Cell Biology & Molecular Genetics Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2750
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Item POLLEN TUBES FAIL TO TARGET OVULE IN THE ABSENCE OF TWO CATION/PROTON EXCHANGERS IN ARABIDOPSIS(2010) Lu, Yongxian; Sze, Heven; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Flowering plant reproduction requires precise delivery of the sperm cells to the ovule by a pollen tube. Guiding signals from female cells are being identified, though how pollen senses and responds to those cues are largely unknown. Here I provide genetic evidence that two predicted cation/proton exchangers expressed in Arabidopsis pollen play essential roles in pollen targeting of ovules. Male fertility was unchanged in single chx21 or chx23 mutant pollen; however, male-specific gene transmission was blocked in chx21chx23 double mutant. Wild-type pistil provided with a limited amount of pollen containing a mixture of single and double mutant produced ~60% less seeds compared to that produced with chx23 single mutant pollen, indicating that chx21chx23 pollen is infertile. The double mutant pollen, visualized by a pollen-specific promoter-driven GUS activity, germinated and extended a tube down the transmitting tract, but the tube failed to turn and target an ovule. Unlike wild-type pollen that targeted isolated ovules in a semi-in vivo assay, tube guidance in chx21chx23 pollen was compromised. As a first step to understand the cellular and molecular bases of tube guidance, membrane localization and activity of CHX23 was determined. GFP-tagged CHX23 was localized to endomembranes, predominantly endoplasmic reticulum (ER), in elongating pollen tubes. Furthermore, expression of CHX23 in E. coli resulted in enhanced K+ accumulation at alkaline pH, suggesting a role for CHX23 in K+ acquisition and pH homeostasis. Based on these studies and observations by others that ER oscillates and enters the apex, a simple model is proposed: Modification of localized pH by CHX21 or CHX23 enables pollen tube to sense female signals and respond by shifting directional growth at the funiculus and micropyle to target pollen tip growth towards the ovule.Item Geometrical Relationships Specifying the Phyllotactic Pattern of Aquatic Plants(2008-04-15) Kelly, Wanda Jean; Cooke, Todd J; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Aquatic plants provide an opportunity to characterize the geometrical relationships of leaf patterning. A new polar-coordinate model was used to correlate meristem shape and leaf arrangement in aquatic plants. In aquatic plants, the primary geometrical relationship specifying spiral vs. whorled phyllotaxis is primordial position: primordia arising on the apical dome (as defined by displacement angles θ ≤ 90° during maximal phase) are often positioned in spiral patterns, whereas primordia arising on the subtending axis (as defined by displacement angles of θ ≥ 90° during maximal phase) are arranged in whorled patterns. A secondary geometrical relationship shows an inverse correlation between the primordial size : available space ratio and the magnitude of the Fibonacci numbers in spiral phyllotaxis or the number of leaves per whorl in whorled phyllotaxis. This geometrical analysis provides the morphological context for interpreting phyllotaxis mutants and for constructing realistic models phyllotactic patterns.Item Diversity of conjugating green algae; phylogenetic studies of a species-rich eukaryotic lineage(2007-12-21) Hall, John David; Delwiche, Charles F; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)This thesis used molecular phylogenetic techniques to investigate diversity in the conjugating green algae. The conjugating green algae are closely related to land plants and evolution within the group provides a good analogy of how evolution may have proceeded in the lineage that gave rise to land plants. I developed a dataset of the genes coxIII, psaA and rbcL with 109 taxa to determine phylogenetic relationships of the families and genera. I found that the order Zygnematales is not monophyletic and that Spirogyra was the first to branch. The order Desmidiales is monophyletic if one includes the genus Roya. The family Peniaceae is not monophyletic. The genera Cosmarium, Cylindrocystis, Mesotaenium, Penium, Spondylosium, Staurodesmus and in later studies Desmidium and Hyalotheca were found to be paraphyletic or polyphyletic. Investigation of cell division syndromes among filamentous Desmidiaceae revealed greater diversity than was previously reported. Notable among these discoveries is that Spondylosium pulchrum displays the Desmidium-type