil conditions. Heirloom control Bonnie Best under intermediate P conditions had a higher fresh, dry, and Total P (μg/plant) than hybrid genotypes. Overall, the lack of variability between control and inoculated plants and between genotypes implies that AMF association and phosphorus acquisition may not be affected by plant genotype.
Name: Lonnen, Jennifer J.
Date: Spring, 2011
Major: Biology
Thesis Committee: Dr. Catharina Coenen, Dr. Tricia Humphreys
Title: Cinnamon Oil Effects on Survival and pH Dependent Biofilm Formation ofStreptococcus mutans
Streptococcus mutans, the main bacterium in plaque (making up 60-90%), is the primary cause of dental cavities. S. mutans can metabolize sugars in the oral cavity into organic acids via glycolysis and fermentation; the acids then dissolve the calcium phosphate from tooth enamel, causing cavities. Cinnamon oil (active ingredient: cinnamaldehyde) is a regular additive to oral hygiene products because of its antimicrobial properties against many oral bacteria. Cinnamon oil has been shown to inhibit bacterial growth, prevent biofilm formation, and degrade preformed plaque. Here, a novel assay was used to demonstrate that a 1 hour exposure to 3% cinnamon oil inhibits growth of S. mutans.Inhibition of S. mutans biofilm formation by cinnamon oil was assed at an altered pH through the addition of sucrose to the growth medium. Sucrose addition induced a concentration-dependent decrease in pH with acids produced by S. mutans. Both biofilm formation and growth medium acidification were completely inhibited at cinnamon oil concentrations greater than 250 ppm. At 250 ppm, plaque formation by S. mutans was inhibited significantly more in treatments containing excess sucrose compared to limited sucrose. Treatment with cinnamon oil resulted in significantly less biofilm formation and there was also a significant interaction effect between sucrose and cinnamon oil treatment. Cinnamon oil is an effective in vitro treatment for plaque inhibition and pH stabilization. The potential of cinnamon oil for use as an oral anticavity agent will need to be further assessed through in vivo experiments.
Name: McGrogan, Kaitlin A.
Date: Spring, 2011
Major: Biology
Thesis Committee: Dr. Catharina Coenen, Dr. Margaret Nelson
Title: Influence of phosphate-solubilizing bacteria on auxin-dependent growth promotion in tomato seedlings
Phosphorus is an important plant nutrient that is highly unavailable to plants due to its insoluble form predominately found in soil. Phosphate solubilizing bacteria (PSB) are essential for plant growth because they convert insoluble phosphorus into a form useable by plants. Auxin is a necessary growth hormone utilized by the plant for regulating cellular and developmental responses. When a plant experiences phosphate deficiency, there is an increase in auxin sensitivity however that sensitivity could be negated through increasing the phosphate concentration via inoculation with PSB. By inoculating wild type and diageotropica tomato (Solanum lycopersicum) seedlings with Pseudomonas fluorescens strain Pf-5, a PSB, I was able to observe the effects of inoculation on plant growth. Further by examining the levels of total phosphorus in the root and hypocotyl tissue I determined where the solubilized phosphate was located in the plant. Auxin-dependent growth effects were noted through lateral root formation and length of the primary root. Inoculation was ineffective in increasing plant growth and mass; however inoculated wild type roots had a much higher total P concentration and content. Inoculation also stimulated lateral roots in dgt seedlings. Overall, the high variability between experiments indicates that further parameters contribute to the regulation of phosphate-auxin interactions.
Name: Narney, Stephanie M.
Date: Spring, 2011
Major: Biology
Thesis Committee: Dr. Catharina Coenen, Dr. Tricia Humphreys
Title: The Effects of the plant hormone auxin on Pseudomonas fluorescensresistance to ultraviolet radiation and hydrogen peroxide
Indole-3-acetic acid is a molecule with regulatory roles in many living organisms. Increased resistance to several stress conditions (heat and cold shock, UV-irradiation, osmotic and acid shock and oxidative stress) and several toxic compounds (antibiotics, detergents and dyes) has been found in E. coli, when treated with indole-3-acetic acid, a plant hormone involved in growth and development. To better understand if indole-3-acetic acid-induced stress resistance is unique to E. coli, it is necessary to perform these types of experiments on other types of biologically relevant bacteria. Pseudomonas is a soil dwelling genus of bacteria that is also frequently found on and within the human body. In this study, Pseudomonas fluorescens strain Pf-5 UV and oxidative responses were monitored in the presence of indole-3-acetic acid. There was no interaction found between indole-3-acetic acid and UV exposure survival. An increased resistance to oxidative stress in response to IAA was demonstrated, perhaps due to increased DnaK chaperone accumulation caused by indole-3-acetic acid.
Name: Nicolau-Raducu, Astrid
Date: Spring, 2011
Major: Biology
Thesis Committee: Dr. Catharina Coenen, Dr. Margaret Nelson
Title: 4-Hydroxybenzoic Acid Enhances Auxin Responses in Nicotiana plumbaginifolia
Interactions between the hormones auxin (indole-3-acetic acid, IAA) and salicylic acid (SA) regulate growth and defense responses in plants. The SA analogue 4-hydroxybenzoic acid (4-HBA) is usually referred to as a biologically inactive compound. When used as a negative control to characterize the mechanism through which SA regulates auxin responses of the auxin-induced GH3 promoter, HBA at a concentration of 100 μM increased promoter activity by nearly 2-fold compared to IAA-only treatment. This indicates that HBA acts as a biologically active compound. Activity was monitored using the GH3::Luc reporter complex, allowing for quantitative in vivo data to be obtained. It was originally hypothesized that HBA acts as a SA antagonist on IAA-induced promoter activity and that HBA action is dose dependent. Due to high variability among treatments and poor reproducibility across assays this hypothesis was not supported. Because HBA is biologically active more investigation is required to determine the effects of substituted benzoates on IAA signaling. Understanding these relationships opens up possibilities to modulate trade-offs between plant growth rate and disease resistance.
Name: Paul, Daniel
Date: Spring, 2011
Majors: Biology / Environmental Science
Thesis Committee: Dr. Catharina Coenen, Dr. Milt Ostrofsky
Title: Characterization of Root Exudates of pct1, dgt and Wild-Type Tomato Plants and their Effects on Root Colonization by Biological Control Bacteria
Pathogenic infections cause detrimental effects on crop plants. Potentially hazardous and expensive agricultural chemicals are used to protect crops from pathogenic infection. Inoculation of crop roots with biological control bacteria shows great promise as a less harmful alternative to agricultural chemical use in protecting crops. Root colonization is influenced by plant root exudation and H+-ATPase activity, which are possibly regulated by the plant hormone auxin. This experiment analyzed auxin’s influence on medium acidification as an indirect measure of H+-ATPase activity, root exudation, and bacterial root colonization through the use of wild-type tomato roots as well as the roots of the related auxin mutants. Diageotropica is an auxin resistant mutant, and polycotyledon is an auxin-hyper transporting mutant. Bacterial colonization was greater in the wild-type roots compared to both the auxin mutants. Root exudation and medium acidification were not greater for wild-type roots compared to the auxin mutants, however. The difference in root colonization between the auxin mutants and the wild type roots, therefore, are not explained by differences in H+ secretion or exudation. Regardless, the colonization results call to question the feasibility of transitioning from widespread agricultural chemical use to biological control bacterial inoculation for pathogenic protection in auxin mutant crops.
Name: Wargo, Catherine A.
