UMD Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/3
New submissions to the thesis/dissertation collections are added automatically as they are received from the Graduate School. Currently, the Graduate School deposits all theses and dissertations from a given semester after the official graduation date. This means that there may be up to a 4 month delay in the appearance of a given thesis/dissertation in DRUM.
More information is available at Theses and Dissertations at University of Maryland Libraries.
Browse
10 results
Search Results
Item INVESTIGATION INTO THE ROLE OF UVR8 IN BALANCING GROWTH AND ACCLIMATION TO UV-B RADIATION IN NATURAL AND TRANSGENIC POPULUS VARIANTS(2021) Wong, Tiffany Marie; Eisenstein, Edward; Sullivan, Joseph; Plant Science and Landscape Architecture (PSLA); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Research on woody plants offers promise for the development of next-generation biofuel feedstocks with reduced lignin recalcitrance and enhanced saccharification for ethanol production. Natural variants of Populus trichocarpa with diverse lignin content and saccharification differences, and transgenic Populus deltoides constructed for reduced lignin levels for improved cellulose extraction, offer clues to enhance biofuel production but with a tradeoff to overall fitness and biomass. One concern of engineering lignin relates to the protection of plants against environmental stress such as UV-B radiation. Secondary metabolite biosynthesis initiated by UV-B, particularly phenylpropanoids (lignin precursors) and flavonoids, plays an important role in managing and protection of UV stress. Genetic modifications affecting the production of these compounds may have significant physiological consequences. Thus, the goal of this research was to develop a model for biosynthetic compensation of low-lignin Populus to UV-B stress. The effect of UV-B on Populus was evaluated by spectroscopic and metabolomic measurements on leaves. UV-B promoted shifts in physiological and metabolomic responses of natural and transgenic Populus with varying levels of lignin were complex, reflecting compensation from variety of biosynthetic alterations. Therefore, the impact of modulating the expression of the photoreceptor, UVR8, in regulating the response of Populus to UV-B was pursued. Modulation of UVR8 expression in Populus hybrid was achieved by constructing transgenic plants using CRISPR and RNAi, in wild-type, and an RNAi-constructed cinnamyl alcohol dehydrogenase knockdown line. UV-B response of UVR8 modulated Populus indicated that flavonoids were upregulated in UVR8 overexpression lines, and that in a CAD knockdown background, these effects were slightly enhanced. Salicylates were upregulated in UVR8 knockout poplars, suggesting metabolic flux in the pathway, but little difference was seen relative to wild-type plants in CAD lines, and UV-B treatment had little effect. An interesting and unexpected finding was that UVR8 modulated Populus exhibited more rapid growth than wild-type plants. The findings underscore the key role of UVR8 in synchronizing protective metabolic responses to UV-B and suggest an additional function of the photoreceptor in regulating growth and development of Populus through shifts in the chemical equilibria of UVR8 monomers and dimers and interactions with other regulatory factors.Item The Ecology of Early Cretaceous Angiosperms: Insights from the Fossil Record(2014) Jud, Nathan A.; Neel, Maile C.; Wing, Scott L.; Behavior, Ecology, Evolution and Systematics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The Early Cretaceous diversification of flowering plants was not preceded by a mass extinction event. This suggests that biotic factors intrinsic to flowering plants played an important role promoting diversification, but the rarity of fossils of early flowering plants makes identifying the important features difficult. Here, I present the results of my specimen-based analysis of plant megafossil collections from Lower Cretaceous deposits of the United States. First, I describe previously unrecognized eudicot leaf fossils from a historically important Aptian (Lower Cretaceous) plant fossil site in the Potomac Group, and I provide a set of characters for recognizing the fossil leaves of these plants. Then, I present a morphotype catalog for the fossil plants from and Aptian-early Albian (Lower Cretaceous) site in the Potomac Group. This collection includes one angiosperm morphotype. Next I describe the angiosperm morphotype identified in the previous chapter. I show that it is widely distributed among coeval collections of the Potomac Group and some specimens were previously described as ferns. The preservation of attached stems leaves and root provides direct evidence of weedy, fast-growing, herbaceous angiosperms in the Aptian-early Albian. In the following chapter, I use megafossil data from the literature and museum collections to test the hypothesis that the diversification of flowering plants is associated with an increase in alpha diversity during the Early Cretaceous. Despite the evidence for a high diversification rate among early flowering plants, I found no relationship between collection age and collection