Theses and Dissertations from UMD
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Item INVESTIGATING MECHANISMS UNDERLYING MLO’S ROLE AS A HOST FACTOR ESSENTIAL FOR PATHOGENESIS OF POWDERY MILDEW FUNGI(2024) Bloodgood, David; Xiao, Shunyuan; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Loss-of-function mutations in Mildew Locus O (MLO) family genes confer broad-spectrum resistance to powdery mildew (PM) fungi in various plant species. mlo-mediated resistance is invariably coupled with increased defense responses and early leaf senescence; hence the molecular basis of mlo-mediated resistance remains unresolved. A saturated genetic screen in the background of an Arabidopsis triple mutant where three essential immune components, EDS1, PAD4 and SID2 are mutated, led to the identification of five allelic mutations in MLO2, each of which results in compromised immunity yet poor infection (cipi) to PM. Further CRISPR-targeted mutagenesis of two functional homologs, MLO6 and MLO12 in a cipi mutant background result in complete lack of infection from PM fungi. The sextuple mutant, eds1pad4sid2mlo2mlo6mlo12 (epsm3) showed no early leaf senescence, ROS accumulation or expression of defense genes, indicating that MLO2, MLO6 and MLO12 are bona fide host susceptibility factors for PM. Expression of MLO2-GFP as a transgene in epsm3 restores susceptibility to PM and MLO2-GFP focally accumulates at the fungal penetration site. Thus, restoration of susceptibility to PM in the epsm3 background can be used as a sensitive reporter to assess whether other MLO family members share a conserved molecular function when expressed in leaf epidermal cells. The Barley MLO and Arabidopsis MLO7 enabled PM pathogenesis whereas MLO1, MLO3 and MLO4 could not, suggesting the existence of two distinct classes of MLO family members. Sequence alignment identified three conserved amino acid residues in the C terminal calmodulin-binding domain of MLO2, and MLO7, which are absent in MLO1, MLO3 and MLO4. This observation suggests that the C-terminal domain of MLO proteins could contribute to their functional divergence. Creation and functional assays of chimeric MLO2/MLO1 proteins by swapping their C terminal domains revealed that the C terminus determines the localization pattern of MLO proteins. The Feronia (FER) receptor-like kinase is required for localization of MLO7 in synergid cells; however, CRISPR-targeted mutagenesis of FER did not disrupt the localization of MLO2 to the fungal penetration site. Based on the results described above, it can be inferred that MLO2 localization to and possible stabilization of the plasma membrane at the fungal penetration site is essential for allowing PM fungi to penetrate the host cell and subsequently differentiate the haustorium. Further multiplexed CRISPR mutagenesis of other gene families suggests that SYP121 and SYP122, two closely related SNARE genes play essential roles in focal accumulation of MLO2 at the fungal penetration site.Item The MEIOTIC PROPHASE AMINOPEPTIDASE 1 regulates polyploidy in Arabidopsis thaliana(2017) Wattarantenne, Kasuni Vishwaprabha; Peer, Wendy A; Plant Science and Landscape Architecture (PSLA); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Growth and development in plants is dependent on cellular functions such as cell cycle progression. M1 aminopeptidases have been shown to regulate mitosis and meiosis in animals. MEIOTIC PROPHASE AMINOPEPTIDASE M1 (MPA1) in Arabidopsis thaliana was previously shown to regulate cell cycle progression during prophase I in meiosis I in both female and male gametophytes and be essential for homologous recombination. mpa1 homozygous embryos are lethal due to chromosome de-synapsis resulting in uneven distribution of chromosomes in daughter cells and massive decrease in homologous crossovers reduces independent assortment. Here, I show that MPA1 is a soluble protein and is expressed throughout the seedling: in the primary root, hypocotyl, cotyledons, petioles and root and shoot apical meristem. I isolated and characterized four mpa1 alleles, and I showed that MPA1 loss-of-function mutants exhibited three significant phenotypes corresponding to development in seedlings and adult plants in Arabidopsis: non-disjunction in mitotic cells, altered polyploidy, and temporary arrest of primary root growth during seedling establishment.Item INVESTIGATION OF ARABIDOPSIS TSO1, A REGULATOR OF CELL PROLIFERATION AND DIFFERENTIATION(2017) WANG, WANPENG; Liu, Zhongchi; Plant Science and Landscape Architecture (PSLA); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Multicellular eukaryotic organisms build complex body structures from a single cell. Through coordinated cell proliferation and differentiation, the collective behavior of cells forms organs that achieve physiological functions. Underlying the developmental processes are the molecular machineries that integrate cell cycle regulation with cell fate acquisition. While animal organogenesis occurs early during embryogenesis, plants maintain pluripotent stem cells at the growing tips (meristems) and generate organs iteratively throughout lifespan. The amazing ability to balance stem cell self-renewal and differentiation underlies the extreme longevity of some plants species. Despite the differences, common mechanisms exist across plant and animal developmental regulation. Understanding both unique and common mechanisms of plant development has broad implications on basic science as well as agriculture and medicine. The Arabidopsis TSO1 gene is a regulator of cell proliferation and differentiation at the shoot and root meristems. TSO1 encodes a CXC domain protein and its animal homologs encode core components of a cell cycle regulatory complex, the DREAM complex. To investigate TSO1 function and identify