Theses and Dissertations from UMD

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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 give thesis/dissertation in DRUM

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Subject: search.filters.subject.Biology, Plant Physiology

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Now showing 1 - 10 of 21
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    Suppressors of etr1-2: I. etr1-11 is a loss-of-function mutation of the ETR1 ethylene receptor. II. REVERSION TO ETHYLENE-SENSITIVITY3 is a regulator of seedling growth.
    (2009) McClellan, Christopher Alan; Chang, Caren; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The plant hormone ethylene is an important regulator of plant growth and development, including senescence, abcission, fruit ripening, and responses to biotic and abiotic stresses. To find new members of the ethylene signaling pathway, a genetic screen for suppressors of the ethylene-insensitive mutant etr1-2 was performed. One mutant identified in this screen, etr1-11, is an intragenic mutation within ETR1. etr1-11 is a unique missense mutation that appears to eliminate ETR1-2 signaling. Mutant analysis further revealed that etr1-11 is a partial loss-of-function allele. The rte3 (reversion to ethylene sensitivity3) mutant was another mutant isolated in a genetic screen for suppressors of etr1-2. After testing other ethylene responses, such as leaf senescence, and performing epistasis analysis with other ethylene signaling mutants, it was determined that RTE3 is unlikely to play a direct role in the ethylene signaling pathway. Instead, RTE3 appears to be responsible for promoting hypocotyl elongation in etiolated seedlings in the ethylene triple response assay. The RTE3 gene was identified by positional cloning, and is predicted to encode a protein with an annotated SAC3/GANP domain. SAC3/GANP domains are present in proteins that participate in large multi-peptide complexes, such as the 26S proteasome regulatory subunit and the eIF3 translation initiation complex. Similarities in protein composition between these two complexes and the COP9 signalosome (CSN) suggest that a SAC3/GANP domain-containing protein may interact with members of the CSN. Interestingly, yeast two-hybrid analysis reveals that RTE3 interacts with EER5 and EIN2, proteins that have been shown to interact with members of the CSN. In addition, rte3-1 ein2-1 seedlings show a synthetic phenotype of delayed growth. Protein localization using a GFP tag reveals that RTE3 and EER5 both localize to the nucleus. These interactions suggest that RTE3, EER5, EIN2, and the CSN form a protein complex that regulates seedling growth.
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    LEUNIG, LEUNIG HOMOLOG, AND SEUSS ARE TRANSCRIPTIONAL CO-REPRESSORS THAT REGULATE FLOWER DEVELOPMENT, MUCILAGE SECRETION, AND PATHOGEN RESISTANCE
    (2009) Bui, Minh; Higgins, William J; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Transcriptional repression is an important regulatory mechanism for development. My thesis focuses on dissecting the function of Groucho (Gro)/Transducin-Like Enhancer of split (TLE) family of transcriptional repressors in plant development. My work characterizes two Arabidopsis thaliana genes, LEUNIG (LUG), first discovered to repress transcription of the floral homeotic gene AGAMOUS (AG), and LEUNIG_HOMOLOG (LUH), a gene with the highest sequence similarity to LUG. To investigate the functional redundancy between LUG and LUH, I constructed and analyzed lug; luh double mutants, and concluded that both LUG and LUH repress AG expression in the flower, with LUG playing a more prominent role than LUH. The double mutant also revealed a previously unknown function of LUG and LUH in embryogenesis because lug-3; luh-1 double mutants are embryo lethal, while the single mutants develop normal embryos. During the course of this study, I developed a new genotyping method called Simple Allele-discriminating PCR (SAP), which is cost-effective, quick, and easy to perform. This method has greatly facilitated my research as well as others in the lab. A second part of my thesis addresses the role of LUG and LUH in other developmental processes besides flower development. My data indicate that these two genes, like their counter parts in fungi and animals, act as "global co-repressors" in various developmental and physiological processes. My thesis work revealed that both co-repressors, together with its interacting protein SEUSS (SEU), repress the Salicylic Acid (SA) pathogen defense pathway. Although lug-3, luh-1, and seu-1> mutants induced PR1 expression at higher levels than wild-type, only lug-3 and seu-1 mutants were pathogen resistant. Furthermore, LUH functions as a positive regulator in seed mucilage secretion, a process important for proper seed germination, hydration, and dispersal. I propose a possible connection between the defect in mucilage secretion and pathogen defense in luh-1 mutant plants and seeds, which places the foundation for further investigation and may uncover mucilage secretion as a major defense mechanism. My thesis has provided important insights into how transcriptional co-repressors regulate diverse developmental and physiological pathways in higher plants.