richness, but I found strong evidence that angiosperms were consistently rare during the Aptan-middle Albian, and that locally abundant angiosperms became common during the late Albian, long after the initial diversification. Finally, I use new plant megafossil collections that I made from the Cloverly and Sykes Mountain Formations in Wyoming, USA, for a more high-resolution study of early angiosperm diversity, distribution, and abundance. I show that the Cloverly Formation records the appeareance of flowering plants in North America, and that by the Albian angiosperms were widely distributed among available habitats. I test the hypothesis that variation in community composition (beta diversity) increased with the appearance of angiosperms. I did not find strong support for the hypothesis that angiosperms increased beta diversity; however, rarefaction analysis shows that the rate of morphotype discovery in both the pre-angiosperm and the angiosperm interval is high, which means that additional sampling may reveal a difference in beta diversity between the two intervals. Together my findings indicate that flowering plants diversified during the Early Cretaceous not because they had features that allowed them to displace other plant groups, but because they were uniquely able to maintain high diversification rates in the face of rarity and dispersed populations.Item AN INVESTIGATION OF MECHANISMS OF AN INTRON-MEDIATED GENE SILENCING(2014) Li, Dandan; Liu, Zhongchi; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Introns, non-coding regions between exons, exist in most eukaryotic genes. Many studies have shown that introns regulate gene expression through both transcriptional and posttranscriptional mechanisms. I revealed that in Arabidopsis thaliana, over-expression of the first and longest intron of CAULIFLOWER (CAL) gene led to the silence of the endogenous CAL gene through DNA methylation, which is mediated by the intron-derived 24 nt siRNAs. I investigated mechanisms of this intron-mediated gene silencing phenomenon through several different approaches, including northern blot, qRT-PCR, small RNA sequencing, bisulfite sequencing, McrBC-PCR, and bioinformatics. The CAL first intron does not show evolutionary conversation among different species in Rosid family. A distinctly folded stable secondary structure was found in the CAL first intron but its relevance to the silencing remains to be determined. Further, the CAL first intron likely possesses regulatory sequences demonstrated by the intron's ability to induce GUS reporter gene expression when fused upstream of a TATA box and the GUS gene. Antisense long non-coding RNAs (lncRNAs) from the intron was detected by qRT-PCR, which may pair with the over-expressed sense CAL first intron transcript from the transgene to from double stranded RNAs and subsequently generate 24 nt siRNAs. Therefore, this study provides a potentially novel and simple method to silence target genes by over-expressing cis regulatory elements either in introns or in promoters. I investigated how the intron-mediated silencing is inherited and showed that the silencing of CAL occurs in seeds and the silencing efficiency is dependent on the length of seed storage time. The extent of methylation in the CAL first intron increases when the seed age increases. This work has important biotechnology implications. Combined, my research not only describes a novel phenomenon of intron-mediated silencing but also sheds light in the mechanism of intron- or cis regulatory element-mediated gene silencing. Hence my research work will have broad implications both in basic research and in biomedical and agricultural applications.Item The comparative effects of three Sedum species on green roof stormwater retention(2013) Starry, Olyssa; Lea-Cox, John; Plant Science and Landscape Architecture (PSLA); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Green roofs are typically dominated by Sedum species because they can tolerate hot, xeric environments. However, due to their high water use efficiency, some have questioned the selection of these species for stormwater management. We investigated (1) how three common Sedum species contribute to overall stormwater retention efficiency by green roofs in the mid-Atlantic region, and (2) whether species-specific differences in water use could be explained by morphological and physiological characteristics. Water use and CO2 exchange were continuously monitored in growth chamber studies under increasing drought stress for S. album, and S. kamtschaticum, two species known to variably cycle between CAM and C3 metabolisms. Under fall temperature conditions, S. kamtschaticum had gas exchange rates akin to C3 photosynthesis and used 35% more water compared to S. album. Interestingly, S. album conserved water and had malic acid accumulation confirming CAM metabolism for the duration of the experiment, even under well-watered conditions. In field studies, sixteen replicate green roof platforms (n=4 per species) were planted with S. album, S. kamtschaticum, S. sexangulare, or left unplanted during summer 2010. The platforms were monitored intensively for canopy growth, leaf area, root biomass, substrate moisture and