Studies of Ca²⁺-ATPase involvement in the gravity-directed calcium current and polar axis alignment of germinating Ceratopteris richardii spores
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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.