Doctor of Philosophy (PhD), Ohio University, 2024, Plant Biology (Arts and Sciences)

Plants encounter various biotic and abiotic stresses daily and have developed defense mechanisms to overcome these challenges. One key system involved in these defense mechanisms is the ubiquitin (Ub)-26S proteasome system (UPS), which targets malfunctioning proteins for degradation through Ub-tagged proteasomal pathways. The E3 ligases, specifically S-phase kinase-associated protein 1 (SKP1), Cullin 1 (CUL1), and F-box (SCF) complexes, play crucial roles in this process by recognizing and tagging specific protein substrates. Arabidopsis thaliana, with over 700 F-box proteins, has the largest group of E3 ligases, yet only 5% have been functionally characterized. Phylogenetic relationships among 111 plant species have identified four clusters of F-box genes, including a cluster with more conserved F-boxes, referred to as core Arabidopsis F-box (CAF) genes. Given that CAF genes have more known functions compared to other clusters, this dissertation hypothesizes significant potential for discovering new functions among the uncharacterized F-boxes within this group. Considering the evolutionary conservation of most CAFs, I adopted a genetic approach to investigate the roles of CAFs during seed germination and seed development. To address the challenges posed by functional redundancy of duplicated CAF genes and the lethality associated with constitutive F-box overexpression in transgenic plants, I created a library of inducible overexpression lines for 40 CAF genes, many of which lacked known biological functions. By systematically examining the effects of conditional overexpression of these 40 CAFs, I found that CAF overexpression during seed germination and seed development can positively or negatively regulate radicle rupture growth, thus controlling the germination process. Specifically, I identified 24 CAFs that enhance radicle rupture and two that inhibited it by interfering with abscisic acid (ABA)-mediated germination suppression. Induction of CAFs during seed (open full item for complete abstract)

Committee: Zhihua Hua (Advisor); Yang Li (Committee Member); John Schenk (Committee Member); Morgan Vis (Committee Member) Subjects: Biology; Genetics; Molecular Biology; Plant Biology; Plant Sciences

Doctor of Philosophy, The Ohio State University, 2024, Molecular Genetics

Plant reproduction has been of interest to humans since the advent of agriculture over 10,000 years ago in ancient Mesopotamia. Knowing when and how plants reproduced allowed humans to cultivate them and gradually over time increase crop yields through unguided selective breeding. In our present day, the cycles of plant reproduction are still of paramount importance. The reproductive stage of plants is often the most susceptible to environmental fluctuations. Thus, as our climate changes, it is becoming increasingly important to understand the core molecular mechanisms of plant reproduction. For genetically engineered plants to withstand the effects of climate change, we must first understand the molecular mechanisms of plant reproduction. Unlike in animals, where each individual sperm cell is self-motile via the use of a flagella, plant sperm cells are not self-motile. They possess no flagella or means of independent motion. Instead, the sperm of plants relies on the specialized pollen tube structure to deliver plant sperm from the pollen grain to the ovule for fertilization. The pollen tube is a single cell that grows from a pollen grain in a tip-growing manner. It grows down into the female tissue of the flower. Within each pollen tube are two sperm cells. These two sperm cells connect to the vegetative nucleus of the pollen tube itself to form a unit collectively called the male germ unit. This unit traffics together through the cytoplasm of the pollen tube as it grows. The mechanism of this motion is not understood. For over 30 years, it has been proposed that this motion is achieved by motor proteins trafficking the male germ unit along the cytoskeleton of the pollen tube. However, the underlying molecular mechanisms involved in this process have remained obscure. In this work, I investigate the 61 kinesins present in the model organism Arabidopsis thaliana to identify motor proteins that play a role in the trafficking of the male germ unit and in male-der (open full item for complete abstract)

Committee: Iris Meier (Advisor); Anna Dobritsa (Committee Member); Adriana Dawes (Committee Member); Patrice Hamel (Committee Member) Subjects: Cellular Biology

Doctor of Philosophy, Miami University, 2011, Chemistry and Biochemistry

Although the glyoxalase system was discovered more than seven decades ago, its specific functional roles are still unclear. It is believed that the major role for this system is the chemical detoxification of methylglyoxal. However, the identification of isoforms that are non-catalytic with methylglyoxal and S-lactoylglutathione, a known substrate for the enzyme, has suggested that it may play additional roles. Also the observation of increased glyoxalase expression during stress conditions and diseased state, as well as the ability to introduce stress tolerance in plants by the over-expression of glyoxalase enzymes indicates that the system may be responsible for more than just methylglyoxal detoxification. The fact that the glyoxalase system is present across a range of species, and is expressed in almost all tissues highlights the significance of this system, and why it is an important topic to study. It is with this larger question that the research described herein was conducted. Specifically, the questions that this dissertation addresses are - what are the likely roles of two glyoxalase II-like enzymes (GLX2-1 and ETHE1) in Arabidopsis thaliana? To address this question we have used genetic, cellular, transcriptomic, and metabolomic tools to observe changes in the model plant Arabidopsis when expression levels of these genes are altered. The results of the study establish that GLX2-1 has a role in stress response in Arabidopsis thaliana, and that the absence of GLX2-1 increases the sensitivity of plants to stresses like anoxia (lack of oxygen) and high salt conditions. In case of ETHE1, our results suggest that perturbation of this gene causes pleiotropic effects on plant growth and survival, and possibly due to alterations in mitochondrial function.

