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University of Oklahoma Graduate College Systems UNIVERSITY OF OKLAHOMA GRADUATE COLLEGE SYSTEMS ANALYSIS OF GRASS CELL WALL BIOSYNTHESIS FOR BIOFUEL PRODUCTION A DISSERTATION SUBMITTED TO THE GRADUATE FACULTY in partial fulfillment of the requirements for the Degree of DOCTOR OF PHILOSOPHY By FAN LIN Norman, Oklahoma 2017 SYSTEMS ANALYSIS OF GRASS CELL WALL BIOSYNTHESIS FOR BIOFUEL PRODUCTION A DISSERTATION APPROVED FOR THE DEPARTMENT OF MICROBIOLOGY AND PLANT BIOLOGY BY ______________________________ Dr. Laura E. Bartley, Chair ______________________________ Dr. Ben F. Holt III ______________________________ Dr. Scott D. Russell ______________________________ Dr. Marc Libault ______________________________ Dr. Randall S. Hewes © Copyright by FAN LIN 2017 All Rights Reserved. Acknowledgements I would like to express my deepest appreciation to my supervisor, Dr. Laura Bartley, for her guidance and advice throughout all projects. She is always encouraging and willing to help me with patience. By working with her, I learned more than anything I learned in a classroom. I would like to acknowledge my collaborators, their significant contributions to the data collection process and their useful discussion were essential for the completion of these projects. Especially thanks to Dr. Lobban, Dr. Mallinson and Dr. Waters for working together as an excellent interdisciplinary team on the thermal conversion project. I enjoy the useful meetings and discussions. I also would like to thank my committee members, Dr. Holt, Dr. Libault, Dr. Russell and Dr. Hewes. They spent their valuable time to meet with me and discuss about these projects. I also thank members of the Bartley lab, Russell lab, and Libault lab for useful discussions. Especially thanks to Dr. Zhao for the discussion on statistical analysis and network analysis. Last but not least, my deepest gratitude is to my parents, Wanshu Mu and Song Lin, I appreciate their sincere love and support. Projects in this dissertation were funded by U.S. Department of Energy, National Science Foundation, U.S. Department of Agriculture. Also, thank OU Student Travel Assistance Program for funding the travels to present these projects. iv Table of Contents Acknowledgements .......................................................................................................... iv Table of Contents .............................................................................................................. v List of Tables ................................................................................................................. viii List of Figures .................................................................................................................. ix Abstract ............................................................................................................................ xi Chapter 1: Background ..................................................................................................... 1 Biological Functions of Cell Walls ............................................................................. 1 Cellulosic Biofuel ....................................................................................................... 4 Grass Cell Wall Composition and Biosynthesis ......................................................... 9 Chapter 2: Cell Wall Composition and Candidate Biosynthesis Gene Expression During Rice Development ............................................................................................... 16 Abstract ..................................................................................................................... 17 Introduction ............................................................................................................... 18 Results. ...................................................................................................................... 25 Cell Wall Content Changes During Rice Development ..................................... 26 Glucuronoarabinoxylans ..................................................................................... 28 Mixed Linkage Glucan ....................................................................................... 29 Pectins. ................................................................................................................ 30 Arabinogalactan Proteins .................................................................................... 30 Cellulose ............................................................................................................. 31 Lignin and pCA ................................................................................................... 31 Enzymatic Digestibility ...................................................................................... 32 v Identification of Genes Involved in Cell Wall Synthesis by Correlation Analysis ................................................................................................................. 33 Gene Expression Clustering and Correlation Analysis ....................................... 34 Discussion ................................................................................................................. 36 Cell Wall Composition Relationships ................................................................. 36 Identification of Genes Involved in Cell Wall Synthesis by Correlation Analysis ................................................................................................................. 40 Conclusion ................................................................................................................ 45 Materials and Methods .............................................................................................. 47 Figures and Tables .................................................................................................... 50 Chapter 3: Proteome, Metabolite, and Cell Wall Profiling of an Elongating Rice Stem Internode, a Secondary Cell Wall Biosynthesis Model ...................................... 66 Abstract ..................................................................................................................... 67 Introduction ............................................................................................................... 69 Results. ...................................................................................................................... 73 Cell Wall Polysaccharides and Lignin Varies along the Elongating Rice Internode ................................................................................................. 73 Protein Catalog of Elongating Internode ............................................................ 76 Proteome Gene Ontology (GO) and KEGG Ontology (KO) Analysis ............... 77 Cell Wall Synthesis and Remodeling Proteins, and Extracellular Proteins ........ 78 Kinases and Transcription Factors ...................................................................... 80 Metabolite Profiles of the Elongating Internode, Mature Internodes, Leaf and Root ......................................................................................................... 81 vi Discussion ................................................................................................................. 84 Cell Wall Changes Associated with Stem Development .................................... 84 Cell Wall Synthesis, Remodeling, and Protein Phosphorylation are Important During Stem Elongation ......................................................................... 85 Secondary Metabolites Vary During Stem Maturation ...................................... 89 Materials and Methods .............................................................................................. 91 Figures and Tables .................................................................................................. 107 Chapter 4: The Effect of Genetic Modification of The Lignin Biosynthesis Pathway on Low Temperature Pyrolysis Product Yields ..................................................... 134 Abstract ................................................................................................................... 135 Introduction ............................................................................................................. 137 Results and Discussion ........................................................................................... 142 Materials and Methods ............................................................................................ 146 Figures and Tables .................................................................................................. 149 Chapter 5: Conclusions and Future Directions ............................................................. 154 References ..................................................................................................................... 162 vii List of Tables Table 3-1. Samples and methods for the three proteomics experiments in this study. 119 Table 3-2. Rice phenylpropanoid enzymes observed in the elongating internode. ...... 119 Table 3-3. Rice cell wall-related acyltransferases and glycosyltransferases observed in the elongating internode. ............................................................................................... 123 Table 3-4. Metabolites enriched at different stages of rice stem internode development ......................................................................................................................................
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