Date: Spring, 2011
Major: Biology
Thesis Committee: Dr. Catharina Coenen, Dr. Ann Kleinschmidt
Title: Glow with the Flow: Monitoring Auxin Response Patterns through Luciferase Activity in Nicotiana plumbaginafolia
Auxin is an important phytohormone used in plant growth, development, and defense. It is transported through plant tissues using passive diffusion and active, polar auxin transport (PAT). In PAT, auxin uses auxin uptake carriers to enter cells and auxin efflux carriers to exit cells. I used tobacco, Nicotiana plumbaginafolia, seedlings carrying the gene construct GH3::Luc, consisting of the auxin-inducible GH3 promoter and a luciferase reporter gene, to study auxin activity patterns in N. plumbaginafolia. Auxin-induced luciferase expression was monitored with the help of a 4.2 megapixel, 16 bit imaging, cooled, charge-coupled device (CCD) camera system, which permitted monitoring of luciferase activity at high temporal (5 min) and spatial (~1 mm) resolution. The system proved sufficiently sensitive to detect luminescence in response to the weak auxin indole-3-methanesulfonic acid (IMS), which was undetectable with a GUS reporter. Polar application of the native auxin indole-3-acetic acid (IAA) to hypocotyl segments did not produce different luminescence patterns, however, the polar auxin transport inhibitors N-1-naphthylphthalamic acid (NPA), 2,3,5-triiodobenzoic acid (TIBA), and 1-naphthoxyacetic acid (1-NOA) changed auxin activity patterns in whole seedlings. In summary, luciferase expression driven by the auxin-responsive GH3 promoter can be monitored at the whole seedling and organ level using a commonly available cooled, CCD camera system. Luminescence patterns in response to auxin pulses and endogenous auxin demonstrate PAT-dependent decay.
Name: Gumpper, Kristyn N.
Date: Spring, 2010
Major: Biology
Thesis Committee: Dr. Catharina Coenen, Dr. Tricia Humphreys
Title: Production of Indolic Compounds in DAPG-Producing Pseudomonas fluorescens
Pseudomonas fluorescens is a plant-associated bacterium that can enhance root growth through the production of the plant hormone auxin, indole-3-acetic acid or IAA. Recent research, however, has shown they also produce another unknown secondary metabolite. The purpose of this research was to determine the requirements for the production of the unknown secondary metabolite using a colorimetric assay called a Salkowski assay. M9 Minimal media produces the unknown metabolite; however, Castric minimal media does not. IAA production occurred in the presence of tryptophan in the Castric minimal media. Production of the unknown metabolite occurred in the Castric media with added glucose; however, IAA was produced in all other trials. This indicates glucose is either up regulating the production of the unknown metabolite, which down-regulates the production of IAA, or the glucose down-regulates the production of IAA, which stimulates the production of the unknown metabolite.
Name: Lambert, Laci E.
Date: Spring, 2010
Major: Biology
Thesis Committee: Dr. Catharina Coenen, Dr. Milt Ostrofsky
Title: The Effect of Endomycorrhizal Colonization on Hyperaccumulating Plants used for Phytoremediation of Lead-contaminated Residential Land
Lead contamination is a problem in residential areas because it poses a threat to humans interacting with the soil. Children are especially susceptible to the degenerative neurological effects and thus ridding residential areas of lead contamination is a priority for families with young children. Traditional remediation techniques are expensive and require extensive land excavation. Phytoremedation is an alternative option that involves using hyperaccumulating plants to remove lead from soil and accumulate it in their biomass. Mycorrhizal fungi is able to form a symbiotic relationship with plant roots and increase the amount of nutrient and metal uptake. This study focused on the effects of mycorrhiza on the hyperaccumulating ability of four hyperaccumulators, Schizachyrium scoparium, Solidago rigida, Zea mays, and Medicago sativa. It was hypothesized that samples grown in mycorrhiza inoculated soil would accumulate more lead than non-inoculated samples. Plants were grown for a total of 8 weeks with analysis taking place at time points of 4 weeks, 6 weeks, and 8 weeks. At each time point, lead concentration and percent mycorrhiza root colonization were determined. Analysis revealed differences in the amount of lead accumulated by control and mycorrhiza samples were not significant for this study. However, differences in the amount of lead accumulated was significant with regard to species type and time grown.
Name: Miller, Hayley J.
Date: Spring, 2010
Major: Biology
Thesis Committee: Dr. Catharina Coenen, Dr. Tricia Humphreys
Title: The Effects of Auxin on Pseudomonas fluorescens Antibiotic Responses
Catheter-associated urinary tract infections are a large reservoir of antibiotic-resistant bacteria that render many drug therapies ineffective and have fueled a search for solutions. Increased antibiotic resistance has been observed in E. coli cells whenever treated with auxin, a plant hormone involved in growth and development. To understand if auxin-induced antibiotic resistance is unique E. coli, it is necessary to extend these types of experiments to other types of bacteria. Pseudomonas is the most common bacteria genus found on human skin and may be a source of catheter-associated urinary tract infections. In this study, Pseudomonas fluorescens Pf-5 antibiotic responses were observed in the presence of auxin. There was no correlation found between auxin concentrations and antibiotic resistance. The increased antibiotic resistance observed in E. coli may have been a specific response for the bacterium.
Name: Schneider, Brittany N.
Date: Spring, 2010
Major: Biology
Thesis Committee: Dr. Catharina Coenen, Dr. Christy Donmoyer
Title: Auxin Induced Gene Expression in Pseudomonas fluorescens: A Bioinformatics Approach
There is much information detailing the auxin signal transduction pathways in plants, but very little is known about these same pathways in bacteria. The purpose of this study was to determine if genes that are known to be auxin inducible in other bacteria have homologies in the Pseudomonas fluorescens Pf-5 genome, and to investigate the promoters of these homologs for putative auxin responsive elements. Homology searches were conducted between genes known to be auxin-inducible in other bacteria and the Pf-5 genome, and the promoter regions were analyzed for auxin-inducible elements. Of the 26 genes explored, 7 had no significant homologs in the Pf-5 genome and 5 had no auxin responsive elements (AuxRE) in their promoter regions. There are many different auxin-inducible elements in plant promoters, and there are many variations of these sequences that are also auxin-responsive. Two genes from the Pf-5 genome, PFL_0343 and YP_262762.1, were chosen for PCR analysis to determine whether they are auxin-inducible in this promoter. The two genes that were chosen are the best candidates of the genes that I searched because the upstream region of PFL_0343 contains an entire auxin responsive element, and YP_262762.1 contains two AuxRE homologs in its promoter region. Both of the genes examined with PCR showed increased levels of expression in the presence of IAA when compared to their expression levels in the absence of IAA.
Name: Wusylko, Christine A.
Date: Spring, 2010
Major: Biology
Thesis Committee: Dr. Catharina Coenen, Dr. Ann Kleinschmidt
Title: Caffeine effects on peroxidase expression in Nicotiana tabacum
Caffeine (1,3,7-trimethylxanthine), is a common secondary metabolite and it has alleopathic, antiherbivory, and plant defense properties that could be used to deter pathogens in agricultural plants. Plant heme peroxidases (class III peroxidases) are enzymes that are present during all phases of plant life that protect the plant from abiotic and biotic stress. I hypothesized that if caffeine induces a defense response in plants, then peroxidase activity may be increased in plants that are given exogenous caffeine and in plants that make caffeine endogenously. In this study I used wild type Nicotiana tabacum and transgenic N. tabacum that produced endogenous caffeine. Exogenous caffeine was applied to wild type N. tabacum in varied concentration and I found increased peroxidase activity in plants given 1 μM caffeine but decreased peroxidase activity in plants given 10 μM, 100 μM, and 1000 μM caffeine. Exogenous caffeine was then applied to wild type N. tabacum but harvest time was varied. I found an up-regulation in peroxidase activity over time for the control and plants given no treatment, and an up-regulation in peroxidase activity during the dark cycle and a down-regulation in peroxidase activity during the light cycle of plants given 1000 μM caffeine. Transgenic N. tabacum plants that produced a low amount of caffeine (10 μM) were found to have a mix of up-regulation and down-regulation of peroxidase isoenzymes. Plants that produced high amount of caffeine (13 μM) had decreased peroxidase isoenzymes. All of the plants were grown in cramped and moldy environments that could have increased their susceptibility to stress. Although my experiment only estimated protein activity, at high concentrations of caffeine (1000 μM), caffeine may interfere with nucleic acids, transcription and translation, and protein synthesis. However, at low concentrations of caffeine (1 μM of exogenously applied caffeine, and 10 μM of endogenously produced caffeine), caffeine can turn on defense genes.
