Cell Biology & Molecular Genetics Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2750
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Item THE PHYTOHORMONE ETHYLENE: (I) INVESTIGATING THE MOLECULAR FUNCTION OF RTE1 AND (II) INSIGHTS ON THE EVOLUTION OF THE ETHYLENE BIOSYNTHESIS AND SIGNALING PATHWAYS(2017) Clay, John; Chang, Caren; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Ethylene is an important phytohormone that regulates growth, development and stress responses in land plants and charophycean green algae. In Arabidopsis thaliana, ethylene is perceived by a family of five receptors. One of these five receptors, ETR1, is dependent on REVERSION-TO-ETHYLENE1 (RTE1) and Cytochrome B5 (Cb5) while the other four receptors are not. We found that RTE1 and Cb5 interact in planta and used genetic analyses to place Cb5 upstream of RTE1 in the ethylene signaling pathway. After comparing different ethylene receptors we identified an N-terminally localized proline that is important in determining whether a receptor is RTE1-dependent. Our results suggest that Cb5 receives electrons from upstream redox molecules, passes these electrons to RTE1; RTE1 is then able to activate the ETR1 receptor possibly by acting a molecular chaperone that refolds the ETR1 receptor into an active conformation. The ethylene signal transduction pathway is functionally conserved in the charophycean green algae such as Spirogyra pratensis, suggesting that this signaling pathway was present in the common ancestor of charophytes and land plants over 450 million years ago. However, it is unclear whether the central regulator of ethylene response, EIN2, was conserved in charophytes. Furthermore, the details of ethylene biosynthesis in charophytes were unresolved. After examining the genomes and transcriptomes of many green algae we are able to report that EIN2 is conserved in most charophytes and even some of the more distantly related chlorophycean green algae. Moreover, the Spirogyra EIN2 is functionally conserved and able to activate ethylene responses in Arabidopsis. Ethylene is synthesized via a two-step reaction involving the conversion of S-adenosyl-L-methionine (SAM) to 1-aminocyclopropane-1-carboxylic acid (ACC) by the enzyme ACC synthase (ACS), followed by oxidation of ACC to ethylene gas by the enzyme ACC oxidase (ACO). We identified S. pratensis ACS homologs and demonstrated that S. pratensis can synthesize ACC. S. pratensis lacks ACO homologs but we find it is still capable of producing low levels of ethylene. From our results we conclude that the ethylene biosynthesis and signaling pathways were established in early charophytes allowing these algae to establish ethylene as an important signalling molecule.Item Dinoflagellate Genomic Organization and Phylogenetic Marker Discovery Utilizing Deep Sequencing Data(2016) Mendez, Gregory Scott; Delwiche, Charles F; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Dinoflagellates possess large genomes in which most genes are present in many copies. This has made studies of their genomic organization and phylogenetics challenging. Recent advances in sequencing technology have made deep sequencing of dinoflagellate transcriptomes feasible. This dissertation investigates the genomic organization of dinoflagellates to better understand the challenges of assembling dinoflagellate transcriptomic and genomic data from short read sequencing methods, and develops new techniques that utilize deep sequencing data to identify orthologous genes across a diverse set of taxa. To better understand the genomic organization of dinoflagellates, a genomic cosmid clone of the tandemly repeated gene Alchohol Dehydrogenase (AHD) was sequenced and analyzed. The organization of this clone was found to be counter to prevailing hypotheses of genomic organization in dinoflagellates. Further, a new non-canonical splicing motif was described that could greatly improve the automated modeling and annotation of genomic data. A custom phylogenetic marker discovery pipeline, incorporating methods that leverage the statistical power of large data sets was written. A case study on Stramenopiles was undertaken to test the utility in resolving relationships between known groups as well as the phylogenetic affinity of seven unknown taxa. The pipeline generated a set of 373 genes useful as phylogenetic markers that successfully resolved relationships among the major groups of Stramenopiles, and placed all unknown taxa on the tree with strong bootstrap support. This pipeline was then used to discover 668 genes useful as phylogenetic markers in dinoflagellates. Phylogenetic analysis of 58 dinoflagellates, using this set of markers, produced a phylogeny with good support of all branches. The Suessiales were found to be sister to the Peridinales. The Prorocentrales formed a monophyletic group with the Dinophysiales that was sister to the Gonyaulacales. The Gymnodinales was found to be paraphyletic, forming three monophyletic groups. While this pipeline was used to find phylogenetic markers, it will likely also be useful for finding orthologs of interest for other purposes, for the discovery of horizontally transferred genes, and for the separation of sequences in metagenomic data sets.Item Genomic studies of the evolution of haptophytes and dinoflagellates with emphasis on the chromalveolate hypothesis(2006-06-08) Sanchez Puerta, Maria Virginia; Delwiche, Charles F; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)All photosynthetic eukaryotes rely, partially or totally, on their plastids to live. The plastids, which ultimately are highly modified cyanobacteria, were acquired through a process of primary, secondary, or tertiary endosymbiosis. Four photosynthetic lineages, including haptophytes, dinoflagellates, cryptophytes, and heterokonts, contain secondary plastids with chlorophyll c as a main photosynthetic pigment. These four lineages were grouped together, along with their heterotrophic relatives, on the basis of their pigmentation and called chromalveolates by Cavalier-Smith. However, the phylogenetic relationships among these algae are unknown and the chromalveolate hypothesis remains very controversial. This study focuses on increasing the amount of genomic data from a poorly studied chromalveolate lineage, the haptophytes, and understanding plastid evolution in chromalveolates. Both the chloroplast and mitochondrial genomes of the haptophyte <em>Emiliania huxleyi</em> were sequenced and examined to describe basic genomic properties, as well as perform comparative studies. Phylogenetic analyses, including data acquired from haptophytes, support a monophyletic chl c containing plastid clade derived from the red algae, after the divergence of Cyanidiales, with the cryptophyte plastid basal or sister to the haptophyte plastid. In addition, phylogenetic analyses using mitochondrial data suggest a relationship of haptophytes and cryptophytes. The chromalveolate clade as a whole is not recovered nor rejected by the data. Analysis of an EST project from the heterotrophic dinoflagellate <em>Crypthecodinium cohnii</em> indicates that <em>C. cohnii</em> is not only derived from a photosynthetic ancestor, but very likely retains a non-photosynthetic plastid. Analyses of putative gene function suggest that heme biosynthesis, non-mevalonate isoprenoid biosynthesis, amino-acid metabolism, and Fe-S cluster assembly may occur in the plastid. These observations are also consistent with the chromalveolate hypothesis, which proposes that several major groups of eukaryotes, including alveolates, haptophytes, cryptophytes, and heterokonts, may form a monophyletic group with a photosynthetic common ancestor, and that nonphotosynthetic members are secondarily so.