Committee: Christopher A. Makaroff Dr. (Advisor); Michael W. Crowder Dr. (Committee Chair); Ann E. Hagerman Dr. (Committee Member); Carole Dabney Smith Dr. (Committee Member); Qingshun Quinn Li Dr. (Committee Member) Subjects: Biochemistry

Bachelor of Science (BS), Ohio University, 2024, Environmental and Plant Biology

A plant's survival is largely impacted by its ability to sense and respond to gravitational changes, such as the changes that occur in spaceflight. However, the gravitropic signaling pathway is not fully understood. In the BRIC-20 experiment aboard the International Space Station, the Arabidopsis proton pump AHA2 was found to be differentially phosphorylated in microgravity compared to ground controls. AHA2 is hypothesized to be involved between plant hormone movement and differential growth during gravity signaling. When subject to reorientation, aha2 mutant seedlings had increased root curvature compared to wild type. Furthermore, wild type seedlings treated with fusicoccin, a phytotoxin that increases AHA2 phosphorylation and activity, had increased root curvature compared to untreated plants. To determine the role of AHA2 phosphorylation in gravitropic signaling, several lines of transgenic Arabidopsis with modifications in phosphorylation sites are being developed. The transformants will then be phenotyped for altered gravity response, showing if the phosphorylation of the AHA2 protein is involved in a plant's gravity response. These findings will further knowledge on the molecular mechanisms of gravitropism to develop space-tolerant plants for life support in spaceflight and for habitation of the moon, Mars, and beyond.

Committee: Rebecca Snell (Advisor); Sarah Wyatt (Advisor) Subjects: Plant Biology

Doctor of Philosophy (PhD), Ohio University, 2024, Molecular and Cellular Biology (Arts and Sciences)

Functional studies of the ubiquitin (Ub)-26S proteasome system (UPS) have demonstrated that virtually all aspects of the plant's life involve UPS-mediated turnover of abnormal or short-lived proteins. However, developmental characterization of the UPS, including in seeds and fruits, remains scarce. Unfortunately, early termination of embryogenesis limits the scope for characterizing the UPS activities in reproductive organs. In this dissertation, I utilized a biochemical approach to tackle the developmental role of UPS in plants, using Arabidopsis thaliana (Arabidopsis hereafter) as a model. The overarching goal of my research is to unravel the molecular mechanisms of UPS underpinning seed development so that new molecular breeding technologies could be developed to promote seed production. First, I systematically compared expression changes of multiple 26S proteasome subunits along with the dynamics of proteasome activity and total protein ubiquitylation in seedlings and developing siliques of Arabidopsis. Because autophagy plays the second largest role in maintaining proteome stability, I parallelly studied three late-limiting enzymes that are involved in autophagy influx. My experiments unexpectedly discovered that, in opposite to the activities in seedlings, both protein and transcript levels of six selected 26S proteasome subunits gradually decline in immature siliques toward maturation while the autophagy influx rises, albeit in a nutrient-rich condition. I also discovered a reciprocal turnover pathway between the proteasome and autophagy. While the autophagy influx is suppressed in seedlings by UPS-mediated degradation of its three key enzymes, transcriptional reprogramming dampens this process in siliques that in turn stimulates a bulk autophagy degradation of proteasomes. Collectively, my discovery about the developmental changes of the UPS and autophagy activities suggests that they relay the proteome homeostasis regulation in early seed development, w (open full item for complete abstract)

Committee: Zhihua Hua (Advisor) Subjects: Biology; Plant Biology; Plant Sciences

Doctor of Philosophy, The Ohio State University, 2023, Plant Pathology

Plants exist in a constantly evolving microbial environment that significantly influences their growth, development, and overall well-being. Within this microbial milieu, certain bacteria play a pivotal role in enhancing plant health and growth. These beneficial bacteria are collectively referred to as plant growth-promoting bacteria (PGPB). They offer valuable services to plants, including improved nutrient absorption, heightened growth stimulation, and increased resilience against pathogens and the other environmental adversities. PGPB engage with plants through diverse modes of interaction, such as root colonization, endophytic association, or rhizosphere competence. An in-depth comprehension of the molecular mechanisms and ecological dynamics governing these interactions is essential for unlocking the potential of PGPB in promoting sustainable agriculture and environmental remediation. In Chapter 1, I provide an overview of current methods used to detect and diagnose Pseudomonas syringae. This encompasses traditional approaches like culture isolation and microscopy, as well as modern techniques such as PCR and ELISA. Furthermore, I explore the upcoming advancements in this domain, emphasizing the necessity for highly sensitive and specific methods to detect pathogens even at low concentrations. Additionally, I delve into approaches for diagnosing P. syringae infections when they coexist with other pathogens. Chapter 1 Figures can be found in Appendix A. In Chapter 2, I present a significant protocol for monitoring the progression of gray mold fungal infection at various developmental stages of strawberries. I detail three distinct in vivo inoculation methods for Botrytis cinerea on strawberry plants, focusing on early, middle, and late stages of strawberry growth. Chapter 2 Figures can be found in Appendix B. In Chapter 3, I introduce Bacillus proteolyticus OSUB18 as a novel inducer of ISR (Induced Systemic Resistance). This bacterium enhances plants' r (open full item for complete abstract)