factors that act together with TSO1, I carried out two genetic screens for suppressors and enhancers of tso1 mutants. I discovered that loss-of-function mutations in MYB3R1, which encodes the Arabidopsis ortholog of human B-Myb, can suppress tso1 mutant defects at both the shoot and root meristems. In tso1-1 mutant, MYB3R1 is over and ectopically expressed at the shoot and root meristems. Furthermore, MYB3R1 phospho mimic enhanced the tso1-3 phenotype, indicating that hyper-active MYB3R1 may mediate the tso1-1 phenotype. TSO1 physically interacts with MYB3R1 and likely forms a plant DREAM-like complex that operates in the plant meristems to balance cell proliferation with differentiation. A gain-of-function mutation of a HD-ZIP III transcription factor, REVOLUTA (REV), was identified as an enhancer of tso1 mutants. TSO1 directly represses REV transcription to balance adaxial and abaxial polarity of lateral organs and maintains the shoot apical meristem. This genetic and molecular interaction between TSO1 and the adaxial factor REV presents an integration point of cell cycle, lateral organ polarity, and meristem regulation. Together, our findings demonstrate a cell cycle regulatory module conserved across plants and animals and describe its integration into plant specific developmental context.Item BOLT, AN AP2/ERF TRANSCRIPTION FACTOR, REGULATES ABIOTIC STRESS AND DEFENSE RESPONSES IN ARABIODPSIS THALIANA(2016) Bouten, Roxane; Chang, Caren; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Biotic and abiotic stresses negatively affect plant growth and development, hence decrease productivity. Many AP2/ERF family transcription factors in plants have important roles in stress response signaling although most have not yet been studied. Here I show that expression of an Arabidopsis thaliana AP2/ERF family member, which I call BOLT, is regulated by a MAPK pathway that includes MEKK1, MKK1, MKK2, and MPK4, and has roles in both biotic and abiotic stress response as well influencing growth and development. In this thesis, I examined BOLT’s gene expression pattern and protein localization, using GUS and YFP reporter genes respectively, measured its expression in response to biotic and abiotic stress and plant hormones using RT-qPCR, examined phenotypes by generating overexpressing and RNAi lines, and analyzed its effect on downstream gene expression using a microarray at time points after inducing BOLT expression. I found that BOLT is expressed in various plant tissues and the protein localizes to nuclear bodies as demonstrated in onion epidermal cells. Knockdown (RNAi) plants exhibit greater drought tolerance and are larger than wild type under low light conditions, while the overexpressors exhibit a dramatic early flowering phenotype and are small and weak under low light. Gene expression analysis suggests BOLT regulates genes involved in photosynthesis, hormone biosynthesis and signaling, and defense, many of which are also regulated in the MAPK pathway. Increased BOLT expression downregulates two discreet systems, cyclic electron flow and glycine cleavage, components of photosynthesis and photorespiration, respectively, which are two systems that are important under low light conditions. Taking these results together, I conclude that BOLT functions downstream of a stress responsive MAPK pathway and regulates a variety of growth- and stress-related genes necessary to balance growth and defense in response to biotic or abiotic stresses, or low light conditions.Item IDENTIFICATION AND CHARACTERIZATION OF IAA OXIDASES AND THEIR ROLE IN IAA HOMEOSTATIC REGULATION IN ARABIDOPSIS(2016) Zhang, Jun; Peer, Wendy Ann; Plant Science and Landscape Architecture (PSLA); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Auxin is a crucial plant hormone that shapes and directs plant growth. Indole-3-acetic acid (IAA) is the predominant auxin in nature. Auxin regulates cell expansion and cell division in a dose dependent way. Therefore, plants evolved an extremely complex yet highly coordinated network to maintain auxin homeostasis, including IAA biosynthesis, transport, conjugation and oxidation. Among these, the least known process is IAA oxidation. Discovering how IAA is terminated is very important in completing the whole picture of IAA homeostatic regulation. By partial purification of IAA oxidases from Arabidopsis, we detected IAA oxidation activity from both microsomal fractions and soluble fractions. We first investigated the protein in microsomal fraction and identified one oxidase named as ACC oxidase 2 (ACO2), an ethylene synthetase that belongs to 2-oxoglutarate and iron (II) [2OG(Fe)] dependent dioxygenase family. In vitro enzyme assays with IAA showed that ACO2 could catabolize IAA and that the product had the same retention time as indole-3-carbinal (ICA), an decarboxylative IAA oxidation product. The same enzyme assay with the ACO2 homologues ACO3 was conducted, and ACO3 showed similar activity. An ACO2 loss-of-function allele showed ethylene related phenotypes, including longer hypocotyls and reduced apical hook angle in etiolated seedlings, and delayed bolting. Further, null aco2 mutants also showed reduced phototropic bending, a typical auxin related phenotype. These results indicate that ACO2 might be involved in both ethylene and auxin signaling. We also investigated the soluble IAA oxidases, AtDAO1 (DAO1) and AtDAO2 (DAO2). In vitro enzyme assays showed that both recombinant DAO1 and DAO2 have IAA oxidation activity and the product is the non-decarboxylated 2-oxindole-3-acetic acid (oxIAA), the major IAA metabolite observed under normal growth conditions. Analysis of the loss-of-function null allele dao1-1 showed that DAO1 is the predominant IAA oxidase and is responsible for 95% of oxIAA production in Arabidopsis seedlings. Dysregulation of IAA oxidation altered the IAA metabolism profile and causes accumulation of other IAA conjugates and a series of morphological alteration, including elongation of organs, increased lateral roots and delayed sepal opening. Investigation of expression patterns shows that DAO1 is a cytosolic protein that widely expressed throughout the plant, especially in the root tip, the pericycle of root, the cotyledon, and the sepal, highly correlating to the phenotypes of dao1-1. These results suggest that IAA oxidation plays an important role in IAA homeostasis during the whole life of Arabidopsis.Item MOVING TOWARD AN OPTIMUM: THE ADAPTATION GENETICS OF ARABIDOPSIS THALIANA.