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    Investigation of Ethylene Signal Transduction Mechanisms: Characterizing the Novel Gene AWE1 and Testing Hypothesis of Raf-like CTR1's Function In Vivo
    (2009) Kendrick, Mandy Danielle; Chang, Caren; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Ethylene is a gaseous plant hormone affecting multiple plant processes. Sixteen years ago the first components of the ethylene signaling pathway, the receptor ETR1 and Raf-like kinase CTR1, were identified. Since then many additional components of the pathway have been elucidated through genetic screens. Recent discoveries suggest ethylene signaling, once thought to be a linear pathway from ethylene perception at the endoplasmic reticulum to transcriptional activation at the nucleus, is more complex with multiple auto-feedback loops and potential parallel kinase cascades downstream of the receptors. Although the genetic backbone of the pathway is well established, the signaling mechanisms of the components remain unclear. ETR1 displays histidine kinase activity in vitro and physically interacts with the next-known downstream component of the pathway, CTR1. However the histidine kinase activity of ETR1 is mostly dispensable for signaling to CTR1. How then is CTR1 activated? I proposed that additional proteins, like AWE1, play a role in ETR1 to CTR1 signaling, and that the non-catalytic, amino-terminal region of CTR1 is required both for activation through direct interaction with the ETR1 receptor complex and for auto-inhibition of CTR1 kinase activity. ASSOCIATES-WITH-ETR1 (AWE1) was isolated in a yeast-two-hybrid screen for ETR1-interacting proteins and was of specific interest because the AWE1 clone also interacted with a portion of CTR1. Protein-protein interaction studies and genetic analysis of an awe1 mutant support a role of AWE1 in repressing ethylene responses. However double mutant analysis, over-expression analysis, and protein sub-cellular localization studies suggest that AWE1's function in hypocotyl elongation and cell expansion is more general. AWE1's function may require ETR1 for proper regulation but is likely to lie outside of the direct step from ETR1 to CTR1. To investigate a role of the CTR1 amino-terminal region in CTR1 regulation, I constructed transgenes consisting of truncated ETR1 receptors fused to truncated or full length CTR1 and examined how those transgenes carrying the truncated CTR1 (kinase domain only) affected Arabidopsis seedling growth compared to those transgenes expressing full length CTR1. I concluded that the CTR1 amino-terminal region may have a role in autoregulation, but additional components are required for regulation of CTR1 signaling.
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    Acclimation of marine macrophytes (Saccharina latissima and Zostera marina) to water flow
    (2008-05-12) Jordan, Terry Lynn; Koch, Evamaria; Davison, Ian; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    I examined the physiological response of two marine macrophytes, the brown alga Saccharina latissima and the angiosperm Zostera marina, to water flow in nature and in controlled experiments. Limitation of photosynthesis of both species by the availability of dissolved inorganic carbon (DIC) was increased under low current velocities. Physiological acclimation to low water flow occurred via upregulation of DIC uptake mechanisms in both S. latissima and Z. marina. Both species increased their ability to generate CO2 in the boundary layer by increasing external carbonic anhydrase and in Z. marina by also increasing proton extrusion and photosynthetic capacity. Changes in the xanthophyll-cycle in low-flow grown S. latissima increased non-photochemical quenching, thus reducing photodamage when photosynthesis was limited by DIC uptake. Water flow also affected root length in Z. marina but root length and below ground biomass were also significantly affected by sediment type, an indirect effect of water flow.