Committee: Ye Xia (Advisor); Christopher Taylor (Committee Member); Yu (Gary) Gao (Committee Member); Lisa (Beck) Burris (Committee Member); Jonathan Jacobs (Committee Member) Subjects: Agriculture; Agronomy; Biochemistry; Bioinformatics; Biology; Botany; Cellular Biology; Plant Biology; Plant Pathology; Plant Sciences

Doctor of Philosophy, The Ohio State University, 2022, Molecular Genetics

StARkin domains are a superfamily of structurally conserved ligand binding domains found in all the domains of life. These domains use a diverse set of mechanisms to regulate the activity of multidomain proteins they are integrated into, will identified roles in mediating homo/heteromeric interactions, subcellular localization, protein stability, and protein turnover. One subfamily of StARkin domains, StAR-related lipid transfer (START) domains, is found in both plant CLASS III HOMEODOMAIN-LEUCINE ZIPPER (HD-ZIPIII) transcription factors (TFs) and human deleted in liver cancer (DLC) tumor suppressor genes. Both families of proteins have critical roles in development, however, roles of the START domain in regulating the activity of either of these protein families is largely unknown. In Arabidopsis thaliana, phylogenetic analysis of HD-ZIPIII paralogs shows divergent activity stemming from coding sequence divergence, separating the five HD-ZIPIII paralogs into two sister clades, the REVOLUTA clade and the CORONA clade. HD-ZIPIII paralogs have redundant developmental activities, with distinct and antagonist effects between these sister clades. Preliminary work in our lab investigating the START domain of a representative REVOLUTA clade paralog, PHABULOSA (PHB), shows that the START domain is required for the developmental activity of PHB and regulates homodimerization, DNA-binding, and transactivation potential. In this study, I present the divergent activity of a representative CORONA clade paralog, CORONA (CNA), mediated by its START domain. We identify the target genes of PHB and CNA, highlighting overlapping, distinct, and antagonist regulation of target genes representative of their redundant developmental roles with distinct, and antagonist effects on each other. Additionally, using domain swap experiments, I identify two new properties of the CNA START domain in modulating the behavior of the binding sites of CNA, as well as modulating transactivation / tra (open full item for complete abstract)

Committee: Aman Husbands (Advisor); Jay Hollick (Committee Member); Mark Seeger (Committee Member); Ruben Petreaca (Committee Member); Amanda Bird (Committee Member) Subjects: Biology; Molecular Biology; Plant Biology

Doctor of Philosophy (PhD), Ohio University, 2022, Molecular and Cellular Biology (Arts and Sciences)

Arabinogalactan-proteins (AGPs) are a family of structurally complex hydroxyproline-rich cell wall glycoproteins found throughout the plant kingdom. Arabinogalactan (AG) sugar chains rich in arabinose and galactose are attached to hydroxyproline (Hyp) residues in the AGP protein cores and constitute 90-98% of the overall weight of AGPs. Although much is known about AGP structure and function, little is known about the contributions of the AG sugar moieties to AGP function. To date, eight Hyp-galactosyltransferases (Hyp-GALTs), named GALT2-GALT9, are known to initiate AGP glycosylation by catalyzing the addition of galactose sugars to Hyp residues in AGP protein core and thereby enable subsequent sugar additions. The extent of Hyp-GALT genetic redundancy, however, remains to be elucidated. To address this issue, I generated various multiple (T-DNA) galt gene knockouts, including a triple mutant (galt5galt8galt9), two quadruple mutants (galt2galt5galt7galt8, galt2galt5galt7galt9), and one quintuple mutant (galt2galt5galt7galt8galt9, referred to as galt25789), and comprehensively examined their biochemical and physiological phenotypes. The mutants had reduced AGP precipitations with β-Yariv reagent in leaves, stems, siliques, and flowers. Monosaccharide composition analysis of the silique and root AGPs in all mutants showed a decreased galactose and arabinose content. The transmission electron microscopy (TEM) analysis of the galt25789 quintuple mutant stems indicated cell wall defects coincident with observed growth impairment. As more and more Hyp-GALT genes were knocked out, the quadruple and quintuple mutants displayed additive effects on insensitivity to β-Yariv-induced growth inhibition, silique length, plant height, and pollen viability. Interestingly, GALT7, GALT8, and GALT9 contributed more to primary root growth and root tip swelling under salt stress, whereas GALT2 and GALT5 played more of a role in seed morphology, germination, and seed set. Since the gal (open full item for complete abstract)

