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University of Nevada, Reno Lipid Flippases and Elemental University of Nevada, Reno Lipid Flippases and Elemental Homeostasis Systems in Arabidopsis thaliana A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry By Stephen C. McDowell Dr. Jeffrey F. Harper/Dissertation Advisor May, 2013 Copyright © by Stephen C. McDowell 2013 All Rights Reserved THE GRADUATE SCHOOL We recommend that the dissertation prepared under our supervision by STEPHEN C. MCDOWELL entitled Lipid Flippases and Elemental Homeostasis Systems in Arabidopsis thaliana be accepted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Jeffrey F. Harper, Ph.D., Advisor John C. Cushman, Ph.D., Committee Member David K. Shintani, Ph.D., Committee Member Grant R. Cramer, Ph.D., Committee Member Patricia M. Berninsone, Ph.D., Graduate School Representative Marsha H. Read, Ph. D., Dean, Graduate School May, 2013 i Abstract Many molecules in living systems are present in charged forms, and these molecules are often highly regulated. The work presented in the following chapters addresses two main topics involving charged molecules using the model plant Arabidopsis thaliana: elemental homeostasis and lipid flippases. The study of elemental homeostasis is referred to as ionomics and is the topic of Chapter II. P4-ATPases are thought to be the principle class of proteins with lipid flippase activity and are the topics of Chapter III and Chapter IV. Plants, especially seed crops, are an important source of mineral nutrition in the human diet and are thus important targets for biofortification and toxic element exclusion. Here, we report the results of a pilot ionomic screen in which we quantified the concentrations of 14 elements in Arabidopsis seeds. To identify conditional ionomic phenotypes, plants were grown under four different soil conditions: standard, or modified with NaCl, heavy metals, or alkali. To help identify the genetic networks regulating the seed ionome, elemental concentrations were evaluated in mutants corresponding to 760 genes as well as 10 naturally occurring accessions. The frequency of ionomic phenotypes observed in the mutant screen supports an estimate that up to 11% of the Arabidopsis genome encodes proteins of functional relevance to the seed ionome. A subset of mutants were analyzed with two independent alleles, providing five examples of genes important for regulation of the seed ionome: SOS2, ABH1, CCC, At3g14280, and CNGC2. Reproducible ionomic differences were also observed between the Col-0 reference accession and eight of the other nine accessions screened. Significantly, all 15 mutants with reproducible ionomic phenotypes showed at least one change under ii standard soil conditions. This suggests that the sole use of a standard growth environment might be the most effective strategy for continued reverse-genetic efforts to identify genes that impact the Arabidopsis seed ionome. Nonetheless, each soil modification had a unique impact on the Col-0 seed ionome and elicited several conditional phenotypes in both the mutant and accession screens, indicating that seed elemental homeostasis is sensitive to soil conditions. Together, the results of this study establish that elemental analysis is a sensitive approach to identify genes and environmental conditions that impact elemental accumulation in Arabidopsis seed. By flipping lipids between membrane leaflets, P4-ATPases are thought to help create and maintain asymmetry in biological membranes. Lipid asymmetry between membrane leaflets has been implicated in a wide range of biological processes including: vesicular trafficking, cell signaling, modulation of membrane permeability, protein recruitment, and regulation of protein activity. Additionally, one P4-ATPase, Neo1p, is essential in yeast. In Arabidopsis thaliana, 12 P4-ATPases have been identified: Aminophospholipid ATPase 1 (ALA1) to ALA12. However, very little is known about P4-ATPases in the context of plant systems. Of the 12 ALA isoforms, only ALA3 has been extensively studied. Previous studies have shown that loss of ALA3 results in pleiotropic phenotypes affecting root, shoot, and reproductive development. Here, we expand on the previous studies by showing that multiple phenotypes for ala3 mutants are strongly sensitive to growth conditions. We also expand on the ala3 pollen phenotype by identifying three points of defect in ala3 pollen tubes: delayed germination, slow growth, and reduced overall length. Furthermore, we show that ala3 pistils have reduced ovule production, thus iii providing the first evidence of a female reproductive defect in ala3 mutants. Together, these results support a model in which ALA3 functions in multiple cell types and is critical to plants for development and adaptation to varied growth conditions. Two other