Name: Green, Ryan J.
Date: Spring 2009
Major(s): Biology
Thesis Committee: Dr. Catharina Coenen, Dr. Scott Wissinger
Title: Using Pennsylvania Native And Ornamental Hyperaccumulating Plants for Phytoremediation of Lead-Contaminated Residential Land
This study used two components, a residential field study and a greenhouse study, to examine the hyperaccumulating ability of Pennsylvania native and ornamental plant species for the purpose of phytoremediating lead(Pb)- contaminated soil around residential homes. The field study focused on pre- germinated plants grown for a full growing season, in order to achieve the greatest biomass and lead accumulation possible. The greenhouse study focused on plants grown from seed for 6 weeks in soil of lead concentrations commonly found around Pennsylvania homes. It was concluded that the native species S. scoparium and S. rigida are the most successful at accumulating lead, followed by M. sativa, Z. mays, R. hirta, B. nigra, M. fistulosa, and A. novae-angliae. Based on these species, a trend of good hyperaccumulation was found among the Asteraceae and Poaceae families. These findings were then directed at homeowners concerned about contaminated soil that could endanger children. The recommended course of action is to plant these species, if not already planted, and take proper care to dispose of the harvested plant material off site after the growing season has ended.
Name: Horning, Erin L.
Date: Spring 2009
Major(s): Biology
Thesis Committee: Dr. Catharina Coenen, Dr. Tricia Humphreys
Title: 2,4-diacetylphloroglucinol producing biocontrol Pseudomonads stimulate root branching through altering auxin distribution
2,4-diacetylphloroglucinol (DAPG) is an antibiotic produced by strains of Pseudomonas fluorescens that are able to suppress fungal diseases in crop plants. To understand the effects of DAPG on Nicotiana plumbaginafolia root development, DAPG effects on root morphology and auxin distribution were compared to the auxin 1-naptheleneacetic acid (1-NAA), the anti-auxin 2–napthaleneacetic acid (2-NAA), and the auxin uptake inhibitor 1-naphthoxyacetic acid (1-NOA). None of the auxin effects were directly comparable to the DAPG effects, suggesting that DAPG acts in a different mechanism than the tested auxins and auxin analogues. A novel in vitro method of colonizing N. plumbaginafolia withP. fluorescens was developed to assess effects of DAPG production on host plants. A comparison of plants colonized by the DAPG-producing strain Pf-5 and the non-producing DAPG mutant phlD- showed that DAPG does stimulate root branching via a change in auxin distribution in Pseudomonas colonized plants.
Name: Lewish, Erik A.
Date: Spring 2009
Major(s): Biology
Thesis Committee: Dr. Catharina Coenen, Dr. Milt Ostrofsky
Title: Effect of Polar Auxin Transport on Arbuscular Mycorrhiza in Zea mays
Polar Auxin Transport (PAT) is an essential function of plants in order for correct growth and development but little is known about its effect on Arbuscular Mycorrhiza. Brachytic-2 maize and wild-type maize were grown in a greenhouse for 50 days and were either treated with Arbuscular Mycorrhiza, Tri-iodo-benzoic Acid (TIBA) or were left as controls. It was found that neither the Brachytic-2 mutants nor the wild-type maize plants differed in colonization due to disrupted polar auxin transport as was originally hypothesized. It was concluded that an increase in mycorrhizal colonization of roots due to disrupted auxin transport maybe a property unique to legumes. Flavonoids as root exudates, which stimulate mycorrhiza growth and inhibit PAT are speculated as possible chemical signal involved in the symbiosis. Although AM colonization was not different between plant genotypes, there was an interaction between genotype and mycorrhiza colonization. The differential effects on plant growth due to the AM colonization are explained in terms of the physiology of the mutants.
Name: Maxwell, Alaina C.
Date: Spring 2009
Major(s): Biology
Thesis Committee: Dr. Catharina Coenen, Dr. Tricia Humphreys
Title: Auxin as a potential quorum sensing signal in Pseudomonas fluorescens
The soil bacterium Pseudomonas fluorescens can act as a biocontrol agent because of its antibacterial and antifungal properties. One method used by bacteria to grow on the plant surface is quorum sensing via autoinducer chemicals. Auxin is known to act as a communication signal between bacteria and plants, and it is possible that it may also act as a quorum sensing signal in P. fluorescens. In the Clinton 1R strain, auxin inhibited the production of an unknown secondary metabolite, which may be involved in cell to cell signaling. To test whether applying auxin to the Pf-5 strain resulted in coordinated behavior associated with quorum sensing, a series of swarming motility assays were performed. In every assay performed, there was a definite increase in swarming motility with auxin compared to controls. Other chemicals were also tested in order to determine which characteristics of auxin were causing the response. Compounds that are known to be chemoattractants or chemorepellants also caused an increased swarming response. These results indicate that auxin is being used by the bacteria for intercellular communication and that it may be acting as a quorum sensing signal in P. fluorescens. Defining auxin as a quorum sensing signal furthers our understanding of the quorum sensing mechanism and may lead to the better use of Pseudomonas fluorescens as a biocontrol bacterium.
Name: Alonso-Hohmann, Erinn J.
Date: Spring 2008
Major(s): Biology
Thesis Committee: Dr. Catharina Coenen, Dr. Ann Kleinschmidt
Title: 2,4-Diacetylphloroglucinol Effects on Lycopersicon esculentum Root Peroxidase Activity
Certain biocontrol strains of the soil bacterium Pseudomonas fluorescens are known for their ability to suppress plant root disease and to produce the antibiotic 2,4-diacetylphloroglucinol (DAPG). DAPG is frequently studied due to its antibacterial, antifungal, and phytotoxic activity. Its recently discovered effects on tomato roots include stunted root growth and root tip necrosis. A root peroxidase with isoelectric point 5.4 was strongly induced in primary roots of 5 day old tomato seedlings treated with 20 and 30 µM DAPG for 24 hours. Five day old seedlings transferred to DAPG plates exhibited strong peroxidase activity and root tip browning in primary roots after a 96 hour exposure, but lateral roots did not display peroxidase induction or necrosis. This apparent lateral root resistance to DAPG could be explained by lateral root formation occurring a few days after seedlings were transferred to DAPG plates, because DAPG is light-sensitive. When seedlings were transferred to DAPG plates after lateral roots had formed, peroxidase activity and root tip necrosis in primary and lateral roots was very similar. Peroxidase 5.4 responds the same to wounding and 30 µM DAPG exposure, suggesting that the plant’s wound response is enacted upon DAPG exposure. However, peroxidase 6.8 is only responsive to wounding and is not DAPG-inducible, indicating that DAPG does not exclusively initiate a wound-like response.
Name: Pfeufer, Emily E.