Committee: Allan Showalter Dr. (Advisor); Sarah Wyatt Dr. (Committee Member); Morgan Vis-Chiasson Dr. (Committee Member); Michael Held Dr. (Committee Member) Subjects: Biochemistry; Cellular Biology; Genetics; Molecular Biology; Plant Biology

Doctor of Philosophy, The Ohio State University, 2021, Evolution, Ecology and Organismal Biology

Humans engineer their environments by transporting species around the planet. In a new environment, most introduced species will perish, but a small proportion can become invasive, spreading widely and impacting their environments. My dissertation explores how evolutionary processes shape invasive species. I studied two mechanisms of invasive species evolution that can induce rapid evolutionary change: hybridization (mating between genetically distinct individuals) and whole genome duplication (WGD, when offspring inherit an extra set of chromosome pairs). In Chapters 1 and 2, I describe experiments with members of the model plant genus Arabidopsis differing only in genome size and status as either parent or hybrid, effectively isolating the independent effects of WGD and hybridization on traits. I grew plants together under controlled conditions and measured traits and phenotypic plasticity (the change in trait values across imposed environmental gradients). For the handful of traits and gradients in which WGD shifted plasticity values, WGD consistently increased plasticity (Chapter 1). This study provides the most controlled experimental evidence to date in support of the hypothesis that WGD increases plasticity, a hypothesis invoked to help explain how WGD has driven evolution. In contrast to WGD, I found that hybridization produced larger effects on both mean traits and plasticity (Chapter 2). This experiment is the first to fully isolate hybridization and WGD effects on plasticity. In nature, genetic and trait variation provide the raw material allowing invasive species to initially prevail in and, potentially, adapt to the introduced environment. I examined patterns of variation related to hybridization and WGD for two invasive plant systems (Chapters 3 and 4). Chapter 3 focuses on purple loosestrife (Lythrum salicaria), a well-studied species for which other authors have documented post-introduction changes in traits and genetics. A little-studied, mo (open full item for complete abstract)

Committee: Stephen Hovick (Advisor); Alison Bennett (Committee Member); Andrea Wolfe (Committee Member); Kristin Mercer (Committee Member); Amanda Simcox (Committee Member); Robert Klips (Committee Member) Subjects: Biology; Botany; Conservation; Ecology; Evolution and Development; Genetics; Horticulture; Morphology; Organismal Biology

Doctor of Philosophy, The Ohio State University, 2021, Translational Plant Sciences

While the overwhelming majority of bacteria in the plant phyllosphere are commensal and benign to their host, a fraction of these have the capacity to aggressively multiply at the expense of their host and cause devastating disease. This capacity is largely dependent on the pathogen's virulence arsenal of effector proteins and toxins. Many elements of these arsenals are specialized and conserved only among closely related pathogens, yet some are both widespread and essential to the pathogen's ability to cause disease. Members of the AvrE family of type-III effectors generally belong to the latter category. By mechanisms that remain unclear, AvrE effectors suppress host immunity and cause water-soaking, a syndrome at the earliest stages of disease when tissues appear waterlogged. Recent investigations indicated a set of leucine-rich repeat receptor-like kinases (LRR-RLK) in Arabidopsis thaliana Col-0 that are likely to interact with the AvrE1 effector of Pseudomonas syringae pathovar tomato (Pto) DC3000. Thus, these LRR-RLKs were tested for AvrE1-dependent susceptibility, which identified a possible role in the host defense response for a subset of these largely unstudied receptors. Future directions regarding construction of LRR-RLK polymutant host lines are discussed. Experiments also explored the virulence pathway of the toxin coronatine (COR). While primarily understood to mimic the plant hormone jasmonoyl-isoleucine to suppress the host immune response to Pto DC3000, an experimental condition was identified wherein COR induces host resistance. In order to explore the mechanism underlying COR-induced resistance, infected host transcriptomes were sequenced, which revealed a transcriptional profile enriched with COR-dependent activation of the immune response and signaling pathways.

