Senior Project Abstracts 2013

The effect of 5-methyl cytosine on RNA duplex stability
Dishler, Abigael, 2013 (Advisor: Dr. Marty Serra)

Synthesis of Sodium Sulfonate Auxin Derivatives
Zachary Einwag, 2013 (Advisor: Dr. Shaun Murphree)

Auxin effects on root exudation in tomato (Solanum lycopersicum)
Colleen Friel, 2013 (Advisor:  Dr. Catharina Coenen)

Plant roots exude a wide range of chemicals into the rhizopshere.  These exudates are important for mediating interactions between plants and soil microorganisms and constitute a significant fraction of global carbon transfer into soils.  Despite this importance, the regulation of root exudation is currently poorly understood.  To test the hypothesis that the plant hormone auxin, which regulates sugar flow between different plant tissues, also regulates root exudation, we are characterizing root exudates in tomato.  Exudates from wild-type tomato root seedlings were compared to those of the auxin-resistant tomato mutant diageotropica (dgt).  Sugars in exudates were identified and quantified by HPLC with refractive index detection, and organic acids were identified and quantified by HPLC with UV detection.  Exudate profiles suggest that auxin stimulates the exudation of oxalic and citric acid, but does not affect exudation of succinic and fumar ic acid, or sugars.  Root colonization  of WT and dgt seedlings by two strains of the biocontrol bacterium Pseudomonas fluorsecens  was  also quantified.  P. fluorescens Pf-5 colonized WT roots at a higher level than dgt roots, while there was no difference in colonization between WT and dgt by P. fluorescens Wayne 1R.  When this data is analyzed along with results from similar experiments in root organ culture (ROC), a model for auxin regulation of root exudation can be developed.  Auxin appears to upregulate the synthesis or export of specific organic acids that accumulate in the tricarboxylic acid (TCA) cycle.  Auxin also appears to promote sugar exudation via invertase stimulation only in high carbon environments, such as ROC.  Finally, auxin effects on colonization by biocontrol bacteria appear to be strain-dependent.  These results highlight the complexity of auxin effects on carbon flux to the rhizosphere.  Auxin effects on root exudation are of major importance for carbon transfer to agricultural soils, because many green revolution crops carry mutations in auxin transport proteins, suggesting that use of these crops on large land areas may affect soil microbial communities and patterns of carbon flow.

Comparison of Spectral and Kinetic Characteristics of the Brassica rapa and Pisum sativum Cytoplasmic Ascorbate Peroxidases
Silas Hartley, 2013 (Advisor:  Dr. Ann Kleinschmidt)

Ascorbate peroxidases are important for regulating hydrogen peroxide in plants. Soybean and pea ascorbate peroxidases (APXs) have been extensively studied, whereas APXs from other plant families have not. To determine whether peroxidases from different plant families share similar spectral and kinetic properties, Pisum sativum (Ps) and Brassica rapa (Br) APXs were compared. Using second order conditions, the APX reaction with hydrogen peroxide, the oxidation from resting state enzyme to compound I, was monitored using stopped-flow methods. Using a derived second-order equation, values were determined for the reaction rate constant, k1, to be (3.44±0.346)x107M-1s-1 for Ps APX and (3.97±0.324)x107M-1s-1 for Br APX, when only observing the decrease in absorbance. When the enzyme was reacted with hydrogen peroxide while observing the decrease and increase in absorbance, the k1 values were (3.52±0.0975)x107M-1s-1 and (3.88±0.144)x107M-1s-1 for Ps and Br APX respectively. The k2 values, without the addition of substrate, were (0.646±0.0204)s-1 for Ps APX and (0.698±0.00705)s-1 for Br APX. The k1 value for Ps APX was lower than the previously found value for recombinant APX (rAPX). When ascorbate was used as a reductant, Br APX showed a higher steady-state reaction rate than Ps APX. However, when pyrogallol was used as the reductant, Br APX displayed lower kcat values compared to Ps APX. Both Ps and Br APXs had similar Km and n values when reduced by pyrogallol, but Ps APX had a vmax rate nearly twice as large as the rate found for Br APX. These data suggested that Br APX used hydrogen peroxide and ascorbate at faster rates than Ps APX, while Ps APX uses pyrogallol at faster rates than Br APX. These results appear to reinforce the idea of two different binding sites for ionic compounds, such as ascorbate, and non-ionic phenyl reducing agents, such as pyrogallol.