Date: Spring 2008
Major(s): Biology
Thesis Committee: Dr. Catharina Coenen, Dr. Ann Kleinschmidt
Title: Effects of the Pseudomonas fluorescens Secondary Metabolites 2,4-Diacetylphloroglucinol and Hydrogen Cyanide on the Auxin-Inducible Genes
Pseudomonas fluorescens is a rhizobacterium that enhances the overall health of its host plant by controlling pathogens and fungi in the rhizosphere or by augmenting root phytohormone levels. P. fluorescens synthesizes a number of secondary metabolites, including the antibiotic 2,4-diacetylphloroglucinol (DAPG) and hydrogen cyanide (HCN), which disrupts cellular respiration. The wild-type P. fluorescens strain Pf-5 and its phlD- mutant, whose DAPG-producing ability is disabled, were investigated in the transgenic GH3::luciferase promoter::reporter system in tobacco. Supernatants of both strains inhibited auxin-inducible genes after exogenous auxin was added, regardless of DAPG-producing ability. However, the supernatants of DAPG-producing Pf-5 caused a faster exhaustion of luciferase activity (generated by preexisting auxin levels). Both Pf-5 and phlD- broth cultures produced HCN. When this HCN production was successfully restricted, supernatants of both strains again inhibited auxin-inducible genes. This suggests that DAPG is the component of P. fluorescens supernatants that acts the most quickly to destroy GH3 inducibility, although additional components of Pf-5 supernatants inhibit luciferase activity or auxin signaling. HCN appears to take no part in destroying the inducibility of the GH3 promoter. The main disruptor of auxin-inducibility remains unknown.
Name: Brazelton, Jessica N.
Date: Spring 2007
Major(s): Biology
Thesis Committee: Dr. Catharina Coenen, Dr. Margaret Nelson
Title: 2,4-Diacetylphloroglucinol Effects on Plant Root Development
Naturally occurring biocontrol strains of the soil bacterium Pseudomonas fluorescens can reduce the need for fungicides by excluding pathogens from the rhizosphere of crops and also stimulate root growth. Root growth stimulation has been attributed to the production of the plant hormone auxin, whereas biocontrol activity against pathogens depends in part on production of the antibiotic compound 2,4-diacetylphloroglucinol (DAPG). Auxin production was not detected in either the colorimetric Salkowsky assays or in a sensitive bioassay based on activation of the auxin-responsive GH3 promoter, in any of the phlD+ strains of P. fluorescens tested. Supernatants from all strains inhibited GH3 promoter activity, suggesting that DAPG produced by these strains might inhibit auxin action. To test this hypothesis, auxin-inducibility of the GH3::luciferase construct in tobacco hypocotyls was analyzed in the presence of synthetic DAPG and DAPG was applied to tomato seedlings grown on agar media. DAPG concentrations inhibitory to tomato root growth also inhibited auxin-induction of GH3-promoter activity in tobacco hypocotyls, with inhibitory effects detectable as early as 8 minutes after addition. By contrast, lower concentrations of DAPG increased the number and length of lateral roots in tomato seedlings. This stimulation of lateral root development by DAPG is auxin-dependent because DAPG failed to elicit lateral root formation in the auxin-resistant diageotropica mutant. Although the molecular mechanisms of DAPG action remain unresolved, these results indicate that DAPG may act as a regulator of plant root development.
Name: Restori, Katherine H.
Date: Spring 2007
Major(s): Biology
Thesis Committee: Dr. Catharina Coenen, Dr. Ann Kleinschmidt
Title: Arbuscular Mycorrhiza Formation by Nicotiana plumbaginifolia and Glomus intraradices in vitro—A Role for Auxin
Arbuscular mycorrhiza (AM) is an obligate symbiotic relationship in which fungi of the order Glomales exchange nutrients and phosphorus with the roots of land plants. The goal of this study was to characterize the in vitro interaction between Nicotiana plumbaginifolia and Glomus intraradices as an experimental system to study the role of the plant hormone auxin in AM formation. To provide G. intraradices spores for this study, I characterized the influence of inoculum mass on spore production in a two-compartment co-culture system containing Daucus carota explants and G. intraradices. Liquid media from a root-free compartment of the cultures, which contained fungal mycelia, were then tested for auxin activity in a bioassay system. Bioassays were based on activation of the auxin-responsive GH3 promoter linked to a luciferase reporter gene and transformed into N. plumbaginifolia. While this assay system reliably detects auxin concentrations down to one micromolar, no auxin production was detected in fungal culture media. Seedlings expressing a beta-glucuronidase reporter gene demonstrated a down-regulation of GH3 promoter activation in lateral roots of seedlings inoculated with G. intraradices.
Name: Bartlett, Tyler G.
Date: Spring 2006
Major(s): Biology
Thesis Committee: Dr. Catharina Coenen, Dr. Ann Kleinschmidt
Title: Auxin-Induced SAUR Gene Expression in R15, R25, and R28 Auxin-resistant mutants of Nicotiania plumbaginifolia
The SAUR (Small Auxin Up-Regulated RNAs) gene family is rapidly transcribed in the presence of auxin, one of the five “classic” plant hormones. SAUR expression was investigated in 15-day old seedling of the wild-type, r15, r25 and r28 auxin-resistant mutants of Nicotiana plumbaginifolia. RNA was extracted from seedlings treated with various amounts of NAA for 6 hours. RT-PCR was performed using degenerate SAURprimers to amplify the desired approximate 140 bp product. Semi-quantitative analysis was used to measure the SAUR gene expression. The r15 auxin-resistant mutant had an over-expression of SAUR genes in the absence of NAA with an increase in SAUR gene product at 10-5 M NAA in comparison to the wild-type auxin dose response curve. The r25auxin-resistant mutant had an over-expression of SAUR genes in the absence of NAA with an increase in SAUR gene product at 10-4 M NAA in comparison to the wild-type auxin dose response curve. The r28 mutant had an overall under-expression of SAUR genes and required a higher concentration at 10-5 M NAA to have an increase in SAUR gene product in comparison to the wild-type auxin dose response curve. The response of the auxin-resistant mutants suggests that SAUR gene product could play a role in a negative feedback loop of the AUX/IAA signal transduction pathway.
Name: Braun, Audra J.
Date: Spring 2006
Major(s): Biology
Thesis Committee: Dr. Catharina Coenen, Dr. Ann Kleinschmidt
Title: Roles of Peroxidase Overexpression in Hypocotyls of Etiolated Seedlings of the Auxin-resistant diageotropica Mutant of Lycopersicon esculentum
Reactive oxygen species (ROS) are proposed to play a role in auxin-incuded cell wall loosening, which results in cellular elongation. The auxin-resistant tomato mutant dgt, a spontaneous single gene recessive mutant of the tomato cultivar VFN8, does not increase cell wall extensibility in response to auxin. To determine if a lack of ROS production is responsible for the lack of cell wall extensibility in dgt, ROS in the apoplastic fluid of hypocotyls were measured using 2′,7′-dichlorofluoroescein diacetate and fluorescence was read in a spectrofluorometer. It was concluded that after 60 min of 2,4D treatment, dgtdid not increase ROS production in comparison to the wild type. Peroxidases are enzymes that catalyze the oxidation of various substrates at the expense of hydrogen peroxide and have been implicated in the response of plant cells to auxin. To determine if peroxidases may play a role in auxin-induced hypocotyl elongation, peroxidase activity was analyzed. Fluid was extracted from the cells and cell walls of hypocotyl segments and analyzed using gel electrophoresis. Both wild type and dgt hypocotyls were found to express two main peroxidases with isoeletric points of 5.3 and 6.1; however, dgt was found to have more peroxidase activity than WT. Because dgt shows an overexpression of peroxidases and a lack of ROS accumulation, it is hypothesized that these peroxidases contribute todgt’s inability to elongate by scavenging H2O2, a contributor to ROS production.
Name: Jordanhazy, Ryan A.