Committee: David Mackey (Advisor); Jonathan Jacobs (Committee Co-Chair); Pierluigi (Enrico) Bonello (Committee Member); Leah McHale (Committee Member); Jason Slot (Committee Member); Fumiaki Katagiri (Committee Member) Subjects: Genetics; Microbiology; Molecular Biology; Plant Sciences

Doctor of Philosophy (PhD), Ohio University, 2021, Molecular and Cellular Biology (Arts and Sciences)

Arabinogalactan-proteins (AGPs) are glycoproteins that function in plant growth and developmental processes, mainly through the actions of the heterogenous glycan chain attached to their protein cores. Glucuronic acid, which is transferred to the AGP glycan by β-glucuronosyltransferases (GLCATs), is the only acidic sugar in AGPs that binds calcium. Despite the importance of type II arabinogalactans (AGs), our understanding of the underlying biological role of such glycans and their sugar residues in plant growth is incomplete. To better understand the GLCATs, I carried out a comprehensive genomewide analysis of putative GLCAT gene family members belonging to the GlycosylTransferase14 (GT14) family in the Carbohydrate-Active enZYmes (CAZy) database by examining their sequence diversity, genetic architecture, phylogenetic and motif characteristics, selection pressure and gene expression in plants. I found 161 putative GLCAT genes distributed across 14 plant genomes with a widely conserved GLCAT catalytic domain. I discovered a phylogenetic clade shared between bryophytes and higher land plants of monocot grass and dicot lineages and identified positively selected sites that do not result in functional divergence of GLCATs. Also, RNA-seq and microarray data analyses of the putative GLCAT genes revealed gene expression signatures that likely influence the assembly of plant cell wall polymers. Among the eleven GLCAT genes found in Arabidopsis, I focused on identifying the biochemical and physiological roles of three of them, namely GLCAT14A, GLCAT14B and GLCAT14C. Based on in-silico analyses, I discovered that the GLCAT14A and GLCAT14C genes are highly expressed in both the seed coat and micropylar endosperm. Using a reverse genetics approach, I observed that glcat14a-1 mutants displayed subtle alterations in mucilage pectin homogalacturonan (HG) compared to WT, while glcat14a-1 glcat14c-1 double mutants displayed much more severe mucilage phenotypes, including loss of (open full item for complete abstract)

Committee: Allan Showalter Dr (Advisor); Sarah Wyatt Dr. (Committee Member); Michael Held Dr. (Committee Member); Ahmed Faik Dr. (Committee Member) Subjects: Biochemistry; Biology; Plant Biology; Plant Sciences

Doctor of Philosophy, The Ohio State University, 2021, Molecular, Cellular and Developmental Biology

The nucleus is a dynamic organelle whose nuclear envelope (NE) is composed of a double membrane with a distinct but continuous inner nuclear membrane (INM) and outer nuclear membrane (ONM). Linker of the nucleoskeleton and cytoskeleton (LINC) complexes are scattered throughout the NE. LINC complexes are composed of Klarsicht/ANC-1/Syne Homology (KASH) ONM proteins and Sad1/UNC-84 (SUN) INM proteins that interact in the NE lumen, forming a bridge between the nucleoplasm and cytoplasm. Unlike for the case of homologous SUN proteins, plants and opisthokonts (animals and fungi) do not share sequence similarity between their KASH proteins and plant LINC complexes are functionally less well understood. SINE1 and SINE2 were recently discovered as plant-specific KASH proteins that have similar expression levels in roots, but are differentially expressed in leaves: SINE1 only in developing and mature guard cells and SINE2 in trichomes, epidermal and mesophyll cells, and weakly in mature guard cells. Stomatal complexes are composed of two guard cells that form a pore in the epidermal layer of leaves, through which gas exchange between the plant and its environment occurs. Stomata open and close in a highly coordinated and controlled manner, being mediated though several complex signal transduction pathways from changes in light conditions to various fluctuations in environmental stresses. I show here that SINE1 and SINE2 are required for efficient stomatal opening and closing during changes in light-dark conditions, with links to a role in the regulation of K+ and Ca2+ during light-induced opening. Additionally, loss of SINE1 and SINE2 results in increased drought susceptibility. ABA is a plant hormone that induces stomatal closure as a short-term response to stress, involving the activation of guard cell anion channels, cytoskeleton reorganization, and changes in vacuolar morphology. ABA hyposensitivity was seen in sine1-1 and sine2-1 mutants during stomatal opening and (open full item for complete abstract)

Committee: Iris Meier Dr. (Advisor); Hay-Oak Park Dr. (Committee Member); David Mackey Dr. (Committee Member); Anna Dobritsa Dr. (Committee Member) Subjects: Biology; Cellular Biology; Genetics; Molecular Biology; Plant Biology

Master of Science (MS), Bowling Green State University, 2020, Biological Sciences