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    TYPE II MADS-BOX GENES ASSOCIATED WITH POPLAR APICAL BUD DEVELOPMENT AND DORMANCY
    (2008-04-25) Chen, Kuang-Yu; Coleman, Gary D; Plant Science and Landscape Architecture (PSLA); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    MADS-box transcription factors regulate the development of vegetative and reproductive organs in plants. Little is known about the role of MADS-box genes in tree development. Using phylogenetic analysis, 57 putative type II MADS-box genes representing 14 functional classes were identified in the Populus trichocarpa genome. cDNA sequencing of the poplar type II MADS-box genes indicates that 28.1% of the transcripts differed in the intron-exon structures predicted in the genome database and 19.3% of the transcripts appear to be alternatively spliced. The majority of the poplar type II MADS-box genes were expressed in a wide variety of tissues including shoot apices, leaves, bark, xylem, root, and floral tissues and in shoot apices during bud development and dormancy. These results indicate that poplar MADS-box genes have diverse regulatory roles in a broad range of tissues and developmental processes. Six poplar FLC-like genes, PtFLC1-PtFLC6, were identified in the poplar genome and expression of all six genes was detected in poplar shoot apices. The expression of one gene, PtFLC2, declined in apical buds during SD photoperiod and low temperature induced dormancy development suggesting a role in bud dormancy and may represent an analogous regulatory mechanism to the down-regulation of FLC during vernalization in Arabidopsis. In addition, several PtFLC2 splice isoforms (PtFLC2as1-9) were identified that were associated with the later stages of bud dormancy. Overexpression of the PtFLC2as1 isoform delayed photoperiod induced apical bud development and bud dormancy, growth cessation, and leaf senescence while overexpression of the PtFLC2as2 isoform appeared to accelerate bud development and dormancy and reduce the amount of chilling required to overcome dormancy. These findings suggest that PtFLC2, unlike Arabidopsis FLC, could be an integration point for both photoperiod and cold signals that regulate bud development and dormancy. These results also suggest that in addition to transcriptional regulation, that cold-mediated production of PtFLC2 splicing isoforms may have an important regulatory role in bud dormancy. The regulated production of splicing isoforms could regulate bud dormancy either by dominate negative interactions, by forming different protein complexes or regulating different pathways that regulate growth, dormancy, and dormancy release.
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    Functions of the Tobacco mosaic virus helicase domain: regulating formation of the virus replication complex and altering the activity of a host-encoded transcription factor
    (2008-04-23) Wang, Xiao; Culver, James N; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Tobacco mosaic virus (TMV)-encoded 126-kDa and 183-kDa replicases are multidomain and multifunctional proteins. The helicase domain shared by both replicases has been shown to perform multiple tasks during the virus life cycle. In vitro structural and functional analyses demonstrated that monomers and dimers of the TMV helicase domain were the active forms for ATP hydrolysis. However, self-interaction of the helicase polypeptides resulted in the formation of higher-order structures that likely serve as structural scaffolds for the assembly of virus replication complexes (VRCs). Mutagenesis studies of the TMV helicase motifs showed that conserved amino acid residues played important roles in protein ATPase and/or RNA binding activities. A close correlation between ATPase activity of the helicase domain and assembly of wild-type VRC-like vesicles by the 126-kDa replicase further suggests that ATPase activity of the TMV helicase domain may modulate proper VRC assembly. In addition to helicase self-interaction, a novel virus-host interaction involving ATAF2, a NAC domain transcription factor was identified. Members within the NAC domain family are involved in plant developmental processes and stress/defense responses. In this study, transgenic plants overexpressing ATAF2 showed a strong developmental phenotype. Inoculation of TMV in these transgenic plants resulted in reduced virus accumulations. Additionally, transcriptional induction of ATAF2 occurred in response to TMV infection and salicylic acid treatment. Combined, these results suggest that ATAF2 is involved in a host defense response. One interesting finding was that in susceptible hosts, virus-directed induction of ATAF2 and PR1, a well-defined pathogenesis-related (PR) marker gene for host defense system, occurred only in locally-infected but not in systemically-infected tissues. Dynamic changes in the expression of host defense genes suggest that viruses have evolved certain mechanisms to actively modulate host gene expression. Interaction between the TMV helicase domain and ATAF2 may provide one way to suppress the ATAF2-mediated host defense signaling pathway. Combined these studies investigated the importance of the TMV helicase domain in VRC formation and in manipulating the host defense system. The results confirmed the functional versatility of the TMV helicase domain in establishing a successful virus life cycle.