Date: Spring 2006
Major(s): Biology
Thesis Committee: Dr. Catharina Coenen, Professor Chris Lundberg
Title: Auxin and Fibonacci Phyllotaxis
Auxin is a plant hormone that controls nearly all aspects of growth including leaf formation. Phyllotaxis, or leaf arrangement about the stem of plants, is therefore controlled by auxin. Interestingly, most plants display a phyllotaxis that incorporates the ancient mathematical sequence known as the Fibonacci sequence. However, the relationship between auxin and Fibonacci phyllotaxis remains unclear. To help explain this relationship, I measured leaf angles in multiple auxin-resistant mutants in three plant species: tomato (Lycopersicon esculentum), tobacco (Nicotiana plumbaginifolia), and Arabidopsis thaliana. Wild type tomato plants exhibited Fibonacci phyllotaxis, while the auxin-resistant diageotropica mutant and the auxin-hyperresponsive polycotyledon mutant showed different leaf angle frequency patterns. Polycotyledon mutants displayed leaf angles lower than the Fibonacci sequence would predict, while diageotropica mutants displayed two different leaf angles. However, tobacco and Arabidopsis data was inconclusive, mainly due to the limited quantity of data obtained.
Name: Kohan, Matthew W.
Date: Spring 2006
Major(s): Biology
Thesis Committee: Dr. Catharina Coenen, Dr. Margaret Nelson
Title: Induction kinetics of GH3 promoter activity in transgenic Nicotiana plumbaginifolia by the auxins IBA, 3-IPA, and HNC
Auxin, the endogenous plant hormone responsible for vasculature development, shoot elongation and a multitude of other responses, induces the rapid expression of an auxin-inducible gene through the activation of the GH3 promoter. The response elicited is dependent upon the concentration of the auxin, the type of homologue used, and the way in which that homologue interacts with the auxin efflux carrier. To study the activation of the GH3 promoter by various auxin homologues, excised hypocotyls of 5-day old etiolated seedlings of Nicotiana plumbaginifolia transgenic with the GH3::LUCpromoter::reporter construct were exposed to various concentrations of the auxins indole-3-butyric acid (IBA), indole-3-propionic acid (3-IPA), and 2-hydroxy-1-naphthoic acid (HNC). All three homologues were found to elicit significantly weaker responses than the natural auxin indole-3-acetic acid (IAA), suggesting that the auxin receptor site inhibits the effectiveness of sterically bulky auxins such as 3-IPA and IBA. Inhibition of the efflux carrier using auxin transport inhibitors revealed that all three homologues were inhibited by the competitive inhibitor TIBA and that the gene activation elicited by neither IBA nor 3-IPA was affected at the efflux carrier by the allosteric inhibitor, NPA. These results may suggest a role for the efflux carrier as an intermediary to the auxin receptor.
Name: Howe, R. Charles
Date: Spring 2005
Major(s): Biology, Environmental Science
Thesis Committee: Dr. Catharina Coenen, Dr. Jim Palmer
Title: Effects of Ethylene on germination rates of Pennsylvania milkweed seeds
As demand for native plant seed grows there is increased focus on the propagation techniques used to commercially produce seed. However, many native species do not germinate even under optimal conditions. Traditional techniques for breaking dormancy and encouraging germination often require much time. These techniques mimic natural environmental stimuli, promoting germination by altering the hormone concentration or physical characteristics of the seed. In many species, exogenous hormones have been used to artificially end dormancy. This study tested the germination rate response of Pennsylvania native milkweed seed to exogenous ethylene treatment. Three species of Pennsylvania native milkweed were treated with water, 1 mM, 5 mM, and 30 mM ethephon concentrations to measure positive effects of this compound on germination percentage. Ethephon yields ethylene in solution, which has been shown to break dormancy in a number of plant species. A significant increase in germination was observed in the 1 mM ethephon treatment. Furthermore this treatment procedure aimed to describe the relationship between ethephon response and traditional treatment requirements of specific species. No significant interaction was found between species and ethephon response.
Name: Macurdy, Krystle M.
Date: Spring 2005
Major(s): Biology
Thesis Committee: Dr. Catharina Coenen, Dr. Brandi Baros
Title: Auxin-Induced SAUR Gene Expression in Auxin-Resistant Mutants of Nicotiana plumbaginifolia
Transcription of the SAUR (Small Auxin Up-Regulated RNAs) gene family is induced by auxin, a naturally occurring plant hormone. SAUR gene expression was investigated in 15-day-old seedlings of wild-type and auxin-resistant r15 and r28 mutants of Nicotiana plumbaginifolia. RT-PCR was performed using RNA extracted from seedlings treated with various synthetic auxin, NAA, concentrations. Degenerate SAURprimers achieved SAUR gene product amplification during PCR and semi-quantitative analysis was used to measure SAUR gene expression. The expected linear response of wild-type seedlings to increasing auxin concentrations was not obtained, likely due to the presence of endogenus auxin. The auxin-resistant r15mutant showed an auxin hypersensitive response in that the optimum response to auxin in tissue treated with 10-5 M NAA rather than 10-6 M NAA as in wild-type. The r28 mutant shows the same auxin response pattern as wild-type, however with an exaggerated response to the optimum NAA concentration of 10-6 M. The responses of both mutants suggest that SAUR gene product may play a role in a negative feedback loop regulating the auxin response.
Name: Young, Suzanne J.
Date: Spring 2005
Major(s): Biology, Environmental Science
Thesis Committee: Dr. Catharina Coenen, Dr. Jim Palmer
Title: A Risk Assessment of Mercury in Northwestern Pennsylvania and the Potential for Phytoremediation of Mercury in Aquatic Systems Using Elodea canadensis and Lemna minor
Mercury (Hg) in aquatic ecosystems presents a major public health risk due to this pollutant’s persistence in the environment and ability to bioaccumulate in the food web. Over the past several decades, the impacts of environmental pollutants have received increasing attention with the use of risk assessments to monitor pollutants becoming a common tool for environmental policymaking. Here, the risks of Hg pollution and exposure were examined, looking specifically at Northwestern Pennsylvania. This region displayed several high risk characteristics of Hg exposure, which may potentially lead to unsafe levels of Hg for local residents. Consequently, the use of two native, aquatic plant species, Elodea canadensis and Lemna minorhave been proposed as potential species for phytoremediation or biomonitoring, due to their wide distribution and previous ability to accumulate pollutants in aquatic ecosystems. Laboratory experiments were carried out to determine Hg accumulating potential and toxicity for each species. Plants were subjected to different concentrations (0.5, 1.0, 5.0, 10.0, and 20.0µM) of Hg for 14 d, and shoot growth, chlorophyll, and protein content were examined. The Hg concentration sustaining ~50% growth was determined and then compared to controls (0µM Hg) after 24, 48, 96, and 168h of exposure. E. canadensisshowed an Hg accumulation of 0.425 µmol/g dry weight in leaf and shoots, after 14 d at 5µM Hg, and L. minor showed an Hg accumulation of 0.417 µmol/g dry weight in fronds, after 7 d at 5µM Hg. Shoot growth for E. canadensis, and chlorophyll and protein contents for both species decreased significantly with increasing Hg concentrations but varied for treatment durations. While the results suggested a strong uptake of Hg by both species, the severe reductions in growth, chlorophyll levels and protein contents may affect both species potential for phytoremediation.
Name: Cortese, Bethany H.
