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PURDUE UNIVERSITY GRADUATE SCHOOL Thesis/Dissertation Acceptance Graduate School ETD Form 9 (Revised 12/07) PURDUE UNIVERSITY GRADUATE SCHOOL Thesis/Dissertation Acceptance This is to certify that the thesis/dissertation prepared By Gage Koehler Entitled Overwintering Survival of Strawberry (Fragaria x ananassa): Proteins Associated with Low Temperature Stress Tolerance during Cold Acclimation in Cultivars Doctor of Philosophy For the degree of Is approved by the final examining committee: Dr. Christopher Staiger Dr. Paul M. Hasegawa Chair Dr. Stephen Randall Dr. Jonn Watson Dr. Bonnie Blazer-Yost To the best of my knowledge and as understood by the student in the Research Integrity and Copyright Disclaimer (Graduate School Form 20), this thesis/dissertation adheres to the provisions of Purdue University’s “Policy on Integrity in Research” and the use of copyrighted material. Approved by Major Professor(s): ____________________________________Dr. Stephen Randall ____________________________________ Approved by: Dr. Simon Atkinson 11/16/2011 Head of the Graduate Program Date Graduate School Form 20 (Revised 9/10) PURDUE UNIVERSITY GRADUATE SCHOOL Research Integrity and Copyright Disclaimer Title of Thesis/Dissertation: Overwintering Survival of Strawberry (Fragaria x ananassa): Proteins Associated with Low Temperature Stress Tolerance during Cold Acclimation in Cultivars For the degree of DoctorChoose ofyour Philosophy degree I certify that in the preparation of this thesis, I have observed the provisions of Purdue University Executive Memorandum No. C-22, September 6, 1991, Policy on Integrity in Research.* Further, I certify that this work is free of plagiarism and all materials appearing in this thesis/dissertation have been properly quoted and attributed. I certify that all copyrighted material incorporated into this thesis/dissertation is in compliance with the United States’ copyright law and that I have received written permission from the copyright owners for my use of their work, which is beyond the scope of the law. I agree to indemnify and save harmless Purdue University from any and all claims that may be asserted or that may arise from any copyright violation. Gage Koehler ______________________________________ Printed Name and Signature of Candidate 11/16/2011 ______________________________________ Date (month/day/year) *Located at http://www.purdue.edu/policies/pages/teach_res_outreach/c_22.html OVERWINTERING SURVIVAL OF STRAWBERRY (FRAGARIA X ANANASSA): PROTEINS ASSOCIATED WITH LOW TEMPERATURE STRESS TOLERANCE DURING COLD ACCLIMATION IN CULTIVARS A Dissertation Submitted to the Faculty of Purdue University by Gage Koehler In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy December 2011 Purdue University Indianapolis, Indiana ii ACKNOWLEDGMENTS Several people have made this thesis possible. I am especially thankful to Dr. Stephen Randall for his help and support in guiding me through to its successful completion. My deep gratitude is expressed to Dr. Muath Alsheikh for his support and encouragement to participate in this project. I also warmly acknowledge Dr. John Watson for his inspiring guidance and insightful input throughout the process of this research, as well as Dr. Staiger, Dr. Hasegawa, and Dr. Bonnie Blazer-Yost for their time and valuable feedback during the investigation of this project. Achieving the goals of this project was facilitated by the use of resources and equipment, generously provided by Dr. Frank Witzmann. This project also benefitted from the expert advice and experience of many colleagues to whom I am grateful, including the valuable contributions made by Xianyin Lai to the LC-MS/MS peptide identifications and the statistical analysis performed by Dr. John Goodpaster. I am also deeply grateful for Howard Creveling, for generously making the Elizabeth Steele Creveling Memorial Scholarship available to me and other students who have received this honor. A very special recognition is given to Yuji Yamasaki for his support, exemplary professionalism, and excellent sense of humor. iii TABLE OF CONTENTS Page LIST OF TABLES ............................................................................................................................... v LIST OF FIGURES ........................................................................................................................... vi LIST OF ABBREVIATIONS ........................................................................................................ viii ABSTRACT ....................................................................................................................................... xiii CHAPTER 1. INTRODUCTION ................................................................................................... 1 1.1. Processes Associated with Reliable Overwintering Survival .............................................. 1 1.1.1. Membrane Modifications and Lipid Biosynthesis ......................................................... 2 1.1.2. Cytoskeleton in Response to Cold Exposure ................................................................ 2 1.1.3. Reactive Oxygen Species................................................................................................... 3 1.1.3.1. Antioxidant ................................................................................................................... 5 1.1.3.2. Detoxification .............................................................................................................. 6 1.1.4. Chaperones ......................................................................................................................... 7 1.1.5. Pathogenesis-Related Proteins ......................................................................................... 8 1.1.6. Dehydrins ............................................................................................................................ 9 1.2. Significance Aspects from this Study ................................................................................... 10 1.3. Explanation of Interrelatedness of Chapters ...................................................................... 11 CHAPTER 2. PROTEOME ANALYSIS OF CROWNS OF FRAGARIA ANANASSA CULTIVARS WITH DIFFERENT FREEZING TOLERANCE ................................................................................................................................... 14 2.1. Introduction ............................................................................................................................. 14 2.2. Methods .................................................................................................................................... 16 2.2.1. Plant Material and Experimental Design for Freezing Experiment ......................... 16 2.2.2. Plant Material for Protein Analysis................................................................................ 18 2.2.3. Sample Preparation for 2DE .......................................................................................... 18 2.2.4. 2DE (Two-Dimensional Gel Electrophoresis) ........................................................... 19 2.2.5. 2DE Gel Imaging and Data Analysis ............................................................................ 20 2.2.6. 2DE Protein Identification by LC-MS/MS ................................................................. 21 2.2.6.1. Protein Confidence Values Listed as Protein Probability ................................... 21 2.2.6.2. Protein Confidence Values Listed as q-values ...................................................... 22 2.2.7. Shotgun Proteomics ........................................................................................................ 22 2.2.8. Western Blots ................................................................................................................... 23 2.3. 2DE Results ............................................................................................................................. 24 2.3.1. 2DE Maps of F. × ananassa Crown Tissue .................................................................. 25 2.3.2. Agglomerative Hierarchical Clustering (AHC) of 2DE Data .................................... 30 2.3.3. Principal Component Analysis (PCA) of ‘Jonsok’ and ‘Frida’ .................................. 30 2.3.4. 2DE Protein Spot Comparison for ‘Jonsok’ and ‘Frida’ ............................................ 33 iv Page 2.3.5. Functional Categories of Identified Proteins from 2DE ........................................... 40 2.4. Shotgun Results ....................................................................................................................... 43 2.5. Discussion ................................................................................................................................ 47 2.5.1. Comparison of 2DE Protein Expression in ‘Jonsok’ and ‘Frida’ ............................. 47 2.5.1.1. Proteins Involved in the Phenylpropanoid Biosynthetic Pathway .................... 47 2.5.1.2. Proteins Associated with Pathogen Resistance ..................................................... 50 2.5.1.3. Antioxidative and Detoxification Proteins ............................................................ 51 2.5.1.4. Anoxia/Hypoxia Related Proteins .......................................................................... 55 2.5.1.5. Other Proteins Associated with Freezing Stress Tolerance ................................ 56 2.5.2. Comparison of 2DE and Shotgun-based Approaches
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