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    High-Throughput Time Series Metabolomic Analysis of a Systematically Perturbed Plant System
    (2007-04-27) Kanani, Harin H; Klapa, Maria I; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    In the post-genomic era, availability of high-throughput profiling techniques enabled the measurement of entire cellular molecular fingerprints. Major characteristics of the high-throughput revolution were that (a) studying biological problems did not have to rely on prior hypotheses, while (b) parallel occurring phenomena, previously assumed disconnected, could now be simultaneously observed. Metabolomics is the newest of the "omics" techniques. It enables the quantification of hundreds of free metabolite pools, providing a metabolic fingerprint. Considering the importance of cellular metabolism, which is the net effect of changes at the genomic, transcriptomic and proteomic levels and of the cell with its environment, the metabolomic profile, is a fundamental determinant of cellular physiology. Obtaining accurate and complete metabolomic profiles is thus of great importance. However, being recent technology, metabolomics is currently at its standardization phase. As part of my PhD thesis research, I focused on addressing several current challenges in metabolomics technology development. Specifically a novel data correction, validation and normalization strategy for gas chromatography-mass spectrometry (GC-MS) metabolomic profiling analysis was developed, which dramatically increased the accuracy and reliability of GC-MS metabolomic profiles. The optimized metabolomics protocol was applied to study the short-term dynamic response of systematically perturbed Arabidopsis thaliana liquid culture system to study regulation of its primary metabolism. The biological system was studied under conditions of elevated CO2 stress, salt (NaCl) stress, sugar (trehalose) signal, and hormone (ethylene) signal, applied individually; the latter three stresses also applied in combination with the CO2 stress. Analysis of the obtained results required the appropriate application of multivariate statistical analysis techniques, which are developed mainly in transcriptomic analysis, into metabolomics analysis for the first time. The acquired results identified important new regulatory information about the biological systems resulting in new targets for metabolic engineering of plants. The large number of dynamic perturbation allowed re-construction of metabolic networks to identify possible novel metabolic pathways based on correlations between metabolic profiles. In addition, it demonstrates the advantages of dynamic, multiple-stress "omic" analysis for the elucidation of plant systems function. In this sense, it contributes in further advancing the computational and experimental metabolic engineering and systems biology toolbox.
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    The effects of solar ultraviolet-b on the proteome and antioxidant defense systems in soybean leaves
    (2007-04-24) Xu, Chenping; Sullivan, Joe H.; Plant Science and Landscape Architecture (PSLA); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Stratospheric ozone depletion has caused an increase in the amount of ultraviolet-B (UV-B) radiation reaching the earth's surface. Some investigations have demonstrated that UV-B has effects on protein accumulation and active oxygen species (AOS) metabolism in plants. Because of the unrealistically high UV-B level and low levels of ultraviolet-A (UV-A) and photosythetically active radiation (PAR) in indoor studies it has been questioned whether results from these studies can be extrapolated to field responses. In the present study two isolines of the soybean cultivar Clark with different flavonoid contents were grown in the field with or without natural levels of UV-B. The leaf proteome and AOS metabolism were examined. Ambient solar UV-B radiation changed AOS metabolism by decreasing superoxide dismutase activity and increasing the activities of ascorbate peroxidase, catalase and glutathione reductase relative to UV-B exclusion treatments. This resulted in decreased ascorbic acid and increased dehydroascorbate content. Proteomic analysis showed that the accumulations of 67 protein spots were significantly affected by solar UV-B. Proteins related to photosystems in photosynthesis were increased in abundance while enzymes involved in the primary carbon and nitrogen metabolism were decreased. This could lead to overreduction of the photosynthetic electron transport chain and enhance the formation of superoxide radicals and singlet oxygen. The magenta line, which has reduced flavonoid levels, had greater proteomic and oxidative responses than the standard line, suggesting that flavonoids act as screening compounds and antioxidants in protecting plants from UV-B radiation. These line-specific differences occurred even under UV-B exclusion, which may be due to high UV-A, PAR or temperature. More detailed studies are needed to elucidate the effects of other environmental factors on the soybean leaf proteome and AOS metabolism under field conditions.