Date: Spring 2004
Major(s): Biology
Thesis Committee: Dr. Catharina Coenen, Dr. Ann Kleinschmidt
Title: The Determination of Secreted Auxins from Symbiotic Arbuscular Mycorrhizal Fungi
Split-plate cultures of arbuscular mycorrhizal (AM) fungus Glomus intraradices and transformed Ri T-DNA carrot roots were grown over a five-month period. A bioassay was developed in order to detect the secretion of auxin or an auxin-like substance produced by the fungus. To establish a bioassay system for auxin in a mycorrhizal host plant, various concentrations of IAA, IBA, NAA, and 2,4 D were applied to a luciferase assay using wild-type transgenic tobacco roots containing a GH3 promoter linked to a luciferase reporter gene. The lowest detectable auxin concentration that elicited a response statistically significant from an ethanol control for IAA, IBA, NAA, and 2,4D was 10-6 M auxin. Fungus-colonized M-media was applied in a luciferase assay using wild-type transgenic tobacco roots. Liquid M-media harvested from plates containing fungal growth appeared to elicit a greater response when compared to the response elicited by liquid M-media harvested from plates not containing fungal growth. The results indicate the presence of an auxin or auxin-like substance in the liquid M-media containing the fungus.
Name: DeSavigny, Travis C.
Date: Spring 2004
Major(s): Biology
Thesis Committee: Dr. Catharina Coenen, Dr. Ann Kleinschmidt
Title: Isolation of SAUR genes in Ceratopteris richardii her-1 mutant fern gametophytes
SAUR genes are small-auxin-up-RNA’s that are thought to play a role in the plants hormone response, however their exact function is unknown. Additionally, the Ceratopertis richardii her-1 mutant fern serves as a simple vascular plant for study. By isolating a SAUR gene in the fern we expect that it may lead to an evolutionary simple model system for SAUR gene study. Furthermore, because of its simplicity we expect that C. richardii has few homologs of the SAUR gene, allowing for a less complex SAUR library to study. Through a series of molecular techniques we have successfully isolated a gene that, by homology and size, can be classified as a SAUR gene. Alignment of a group of known SAUR genes showed an overall conservation of 55.1 % whereas; the new SAUR gene isolated in C. richardii reduced the overall conservation to 26.6 %. However, this decrease in conservation is hypothesized to be a result of evolutionary divergence among the species of plants examined. This discovery in conjunction with further research techniques such as gene silencing may allow researchers suppress and isolate the SAUR gene in C. richardii revealing a way to determine their function(s).
Name: Hayden, Joshua A.
Date: Spring 2004
Major(s): Biology
Thesis Committee: Dr. Catharina Coenen, Dr. Brandi Baros
Title: Auxin-induced GH3 promoter activity in Nicotiana plumbaginifolia seedlings.
Auxin, an endogenous plant hormone, induces rapid changes in the expression of auxin-inducible genes, in part through activating auxin-responsive promoters. These promoters, such as GH3, are known to respond to a variety of auxins, though a detailed comparison of promoter activation by various auxins has not yet been made. To study activation of the GH3 promoter in live tissues, excised hypocotyl segments from 5-day-old, dark-grown Nicotiana plumbaginifolia seedlings transgenic for a GH3::luc+ promoter::reporter gene construct were used. The resulting luciferase reporter gene data were fit to a sigmoidal logistic regression, which allowed estimates of maximum promoter activity, time at half-maximum activity, and the rate at half-maximum activity. This technique showed differential dose-responses and time-courses for indole-3-acetic acid (IAA), 1-naphthylacetic acid (NAA), and 2,4-dichlorphenoxyacetic acid (2,4-D). In addition, this system was used to examine a potential feedback inhibition to secondary auxin addition. Potential interactions of cytokinin and gibberellic acid on auxin-induced GH3 activity were explored as well. Novel data were also gathered showing that 10 µM NPA, an inhibitor of polar auxin transport, had no effect on IAA- and NAA-induced promoter activation and an inhibitory effect on 2,4-D-induced promoter activity. From the results of this study a novel model for NPA action and auxin perception is proposed.
Name: McMillen, Andrea D.
Date: Spring 2004
Major(s): Biology
Thesis Committee: Dr. Catharina Coenen, Dr. Milt Ostrofsky
Title: Characterization of Nicotiana plumbaginifolia R25 mutant with Respect to Auxin and Auxin Transport Inhibitors sensitivity and gravity perception in the Roots.
The plant hormone, auxin is central to the regulation of growth and development in plants, including their response to gravity. To study physiological mechanisms including those governing gravitropism, genetic mutants are useful tools. R25 is a monogenic nuclear mutant of Nicotiania plumbaginifolia, identified by its resistance to auxin (Blonstein 1991). This study looks at the effect of auxin, IAA and NAA as well as auxin transport inhibitors, TIBA and NPA on the gravitropic response of R25 roots. These results characterize the unique combination of auxin and auxin transport inhibitor sensitivity in response to gravity and supply further insight to the function of the R25 protein.
Name: Todaro, Nicole L.
Date: Spring 2004
Major(s): Biology
Thesis Committee: Dr. Catharina Coenen, Dr. Lauren French
Title: Auxin-Inducible Expression of SAUR genes in the Auxin-Resistant R15 and R28 mutants of Nicotiana plumbaginifolia
The auxin-responsive SAUR genes (Small Auxin Up RNAs) are a family of genes, which are rapidly transcribed in the presence of auxin. While the exact function of the SAURs is still unknown, investigating the regulation of SAUR gene expression using the auxin-resistant mutants of Nicotiana plumbaginifolia that displayed altered auxin response can provide insight into the role of mutant genes in auxin response. Because SAUR genes are rapidly induced, any mutation that changes their expression defines a gene with a function in early steps of the auxin response. In this study, amplification of the expected 140 bp SAUR product using reactions containing cDNA from wild-type seedlings incubated in the presence of 10-4M and 10-5M NAA was achieved. The amplification of the SAUR product was attained by conducting semi-quantitative RT-PCR using a specific primer pair designed from a SAUR gene fragment (S4) cloned from Nicotiana plumbaginifolia. PCR optimization techniques including altering the annealing temperature, adding formamide to reactions, using a hot start method, and using various cDNA, primer, and MgCl2 concentrations were utilized to increase annealing specificity of the SAUR primer.
Name: Wilson, Matthew M.
Date: Spring 2004
Major(s): Biology
Thesis Committee: Dr. Catharina Coenen, Dr. Ann Kleinschmidt
Title: A Proteomic Approach for Studying Humoral Autoimmunity in Type 1 Diabetes: Analysis of Immunoreactivity by Two-dimensional Electrophoresis
Type 1 Diabetes mellitus (T1DM) is an autoimmune disease in which the body’s immune system attacks its own insulin-producing beta cells. Currently, using a combination of conventional assays detecting these autoantibodies, only 56% of first degree relatives who convert to T1DM would be identified to enroll in clinical trials aimed at preventing T1DM. Here, I report the use a proteomic approach for identification of putative novel autoantigens. Sera from 20 diabetic patients and 18 non-diabetic controls were screened in this study and, optimal concentrations of patient antibody and rat insulinoma INS-1E cell line total protein lysate were determined using single dimension SDS-PAGE and standard Western blotting techniques. An INS-1E protein load of 100 µg/gel and patient antibody dilution of 1:2000 were found to be optimal for providing a high signal:background ratio. Two-dimensional electrophoresis was then performed with INS-1E lysates, and selected sera from the patient screening were used for western blotting in addition to silver staining of the 2D gels. Silver stains demonstrated effective 2D separation showing representation of both high and low molecular weight proteins with no distortions in the 2D pattern. Western blots of 2D gels provided preliminary evidence of immuno-specific reactivity of patient serum to two presumably previously unidentified INS 1E autoantigens. This experiment was repeated with an increase in patient sera number used to probe the Western blot and a lower protein load (24 µg/gel) due to a difference in INS-1E lysates. The second experiment indicated 2-4 additional immunoreactive proteins, though it is unclear whether the two proteins identified in the preliminary experiment were observed in this second experiment. These results warrant further research into the identification and characterization of the proteins identified in this paper.
