University of Nevada, Reno Use of synthetic sugar analogs to probe plant cell wall function in Arabidopsis thaliana A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry by Jose A. Villalobos Jr Ian S. Wallace / Dissertation Advisor December 2020 THE GRADUATE• SCHOOL We recommend that the dissertation prepared under our supervision by entitled be accepted in partial fulfillment of the requirements for the degree of Advisor Committee Member Committee Member Committee Member Graduate School Representative David W. Zeh, Ph.D., Dean Graduate School i Abstract: Plants are terrestrial photosynthetic multicellular organisms responsible for producing most of the world’s oxygen. Plants are also responsible for fixating approximately 1011 metric tones of carbon dioxide into reduced forms of carbon that are used for plant metabolism as well as human and animal nutrition. Approximately 70% of plant biomass is derived from plant cell walls. Plant cell walls are polysaccharide-rich extracellular matrices that encapsulate nearly all plant cells and collectively, these polysaccharides are the most abundant biopolymers on the planet. The evolution of cell wall polysaccharides was integral for plants to populate terrestrial environments. Functionally, cell wall polysaccharides are essential for normal plant growth and development, and compromises to the structure or biosynthesis causes severe developmental defects and are often lethal. The essential nature of cell wall polysaccharides creates challenges in conducting genetic studies to elucidate the biosynthesis and function cell wall polysaccharides. The goal of this work was to implement a chemical biology strategy to probe cell wall function in a spatial, temporal, and dose-dependent manner. Our strategy was to use semi-rationally designed monosaccharide analogs that may inhibit glycosyltransferases by competitively binding to their active sites. We screened a small library of monosaccharide analogs and found three analogs: 2-deoxy-2-fluoro-L-fucose (2F-Fuc), 2-deoxy-2-fluoro-D-mannose (2F-Man), and N-dodecyldeoxynojirimycin (ND-DNJ) inhibit growth in Arabidopsis thaliana. Only 2F-Fuc repressed growth by inhibiting fucosylation of a pectic cell wall polysaccharide while 2F-Man and ND-DNJ inhibited ii growth in unexpected ways. In 2F-Man, the primary mechanism of inhibition was through the glucose repression pathway in Arabidopsis. The mechanism of 2F-Man toxicity was further supported in yeast as it also inhibited growth through a similar glucose repression pathway. Uniquely, ND-DNJ inhibited glycosylation events in sphingolipids in Arabidopsis and as a result inhibited crystalline cellulose deposition, highlighting a novel connection between cellulose and glycosylated sphingolipids. Finally, we discovered suberin deposition occurs in response to cellulose biosynthesis inhibition. iii Acknowledgments: This dissertation would not be possible without the support and advice from everyone around me over the years. First, I would like to thank my advisor Dr. Ian Wallace for the opportunity to work on various projects and continuously challenging me to think critically and creatively. For believing in me when I didn’t and giving me that extra push when I needed it, none of this would be possible without you. I would also like to thank my committee members: Dr. Dylan K. Kosma, Jeff F. Harper, John C. Cushman, and Laina M. Geary. Your advice and questions continuously helped improve the direction of my research and getting me to always think outside the box. Thank you all for taking the time to answer my random questions, providing me insight into techniques I was not familiar with, and helping me grow as a graduate student. I would like to thank Jeff for backcrossing our resistant lines. I would also like to thank Dylan for showing me the ins and outs of operating a GC. I would also like to thank Zackary Wahrenburg for our suberin analysis and your expertise with the GC-MS. Finally, I would like to thank the Cahoon lab for the sphingolipidomic analysis. A special thank you to the members of the Wallace lab who have come and gone over the years: Devin Smith, Bret Hart, Edward Cruz, Andrew Larson, Celeste Rodriquez, Eli Holschbach, Megan Warner, Tori Speicher, Hans Joseph Struffert, Bo Yi, Timothy Fox, Brett Allen, Sienna Ogawa, Gabriel Aguilar, Jon Lau, KassaDee Herring, Daniel Jones, Daniel Kinder, and Sarah Pennington. From bouncing ideas back and forth, talking science, teaching me new techniques, to the camaraderie we shared made my Ph.D. experience that much more fulfilling. I would like to thank Bret for the imaging iv experiments with ND-DNJ and Devin for analyzing the kymographs that led to our mind- blowing result. A special thanks to Andrew Larson for your help with kinase assays. I’m grateful for the team I had the opportunity to work with. v Table of Contents List of Tables x List of Figures xi Chapter 1: Introduction to plant cell walls and function. 