runoff for two years (2011 and 2012). Plant treatment effects on stormwater runoff were significant, but most discernible for small and intermediate-sized rainfall events less than 62.5mm. The two species with the greatest stormwater retention efficiencies, S. kamtschaticumand S. sexangulare, also had the highest rates of evapotranspiration (ET), and higher ET rates resulted in less total runoff. Because evapotranspiration was identified as important for predicting performance by plants in the field study, I investigated how ET data from this study, combined with environmental data collected from a weather station at the study site, could be used to improve the application of the FAO56 Penman-Monteith evapotranspiration equations to green roofs. The incorporation of specific seasonal crop coefficients were found to improve correlations between predicted and measured rates of ET and these coefficients were related to plant characteristics. The refinement of ET equations can lead to more accurate hydrologic models of green roofs and design and management tools.Item Specific Targeting of RPW8 Family Proteins To and de novo Biogenesis of the Extrahaustorial Membrane in Arabidopsis Cells Invaded By Powdery Mildew Fungus(2013) Berkey, Robert Michael; Xiao, Shunyuan; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The unique plant resistance (R) protein RPW8.1 and RPW8.2 confers broad-spectrum resistance in Arabidopsis to all tested isolates of Golovinomyces spp. fungi, the casual agents of powdery mildew disease in multiple plant species. RPW8.2 is specifically targeted to the extra-haustorial membrane (EHM) that encases the fungal feeding structure named the haustorium and represents the host-pathogen interface. EHM-localization of RPW8.2 correlates with haustorium-targeted host defense, providing subcellular evidence for the broad-spectrum resistance mediated by RPW8.2. RPW8.1 and RPW8.2 belong to a small gene family in the Arabidopsis and Brassica lineages. However, the cellular function of the other family members remains to be functionally characterized. Here, I report that all homologs of RPW8 (designated HR#) examined are EHM-residents, suggesting that the RPW8 family proteins share a common EHM-targeting signal. Moreover, through a reverse genetics approach I show that three Arabidopsis homologs, i.e. AtHR1, AtHR2 and AtHR3, appear to play a role in salicylic acid-dependent basal resistance against powdery mildew and perhaps other biotrophic pathogens. These results support our hypothesis that the two atypical resistance R genes, RPW8.1 and RPW8.2 evolved from duplication and functional diversification (enhancement) of a more ancient component of basal immunity in Arabidopsis (Chapter 2). Furthermore, I provide the first piece of cell biological evidence to suggest that the enigmatic EHM is formed via de novo synthesis rather than simple extension and differentiation of the host plasma membrane in the invaded host cell during the biogenesis of the fungal haustorium (Chapter 3). I also summarize my contribution to a project that aims to utilize RPW8 as a delivery vehicle to confer novel resistance in other crop species against a variety of fungal or oomycete haustorium-forming pathogens (Chapter 4) and ongoing efforts to further dissect the RPW8 defense and trafficking pathways in relation to bioactive phosphoinositides (Chapter 5) and to characterize putative interacting or signaling components of RPW8-mediated defense mechanisms against powdery mildew in Arabidopsis (Chapter 6).Item Post-transcriptional regulation of spermatogenesis through intron retention in the fern Marsilea vestita(2013) Boothby, Thomas Christopher Clark; Wolniak, Stephen M; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Many rapidly developing systems rely on the use of stored transcripts to carry out their developmental program. The microspore of M. vestita transcribes and stores RNA during a requisite period of desiccation. Rehydration of the microspore triggers spermatogenesis to begin, a process that is mediated by the utilization of these stored RNAs. Here I investigate mechanisms controlling the spatial and temporal utilization of these stored transcripts. Next generation Solexa based RNAseq was conducted using poly(A)+ RNA isolates from specific time ranges during spermatogenesis. A reference transcriptome as well as temporally specific transcriptomes were assembled de novo and analyzed for gene ontology enrichments. This analysis revealed an overrepresentation of catalytic splicing and nuclear speckle factors early in development suggesting that some transcripts are not fully mature. An in house Visual Basic for Applications program was used to identify potential intron retaining transcripts (IRTs) within our transcriptomes. A large subset of IRTs was identified and in silico and molecular biological approaches demonstrated that these IRTs are matured in a spliceosome dependent fashion at different times during development. Intron retention appears to confer a translational block to IRTs and splicing of retained introns alleviates this block. IRTs appear to be associated with splicing machinery organized in nuclear speckles. These subnuclear domains aggregate during