Understanding plant-plant communication further elucidates how plants interact with their en-vironment, and how this communication can be manipulated for agricultural and ecological purposes. Part of understanding plant-plant communication is discovering the mechanisms be-hind plant-plant recognition, and whether plants can distinguish between genetically like and unlike neighbors. It has been previously shown that plants can “communicate” with neighbor-ing plants through airborne volatile organic compounds (VOCs), which can act as signals relat-ed to different environmental stressors. This study focused on the interaction among different genotypes of the annual plant Ar-abidopsis thaliana. Specifically, a growth chamber experiment was performed to compare how different genotypes of neighboring plants impacted a focal plant's fitness-related phenotypes and developmental stages. The focal plant genotype was wild type Col-0, and the neighboring genotypes included the wild type Landsberg (Ler-0), and the genetically modified (GM) geno-types: Etr1-1 and Jar1-1. These GM lines have a single point-mutation that impacts their abil-ity to produce a particular VOC. This allows for the evaluation of a particular role that a VOC may have on plant-plant airborne communication. Plants were grown in separate pots to elimi-nate potential belowground interactions through the roots, and distantly positioned to avoid aboveground physical contact between plants. In addition, to avoid potential VOC cross-contamination between different treatments (genotypes), each neighboring plant treatment oc-curred in separate, sealed growth chambers. Results showed that when A. thaliana Col-0 plants were grown alongside neighbors of different genotypes, they exhibited some significant differences in fitness-related traits, such as increased rosette width, stem height, aboveground biomass, and total fruit number. However, these results differed with neighbor identity, and when the experiment was (open full item for complete abstract)

Committee: Maria Bidart Dr. (Advisor); Heidi Appel Dr. (Committee Member); Vipa Phuntumart Dr. (Committee Member) Subjects: Biology

Doctor of Philosophy (PhD), Ohio University, 2020, Molecular and Cellular Biology (Arts and Sciences)

Arabinogalactan-proteins (AGPs) are a diverse family of plant hydroxyproline-rich glycoproteins implicated to function in a number of physiological processes including growth, development, cellular signaling, somatic embryogenesis, programmed cell death, and wounding. AGPs are known for the abundance of sugars present on their molecular surface. Addition of the various sugars to AGPs requires the action of numerous distinct enzymes called glycosyltransferases (GTs). Glucuronic acid (GlcA), which is the only negatively charged sugar on AGPs, is added by the action of three glucuronic acid transferases (GLCATs), namely GLCAT14A, GLCAT14B, and GLCAT14C. Hydroxyproline-Galactosyltransferases (GALTs) are responsible for initiating sugar addition to AGPs by adding galactose (Gal) to hydroxyproline residues in the AGP core protein. To date, eight GALTs, namely GALT2-6 and Hyp-O-galactosyltransferases 1-3 (HPGTs 1-3) have been identified. To overcome gene redundancy within these two GT families, I applied a cutting-edge clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9 ) gene multiplexing approach to produce higher order genetic mutants (i.e., mutants with multiple gene family members being mutated). Seven CRISPR mutants were generated including glcat14b, glcat14c, glcat14a glcat14b, glcat14b glcat14c, and glcat14a glcat14b glcat14c for the GLCAT gene family and galt3 galt4 galt6 and galt2 galt3 galt4 galt5 galt6 for the GALT gene family. These CRISPR mutants along with two existing T-DNA mutants, namely galt2 galt5 and hpgt1 hpgt2 hpgt3, were subjected to extensive biochemical and physiological phenotypic characterization. Biochemical analysis of the glcat mutants revealed that the double and triple mutants generally had small increases of Ara and Gal and concomitant reductions of GlcA, particularly in the glcat14a glcat14b and glcat14a glcat14b glcat14c mutants. Moreover, all the glcat mutants displayed significant reducti (open full item for complete abstract)

Committee: Allan Showalter (Advisor) Subjects: Biochemistry; Genetics; Molecular Biology; Plant Biology

Doctor of Philosophy (PhD), Ohio University, 2020, Chemistry and Biochemistry (Arts and Sciences)

Extensin peroxidases play a critical role in plant cell growth and are believed to play equally important roles in defense from pathogenesis and mechanical stress. By catalyzing the covalent polymerization of extensin proteins, they participate in the formation of the cell plate for cell division and help to reinforce the wall—preventing pathogen infection. Due to it's anionic character and catalytic processivity, TomEP is a particularly unique extensin peroxidase that requires much less time and enzyme than other extensin peroxidases to crosslink extensin substrate. Previous work identified the TomEP gene, and established methods to produce functional enzyme through heterologous expression in E. coli. This work aimed to expand upon these previous efforts by characterizing TomEP expression, TomEP function in vivo, and design a purification scheme to produce milligram-level quantities of pure enzyme for crystallization. An expression profile of TomEP was compiled using both qPCR analysis and promoter-GUS fusion experiments to provide data describing normal expression and response to wounding. Basal TomEP expression was demonstrated to be significantly higher in roots than in flowers, stems, or leaves. Through the same methods, wounding treatments were shown to increase TomEP expression in tomato roots from one to four hours, followed by attenuation for the following sixteen hours. The foundations of gain and loss-of-function experiments were pursued in an attempt to discern TomEP's influence on di-isodityrosine and pulcherosine content in tomato cell walls, using overexpression and CRISPR knock-out strategies. Overexpression lines of tomato and Arabidopsis were generated using Agrobacterium mediated methods, though these efforts failed to produce verifiable protein product, despite expression being observed on the RNA level. Transient expression in tobacco epidermal cells was successful however, allowing for in vivo analysis of TomEP activity, though no clea (open full item for complete abstract)