(2015) Stearns, Frank Warren; Fenster, Charles B; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Adaptation accounts for many of the interesting characteristics of biodiversity. Despite this, the genetic mechanisms underlying the process of adaptation in nature are largely unknown. While general principles are emerging, important questions remain. Although experimental evidence has corroborated theoretical predictions, very few studies have tested macroorganisms in nature, where adaptation is most relevant. My dissertation addresses several important questions in adaptation genetics in the context of fitness landscapes, primarily using the model plant Arabidopsis thaliana. Fitness landscapes are used to visualize the relationship between genetics and fitness (evolutionary success of an individual). Although fitness landscapes have been considered metaphorical, recent work (and this dissertation) suggests they may approximate reality, providing testable predictions. I first assess pleiotropy (when one gene has multiple effects), an important component of fitness landscape models. I examine this concept in historical context and suggest future directions for research. Next I evaluate how well genetic relatedness corresponds to climate adaptation across the native range of A. thaliana and find support for parallel evolution (identical but independent genetic changes), suggesting that fitness landscapes are complex. In my next chapter, using a combination of natural and artificial conditions, I examine how induced mutations impact traits that are fitness indicators as compared to general traits. I find that new mutations tend to reduce fitness, whereas their effect on general traits is bidirectional. This result is more pronounced under stressful field conditions. Finally, I evaluate a mathematical model of adaptation in the field using induced mutations in A. thaliana. I find support for a previous result from laboratory studies - that lineages that are less well adapted to an environment are more likely to benefit from new mutations whereas lineages that are well adapted are more likely to be disrupted by new mutations - and extend that to the wild. Throughout I explore the importance of contingency in evolution, sometimes underscoring how it leads to unpredictable adaptation (chapters one and two), yet also demonstrating that the actions of mutations can be fit to simplifying assumptions (chapters three and four). These studies therefore significantly contribute to the emerging scholarship on adaptation genetics.Item 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.Item 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.Item Insights into the regulation of ethylene receptor signaling by RTE1(2008-10-10) rivarola, maximo lisandro; Chang, Caren; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Ethylene is an important regulator of plant growth, development and responses to environmental stresses. The higher plant Arabidopsis thaliana perceives ethylene through five homologous receptors, which negatively regulate ethylene responses. The molecular mechanism by which these receptors signal to their next downstream component remains elusive. Genetic analyses have shown that the RTE1 locus is a positive regulator of ETR1. RTE1 encodes a novel protein of unknown molecular function, and is conserved in plants, animals and some protists. The goal of this research was to analyze the mechanisms involved in the regulation of ethylene receptor signaling by RTE1 and to enhance our understanding of the conserved cellular role of RTE1. Here we tested hypotheses for how RTE1 affects ETR1 and is specific to only ETR1, not the other ethylene receptor isoforms. We show that ETR1 and RTE1 gene expression patterns partially overlap and that the ETR1 receptor co-localizes with RTE1 within the cell. Moreover, RTE1 has no effect on ETR1 protein abundance or subcellular localization suggesting other mechanisms to regulate ETR1. We provide supporting evidence that RTE1 affects ETR1 signaling by restoring signaling of a non-functional ETR1 in an rte1 null through changes in ETR1 conformation(s). We next addressed the question of RTE1 specificity to ETR1. We discovered that ETR1 is surprisingly distinct from the other four ethylene receptor genes; in that RTE1-dependent mutations only confer insensitivity in ETR1 and not in the other ethylene receptors when the same mutations are introduced. In contrast, the RTE1-independent ETR1 insensitive mutations do give insensitivity in the closest receptor to ETR1, ERS1. Furthermore, we uncover that the ethylene binding domains are not completely interchangeable between ETR1 and ERS1. Our data point to a model in which RTE1 specifically promotes ETR1 signaling via conformational changes in a unique way that does not occur in other ethylene receptors. These findings highlight the importance and uniqueness of ETR1 signaling conformation(s) with respect to the other ethylene receptors, as well as advance our knowledge of RTE1 at the molecular and cellular level.Item 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.