DETECTION AND QUANTITATION OF TREHALOSE AND OTHER SUGARS IN RICE AND ARABIDOPSIS USING GC-MS A Thesis Presented to the Faculty of California State Polytechnic University, Pomona In Partial Fulfillment Of the Requirements for the Degree Master of Science In Chemistry By Elizabeth N. Martinez 2016 SIGNATURE PAGE THESIS: DETECTION AND QUANTITATION OF TREHALOSE AND OTHER SUGARS IN RICE AND ARABIDOPSIS USING GC- MS AUTHOR: Elizabeth N. Martinez DATE SUBMITTED: Spring 2016 Chemistry and Biochemistry Department Gregory A. Barding Jr., Ph.D. Thesis Committee Chair Chemistry and Biochemistry Lisa A. Alex, Ph.D. Chemistry and Biochemistry Yan Liu, Ph.D Chemistry and Biochemistry . ii ACKNOWLEDGEMENTS There are many people I would like to acknowledge for their continuous support of my educational goals. Most importantly, I would like to thank my mom and dad, for always being there and supporting me through my good days and my bad days, and making sure I always had everything I needed. I would also like to thank my siblings, Joseph for dealing with me being crazy and always trying to help me, Anthony for being entertaining, and Linda for being the awesome sister to me. Also, my amazing friends for always being there to burn off the stress on the weekends, but also understanding when I could not make it, thank you Elizabeth. I would also like to thank my fellow grad students for keeping me motivated and reassuring me that it will definitely be worth it one day. Lastly, I would like to thank my advisor, Dr. Gregory Barding Jr. for putting up with my millions of questions, and guiding me to a successful path. I definitely would not have finished without your help and support during this experience. iii ABSTRACT Trehalose, a disaccharide commonly found in many organisms, is an important sugar often associated with increased tolerance to a variety of stressors. Understanding how trehalose levels change in plants during abiotic stress will enhance our understanding of how plants cope with increasing stresses associated with climate change. For this study, trehalose sucrose, glucose and fructose, were quantified in submergence tolerant and intolerant rice varieties to better understand carbon metabolism during complete flooding and subsequent recovery. In plants, trehalose is present in exceedingly low quantities making it difficult to detect and quantify, especially due to its low relative abundance compared with sucrose, fructose and glucose. The analysis was done using Gas chromatography – mass spectrometry (GC-MS) equipped with a quadrupole detector, a powerful technique for the sensitive detection of gas-phase analytes. Because of the low concentration of trehalose present in plants, it was important to clean up the samples by removing anionic metabolites through weak anion exchange (WAX) solid phase extraction (SPE). For data analysis of metabolites were derivatized with a suitable reagent, such as MSTFA, followed by GC-MS experiments carried out using a full scan (60-600 m/z) to identify each sugar using library matching as well as an in-house library generated with standards, and then selected ion monitoring (SIM) was used to further reduce the background associated with unwanted signals by only detecting specific ions associated with each sugar. The ions chosen were 307, 319, 323, and 361, 13 and used to quantify (fructose, glucose, C6 glucose, and trehalose) the specific ions to the sugars. In addition to rice, different variations of Arabidopsis thaliana were evaluated for sugar content using SIM to quantify trehalose and a full scan to quantify sucrose, iv glucose, and fructose. It was determined the LOD and LOQ for trehalose in the rice samples are 0.640 ng, and 1.01 ng of trehalose per g of tissue, respectively, The LOD and LOQ for trehalose in the Arabidopsis samples are 0.744 ng, and 0.825 ng, respectively. This resulted in an average concentration of trehalose in M202 to be 11.5 ng of trehalose per g of tissue, in IR64 to be 1.3 ng of trehalose per g of tissue, and in Arabidopsis thaliana to be 17.9 μg of trehalose per g of tissue. v TABLE OF CONTENTS SIGNATURE PAGE ........................................................................................................... ii ACKNOWLEDGEMENTS ............................................................................................... iii ABSTRACT ....................................................................................................................... iv LIST OF TABLES ........................................................................................................... viii LIST OF FIGURES .......................................................................................................... xii CHAPTER 1: INTRODUCTION ..................................................................................... 15 1.1 Metabolomics .......................................................................................................... 15 1.2 Gas Chromatography (GC) ..................................................................................... 19 1.3 Mass Spectrometry (MS) ........................................................................................ 25 1.4 Growth conditions, extraction, and sample preparation ......................................... 32 1.5 Data analysis ........................................................................................................... 35 1.6 Application of GC-MS in biological systems ......................................................... 37 1.7 Objectives of study ................................................................................................. 40 1.8 REFERENCES ....................................................................................................... 42 CHAPTER 2: METHODS AND MATERIALS .............................................................. 45 2.1 Tissue extraction and preparation of rice tissue samples ........................................ 45 2.2 Tissue extraction and preparation of Arabidopsis tissue sample ............................ 46 2.3 Sample derivatization .............................................................................................. 46 2.4 Sample injection and GC ........................................................................................ 47 2.5 Mass spectrometry data analysis ............................................................................. 47 2.6 Sugar identification ................................................................................................. 49 2.7 Quantitation of sugars ............................................................................................. 49 2.8 Retention indices (RI) ............................................................................................. 50 2.9 Retention time lock (RTL) ...................................................................................... 50 2.10 Rice tissue bulking and Seed bulking ................................................................... 51 2.11 REFERENCES ..................................................................................................... 55 CHAPTER 3: QUANTITATION OF TREHALOSE AND OTHER SUGARS IN SUBMERGENCE RESISTANCE RICE ......................................................................... 56 3.1 M202 and M202(Sub1) Results .............................................................................. 56 3.2 IR64 Results ............................................................................................................ 66 3.2.1 Discussion ............................................................................................................ 74 3.3 Conclusion .............................................................................................................. 85 vi 3.4 REFERENCES ....................................................................................................... 90 CHAPTER 4: QUANTITATION OF TREHALOSE AND OTHER SUGARS IN DROUGHT TOLERANT ARABIDOPSIS PLANTS ..................................................... 91 4.1 Introduction ............................................................................................................. 91 4.2 Results and Discussion ........................................................................................... 92 4.3 Conclusion ............................................................................................................ 111 4.4 REFERENCES ..................................................................................................... 114 CHAPTER 5: SUMMARY AND CONCLUSION ........................................................ 115 5.1 REFERENCES ..................................................................................................... 119 CHAPTER 6: FUTURE WORK .................................................................................... 120 6.1 REFERENCES ..................................................................................................... 122 vii LIST OF TABLES Table 2.1 M202 submergence tolerant and intolerant rice samples used in study ........... 53 Table 2.2 IR64 submergence tolerant and submergence intolerant rice samples ............ 54 Table 2.3 Arabidopsis drought tolerant and intolerant samples ....................................... 54 Table 3.1 Ratios of M202 to M202(Sub1) for control samples. Samples were harvested at noon (t=0 hr post submergence), dusk (t=6 hr post submergence), midnight (t=12 hr post submergence), Dawn (t=18