Investigation of the role of extracellular nucleotide gradients in plant gravity responses
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Extracellular 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.