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Molecular and Chemical Dissection of Cellulose Biosynthesis in Plants

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Molecular and Chemical Dissection of Cellulose Biosynthesis in Plants University of Kentucky UKnowledge Theses and Dissertations--Plant and Soil Sciences Plant and Soil Sciences 2011 MOLECULAR AND CHEMICAL DISSECTION OF CELLULOSE BIOSYNTHESIS IN PLANTS Darby M. Harris University of Kentucky, [email protected] Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Recommended Citation Harris, Darby M., "MOLECULAR AND CHEMICAL DISSECTION OF CELLULOSE BIOSYNTHESIS IN PLANTS" (2011). Theses and Dissertations--Plant and Soil Sciences. 3. https://uknowledge.uky.edu/pss_etds/3 This Doctoral Dissertation is brought to you for free and open access by the Plant and Soil Sciences at UKnowledge. It has been accepted for inclusion in Theses and Dissertations--Plant and Soil Sciences by an authorized administrator of UKnowledge. For more information, please contact [email protected]. STUDENT AGREEMENT: I represent that my thesis or dissertation and abstract are my original work. Proper attribution has been given to all outside sources. I understand that I am solely responsible for obtaining any needed copyright permissions. I have obtained and attached hereto needed written permission statements(s) from the owner(s) of each third-party copyrighted matter to be included in my work, allowing electronic distribution (if such use is not permitted by the fair use doctrine). I hereby grant to The University of Kentucky and its agents the non-exclusive license to archive and make accessible my work in whole or in part in all forms of media, now or hereafter known. I agree that the document mentioned above may be made available immediately for worldwide access unless a preapproved embargo applies. I retain all other ownership rights to the copyright of my work. I also retain the right to use in future works (such as articles or books) all or part of my work. I understand that I am free to register the copyright to my work. REVIEW, APPROVAL AND ACCEPTANCE The document mentioned above has been reviewed and accepted by the student’s advisor, on behalf of the advisory committee, and by the Director of Graduate Studies (DGS), on behalf of the program; we verify that this is the final, approved version of the student’s dissertation including all changes required by the advisory committee. The undersigned agree to abide by the statements above. Darby M. Harris, Student Dr. Seth DeBolt, Major Professor Dr. Arthur G. Hunt, Director of Graduate Studies MOLECULAR AND CHEMICAL DISSECTION OF CELLULOSE BIOSYNTHESIS IN PLANTS _______________________________________________________ DISSERTATION ___________________________________ A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Agriculture at the University of Kentucky By Darby M. Harris Lexington, Kentucky Director: Dr. Seth DeBolt, Associate Professor of Horticulture Lexington, Kentucky 2011 Copyright © Darby M. Harris 2011 ABSTRACT OF DISSERTATION MOLECULAR AND CHEMICAL DISSECTION OF CELLULOSE BIOSYNTHESIS IN PLANTS Plant cell walls are complex structures that must not only constrain cellular turgor pressure but also allow for structural modification during the dynamic processes of cell division and anisotropic expansion. Cell walls are composed of highly glycosylated proteins and polysaccharides, including pectin, hemicellulose and cellulose. The primary cell wall polysaccharide is cellulose, a polymer composed of high molecular weight !- 1,4-glucan chains. Although cellulose is the most abundant biopolymer on Earth, there is still a lot to learn about its biosynthesis and regulation. This research began by applying a variety of analytical techniques in an attempt to understand differences in cell wall composition and cellulose structure within the plant body, between different plant species and as a result of acclimation by the plant to different environmental conditions. Next, a number of different Arabidopsis thaliana lines possessing mutations affecting cell wall biosynthesis were analyzed for changes in cellulose structure (crystallinity) and biomass saccharification efficiency. One of these mutants, isoxaben resistance1-2 (ixr1- 2), which contains a point mutation in the C-terminal transmembrane region (TMR) of cellulose synthase 3 (CESA3), exhibited a 34% lower biomass crystallinity index and a 151% improvement in saccharification efficiency relative to that of wild-type. The culmination of this research began with a chemical screen that identified the molecule quinoxyphen as a primary cell wall cellulose biosynthesis inhibitor. By forward genetics, a semi-dominant mutant showing strong resistance to quinoxyphen named aegeus was identified in A. thaliana and the resistance locus mapped to a point mutation in the TMR of CESA1. cesa1aegeus occurs in a similar location to