Functional Characterization of Lysine-rich Arabinogalactan-Proteins (AGPs) and an AG Peptide in Arabidopsis A dissertation presented to the faculty of the College of Arts and Sciences of Ohio University In partial fulfillment of the requirements for the degree Doctor of Philosophy Yizhu Zhang November 2008 © 2008 Yizhu Zhang. All Rights Reserved. 2 This dissertation titled Functional Characterization of Lysine-rich Arabinogalactan-Proteins (AGPs) and an AG Peptide in Arabidopsis by YIZHU ZHANG 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 ZHANG, YIZHU, Ph.D., November 2008, Environmental and Plant Biology Functional Characterization of Lysine-rich Arabinogalactan-Proteins (AGPs) and an AG Peptide in Arabidopsis (160 pp.) Director of Dissertation: Allan M. Showalter The plant cell wall is composed of complex polysaccharides and a small amount of structural proteins and cell wall enzymes. Arabinogalactan-proteins (AGPs) are highly glycosylated, hydroxyproline-rich structural proteins that play important roles in plant growth and development. AtAGP17, 18 and 19 comprise the lysine-rich classical AGP family in Arabidopsis. They consist of an N-terminal signal peptide, a classical AGP domain disrupted by a short basic lysine-rich subdomain and a C-terminal glycosylphosphatidylinositol (GPI) anchor addition sequence. A previous study showed a null T-DNA insertion mutant of AtAGP19 displayed pleiotropic phenotypes. Here, a microarray approach was employed to elucidate changes in gene expression associated with the atagp19 mutant. The expression of several genes related to cell expansion were found to change significantly. Interestingly, one gene (At1g68720, cytidine/deoxycytidylate deaminase family protein) adjacent to AtAGP19 was found to be down-regulated about 50 fold and RT-PCR showed the absence of mRNA for this gene in the atagp19 mutant. Furthermore, complementation with the 3’ portion of the At1g68720 gene can fully restore all the wild type phenotypes, indicating this region is critical for the functions revealed by the agp19 mutant. To examine cellular localization of the lysine- rich AGPs, GFP-AtAGP17/18/19 fusion proteins as well as a GFP control were 4 overexpressed in Arabidopsis plants and the fusion proteins were present on the plant cell surface. Plasmolysis of leaf trichome cells further determined the localization of the fusion proteins at the plasma membrane. Moreover, in vitro pollen germination showed that AtAGP17, unlike LeAGP-1 (the lysine-rich AGP in tomato), was not associated with pollen tube elongation. To further elucidate AtAGP17/18/19 function(s), transgenic Arabidopsis overexpressing AtAGP17/18/19 without the GFP tag were produced. AtAGP18 overexpressors displayed several phenotypes distinct from the wild type plants: they were short and bushy, had short roots and produced less viable seeds. In contrast, the vector control transformants as well as the AtAGP17/19 overexpressors had the same phenotypes as the wild type plants. Furthermore, AtAGP18 was down-regulated by the plant hormone ABA, indicating ABA may be involved in AtAGP18 function(s). Finally, the expression pattern of AtAGP14 (At5g56540), an AG peptide in Arabidopsis was examined. AtAGP14 was highly expressed in flowers and young roots and moderately expressed in seedlings, stems and rosette leaves. A plate-based phenotypic analysis was also carried out for the T-DNA insertional mutant of AtAGP14 and wild type Arabidopsis but no significant differences were observed with respect to germination rate, true leaf numbers, primary root length and lateral root numbers. Approved: _____________________________________________________________ Allan M. Showalter Professor of Environmental and Plant Biology 5 ACKNOWLEDGMENTS First of all, I express my gratitude to my advisor, Dr. Allan M. Showalter, for his constant guidance, help and encouragement in my research and his patience to help me improve my writing skills. I thank Drs. Ahmed Faik, Marcia J. Kieliszewski and Sarah E. Wyatt for serving on my doctoral advisory committee and their support and valuable advice on my work. The Department of Environmental and Plant Biology, the Molecular and Cellular Biology Program and the Department of Biological Sciences have provided me with financial assistantship and a good research environment so that I can finish my dissertation. Specifically, I thank our department administrative, Connie Pollard, for her assistance in my lab work and dissertation. I thank Jeffrey Thuma for the confocal microscopy orientation, Darron Luesse for his valuable suggestions on Arabidopsis transformation techniques, John Withers for the insightful discussion of constructs design, Li Tan for the GFP control construct and Vijay Nadella for sequencing service from the Ohio University genomics facility. My labmates, Dr. Ming Chen, Dr. Wenxian Sun, Dr. Harjinder S. Sardar, Dr. Jie Yang, Yan Liang and Brian Keppler have offered me generous help in the lab. Working with them is a pleasant and unforgettable experience. I also thank Rebecca Vondrell for her assistance in screening transgenic plants and phenotype analysis. Finally, I am indebted to my relatives and friends for their understanding, help and support. 6 TABLE OF CONTENTS Page ABSTRACT........................................................................................................................3 ACKNOWLEDGEMENTS................................................................................................ 5 LIST OF TABLES............................................................................................................ 11 LIST OF FIGURES .......................................................................................................... 12 LIST OF ABBREVIATIONS........................................................................................... 14 CHAPTER 1 INTRODUCTION ...................................................................................... 16 Plant cell wall proteins.......................................................................................... 16 Arabinogalactan proteins (AGPs)......................................................................... 18 AGP structure........................................................................................................ 19 Protein backbone....................................................................................... 19 Carbohydrate............................................................................................. 21 The Hyp contiguity hypothesis ................................................................. 21 Molecular structure of AGPs .................................................................... 22 AGP classification ................................................................................................ 23 Cellular localization of AGPs ............................................................................... 24 Physiological function of AGPs............................................................................ 25 Role of AGPs in embryogenesis............................................................... 26 Role of AGPs in xylem development ....................................................... 27 Role of AGPs in reproduction................................................................... 28 Role of AGPs in cell division and expansion ........................................... 31 Role of AGPs in cell signaling.................................................................. 32 Lysine-rich AGPs.................................................................................................. 35 Specific aims of this dissertation research............................................................ 37 CHAPTER 2 T-DNA MUTANT STUDY OF atagp19 ................................................... 39 7 Summary............................................................................................................... 39 Introduction........................................................................................................... 40 Materials and methods .......................................................................................... 42 Plant materials and growth conditions...................................................... 42 Microarray experiments............................................................................ 42 Quantitative PCR (Real-time PCR) .......................................................... 45 Breaking force measurement .................................................................... 47 Complementation construct design with the AtAGP19 promoter sequence ................................................................................................................... 47 DNA extraction and amplification of AtAGP19 promoter ....................... 48 Restriction digestion ................................................................................. 49 Ligation and transformation...................................................................... 49 Construct verification by restriction digestion.......................................... 49 Preparation of Agrobacterium competent cells......................................... 50 Transformation of Agrobacterium using electroporation ......................... 50 Floral dip