Name: Herb, Christine M.
Date: Spring 2003
Major(s): Biology
Thesis Committee: Dr. Catharina Coenen, Dr. Brandi Baros
Title: Antibiotic resistance of Campylobacter and Escherichia coli in retail meat products
In the past decade, the increase of antibiotic resistance due to improper and high antibiotic use has been a major public health concern, with most recent focus on antibiotic use in animal husbandry for prophylaxis, chemotherapy, and growth promotion. This study determined contamination rates and antibiotic resistance patterns of Campylobacter coli, Campylobacter jejuni, and Escherichia coli isolated from chicken and beef products purchased at three grocers in Meadville, Pennsylvania. 66.7% of the chicken products were found to be contaminated with Campylobacter and 33.3% were contaminated with E. coli. None of the beef products were contaminated with Campylobacter and 16.7% were contaminated with E. coli. Campylobacter isolates were found to be highly resistant to erythromycin (78.3%), rifampin (73.9%), and azithromycin (60.9%). E. coli isolates were found to be highly resistant to tetracycline (85.7%) and rifampin (100%). These high levels of unnatural resistance suggest that antibiotic overuse in animal husbandry may be contributing to the increase in antibiotic resistance patterns found in Campylobacter and E. coli inhabiting food animals.
Name: Nardozzi, Kristen L.
Date: Spring 2003
Major(s): Biology
Thesis Committee: Dr. Catharina Coenen, Dr. Brandi Baros
Title: Root anatomy comparison of R15 mutants to wild type Nicotiana plumbaginifolia exposed to various concentrations of auxin
Auxin is responsible for much of the growth and development in plants; in roots it plays a major role in tissue differentiation. Auxin-resistant R15 Nicotiana plumbaginifolia roots exhibit altered phenotypic responses from the wild type when exposed to various concentrations of the natural auxin, indole-3-acetic acid, or IAA. After one week of exposure to IAA, both WT and R15 demonstrated a decrease in sensitivity to auxin. Root hair length and number increased with progressive concentrations of auxin for both genotypes, as did primary root thickness. After seven days of IAA treatment, cross sections of R15 roots exposed to 10-6 M IAA exhibited a third cortex layer. One week later, both control and auxin-treated R15 plants possessed this layer. Therefore, it was concluded that independent of auxin treatment, with time, R15 mutants formed a third cortex layer; this change in anatomy was also induced earlier in the developmental process by the application of large amounts of hormone. Auxin regulates division of all cells including the cortex layer, and evidence from this study suggested that the R15 gene product is involved in this process.
Name: David E. Beil
Date: Spring, 2002
Major(s): Biology
Thesis Committee: Dr. Catharina Coenen, Dr. Ann Kleinschmidt
Title: SAUR promoter activity in response to endogenous auxin from germination to maturation of wild type and R15 auxin-resistant mutant transgenic Nicotiana plumbaginifolia
Auxin, a major regulatory hormone of plant growth and development, is frequently studied in plants. By using a mutant plant line with a specific alteration in its auxin pathway along with transgene insertion, auxin related promoters can be spatially or temporally analyzed by the expression of a reporter gene such as ß-glucuronidase, or Gus. By comparing the response to endogenous auxin in wild type to the response of the R15 auxin-resistant mutant of Nicotiana plumbaginifolia, more can be understood about the role of auxin. In order to make direct comparisons of gene expression in the wild type and R15 mutant, a genetic cross has been proposed by using Mendelian genetic theory to establish lines with identical placement of the same transgene. Patterns of expression under the influence of the SAUR promoter have been characterized throughout development in plant tissues and organs by examining staining patterns due to the inserted Gus reporter gene.
Name: Sara C. Carpenter
Date: Spring, 2002
Major(s): Biology
Thesis Committee: Dr. Catharina Coenen, Dr. Maura Meade
Title: Nicotine levels increase in Nicotiana sylvestris exposed to artificially damaged conspecifics
The release of volatile organic compounds from damaged plants may allow nearby conspecifics to prepare defensive mechanisms against herbivores or pathogens attacking the population. This study investigates between-plant communication of artificially damaged Nicotiana sylvestris with unwounded conspecifics by testing the induction of nicotine synthesis, an inducible defense mechanism of tobacco. Unwounded tobacco was incubated in the dark for 12 hours with freshly wounded or with unwounded tobacco. Five days following exposure to the headspace of damaged plants, nicotine levels in leaf tissue of undamaged tobacco were quantified by HPLC. Levels of nicotine in leaf tissue of unwounded tobacco were higher when the plant had been exposed to the headspace of damaged conspecifics than when the plants had been exposed to undamaged plants (Two-tailed paired t-test, P=0.0026).
Name: Andrea C. DeDent
Date: Spring, 2002
Major(s): Biology
Thesis Committee: Dr. Catharina Coenen, Dr. Maura Meade
Title: Investigation of Peroxidase Isoenzyme Function in the Pathogenic Interaction of the Crucifer Brassica rapa with four Common Fungal Pathogens
The Brassicacea family of plants comprises a diverse and economically important agricultural crop. One of the main threats of disease infection to Brassicas comes from pathogenic fungi. Plant peroxidase enzymes have been implicated in fulfilling various physiological roles within plant cells, including defense from pathogen invasion. However, the presence of various isoenzymes as well as the ubiquitous nature of plant peroxidases has left many gaps in the understanding of the roles they may fulfill in defense. To assess the role of individual peroxidase isoenzymes in response to fungal pathogen infection, the rapid cycling Wisconsin Fast Plant©, Brassica rapa, was used as a model system. Peroxidase activity of different isoenzymes was determined by native gel electrophoresis. Differences in isoenzyme patterns, dependent upon the characteristics of the fungal pathogen present (Leptosphaeria maculans, Sclerotinia sclerotiorum, Albugo occidentalis, and Albugo candida), were observed. Methods were tested for use in characterizing individual isoenzymes based on biochemical properties such as substrate specificity and molecular mass. Host’s defenses may be linked to the aggressiveness the pathogen employs in invasion. In addition possible differential functions or specific responses in individual isoenzyme activity to the pathogen were suggested. Patterns of activity in relations to specific pathogens as well as characterizations of some of the specific peroxidase isoenzymes present in these host-pathogen interactions can provide insight into the mechanisms and roles of peroxidases in plant/fungal interactions.
Name: Laura J. Gutierrez
Date: Spring, 2002
Major(s): Biology
Thesis Committee: Dr. Catharina Coenen, Dr. Margaret Nelson
Title: Kinetic analysis of root growth, gravitropism, and auxin induction of the GH3 promoter in the auxin-resistant R15 mutant of Nicotiana plumbaginifolia
The role of the plant growth hormone auxin has been extensively studied in auxin-resistant mutants of the model plant Arabadopsis, but less is known about the genetics of auxin responses in other species. To understand the role of auxin in roots of Nicotiana plumbaginifolia, two auxin-dependent responses, gravitropism and activation of the GH3 promoter, were studied in the R15 mutant, whose root and hypocotyl growth show reduced auxin inhibition. Root response to gravity was observed as the curvature of the root to vertical after placing it in a horizontal position and analyzing root reorientation every 60 minutes. During the first three hours after gravistimulation, mutant roots curved significantly more slowly towards the gravity vector than did the wild-type roots. After three hours, the rate of curvature was similar for mutant and wild-type roots, which suggests that the R15 mutation may slow initial auxin perception or response. The induction of the GH3 promoter for wild-type roots increased within 30 minutes after auxin (NAA) application and continued to rise up to 50-fold within six hours after auxin administration. Dose-response curves suggest a response optimum at 10-7M NAA. These investigations demonstrate that the GH3 promoter can be induced by auxin treatments that are inhibitory to root growth, suggesting that GH3 gene expression in roots is unlikely to be responsible for elongation growth. Extending these investigations to roots of R15 seedlings will clarify whether slowed gravitropism in the mutant is correlated with slowed activation of auxin-inducible genes.