cell division, Spondylosium pulchellum the Cosmarium-type, and Spondylosium tetragonum the newly described Teilingia-type cell division. The relationship among the syndromes was inferred from phylogenetic analysis of the species that revealed a single lineage comprising filamentous and colonial species and multiple modes of cell division. This suggests that even the fundamental process of cell division can be highly modified. Results from this study also resulted in the taxonomic resurrection of the genus Didymoprium, as well as the creation of the new genus Isthmocatena and the combinations Didymoprium grevillei, Desmidium pulchrum, and Isthmocatena pulchella. Investigations of the Gonatozygaceae revealed unexpected diversity in Gonatozygon brebissonii and G. kinahani. Structural measurements were sufficient to distinguish among strains of Gonatozygon species except for Gonatozygon brebissonii. We have probably underestimated genetic and species diversity in this family. In contrast, the structurally distinct species Triploceras gracile, was found to be closely related to Micrasterias. This relationship was confirmed by sequencing and phylogenetic analysis of the nuclear encoded EF1α, EIF4 and TUA. The results of this study indicate that Triploceras is probably actually bilaterally symmetric, although it has been treated as a radially symmetric species.Item The Chemical Ecology of Hydrastis Canadensis L. (Ranunculaceae): Effects of Root Isoquinoline Alkaloids on the Hydrastis Endophyte, Fusarium oxysporum(2006-08-11) Tims, Michael; Bean, George A; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Goldenseal (Hydrastis canadensis L., Ranunculaceae) is a popular medicinal plant and has been listed in Appendix II of the Convention on International Trade in Endangered Species (CITES). The herbaceous perennial is distributed in North America under deciduous forest canopies throughout much of the south and eastern seaboard north into Canada. The rhizome, rootlets and root hairs produce medicinally active alkaloids. Although berberine, one of the Hydrastis alkaloids, has shown anti-fungal activity, the influence of Hydrastis alkaloids on the plant rhizosphere fungal ecology has not been investigated. While a Fusarium spp. was previously isolated from H. canadensis, this is the first report of a F. oxysporum endophyte isolated from Hydrastis root tissue. Additionally, F. solani was isolated from non-rhizosphere soil surrounding Hydrastis root tissue. A bioassay was developed to study the effect of goldenseal isoquinoline alkaloids on three Fusarium isolates, including the two species isolated from Hydrastis. And, in order to accurately detect the alkaloids, a HPLC-MS method was developed. The whole root extract treatment stimulated macroconidia germination and chlamydospore formation, while inducing mycotoxin production in the Hydrastis endophyte. Chlamydospore formation, macroconidial germination and mycotoxin production of PSU isolate, F. commune, was reduced by the whole root extract. The second Hydrastis isolate, F. solani, responded to the whole root extract by increasing germination rates, but with no other effects. The findings suggest that the Hydrastis root extract appears to provide selective advantage for the endophytic isolate and influences the fungal ecology associated with its root system.Item The role of auxin on the evolution of embryo development and axis formation in land plants(2005-03-10) Poli, DorothyBelle; Cooke, Todd J.; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)ABSTRACT Title of Dissertation: THE ROLE OF AUXIN ON THE EVOLUTION OF EMBRYO DEVELOPMENT AND AXIS FORMATION IN LAND PLANTS DorothyBelle Poli, Doctor of Philosophy, 2005 Dissertation directed by: Professor Todd J. Cooke, Cell Biology and Molecular Genetics This thesis examined the role of auxin in the evolution of land plants. Several approaches were used to study how auxin regulates the development in the bryophyte sporophytes. The altered growth of isolated young sporophytes exposed to applied auxin (indole-3-acetic acid) or an auxin antagonist (p-chlorophenoxyisobutyric acid) suggested that endogenous auxin regulates the rates of axial growth in all bryophyte divisions. In the hornwort Phaeoceros personii, auxin moved at very low fluxes, was insensitive to an auxin-transport inhibitor (N-[1-naphthyl]phthalamic acid), and exhibited a polarity ratio close to 1.0, implying that auxin moves by simple diffusion. The liverwort Pellia epiphylla exhibited somewhat higher auxin fluxes, which were sensitive to transport inhibitors but lacked any measurable polarity. Thus, auxin movement in