Theses and Dissertations from UMD
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Item Probing the 3D Structure and Function of a Cation/H+ Exchanger in Plant Reproduction(2015) Czerny, Daniel; Sze, Heven; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Maintaining intracellular pH and K+ homeostasis are necessary for a cell to divide, grow, and communicate with other cell types. How a cell responds to stimuli and subsequently regulates intracellular pH and K+ content are largely unknown. Ion transporters, including cation/H+ exchangers are one potential determinant of intracellular pH and K+ content. A novel family of CHX transporters, predicted to exchange a cation for a H+, is found in all land plants, though their functions in the plant and the mode of transport are mostly unknown. What is the mode of transport of Arabidopsis thaliana CHX17? Model structures of the CHX17 transmembrane domain were built from two crystallized bacterial Na+/H+ antiporters. Based on protein architecture and homology, residues were selected for mutagenesis and CHX17 activity was tested in yeast. Thr170 and Lys383 in the discontinuous α-helices of transmembrane 4 and 11, and Asp201 and Lys355 in the middle of transmembranes 5 and 10 are necessary for CHX17 activity. Results suggest these are core residues that participate in cation binding and/or catalysis. Glu111 near the cytosolic surface of CHX17 was necessary for activity, suggesting CHX17 could be regulated by cytosolic pH. Thus the protein fold and mode of transport of Arabidopsis CHX17 resemble a K+/H+ exchanger. What roles do K+/H+ exchangers play in plant reproduction? chx17/18/19 mutant plants showed a 56%-77% reduction in seed set though the biological basis was unknown. Reciprocal crosses showed reduced seed set was primarily caused by defects in the male gametophyte. Mutant chx17/18/19 pollen grains developed normally and pollen tubes grew and reached most ovules. However, half the ovules receiving a mutant pollen tube failed to develop. Wild-type pistils that received chx17/18/19 pollen showed unfertilized ovules, ovules with single fertilizations, and some embryos that developed similarly to wild-type. Thus, some triple mutant pollen showed failure to complete fertilization. When fertilization was successful, embryos from self-fertilized chx17/18/19 pods showed delays in development. Our findings suggest maintenance of pH and K+ homeostasis in endomembrane compartments by CHX17 and its homologs could regulate membrane trafficking events necessary for pollen tube growth, male gamete fusion, and embryo development.Item Mapping metabolic fluxes in plant cells to understand carbon-nitrogen interactions and nitrogen storage and cycling(2012) Nargund, Shilpa; Sriram, Ganesh; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Plants provide commodities like food, fiber, fuel and chemicals. Understanding plant metabolism will help find genetic engineering targets that enhance production of these commodities. Interactions between the macronutrients - carbon (C) and nitrogen (N) determine growth and developmental functions in plants (Nunes-Nesi, Fernie, and Stitt 2010; Sakakibara, Takei, and Hirose 2006) and are regulated by complex mechanisms that need systems-level analyses. Metabolic fluxes, the rates of C flow in metabolic pathways, provide a system-wide view of metabolism and are quantified by steady state metabolic flux analysis (MFA) wherein isotopic tracers (13C, 15N) are fed to the cells and the resulting labeling patterns of biomass components are used to fit the fluxes. In this study we i) statistically designed isotope labeling experiments (ILEs) in silico to enhance accuracy of flux estimates through the pentose phosphate pathway (PPP) ii) conducted MFA on heterotrophic cell suspensions of Arabidopsis thaliana (Arabidopsis), a model plant, to investigate regulatory role of light in cell metabolism and iii) conducted MFA on cell suspensions of poplar (Populus tremula × Populus alba; clone N 717-B4), a potential biofuel crop, to understand C-N interactions. In silico label design studies determined that accuracy of flux estimates in the PPP improves by ILEs with 1,2-13C glucose and measuring labeling patterns of sugars, especially ribose. Metabolic fluxes, estimated by the designed ILEs on Arabidopsis cells, under continuous light or dark, showed negligible changes between treatments indicating that light does not regulate central carbon metabolism in heterotrophic Arabidopsis cells. The designed ILEs improved confidences of non-oxidative PPP flux estimates by 40-80% from previous studies (Masakapalli et al. 2009a). ILEs on poplar cell suspensions, grown in batch cultures, displayed unexpected back-mixing between unlabeled seed biomass and newly synthesized labeled biomass. Novel metabolic network models were developed that successfully account for observed back-mixing. ILEs on poplar cells, subjected to different C-N supply treatments to understand C-N interactions showed significant differences in phenylalanine labeling which may implicate role of flavonoid biosynthesis pathway in C-N interactions. Design of ILEs and subsequent improvement in flux estimates and the improvements in modeling metabolic networks are the novel contributions of this work.