Browsing by Subject "Ceratopteris"
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Item Expression profile analysis of early development and gravity response of germinating Ceratopteris richardii spores(2005) Salmi, Mari L.; Roux, Stanley J.Item Investigation of the role of extracellular nucleotide gradients in plant gravity responses(2016-12) Cannon, Ashley Elisabeth; Roux, Stanley J.; Browning, Karen S; Huq, Enamul; Levin, Donald A; Mehdy, MonaExtracellular ATP (eATP) was first identified as a neurotransmitter in animal systems decades ago, but has only recently been classified as a signaling molecule in plants. Previous studies have shown that exogenously applied ATP can disrupt gravitropism in roots, depolarize root hairs, and alter auxin distribution. These results support a clear role for this molecule as a regulatory signal in plants. To further define eATP as a signal in plants, Ceratopteris spores, a model system, were used to study gravity-directed cell polarization. This polarization begins with the uptake of Ca2+ through channels at the bottom of the spore, a process required for the cell’s gravity response. Previous data showing that mechanosensitive channels can release ATP and that eATP can induce the opening of Ca2+ channels led to the hypothesis that eATP could play a role in the gravity-directed polarization. Data described in this dissertation show that an eATP gradient, with significantly higher [ATP] outside the bottom of the cell, is present during and promotes gravity-directed polarization. To explore the link between eATP and Ca2+ in gravity-directed polarization of spores, microparticle bombardment was used to transform Ceratopteris cells with a FRET-based Ca2+ sensor, Yellow Cameleon 3.60. The success of this effort has generated a uniquely valuable tool that can be used to analyze intracellular Ca2+ dynamics and rapidly screen transformants in Ceratopteris, a primitive plant system. In addition to studying the role of eATP signaling in the gravity response of single cells, an assessment of its role in the gravity response of a multicellular system, primary roots of Arabidopsis, was carried out. By using ecto-luciferase-expressing seedlings, a gradient of eATP, with the highest concentration being along the bottom of the root, was visualized within 30 min of gravistimulation. When this gradient was disrupted by excess ATP or an eATP receptor antagonist, the gravity response was attenuated. These results characterize the role of eATP gradients in the gravity responses of single spore cells of ferns and multicellular primary roots of a flowering plant. They suggest that the preferential accumulation of eATP along the bottom of gravity-responding cells is an evolutionarily conserved mechanism for promoting gravity-directed development.Item Studies of Ca²⁺-ATPase involvement in the gravity-directed calcium current and polar axis alignment of germinating Ceratopteris richardii spores(2007-05) Bushart, Thomas James; Roux, Stanley J.All organisms have been subjected to and have evolved with the ubiquitous force of gravity, and most exhibit the ability to sense and respond to this stimulus. To simplify an investigation of the molecular components of a cell's gravity response, this dissertation employs the single-celled spores of the fern Ceratopteris richardii. These spores have a polar calcium flux that is determined by the gravity vector, but an understanding of what the molecular components driving this flux are and how they influence subsequent developmental processes is lacking. Of the possible molecular components, available literature pointed to Ca²⁺-ATPase transporters as an obvious key participant and so they were selected as the main molecule of investigation. Our results describe the first cloned Ca²⁺-ATPase from C. richardii, CrACA1. CrACA1 has high similarity to known plant Ca²⁺-ATPases, specifically plasma membrane (PM) Ca²⁺-ATPases from Arabidopsis, and exhibits in vivo Ca²⁺-ATPase activity. An improved method for the statistical analysis and presentation of qualitative RT-PCR data was employed. The RNA, as well as the protein, of CrACA1 is present during the polarity fixation window which supported the need for further analyses of the role of Ca²⁺-ATPases. Our results showing that Ca²⁺-ATPase inhibitors significantly alter the gravity-directed calcium flux of spores are consistent with previous work but offer valuable new insights. The spore PM Ca²⁺-ATPases have large impacts on the calcium flux and rhizoid growth but no appreciable impact on polar axis alignment. The results on endomembrane-type Ca²⁺-ATPases make it clear that this class of pumps has major roles in both axis alignment and tip growth; rhizoid growth is inhibited but alignment to the gravity vector is improved. The updated model for gravity perception responses in C. richardii spores places a strong emphasis on calcium channels and Ca²⁺-ATPases working in concert to result in a bottom-localized calcium pool to align the polar axis with hints of store-operated calcium mobilization. The work presented represents an increase in our knowledge of one way a single cell can respond to the force of gravity, offering testable hypotheses to further refine gravity perception models incorporating calcium localization.