Expression of Genes Involved in the Regulation of the Intrinsic Apoptotic Pathway in IRBP/ Mus musculus
Linnea Homa, 2013 (Advisor: Dr. Christy Donmoyer)

Apoptosis, or programmed cell death, is a vital process for survival and proper development of certain tissues. When apoptosis occurs in adult neuronal tissue it can have detrimental effects. Apoptosis can occur through the intrinsic pathway, which is activated at the mitochondrial level. Mice deficient in interphotoreceptor retinoid-binding protein (IRBP/) experience photoreceptor loss via apoptosis starting around 18 days after birth (P18) and peaking at P23. These mice exhibit elongated eyeballs, and by 30 days after birth, 50% of their photoreceptors have died. The purpose of this study was to investigate whether two genes found in the intrinsic apoptotic pathway (pro-apoptotic, Bax, and anti-apoptotic, Bcl-2) are involved in retinal apoptosis in IRBP/ mice. Mice overexpressing Bcl-2 and mice deficient for Bax have shown that these proteins play important roles as regulators of photoreceptor cell death in retinal degeneration. To investigate the role of these genes in IRBP/ mice, relative gene expression was measured by quantitative real-time PCR 23 days after birth (P23) in IRBP/ mouse retinas and compared with age-matched IRBP/ (wild-type) mice (n=3). To observe the occurrence of apoptosis, DNA fragments were observed on a 1.2% agarose gel at P26 in IRBP/ and P23 in IRBP/ mice. I hypothesized that expression of the pro-apoptotic gene Bax would be higher in the retinas of the IRBP/ mice compared to wild-type mice, and expression of the anti-apoptotic gene Bcl-2 would be higher in wild-type versus IRBP/ retinas. Results of this study showed that there was not a significant difference in expression for either pro-apoptotic or anti-apoptotic gene between strains, indicating that at P23, the expression of Bcl-2 and Bax may not be altered in the IRBP/mice. DNA laddering showed that there were not enough apoptotic cells in retina samples for the IRBP/ mice to observe with this assay.

A Thermodynamic Investigation of the Effects of GU Wobble Base Pairs on Group II Bulges in RNA
Ryan Kotecki, 2013 (Advisor: Dr. Marty Serra)

Comparative Expression of Brassica rapa Peroxidase Ohnologs
Christina Mucci, 2013 (Advisor: Dr. Ann Kleinschmidt)

Peroxidases are a versatile group of enzymes that consist of three classes; the third of which is found in all land plants, including Brassica rapa, which is unique for the genome triplication event in the evolution of B. rapa from A. thaliana that resulted in varying copies of numerous B. rapa peroxidase genes.  Not enough is known about peroxidase functional pathways to be able to classify them in many circumstances.  The quantification of peroxidase ohnolog expression levels in a variety of B. rapa tissue types will help to elucidate the circumstances under which different peroxidase ohnologs function.  Ohnolog specific primers will be designed and used in real time PCR to quantify the levels of four peroxidase ohnolog pairs in the stems, roots, leaves, and flower buds of B. rapa.

Investigation of the Effect of Salicylic Acid on Ascorbate Peroxidase in Brassica rapa Leaves
Ryan Vietmeier, 2013 (Advisor: Dr. Ann Kleinschmidt)

Reactive oxygen species (ROS) play a key role in plant cells as a signaling molecule in stress response, but at high concentrations ROS can cause damage to cells.  Ascorbate peroxidase (APX) is a ROS scavenging enzyme that regulates ROS levels by converting hydrogen peroxide (H2O2) to water, at the expense of oxidation of ascorbate.  During pathogen response, increases in ROS are observed, and it has been shown that one of the major components of induction of this is salicylic acid.  Thus, the effect of salicylic acid (SA) treatment on total APX activity, and cytosolic APX (cAPX) activity, protein levels, and relative mRNA levels in eight-day-old Brassica rapa plants was investigated.  Techniques used were reverse transcription polymerase chain reaction (cAPX relative mRNA levels), AXP spectrophotometric assay (total APX activity), native gel analysis (cAPX activity), and western blot (cAPX protein levels).  While differences in the level of cAPX mRNA were observed in the 30 minute treatment as compared to that at 0 minutes, there were no significant difference between the level of cAPX in control and SA treated plants at those time points. There was also an observed increase in total APX activity at 0 min for leaves treated with SA, and overall an increase at 30 minutes with increased activity for SA treated leaves.  Also, there was an increase in cAPX activity and protein level at the 30-minute time point for SA treated leaves compared to the control.  These results suggest that in response to SA treatment results in an increase in cAPX activity which in due to increased production of cAPX protein.