Committee: Michael Held II (Advisor); Marcia Kieliszewski (Committee Member); Showalter Allan (Committee Member); McMills Lauren (Committee Member) Subjects: Biochemistry; Botany; Plant Biology; Plant Sciences

Doctor of Philosophy (PhD), Ohio University, 2020, Molecular and Cellular Biology (Arts and Sciences)

Plants use a myriad of environmental cues to inform their growth and development. The force of gravity has been a consistent abiotic input throughout plant evolution, and plants utilize gravity sensing mechanisms to maintain proper orientation and architecture. Despite thorough study, the specific mechanics behind plant gravity perception remain largely undefined or unproven. At the center of plant gravitropism are dense, specialized organelles called starch statoliths that sediment in the direction of gravity. Herein I describe a series of experiments in Arabidopsis that leveraged RNA sequencing to probe gravity response mechanisms in plants, utilizing reorientation in Earth's 1g, fractional gravity environments aboard the International Space Station, and simulated fractional and hyper gravity environments within various specialized hardware. Seedlings were examined at organ-level resolution, and the statolith-deficient pgm-1 mutant was subjected to all treatments alongside wildtype seedlings in an effort to resolve the impact of starch statoliths on gravity response. In all, 132 unique genotype/tissue/treatment datasets were collected to help further our understanding of the gravitropic mechanisms in plants.

Committee: Sarah Wyatt Ph.D. (Advisor); Michael Held Ph.D. (Committee Member); Morgan Vis Ph.D. (Committee Member); Ronan Carroll Ph.D. (Committee Member) Subjects: Biology; Plant Biology; Plant Sciences

Doctor of Philosophy (PhD), Ohio University, 2019, Molecular and Cellular Biology (Arts and Sciences)

Gravitropism is a fundamental growth response in plants. The signal transduction process converts the perception of gravity to the response which causes the roots to bend toward and shoots to bend away from the direction of gravity. On Earth, the 1g gravitational force is ubiquitous, but in space, the force of gravity is negligible providing the ultimate control environment for gravity experiments. To identify the proteins and possible biochemical pathways participating in the signal transduction process, a proteomic study was designed to compare the proteins expressed in seedlings germinated on the International Space Station (ISS) to those germinated on Earth. Arabidopsis Col-0 seeds were sterilized and plated on 60mm petri plates, which were packed in spaceflight hardware and flown to the ISS. Duplicate sets of WT Col-0 seeds were kept at the Kennedy Space Center as ground controls and at Ohio University as additional controls for hardware and preservative. After return from the ISS, proteins were extracted and fractionated into membrane and soluble. Both fractions were analyzed using labeled tandem mass spectrometry at the Donald Danforth Plant Science Center. Differential abundance analysis revealed 129 soluble proteins and 137 membrane proteins that differed in seedlings germinated in ISS versus ground control (p < 0.05). Comparing the differentially abundant proteins from the current study with two previous proteomic experiments identified two membrane proteins and twelve soluble proteins that were differentially expressed in the three experiments. Network analysis of the fourteen proteins using STRING identified a potential interaction between PCAP1, a membrane bound protein with a phosphatidyl inositol binding site, and PATL2, a soluble protein that functions in membrane trafficking. PCAP1 and PATL2 were selected for further study as possible candidate genes for gravity signal transduction. Phenotypic analysis of pcap1 knockout mutants showed that infloresc (open full item for complete abstract)

Committee: Sarah Wyatt (Advisor); Morgan Vis-Chiasson (Committee Member); Alan Showalter (Committee Member); Michael Held (Committee Member) Subjects: Cellular Biology; Molecular Biology; Plant Biology

Master of Science (MS), Bowling Green State University, 2019, Biological Sciences

Changes in the level of polyamines enable plants to respond optimally to both biotic and abiotic stresses such as increased salinity, drought, chilling, flooding, heavy metals and increased concentrations of reactive oxygen species. To identify new classes of transporters that mediate movement of polyamine between cells and within organelles, this study focused on the OCT family in the model plant Arabidopsis thaliana. The A. thaliana OCT family contains six members with no splice forms and have been less studied. Because the expression of OCT5 is upregulated during salt stress assays of seedlings, we tested the response of oct5 seedlings under these conditions. In the salt assays, oct5Δ mutants showed faster bleaching of leaves than the wild type, a common phenotype associated with increased sensitivity to salt stress. To determine whether OCT5, could function as a polyamine transporter, OCT5 was heterologously expressed in WT yeast and in tpo5Δ, a yeast strain deficient in polyamine export. The dilution of rapidly growing yeast cells in liquid culture to fresh media containing elevated levels of putrescine, resulted in an inhibition of growth for all strains in response to 150 or 200 mM putrescine. WT cells expressing OCT5 were more sensitive to exogenous putrescine than WT cells alone. As expected, the tpo5Δ yeast strain was more sensitive to exogenous putrescine than WT cells, but the heterologous expression of OCT5 in this strain did not result in further growth inhibition. The inhibition of growth in WT cells expressing OCT5 was unexpected, since this gene is localized to the vacuole in plants and increased expression of this gene was expected to have a protective effect on cell growth by having this transporter sequester putrescine from the cytoplasm in the vacuole. However, in humans, OCT transporters are polyspecific and are believed to mediate substrate transport in either direction. Thus the increased sensitivity to putrescine when this gene was ex (open full item for complete abstract)