that of cesa3ixr1-2, illustrating both subunit specificity and commonality of resistance locus. These drug resistant CESA TMR mutants are dwarfed and have aberrant cellulose deposition. High-resolution synchrotron X-ray diffraction and 13C solid-state nuclear magnetic resonance spectroscopy analysis of cellulose produced from cesa1aegeus, cesa3ixr1-2 and the double mutant shows a reduction in cellulose microfibril width and an increase in mobility of the interior glucan chains of the cellulose microfibril relative to wild-type. These data demonstrate the importance of the TMR region of CESA1 and CESA3 for the arrangement of glucan chains into a crystalline cellulose microfibril in primary cell walls. Keywords: cell wall, cellulose microfibril, cellulose synthase, saccharification efficiency, crystallinity Darby M. Harris ____________________________ December 16, 2011 ____________________________ MOLECULAR AND CHEMICAL DISSECTION OF CELLULOSE BIOSYNTHESIS IN PLANTS By Darby M. Harris Seth DeBolt ____________________________ Director of Dissertation Arthur Hunt ____________________________ Director of Graduate Studies December 16, 2011 ____________________________ DEDICATION In memory of my grandparents, James and Kitty Darby and Tony and Omah Harris, who were members of “The Greatest Generation” in this country and whose hard work and sacrifice have given those of my generation countless opportunities to succeed. ACKNOWLEDGEMENTS This dissertation was completed with the assistance and support of a number of individuals. I wish to thank the members of my Dissertation Committee: Seth DeBolt, Joe Chappell, Bob Houtz and Chris Schardl. I wish to also thank the outside examiner Czarena Crofcheck. The members of my committee provided me with guidance, encouragement, and multiple suggestions of ways in which to improve my approach to scientific research and ways to improve my dissertation. I wish to thank the faculty of the College of Agriculture for their contributions to my professional development. More specifically, I want to thank the faculty in the Department of Horticulture, where I conducted a majority of my research. In addition to those faculty members who instructed courses I completed, I also want to thank Art Hunt, Director of Graduate Studies for the Plant Physiology Ph.D. Program. A special thanks goes to Seth DeBolt, the director of my dissertation research, who led by example and helped greatly to raise the bar of my own research. Through his willingness to allow me freedom to explore the literature, attend conferences and participate in numerous research projects within his laboratory, I have gained the confidence necessary to step up to the next level and continue to develop my research ideas into the future. I would also like to thank members of the DeBolt Lab for their help, encouragment and friendship over the last four years, including Jozsef Stork, Meera Nair, Kendall Corbin, Venu Mendu, Carloalberto Petti, Nihar Nayak, Ye (Summer) Xia, Mizuki Tateno, Chad Brabham, Matthew Simson and last but not least Randy Collins. Most importantly, I thank my family. I thank my parents, Brenda and Jerry Harris, who have supported me in everything that I have done in life and who continue to show enormous excitement and offer relentless encouragement in my future adventures. I thank my sister Kate and my brother Josh who are a source of happiness and security. My wife and soulmate Isabella Jacovino, not only would I have never attempted this dissertation without her, but it would mean nothing without her by my side. We did this together and I know that we can accomplish anything set before us. iii TABLE OF CONTENTS ACKNOWLEDGEMENTS!!!!!!!!!!!!!!!!!!!!!!!!!!! iii List of Tables !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! vii List of Figures !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!..viii Chapter I !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!. 1 The synthesis, regulation and modification of lignocellulosic biomass as a resource for biofuels and bioproducts !!!!!!!!!!!!!!!!!!!!!!!!!!!. 1 Bioenergy agriculture !!!!!!!!!!!!!!!!!!!!!!!!!!!!... 1 Making biofuel from plant cell walls rather than starch !!!!!!!!!!!!!!... 2 Key cell wall components and their functional significance !!!!!!!!!!!!... 3 Synthesis and regulation of key cell wall biopolymers !!!!!!!!!!!!!!! 9 Cellulose !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!.. 9 Regulation of CESA activity !!!!!!!!!!!!!!!!!!!!..11 Feedback between CESA and the cortical microtubule array !!!!!! 18 Hemicellulose !!!!!!!!!!!!!!!!!!!!!!!!!!!!! 21 Lignin !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!. 25 Modification of cell wall structure and synthesis to overcome biomass recalcitrance .... 28 Modification of cell wall structure and synthesis to increase biomass quantity and energy density!!!!!!!!!!!!!!!!!!!!!!!!!!!!...........
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