liverwort sporophytes appears to result from facilitated diffusion. In the moss Polytrichum ohioensis, auxin movement was predominantly basipetal in young sporophytes and occurred at high fluxes exceeding those measured in maize coleoptiles. In older sporophytes, acropetal auxin flux had increased beyond the level measured for basipetal flux in the specimens observed in several, but not all, seasons. The evidence from both inhibitor treatments and isolated tissues is consistent with the interpretation that the cortex carries out basipetal transport in both younger and older sporophytes, whereas the central vascular tissues carries out basipetal transport in younger sporophytes and acropetal flux in older sporophytes. Given the significant differences in fall rainfall in the collection years, the purported sensitivity of vascular tissue development may account for the seasonal variation observed in these experiments. Auxin regulators and polar transport were also used to study the regulation of the embryogenesis of the fern Marsilea vestita. Auxin biosynthesis inhibitors affected initial cell proliferation resulting in the formation of aborted embryos, p-chlorophenoxyisobutyric acid delayed growth and development in all stages of embryogenesis while -naphthaleneacetic acid mediated rapid cell proliferation that caused enlarged disorganized embryos. Polar auxin transport inhibitors caused no significant abnormalities, which suggested a limited role for polar transport in fern embryogenesis. In conclusion, this evidence suggests that auxin is ultimately involved in the establishment of the body plans in all land plant sporophytes.Item EVOLUTIONARY TRANSITIONS BETWEEN STATES OF STRUCTURAL AND DEVELOPMENTAL CHARACTERS AMONG THE ALGAL CHAROPHYTA (VIRIDIPLANTAE)(2004-12-10) Lewandowski, Jeffrey David; Delwiche, Charles F; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The Charophyta comprise green plant representatives ranging from familiar complex-bodied land plants to subtle and simple forms of green algae, presumably the closest phylogenetic relatives of land plants. This biological lineage provides a unique opportunity to investigate evolutionary transition series that likely facilitated once-aquatic green plants to colonize and diversify in terrestrial environments. A literature review summarizes fundamental structural and developmental transitions observed among the major lineages of algal Charophyta. A phylogenetic framework independent of morphological and ontological data is necessary for testing hypotheses about the evolution of structure and development. Thus, to further elucidate the branching order of the algal Charophyta, new DNA sequence data are used to test conflicting hypotheses regarding the phylogenetic placement of several enigmatic taxa, including the algal charophyte genera <i>Mesostigma</i>, <i>Chlorokybus</i>, <i>Coleochaete</i>, and <i>Chaetosphaeridium</i>. Additionally, technical notes on developing RNA methods for use in studying algal Charophyta are included.Item PHYLOGENETIC STUDIES OF THE CHARALES: THE CLOSEST LIVING RELATIVES OF LAND PLANTS(2004-11-24) Karol, Kenneth Gregory; Delwiche, Charles F; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The embryophytes (land plants) consist of organisms such as mosses, ferns, conifers and flowering plants. Although land plants have long been thought to be related to the green algal group Charophyceae, the nature of this relationship has been unresolved for over a century. A four-gene phylogenetic analysis supports the hypothesis that land plants arose from within the Charophyta and unambiguously identifies the Charales as the closest living relatives of plants. With a robust phylogeny, it is now possibly to explore phylogeny-dependant questions that were previously difficult to assess. Estimating the divergence time of the land plant lineage is one such question. A recent time estimate for the colonization of land by plants is 1,061 ± 109 mya and 703 ± 45 mya for the divergence of vascular plants and bryophytes, a result much older than the fossil record suggests (roughly 470 mya). Unlike most algae, a rich fossil record exists for the Charales in the form of calcified oospores. Representative fossils that can be attributed to five extant lineages in the Charales have been identified with reasonable accuracy. These multiple calibration points were used in conjunction with the four-gene DNA data set to estimate the divergence time of the land plant and Charales lineages. The Bayesian relaxed-clock approach estimated divergence of the Charales/land plant common ancestor in the Late Proterozoic (674.10 ± 99.96 MYA), modern land plants in the Cambrian (497.78 ± 75.66 MYA), and modern Characeae at the Paleozoic/Mesozoic boundary (247.75 ± 25.98 MYA). The genus Nitella is one of the most diverse genera in the Charales. Wood and Imahori's worldwide monograph divides Nitella into three subgenera with seventeen sections and radically modified the taxonomy of this group my submerging over 200 Nitella species into 53 loosely defined species. Phylogenetic analyses of rbcL sequence data from 79 Nitella species (plus outgroups) support the monophyly of Nitella and two subgenera (Hyella and Tieffallenia). Subgenus Nitella formed two paraphyletic lineages at the base of the genus. Few sections were monophyletic and species diversity is interpreted as being much higher than proposed by Wood.Item A phylogenetic and biogeographic analysis of Sanguisorbeae (Rosaceae), with emphasis on the Pleistocene radiation of the high Andean genus Polylepis.(2004-06-16) Kerr, Malin Sofia; Delwiche, Charles F; Reveal, James L; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)A phylogenetic and biogeographic analysis of the tribe Sanguisorbeae (Rosaceae) was conducted with emphasis on the radiation of the Andean tree Polylepis. Phylogenetic analyses of coding and non-coding nuclear markers reveal a complex evolutionary history of the tribe including ancient and recent allopolyploid hybridization. Sanguisorba sensu lato is shown to be paraphyletic and split between the allopolyploid hybrid Sanguisorba and the non-hybrid Poterium and Poteridium. A monophyletic origin of the southern hemispheric subtribe Sanguisorbinae is supported, and this clade is given a phylogentic taxon name (Verruchaena). Dating analyses using the penalized likelihood method suggest that this taxon originated in the late Miocene. A biogeographic hypothesis is presented in which Verruchaena originated in the New World with subsequent transoceanic dispersals to southern Africa and Australasia. The paramo genus Polylepis most likely arose from hybridization between two Andean ancestors supporting a "vertical" rather than "horizontal" origin of this taxon.Item THE ORIGIN OF THE DINOFLAGELLATE PLASTID(2004-04-29) Bachvaroff, Tsvetan Radoslavov; Delwiche, Charles F; Cell Biology & Molecular GeneticsThe peridinin pigmented dinoflagellate chloroplasts are the result of a secondary endosymbiotic event between a photosynthetic eukaryote and a dinoflagellate. Dinoflagellate chloroplast and nuclear evolution were independent before this endosymbiotic event. To reconstruct the evolution of the dinoflagellate chloroplast, phylogenies were constructed with a chloroplast gene <i>psbB</i>. The gene phylogeny should reflect the evolution of the chloroplast and indicate the plastid donor lineage. Gene sequences derived from the dinoflagellate chloroplast were extremely divergent but suggested that the plastid donor could have been a haptophyte. In an attempt to find better genes for analysis and to further understand gene transfer about 4900 randomly selected expressed genes were sequenced from two dinoflagellates, <i>Lingulodinium polyedra</i> and <i>Amphidinium carterae</i>. From these genes, thirty typically plastid-encoded genes were found, including eight otherwise known only from plastid genomes. Based on poly-A tails, gene families, and leader sequences these genes appear to be nuclear-encoded in dinoflagellates. This result suggests that dinoflagellate chloroplasts may have the smallest protein-coding potential yet known. These genes and the partially sequenced chloroplast genome of a haptophyte were used in a phylogenetic analysis. There is strong conflict between genes encoded in the chloroplast and those in the nucleus. The chloroplast genes suggest relationship between haptophyte and dinoflagellate plastids, while the nuclear-encoded genes suggest a relationship with heterokonts. Chromophyte plastid monophyly is supported by these data but the single origin of the chromophyte plastid from red algae does not mean that the host lineages are monophyletic. These results are consistent with at least two different scenarios: either dinoflagellates and haptophytes independently acquired a plastid from the heterokonts, or dinoflagellates acquired their plastids from haptophtyes, who in turn acquired their plastids from heterokonts. The evolutionary rate of the remaining plastid-encoded genes was compared with formerly plastid-encoded genes. These relative rate tests revealed strong incongruence between minicircle genes, formerly plastid-encoded genes and genes that were likely to have been acquired from the nucleus of the plastid donor lineage.