Name: Hillary L. Pranga
Date: Spring, 2002
Major(s): Biology
Thesis Committee: Dr. Catharina Coenen, Dr. Jim Palmer
Title: Selective Resistance and the Effects of Pieris rapae Infestation on Peroxidase Expression of Brassica rapa
Over evolutionary time plants have differentiated and developed a variety of specific and general defense mechanisms. In the case of herbivory plants have developed a wide range of strategies including induced defenses, which allow plants to change their physical nature after infestation to prevent or reduce the damage of future attacks. Peroxidases have been found to be involved in induced defenses increasing cell wall strength and protecting cells against cytoxicity of peroxides and free radicals. This study examined the effects of herbivory on the strength and presence of different peroxidases in Brassica rapa fed upon by the larvae of the butterfly Pieris rapae. Peroxidase isoenzymes with the isoelectric points 10.1 and 8.9 were consistently found in the herbivore-damaged experimental group but rarely in the control group and were used as a basis to select for a breeding population of plants with high defense-related peroxidase expression. Because second-generation experimental seeds failed to germinate, the investigation is not complete; however the presence and activity levels of herbivory-induced peroxidase isoenzymes permitted a detailed correlative analysis of the connection between herbivore-induced damage and peroxidase expression. The peroxidase isoenzymes with the isoelectric points 8.5-8.8, 10, and 11 were expressed differently between the control and experimental groups of the first generation. Trends seen in control and experimental samples of the isoenzymes with Ip 8.5-8.8 or 10 were statistically significant (Mann-Whitney test Tied P-Value .0140 and .0482), suggesting that these isoenzymes are involved in inducible plant defenses to herbivory.
Name: Maryann A. Borsick
Date: Spring 2001
Major(s): Biology
Thesis Committee: Dr. Catharina Coenen, Dr. Maura Meade
Title: The Effects of Selection for Brassica rapa Resistance to Leptosphaeria maculans and Albugo candida on Chitinase and Peroxidase Activity
Plant disease resistance is often a function of several mechanisms that operate together, each contributing to the overall defense of the plant. This research project examined costs of artificially selecting for resistance to one pathogen to growth and to resistance levels to a different pathogen. Brassica rapa, a rapid-cycling, agriculturally important plant species, was chosen as a model system to work with two common fungal pathogens, Leptosphaeria maculans and Albugo candida. Seeds were inoculated with one pathogen and resistant plants were chosen to propagate a second generation. Seedlings from both L. maculans and A. candida resistant plants were tested for differences in their response and susceptibility to each pathogen, as well as for activity of two defense-related enzymes, chitinase and peroxidase. Selection for resistance to either pathogen significantly decreased disease severity of A. candida, whereas L. maculans severity did not fluctuate with selection. Chitinase activity was not significantly different across treatments for either pathogen, indicating it may not play a role in defense against L. maculans or A. candida. Of five peroxidase isoenzymes, one showed increased expression after selection for resistance to A. candida but not L. maculans and another showed decreased expression after selection for resistance
Name: Tara P. Staskowski
Date: Spring 2001
Major(s): Biology
Thesis Committee: Dr. Catharina Coenen, Dr. Milt Ostrofsky
Title: Nutrient Resorption in Perennial Beaver Wetland Plant Species of Northwestern Pennsylvania
While the efficiency of nutrient resorption in perennial woody plant species has been studied extensively, there have been very few studies on the nutrient resorption efficiency of wetland plant species. In this investigation, the nutrient levels (N, P, Ca, Mg, Na, and K) in the leaves of wetland plant species were measured throughout the autumn until leaf senescence in 29 plant species to find the nutrient resorption efficiency of plants in a wetland ecosystem. The hypothesis tested was that as nutrient availability increases, it is energetically inefficient for the plant to resorb nutrients to the same extent as in a less nutrient-rich environment. The percent of the nutrients resorbed by wetland plants was then compared to the literature values of the percent of nutrients resorbed in perennial woody plant species to compare the resorption efficiency of perennial plant species in environments with different nutrient availability. Although, N and P concentrations differed significantly in resorption efficiency of wetland and forest plant species, there was no correlation between leaf nutrient concentration and percent of N and P resorbed.
Name: Jennifer L. Fuhr
Date: Spring 2000
Major(s): Biology
Thesis Committee: Dr. Catharina Coenen, Dr. Maura Meade
Title: Competition betweenBradyrhizobium japonicumTA11 Nod+,Bradyrhizobium elkaniUSDA 309, and Sinorhizobium frediiUSDA 205 on Phaseolis vulgaris, Medicago sativa,and Glycine max
Nitrogen fixation by rhizobia has been extensively studied because of its benefits to agriculture. Chemical fertilization has been a source of controversy because of its adverse ecological effects. Therefore, many ways of creating a more efficient system of nitrogen fixation by rhizobia have been studied. Many strains of rhizobia have been genetically altered to produce more efficient nitrogen fixers. Many times, however, these strains cannot compete with the indigenous rhizobia, so competition between different strains of rhizobia is an important area of study.
This study examined the competition of three strains of rhizobia: Bradyrhizobium japonicum (TA11 Nod+), Bradyrhizobium elkani (USDA 309), and Sinorhizobium fredii (USDA 205) on Glycine max, Phaseolis vulgaris, and Medicago sativa. To analyze the nitrogen fixing efficiency of the symbiotic bacteria, the nodules were counted and weighed, the color of the nodules was observed, the dry weight of the plants were measured, the nitrogen content of the plants was analyzed, and the nitrogenase activity was assayed by an acetylene reduction on the nodules. Antibiotic tests were performed to identify the rhizobia that had colonized in the nodules. Medicago sativa and Phaseolis vulgaris did not form a symbiosis with any of the strains. On Glycine max, TA11 out-competed 205 and 309. However, 205 and 309 were likely to be more efficient nitrogen fixers because plants infected with these strains had a higher nitrogen content.
Name: Kelly L. Zakel
Date: Spring 2000
Major(s): Biology
Thesis Committee: Dr. Catharina Coenen, Dr. Maura Meade
Title: The VC Group Characterization of the Chestnut Blight Fungus, Cryphonectria parasitica, at the Cussewago Chestnut Farm in Northwestern Pennsylvania
Cryphonectria parasitica, the Chestnut Blight fungus, has plagued the American Chestnut in North America for nearly a century. Due to dramatic increases in the genetic differentiation of the fungus, current biological control methods have been found to be largely ineffective. Characterizing Blight-infected chestnut stands in terms of the phenotype vegetative compatibility can provide an overview of the genetic diversity of the fungus and lead to effective means of biological control. The goal of this project was to characterize the population of C. parasitica at the Cussewago Chestnut Farm, located in Edinboro, Pennsylvania, by means of vegetative compatibility testing and Southern Blot hybridization. Twenty-nine isolates of C. parasitica were paired for anastomosis experimentation and were further characterized into 8 distinct VC groups. Southern Blot and hybridization analysis on isolates representing seven VC groups revealed that DNA fingerprinting did not support the findings of the anastomosis testing. Banding patterns for the isolates that were classified into separate VC groups were different with the exception of strains 7 and 18. Banding patterns for isolates 6, 9 and 30 which, by VC testing have been classified into the same VC group, showed different patterns at two bands. Results of this study provide a first indication of C. parasitica genetic diversity in Northwestern Pennsylvania.