ALA isoforms, ALA6 and ALA7, were also examined in this study. We provide in-vitro and in-vivo evidence that ALA6 and ALA7 are important for rapid, sustained pollen tube growth. Expression of fluorescently-labeled ALA6 fusion proteins indicates that the subcellular localization of ALA6 includes the plasma membrane and highly mobile endomembrane structures. We also show that staining by lipophilic FM dyes is reduced by ~10-fold in ala6-1/7-2 pollen tubes relative to wild-type, suggesting differences in plasma membrane composition. Furthermore, tandem mass spectroscopy analysis revealed significant differences between the lipid compositions of ala6-1/7-2 and wild-type pollen grains, both in the concentrations of different headgroups and in the average number of double bonds present within acyl side chains. Together, these results support a model in which ALA6 and ALA7 function to directly or indirectly regulate the distribution and concentration of lipids in pollen and are thus critical for pollen fitness. iv Acknowledgements I would first like to thank my advisor, Dr. Jeff Harper, for his guidance and support. I appreciate all of the learning opportunities he has given me and the freedom that I have had in my research. Jeff’s enthusiasm for science is very inspiring and has been a source of immeasurable motivation for me over the years. I would also like to thank all of my committee members for their guidance. Thanks to Dr. John Cushman for his diligence in making sure that my degree was always moving forward. Thanks to Dr. Dave Shintani for his instruction on lipids and lipid metabolism. Thanks to Dr. Grant Cramer for teaching me to write in a concise and cogent manner. Thanks to Dr. Patricia Berninsone for her perspective, regarding both my research and science as a whole. I also owe a great many thanks to my colleagues and collaborators, who have all been very generous with their time and ideas. I am very grateful to Dr. Kathy Schegg for all of the time, encouragement, and guidance she has given me. I would also like to thank our collaborators Dr. Michael Palmgren, Dr. Rosa López-Marqués, and Dr. Lisbeth Poulsen for their time and effort on my behalf. I also owe thanks to Dr. David Schooley for helping me develop my public speaking skills with the BCH 790 seminar series, and to Dr. William Welch for his interest in my work and his good ideas. Finally, thanks to all of the staff at UNR Biochemistry who keep the department running smoothly. I would also like to thank all members of the Harper Lab, both past and present. I would like to thank Kelly Zinn, Norman Groves, Richard Hilleary, Nick Noel, Ing-Feng “Ira” Chang, and Sabine Frietsch for their friendship, and all of the great times we had both in and out of the lab. I also owe a great many thanks to Taylor Cohen, who has been a tremendous help to me over the years. I would also like to thank Liz Brown for being a great technician and keeping the lab running smoothly. I’m sorry I can’t list everyone individually, but let me take this opportunity to thank all of you for being such great labmates. Finally, my deepest thanks go to my friends and family. I would never have gotten through this without all of you. I would like to thank my parents, Chuck and Pauline McDowell, for the love and support they have always given me. I would like to thank my wonderful girlfriend, Caitlin Gallagher, for her love and encouragement, and all of the time she has spent talking out ideas with me, proofreading manuscripts, etc. I am very grateful to Miki Proud, for giving me a place to live while finishing grad school and for being such a great friend. I am also grateful to Tom Soboleski for his friendship and encouragement. I would also like to express my thanks to: JR Tillett, Matt Wheatley, Matt Ford, Maygan Herrin, Laura Proud, Mike Mouradian, Upul Hathwaik, Leyla Hathwaik, Gadi Miller, Sage Hiibel… Thanks again to everyone; none of this would have been possible without each and every one of you. v Table of Contents Chapter I: Introduction – Ionomics and P4-ATPases .....................................................1 Ionomics .......................................................................................................................1 P4-ATPases ...................................................................................................................5 Literature Cited ...........................................................................................................19 Chapter II: Elemental Concentrations in the Seed of Mutants and Natural Variants of Arabidopsis thaliana Grown Under Varying Soil Conditions ..................................31 Abstract ......................................................................................................................32
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