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Characterization of Genetic Mutants Encoding Four Hydroxyproline

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Characterization of Genetic Mutants Encoding Four Hydroxyproline Characterization of Genetic Mutants Encoding Four Hydroxyproline Galactosyltransferases (Hyp-galts) for Arabinogalactan-proteins in Arabidopsis A thesis presented to the faculty of the College of Arts and Sciences of Ohio University In partial fulfillment of the requirements for the degree Master of Science Lu Tian December 2015 © 2015 Lu Tian. All Rights Reserved. 2 This thesis titled Characterization of Genetic Mutants Encoding Four Hydroxyproline Galactosyltransferases (Hyp-galts) for Arabinogalactan-proteins in Arabidopsis by LU TIAN has been approved for the Department of Environmental and Plant Biology and the College of Arts and Sciences by Allan M. Showalter Professor of Environmental and Plant Biology Robert Frank Dean, College of Arts and Sciences 3 ABSTRACT TIAN, LU, M.S., December 2015, Molecular and Cellular Biology Characterization of Genetic Mutants Encoding Four Hydroxyproline Galactosyltransferases (Hyp-galts) for Arabinogalactan-proteins in Arabidopsis Director of Thesis: Allan M. Showalter Arabinogalactan-proteins (AGPs) are a family of highly glycosylated proteoglycans found in all plants as components of the cell wall, plasma membrane, and cellular secretions. AGPs function in a variety of cellular processes including cell proliferation, cell expansion, somatic embryogenesis, and cell death. They also possess valuable adhesive and emulsification properties that are utilized for commercial purposes. Up to 90% of a typical AGP’s mass corresponds to type II arabinogalactan (AG) polysaccharides which are attached to hydroxyproline (Hyp) residues in the protein backbone by glycosylltransferases (GTs). Of all the GTs involved in the O-glycosylation of AGPs, the GALTs that add the first galactose onto hydroxyproline residues in the protein backbone of AGPs are crucial as they initiate the glycosylation process and produce the substrate acceptor for further glycosylation enzymes. Since attempts to solubilize AGP-specific GALTs from Golgi enriched Arabidopsis microsomes were unsuccessful, a bioinformatics approach was adopted to identify and characterize putative GALT genes responsible for synthesizing Hyp-Gal linkages. Six putative GALT genes were selected and named GALT 1-6. All six genes belong to the GT-31 family of the Carbohydrate-Active Enzymes (CAZy) database. Recent studies in the Showalter lab demonstrated that two members of the six GALTs, namely GALT2 and GALT5, encode 4 Hyp-GALTs that are required for AGP biosynthesis. This thesis reports on the characterization the other four candidate GALT genes (i.e., GALT1, GALT3, GALT4, and GALT6). Towards this goal, two allelic mutants for each gene (galt1-1, galt1-2, galt3-1, galt3-2, galt4-1, galt4-2, galt6-1, and galt6-2) in Arabidopsis were obtained, verified by molecular analyses, and subjected to phenotypic analysis. The four candidate GALT genes were heterologously expressed in tobacco epidermal leaves, and all showed Hyp-GALT enzyme activity except GALT1. Biochemically, galt3 and galt6 plants, and to a lesser extent galt4 plants, displayed reduced Hyp-GALT enzyme activity and reduced precipitation of AGPs by β-Gal Yariv reagent compared to wild type and galt1 plants. This is consistent with the report that GALT1 is involved with N-glycosylation, and not O- glylcosylation of AGPs. Under optimal physiological conditions, all four mutants were identical to wild type with the exception that galt4, and to a lesser extent galt6, displayed reduced numbers of seeds per silique. However, under restrictive conditions, such as elevated NaCl, galt3 and galt6, and to a lesser extent galt4, exhibited salt hypersensitive root growth, delayed root bending, and swollen root tips. The galt1 mutants, however, did not show these conditional phenotypes. Primary root growth was also less sensitive to β- Gal Yariv reagent in galt3, galt4, and galt6 plants compared to the wild type and galt1 plants. Thus, characterization of the GALTs by investigating the function of these four genes in their native setting will help to delineate the role of AGPs in plants and in manipulating the structure of AGPs for commercial purposes. Another part of this thesis is devoted to delineating the potential signaling role of AGPs in Arabidopsis root growth. The sos5 (salt overly sensitive 5) mutant, is a plasma 5 membrane-anchored fasciclin-ike AGP mutant, while fei1fei2 is a double mutant affecting two cell wall leucine-rich repeat, receptor-like kinases. These mutants displayed identical phenotypes, a swollen root tip in the presence of moderately high salt (100 mM NaCl). Notably, this phenotype is also displayed by two AGP galt mutants, galt2 and galt5, as well as by galt2galt5 double mutants. It is hypothesized that the extracellular portion of the FEI/FEI2 kinase interacts with sugar residues on AGPs like those presumably added onto SOS5 by the action of GALT2 and GALT5 and other AGP glycosyltransferases. Quintuple mutant plants (fei1fei2sos5galt2galt5) were generated and provided genetic evidence that these five genes act in a single genetic pathway in support of the proposed hypothesis. This work contributes to a better understanding of O-linked glycosylation in plants and more specifically to AGP biosynthesis and function. 