Committee: Paul Morris Dr. (Advisor); Vipaporn Phuntumart Dr. (Committee Member); Helen Michaels Dr. (Committee Member) Subjects: Biology

Doctor of Philosophy, The Ohio State University, 2019, Evolution, Ecology and Organismal Biology

Herbicide resistant weeds are among the greatest threats facing modern agriculture. Glyphosate, the active ingredient in the commercial herbicide RoundUp, is the most widely applied herbicide worldwide, and 43 weed species have evolved resistance to glyphosate to date. My research addressed four key questions about the strength, mechanisms, and fitness effects of glyphosate resistance using Conyza canadensis, a representative glyphosate-resistant weed, and Arabidopsis thaliana, a model plant species. In northcentral Ohio and southern Iowa, maternal biotypes (individual plants) of Conyza canadensis, also known as horseweed or marestail, were collected from 74 locations (both agricultural and non-agricultural) in both states. Dose-response experiments were used to categorize biotypes into resistance categories based on 80% survival at 0x only (Susceptible, S), and up to 1x (equivalent to 840 g ae ha-1; R1), 8x (R2), 20x (R3), and 40x (R4). Extreme glyphosate resistance (R4 biotypes) was quite common in both states and both habitats, and non-agricultural habitats served as a refuge for R4 biotypes. Glyphosate resistance mechanisms are generally presumed to impose a fitness cost due to possible trade-offs in resource allocation to plant defense, growth, and reproduction. Nine S, eight R1, and nine R4 biotypes originally collected in Iowa were grown in a common garden experiment in Iowa over two years and two sites to test for fitness effects. Based on early rosette sizes and days to bolting, nested ANOVAs showed that R4 biotypes grew as large as, if not larger than, both S and R1 biotypes, and bolted significantly earlier. Furthermore, R1 and R4 biotypes were less likely to display disease symptoms than S biotypes. Glyphosate resistance in horseweed appears not to impose an early growth penalty, and possibly no lifetime fitness cost. Specific mechanisms of glyphosate resistance in horseweed include altered translocation and vacuolar sequestration. Recently, a Canadi (open full item for complete abstract)

Committee: Allison Snow (Advisor); Stephen Hovick (Committee Member); Kristin Mercer (Committee Member) Subjects: Agriculture; Agronomy; Biology; Ecology; Evolution and Development

Doctor of Philosophy, The Ohio State University, 2018, Molecular, Cellular and Developmental Biology

Nuclear movement is a widely conserved phenomenon throughout eukaryotes. In animals, it is known to be necessary for developmental events ranging from nematode neurogenesis to fly eye development. Plant nuclei move in a range of cell types during many developmental events and in response to several environmental stimuli. However, the mechanisms of nuclear movement in plants have only recently begun to be characterized. In animals, nuclear movement is dependent on linker of nucleoskeleton and cytoskeleton (LINC) complexes, which are comprised of outer nuclear membrane (ONM) Klarsicht/ANC-1/Syne-1 (KASH) proteins and inner nuclear membrane (INM) Sad/UNC-84 (SUN) proteins. These two proteins bridge the nuclear envelope (NE) via the interaction of KASH proteins and SUN proteins in the NE lumen. While plants do not encode homologs of animal KASH proteins, functional analogs of animal KASH proteins have been identified. The first to be identified were Arabidopsis thaliana WPP domain-interacting proteins (WIPs). These proteins interact in the ONM with their binding partners, the WPP domain-interacting tail-anchored (WIT) proteins, and in the NE lumen with Arabidopsis SUN proteins. Together, a LINC complex comprised of SUN, WIP, and WIT is necessary for proper nuclear movement and nuclear morphology in root hairs and vegetative nuclear movement in pollen tubes. However, many other instances of nuclear movement have been described in plants that have yet to be functionally characterized. One of these as-yet-uncharacterized instances of nuclear movement occurs during the initiation of rhizobial symbiosis, or nodulation. To understand the role of nuclear movement in this legume-specific phenomenon, I bioinformatically identified a host of LINC complex components in the model legume Medicago truncatula. LINC complex components were then verified by determining their localization in both Nicotiana benthamiana and M. truncatula and establishing that the KASH proteins interact w (open full item for complete abstract)

Committee: Iris Meier (Advisor); Anita Hopper (Committee Member); Stephen Osmani (Committee Member); Aman Husbands (Committee Member) Subjects: Biology; Botany; Cellular Biology; Genetics; Plant Biology; Plant Sciences