desiccation and upon rehydration are proportioned asymmetrically to spermatogeneous cells. It appears that intron retention mediates both the association and asymmetric distribution of IRTs with nuclear speckles as well as their temporal utilization through post-transcriptional splicing.Item Genetic consequences of habitat fragmentation and restoration(2012) Lloyd, Michael Warren; Neel, Maile C.; Plant Science and Landscape Architecture (PSLA); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The objective my dissertation was to assess the effects of habitat loss and fragmentation on genetic diversity and landscape connectivity. I focused on Vallisneria americana Michx. (Hydrocharitaceae), a submersed aquatic plant species found in the Chesapeake Bay. Vallisneria americana has undergone dramatic changes in abundance and distribution throughout its range and has been targeted for restoration, which makes it ideal for examining the effects habitat loss and fragmentation. I examined the naturally occurring genetic diversity across the Chesapeake Bay and its major tributaries. Sites were genetically diverse, but had a range of genotypic diversities. There were four genetic regions, corresponding with geographic regions in the Bay. Vallisneria americana has been the target of restoration, and restoration techniques could be influencing genetic diversity and potentially lowering overall success. I examined various restoration techniques across eight restoration sites, and found that technique did not greatly influence genetic diversity. However, small population size, significant inbreeding coefficients, and low overlap of allele composition among sites provide cause for concern. Measures of functional and potential connectivity provide insights into the degree of contemporary gene flow occurring across a landscape. Pollen dispersal distance was measured using indirect paternity analysis, and is spatially restricted to only a few meters. Dispersal at this scale imposes small genetic neighborhoods within sites, evidenced by high seed relatedness within mothers. I used a graph theoretic approach to examine the distribution and potential connectivity of historic and current patches of V. americana. There was a high turnover in the distribution of patches, and connectivity varied through time, but even if all habitat were occupied, increases in overall network connectivity would not necessarily be observed. I developed an individual based model that I used to test the ability of measures of genetic differentiation to detect changes in landscape connectivity. Genetic differentiation measures became significant after two generations, but the magnitude of change in each was small in all cases and extremely small when population sizes are greater than 100 individuals. These results suggest that genetic differentiation measures alone are inadequate to rapidly detect changes in connectivity.Item Molecular and Genetic Analysis of Flower Development in Arabidopsis thaliana and the Diploid Strawberry, Fragaria vesca(2012) Hollender, Courtney Allison; Liu, Zhongchi; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In a world with a warming climate and a rapidly growing population, plant biology is becoming a field of increasing importance. Deciphering the molecular and genetic mechanisms behind the development of the flower, the fruit and seed progenitor, will enhance the agricultural productivity needed to ensure a sustainable food supply. My PhD research ties in with this need by furthering the basic knowledge of the mechanisms underlying flower development in two ways. First, using Arabidopsis thaliana, the classic model plant, I investigated the regulation of a gene, SPATULA (SPT), necessary for the proper development of the gynoecium, the female flower organ that, upon fertilization, directly gives rise to fruit. For flower and fruit to properly develop, the expression of SPT, must be tightly regulated both spatially and temporally. My research examined the mechanism of transcriptional repression of SPT in the sepals and petals by several interacting transcription factors (LEUNIG, SEUSS, APETALA2) and the molecular and genetic interaction between ETTIN and SPT in patterning gynoecium. The second focus of my research was to develop Fragaria vesca (the diploid strawberry), as a model Rosaceae for the study of flower and fruit development. Arabidopsis has much value as a small, fast growing, flowering plant with a multitude of genetic and genomic resources, however the flower of this mustard family weed is not representative of all crop flowers. The Rosaceae family, including many agriculturally important fruit trees such as apple, peach, blackberry, and strawberry, warrants its own model plant to investigate the distinct mechanisms behind their unique reproductive biology. Toward developing F. vesca as the model plant for studying Rosaceae flowers, I characterized and described developmental progression of F. vesca flowers morphologically through scanning electron microscopy and histological analysis as well as molecularly through transcriptomes and in situ hybridization. In addition, I pioneered a small-scale mutagenesis screen of F. vesca that will lead to future genetic