IRX14 and IRX14-LIKE: Two Glycosyl Transferases involved in Glucuronoxylan Biosynthesis 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 Brian D. Keppler March 2010 © 2010 Brian D. Keppler. All Rights Reserved. 2 This thesis titled IRX14 and IRX14-LIKE: Two Glycosyl Transferases involved in Glucuronoxylan Biosynthesis in Arabidopsis by BRIAN D. KEPPLER 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 Benjamin M. Ogles Dean, College of Arts and Sciences 3 ABSTRACT KEPPLER, BRIAN D., M.S., March 2010, Plant Biology IRX14 and IRX14-LIKE: Two Glycosyl Transferases involved in Glucuronoxylan Biosynthesis in Arabidopsis (96 pp.) Director of Thesis: Allan M. Showalter IRX14 and IRX14-LIKE are two closely related glycosyl transferases in the glycosyl transferase 43 (GT43) family of Arabidopsis. IRX14 was initially identified during a coexpression search to identify glycosyl transferases involved in cell wall biosynthesis. A T-DNA insertion mutant for IRX14 results in comparatively minor changes, such as irregular xylem, while a mutation for IRX14L results in no changes. However, an irx14 irx14L double mutant severely affects growth and development with the dwarf plants failing to produce an inflorescence stem. Plants which are homozygous for the mutant version of IRX14 but heterozygous for IRX14L [irx14 irx14L(±)] exhibit an intermediate phenotype, including noticeably smaller leaves, stems, and underdeveloped siliques which contain only a few seeds. Additionally, the T-DNA insertion mutant for IRX14 was found to result in a drought tolerant phenotype. Carbohydrate analysis of total cell wall extracts revealed a reduction in xylose for the irx14 and irx14 irx14L(±) mutants, consistent with a defect in glucuronoxylan biosynthesis. Glucuronoxylan, along with cellulose and lignin, are the major components of secondary cell walls in Arabidopsis. Immunolocalization of xylan with the LM10 antibody revealed a loss of xylan in irx14 mutants and a further reduction in the irx14 4 irx14L(±) mutants. IRX14L likely functions redundantly with IRX14 in glucuronoxylan biosynthesis, with IRX14 having the more important role in the process. Approved: _____________________________________________________________ Allan M. Showalter Professor of Environmental and Plant Biology 5 ACKNOWLEDGMENTS First and foremost, I want to express my gratitude for my advisor, Dr. Allan Showalter, for his constant help, support, and guidance. I have learned so much and developed a much greater interest in research since I first began working in the lab as an undergraduate, largely due to the influence of Dr. Showalter. I also thank Dr. Sarah Wyatt, Dr. Ahmed Faik, and Dr. Marcia Kieliszewski for serving on my advisory committee and for their helpful advice and guidance. I am very grateful to the other members of the Showalter lab who have made my time here such an enjoyable experience including Dr. Harjinder Sardar, Dr. Jie Yang, Dr. Yizhu Zhang, Yan Liang, Dr. Wenliang Xu, Dr. Wei Tang, and Debarati Basu. I am also thankful for the undergraduates who have worked in the lab and assisted me with my work, Shawna Callaghan, Celeste Taylor, and Rebecca Vondrell, particularly for their help in screening mutant plants and phenotypic analysis. I thank Dr. Chris Havran for teaching me the histological techniques and Jeff Thuma for instruction in using the confocal microscope. I am grateful to Daniel Mullendore at Washington State University for capturing images with scanning electron microscopy and the Complex Carbohydrate Research Center in Athens, GA for performing the monosaccharide analysis. 6 TABLE OF CONTENTS Page Abstract ............................................................................................................................... 3 Acknowledgments............................................................................................................... 5 List of Tables ...................................................................................................................... 8 List of Figures ..................................................................................................................... 9 List of Abbreviations ........................................................................................................ 11 Chapter 1: Introduction to the Plant Cell Wall ................................................................. 13 Importance of the Cell Wall .......................................................................................... 13 Cellulose ....................................................................................................................... 13 Hemicelluloses .............................................................................................................. 14 Pectin ............................................................................................................................ 16 Glycoproteins ................................................................................................................ 17 Chapter 2: Identification of Glycosyl Transferases by a Coexpression Approach ........... 18 Introduction ................................................................................................................... 18 Materials and Methods .................................................................................................. 19 The Arabidopsis thaliana Co-response Database ...................................................... 19 Carbohydrate-Active Enzymes (CAZy) ................................................................... 19 Results ........................................................................................................................... 20 Discussion ..................................................................................................................... 33 Effectiveness of a Coexpression Approach .............................................................. 33 Selection of a Glycosyl Transferase for Further Analysis ........................................ 35 Chapter 3: IRX14 and IRX14-Like, Two Glycosyl Transfereases involved in Glucuronoxylan Biosynthesis in Arabidopsis ................................................................... 37 Introduction ................................................................................................................... 37 Glucuronoxylan Biosynthesis ................................................................................... 37 Materials and Methods .................................................................................................. 39 Plant Growth Conditions ........................................................................................... 39 Identification of T-DNA Insertion Lines .................................................................. 40 Bioinformatics ........................................................................................................... 40 RNA Extraction and RT-PCR ................................................................................... 41 7 Histology ................................................................................................................... 42 Drought Stress ........................................................................................................... 42 NaCl Treatment ......................................................................................................... 43 Monosaccharide Analysis ......................................................................................... 43 Xylan Immunolocalization ........................................................................................ 44 Results ........................................................................................................................... 44 The GT43 Family ...................................................................................................... 44 Expression Pattern .................................................................................................... 47 T-DNA Insertion Mutants ......................................................................................... 48 Comprehensive Phenotypic Analysis ....................................................................... 52 irx14 irx14L Double Mutant ..................................................................................... 54 RT-PCR Analysis ...................................................................................................... 56 Irregular Xylem ......................................................................................................... 57 Drought Tolerance .................................................................................................... 61 Salt Tolerance ........................................................................................................... 64 Carbohydrate Analysis .............................................................................................. 65 Xylan Immunolocalization ........................................................................................ 66 Allelic T-DNA Mutants ............................................................................................ 67 Discussion ....................................................................................................................