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    MOLECULAR CHARACTERIZATION OF INTERACTIONS BETWEEN TMV REPLICASE PROTEIN AND AUXIN RESPONSIVE PROTEINS: IMPLICATIONS IN DISEASE DEVELOPMENT
    (2006-11-25) Padmanabhan, Meenu Sreedevi; Culver, James; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Tobacco Mosaic Virus and Arabidopsis thaliana serve as ideal model systems to study the molecular aspects of virus - host interactions. Using this system, an interaction between the helicase domain within TMV replicase protein and an auxin responsive protein, IAA26 was identified. IAA26 is a member of the Aux/IAA family of transcription factors that function as repressors in signaling pathways controlled by the phytohormone auxin. Characterization of the interaction was carried out utilizing a helicase mutant defective in its interaction with IAA26 and by creating transgenic plants silenced for IAA26 expression. These studies suggest that the interaction was not essential for either viral replication or movement but promoted the development of disease symptoms. Cellular co-localization studies revealed that in TMV infected tissue, the nuclear localization and stability of IAA26 was compromised and the protein was relocalized to distinct cytoplasmic vesicles in association with the viral replicase. In keeping with its role as a transcription factor, the alterations in IAA26 function should lead to misregulation of downstream auxin responsive genes and this is supported by the fact that ~ 30% of the Arabidopsis genes displaying transcriptional alterations to TMV could be linked to the auxin signaling pathway. Aux/IAA family members share significant sequence and functional homology, and an additional interaction screen identified two more Arabidopsis Aux/IAA proteins, IAA27 and IAA18 and a putative tomato Aux/IAA protein, LeIAA26 that could interact with TMV helicase. The nuclear localization of these three proteins was disrupted by TMV and alterations in LeIAA26 levels induced virus infection-like symptoms in tomato. Additionally, transgenic plants over-expressing a proteolysis resistant mutant of IAA26 showed abnormal developmental phenotype, the severity of which was abrogated during TMV infection which blocked nuclear accumulation of the protein. Taken together, these findings suggest that TMV induced disease symptoms can partially be explained by the ability of the virus to disrupt the functioning of interacting Aux/IAA proteins within susceptible hosts. The significance of such interactions is yet to be determined but it appears that they may be advantageous to the virus while infecting tissues that are in a developmentally static stage.
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    REVERSION-TO-ETHYLENE-SENSITIVITY1: A Novel Regulator of Ethylene Receptor Function in Arabidopsis thaliana
    (2006-11-25) Resnick, Josephine; Chang, Caren; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Ethylene is a plant hormone that has profound effects on plant growth and development. Genetic analysis has been central in the elucidation of the ethylene-signaling pathway, made possible through the isolation of ethylene-response mutants in Arabidopsis. This thesis focuses on elucidating the function of the Arabidopsis REVERSION-TO-ETHYLENE-SENSITIVITY1 (RTE1) locus, which was identified in a genetic screen for suppressors of the ethylene-insensitive receptor mutant etr1-2. The RTE1 gene was cloned by positional cloning and found to encode a novel integral membrane protein with homologs in plants and animals, but with no known molecular function. Our studies show that RTE1 is a negative regulator of the ethylene-response pathway, specifically acting as a positive regulator of the ETR1 ethylene receptor. Loss-of-function mutations in the RTE1 gene suppress the etr1-2 ethylene-insensitive phenotype, and genetic analysis suggests that loss of RTE1 results in a largely non-functional ETR1-2 mutant receptor. Similarly, wild-type ETR1 function appears to be greatly reduced in the absence of RTE1. Overexpression of the RTE1 gene confers weak ethylene insensitivity that is largely dependent on ETR1. rte1 mutations do not appear to affect the other four ethylene receptors of Arabidopsis, indicating that RTE1 specifically regulates ETR1. Sequence analysis revealed regions of conserved cysteine and histidine residues, and one rte1 loss-of-function mutant contains a point mutation at Cys161. Since such residues are common in metal binding proteins, we explored the possibility that RTE1 may be involved in facilitating the binding of an essential copper cofactor to the ETR1 receptor. However, experimental evidence suggests that this is not the likely role of RTE1. Interestingly, rte1 was unable to suppress the ethylene insensitive mutant etr1-1, indicating that the differences between etr1-2 and etr1-1 may hold a clue as to how RTE1 regulates ETR1. A suppression analysis of eleven additional etr1 insensitive mutants suggests that RTE1 plays a role in regulating signaling by the transmitter domain of ETR1. A possible role for RTE homologs in non-plant systems is also discussed, although more work is required to elucidate a detailed biochemical model for RTE1 action.