resources. My thesis work places the groundwork for future discoveries in F. vesca and Rosaceae and benefits research, education, and agricultural applications for the Rosaceae and the plant biology communities.Item Investigating the molecular mechanism of RTE1 activation of the ethylene receptor ETR1 in Arabidopsis(2011) Chang, Jianhong; Chang, Caren; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The plant hormone ethylene plays a vital role in regulating plant growth and development as well as plant defense to biotic and abiotic stresses during the entire life of the plant. In Arabidopsis, ethylene is perceived by a family of five receptors, one of which is ETR1. The Arabidopsis REVERSION-TO-ETHYLENE SENSITIVITY1 (RTE1) gene is a positive regulator of ETR1. RTE1 encodes a novel integral membrane protein that interacts with ETR1 at the Golgi apparatus and the endoplasmic reticulum (ER). Genetic evidence indicates that RTE1 is required for the formation of a functional ETR1 receptor, whereas the other ethylene receptors in Arabidopsis do not require RTE1. But the molecular mechanism by which RTE1 specifically activates ETR1 remains unknown. I took different approaches to gain insights into the molecular function of RTE1 and the basis for the specificity for activating ETR1. In a library screen for RTE1–interacting proteins using the yeast split–ubiquitin assay, an ER–localized cytochrome b5 isoform (AtCb5–D) was identified. Cb5 is a small hemoprotein that functions in oxidation/reduction reactions. Mutants of three AtCb5 isoforms show phenotypes in ethylene responses that are similar to those of the rte1 mutant, suggesting the functional parallel between AtCb5 and RTE1 in ethylene signaling. Additional genetic analyses suggest that AtCb5 might act in the same pathway as RTE1 and that AtCb5 is specific to ETR1 like RTE1. Moreover, using a hemin–agarose affinity chromatography assay, I found that RTE1 homologs are able to bind heme in vitro, raising the possibility that RTE1 carries out redox with cytochrome b5s. I also found that the specificity for regulating ETR1 by RTE1 is largely due to a unique proline (P9) conserved only in ETR1 orthologs; introduction of P9 into the Arabidopsis ERS1 ethylene receptor was sufficient to convert ERS1 into an RTE1–dependent receptor. I propose that P9 may interfere with the proper folding of ETR1 EBD and formation of the ETR1 homodimer by affecting the conserved disulfide bond–forming cysteines (C4, C6) in the ETR1 homodimer. Taken together, our results suggest a model in which RTE1, together with cytochrome b5, promotes the active conformation of ETR1 through oxidative folding.Item PLANT-SPECIFIC K+ TRANSPORTERS WITH DISTINCT PROPERTIES AND THEIR EMERGING ROLES IN ENDOMEMBRANE TRAFFICKING(2011) Chanroj, Salil; Sze, Heven; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Plant growth, development and survival are dependent on the dynamic endomembrane system that modulates the delivery of various cargos to their destination or removal of components for degradation in time and in space. Yet the molecular determinants that regulate this dynamic and intricate machinery in plants are poorly understood. The multiplicity of Cation/H+ eXchangers (CHX) genes in higher plants, but not in metazoans, raises questions about their transport activity and suggests roles characteristic to plant life. Five CHX proteins from Arabidopsis thaliana were implicated in pH homeostasis as each rescued growth of alkaline-sensitive yeast strains, though with distinct properties. Each CHX improved growth of K+-uptake deficient strain at different pHs. Moreover, CHX17, 18 and 19 conferred hygromycin B resistance when CHX20 could not. Although both CHX17 and CHX20 mediated K+ uptake when expressed in Escherichia coli; their properties suggested differential modes of transport. CHX20, but not CHX17, acidified cytoplasmic pH and alkalinized vacuolar pH in yeast. CHX17 was more effective than CHX20 in reducing secretion of a vacuolar lumen protein in yeast. As these CHXs were localized to yeast endomembranes, results suggested that CHXs differentially modulate pH and K+ homeostasis of intracellular compartments which affect protein sorting. CHX20-tagged with fluorescent protein was localized to ER, whereas CHX16, 17, 18 and 19 localized to prevacuolar compartments (PVC) and to plasma membrane (PM) in planta. Brefeldin A diminished PM-associated CHX17, whereas wortmannin caused formation of ring-like structures of CHX17-bound compartments. When full-length CHX17 (820 residues) was truncated, CHX17(1-472) partially restored yeast growth on alkaline medium. However, the truncated protein was localized to Golgi, suggesting a role of the C tail in sorting CHX17 from Golgi to PVC and PM. These results suggest that CHX17 is associated with a subset of endosomes that traffick among PVC, vacuole and PM in cells of whole plants. Together, the results support a model where CHX17 and its homologs modulate localized pH and K+ environment of distinct endomembrane compartments and so affect membrane trafficking and cargo sorting through the endocytic and/or secretory pathways. CHXs are proposed to facilitate cell wall modifications as plants adapted to dry land.