1 I. Plant Cell walls 1 II. Cell walls: Pectin structure and function 2 a. Homogalacturonan 5 b. Rhamnogalacturonan-I 9 c. Rhamnogalacturonan-II 11 III. Cell walls: Hemicellulose structure and function 14 a. Mannan and Glucomannan 14 b. Xyloglucan 17 c. Xylan 19 IV. Cell walls: Cellulose biosynthesis and regulation 20 a. Cellulose production 22 b. CSC accessory proteins 25 c. Regulation of CSCs 28 V. Non-polysaccharide wall components: lignin and suberin 32 a. Lignin 32 b. Suberin 35 vi VI. Cell walls: Integrity sensing and Cell wall damage 37 a. Cell wall integrity sensing 37 VII. Plant Glycoproteins and Glycolipids: Structure, function and metabolism 40 a. Protein Glycosylation 40 b. Glycosylphosphatidylinositol anchors 45 c. Glucosylceramides 48 d. Glycoinositol phosphoceramides 51 VIII. Use of small molecules, monosaccharide analogs, and monosaccharide 53 mimicking small molecules a. Common cell wall biosynthesis inhibitors 54 b. Monosaccharide analogs as diagnostic tools and inhibitors 57 c. Iminosugars a class of monosaccharide mimicking compounds 60 IX. Screening of monosaccharide analogs as semi-rational inhibitors of plant 61 cell wall biosynthesis References 68 Chapter 2: 2-Deoxy-2-Fluoro-L-Fucose is a metabolically incorporated 114 inhibitor of plant cell wall polysaccharide fucosylation. I. Introduction 114 II. Materials and Methods 117 III. Results 122 IV. Discussion 145 References 149 Chapter 3: 2-Deoxy-2-Fluoro-D-Mannose is an inhibitor of energy 154 metabolism in Arabidopsis thaliana. I. Introduction 154 vii II. Materials and Methods 158 III. Results 167 IV. Discussion 191 References 197 Chapter 4: 2-Deoxy-2-Fluoro-D-Mannose is a potent inhibitor of fungal 212 growth. I. Introduction 212 II. Materials and Methods 214 III. Results 223 IV. Discussion 244 References 248 Chapter 5: Sphingolipids are required for cellulose deposition and cellulose 258 synthase complex motility. I. Introduction 258 II. Materials and Methods 261 III. Results 265 IV. Discussion 283 References 292 Chapter 6: Cellulose biosynthesis inhibitors induce suberin production in 306 Arabidopsis thaliana as part of the Cell Wall Damage response. I. Introduction 306 II. Materials and Methods 310 III. Results 312 IV. Discussion 318 viii References 323 Chapter 7: Discussion 330 I. Potential of fluoro fucose resistant lines 330 a. Current state of the ffr lines and their potential 330 b. Other utilities of 2F-Fuc 333 II. Sugar sensing in plants and fungi in relation to glucose and mannose 337 a. Sugar sensing in plants 338 b. Sugar sensing in yeast 342 III. Implications of sphingolipid biosynthesis on cellulose 346 a. Known glycosylated sphingolipid biosynthesis mutants: do they contain 346 less cellulose b. Non-motile observation in ND-DNJ and DCB 347 c. Utility of other iminosugars 350 IV. Cell wall integrity sensing and differential responses to cell wall damage 352 a. THESIUS1 and STRUBBELIG in response to cellulose defects 352 b. Lignin and suberin, in response to cell wall damage 353 V. Possible strategies to isolate novel GTs 355 a. Capture of GTs with photo-reactive crosslinkers 355 b. Isolation of partial polysaccharide glycans as substrates 359 VI. Implications of sugar analogs as herbicides 361 VII. Other monosaccharide analogs to explore in plant growth 363 VIII Conclusions 365 ix References 367 x List of Tables Chapter 1 Table 1.1: Monosaccharide analog screen on Arabidopsis. 66 Chapter 2 Table 2.1: Primer sequences used in this chapter. 123 Table 2.2: T-DNA lines used in this chapter. 131 Table 2.3: Analysis of potential fluoro fucose resistant lines. 141 Table 2.4: Segregation Ratios of ffr x Col-0 F2 progeny. 144 Chapter 3 Table 3.1: Retesting fmr lines on 50 µM 2F-Man 185 Chapter 4 Table 4.1: Primer sequences used in this study 219 Table 4.2: IC50 Values of hypersensitive deletion strains. 236 Chapter 5 Table 5.1: Nojirimycin analogs screened against Arabidopsis 268 Col-0. xi List of Figures Chapter 1 Figure 1.1: General representation of where cell wall polysaccharides, 4 glycan containing lipid, and proteins are synthesized. Figure 1.2: Structures of common pectin polymers found in plant cell walls. 6 Figure 1.3: Structures of common Hemicellulose polymers found in plant 15 cell walls. Figure 1.4: Schematic of cellulose biosynthesis. 21 Figure 1.5: Structures of Lignin and Suberin monomers. 34 Figure 1.6: Structures of N-linked Glycans and GPI-Anchors found in plant 41 cells. Figure 1.7: Structures of GlcCers and GIPCs found in plant cells. 49 Figure 1.8: Structures of cellulose biosynthesis inhibitors and RG-II 55 biosynthesis inhibitor. Figure 1.9: Hypothetical SN2 mechanisms of deoxy and fluor/Azido 63 monosaccharide analogs in non-retaining GTs. Figure 1.10: Monosaccharide analog screen on Arabidopsis thaliana. 65 Chapter 2 Figure 2.1: Effects of fucose analogs on Arabidopsis root growth. 125 xii Figure 2.2: Effects of short-term 2F-Fuc treatment on Arabidopsis seedlings. 127 Figure 2.3: Matrix polysaccharide analysis of 2F-Fuc treated seedlings.