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 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.Item Time-Series Transcriptomic Analysis of a Systematically Perturbed Arabidopsis thaliana Liquid Culture System: A Systems Biology Perspective(2007-05-16) Dutta, Bhaskar; Klapa, Maria I; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Revealing the gene regulation network has been one of the main objectives of biological research. Studying such a complex, multi-scale and multi-parametric problem requires educated fingerprinting of cellular physiology at different molecular levels under systematically designed perturbations. Conventional biology lacked the means for holistic analysis of biological systems. In the post-genomic era, advances in robotics and biology lead to the development of high-throughput molecular fingerprinting technologies. Transcriptional profiling analysis using DNA microarrays has been the most widely used among them. My Ph.D. thesis concerns the dynamic, transcriptional profiling analysis of a systematically perturbed plant system. Specifically, Arabidopsis thaliana liquid cultures were subjected to three different stresses, i.e. elevated CO2 stress, salt (NaCl) stress and sugar (trehalose) applied individually, while the latter two stresses were also applied in combination with the CO2 stress. The transcriptional profiling of these conditions involved carrying out 320 microarray hybridizations, generating thus a vast amount of transcriptomic data for Arabidopsis thaliana liquid culture system. To upgrade the dynamic information content in the data, I developed a statistical analysis strategy that enables at each time point of a time-series the identification of genes whose expression changes in statistically significant amount due to the applied stress. Additional algorithms allow for further exploration of the dynamic significance analysis results to extract biologically relevant conclusions. All algorithms have been incorporated in a software suite called MiTimeS, written in C++. MiTimeS can be applied accordingly to analyze time-series data from any other high-throughput molecular fingerprint. The experimental design combined with existing multivariate statistical analysis techniques and MiTimeS revealed a wealth of biologically relevant dynamic information that had been unobserved before. Due to the high-throughput nature of the analysis, the study enabled the simultaneous identification and correlation of parallel-occurring phenomena induced by the applied stress. Stress responses comparisons indicated that transcriptional response of the biological system to combined stresses is usually not the cumulative effect of individual responses. In addition to the significance of the study for the analysis of the particular plant system, the experimental and analytical strategies used provide a systems biology methodological framework for any biological system, in general.Item TIME SERIES METABOLIC PROFILING ANALYSIS OF THE SHORT TERM Arabidopsis thaliana RESPONSE TO ELEVATED CO2 USING GAS CHROMATOGRAPHY MASS SPECTROMETRY.(2004-08-30) Kanani, Harin Haridas; Klapa, Maria I; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Metabolic profiling has emerged as a high throughput technique for the quantitative analysis of the cellular physiological state at the metabolic level. It allows for the simultaneous relative quantification of hundreds of low molecular weight intra cellular metabolites. In this analysis, the polar metabolic profiles of A. thaliana liquid cultures (grown for 12 days, under light and 23°C) throughout 1-day treatment with 1% CO2, were measured using gas chromatography-mass spectrometry. Despite the advantages of time series analysis, this is the first plant metabolic profiling study of this type reported in the literature. The time series metabolic profiles were analyzed using multivariate statistical techniques. Data analysis revealed repression of photorespiration, repression of nitrogen assimilation and increase in structural carbohydrates. It is for the first time that the latter phenomenon is observed as a result of elevated CO2 in the plant environment.Item TIME SERIES TRANSCRIPTIONAL PROFILING ANALYSIS OF THE Arabidopsis thaliana USING FULL GENOME DNA MICROARRAY AND METABOLIC INFORMATION(2004-08-26) Dutta, Bhaskar; Klapa, Maria I; Quackenbush, John; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)With the advent of the DNA microarray technology, it became possible to study the expression of entire cellular genomes. Tanscriptional profiling alone can not provide a comprehensive picture of the cellular physiological state and it should be complemented by other cellular fingerprints. Transcriptional profiling combined with metabolic information of a systematically perturbed system can unravel the relationship between gene and metabolic regulation. In this context the transcriptional response of Arabidopsis thaliana liquid cultures (grown for 12 days under light and 23 sup oC) to 1-day treatment with 1% CO sub 2 was measured by full genome cDNA microarrays. The Time series gene expression profiles were analyzed in the context of the known Arabidopsis thaliana physiology using multivariate statistics. Data analysis revealed an increase in the rate of CO sub 2 fixation, biomass production and cell wall growth. The breadth of the information obtained from a single experiment validated the significance of the high throughput transcriptional profiling.