6 ACKNOWLEDGMENTS I am very grateful to my advisor, Dr. Allan Showalter, for giving me the opportunity to finish my MS. I am thankful for his patience, and that he never gave up on me. I am very honored and lucky to be his graduate student. I am grateful to all my committee members for the suggestions related to my thesis. I am very thankful to Debarati Basu, a past member of the Showalter lab who always helped me solve many difficult issues realted to my resreach. I am grateful to Wuda Wang, another member of the Showalter lab who spent much time teaching me so many lab techniques. Lastly, none of this would have been possible without my family. 7 TABLE OF CONTENTS Page Abstract ............................................................................................................................... 3 Acknowledgments ............................................................................................................... 6 List of tables………………………………………………………………………………10 List of figures……………………………………………………………………………..11 List of abbreviations………………………………………………………………………13 Chapter 1: Introduction……………………………………………………………….15 1.1. Introduction……………………………………………………………………….15 1.1.1 Plant cell walls ................................................................................................. 15 1.1.2. Cellulose ......................................................................................................... 15 1.1.3. Hemicellulose ................................................................................................. 16 1.1.4. Pectin ............................................................................................................... 16 1.1.5. Glycoprotein ................................................................................................... 17 1.1.6. Arabinogalactan-proteins (AGPs) .................................................................. 17 1.2. Objectives .............................................................................................................. 20 1.3. Explaination of my contribution and that of others to this thesis research ............ 21 1.4. Organization of the Thesis ..................................................................................... 22 Chapter 2: A small multigene hydroxyproline-O-galactosyltransferase family functions in arabinogalactan-protein Glycosylation .......................................................... 23 2.1. Introduction ............................................................................................................ 23 8 2.2. Materials and Methods ........................................................................................... 25 2.2.1. In silico analysis of temporal and spatial expression method of GALT1, GALT2, GALT3, GALT4, GALT5, GALT6, HPGT1, HPGT2, and HPGT3 .......... 25 2.2.2. Plant lines and plant growth conditions .......................................................... 26 2.2.3. Mutant confirmation by PCR and RT-PCR .................................................... 26 2.2.4. Fluorescent protein fusion and subcellular localization .................................. 30 2.2.5. Galactosyltranferase assay with microsomal preparations from transiently expressed GALT1, GALT3, GALT4, GALT6 in tobacco epidermal cells .............. 30 2.2.6. Extraction of AGPs ......................................................................................... 31 2.2.7. Evaluation of seed set ..................................................................................... 31 2.3. Results .................................................................................................................... 31 2.3.1. In silico analysis of temporal and spatial expression of GALT1, GALT2, GALT3, GALT4, GALT5, GALT6, HPGT1, HPGT2, and HPGT3 ........................ 31 2.3.2. Biochemical characterization of galt1, galt3, galt4, and galt6 genetic mutants……………………………………………………………………………32 2.3.3. Phenotypic characterization of galt1, galt3, galt4, and galt6 genetic mutants under normal (optimal) growth conditions………………………………………. 38 2.3.4. Phenotypic characterization of galt1, galt3, galt4, and galt6 genetic mutants under abnormal (restrictive) growth conditions (i. e., Yariv reagent and NaCl)....43 2.3.5. Subcellular localization of GALT4
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