Identification and Characterization of Galactosyltransferases and Fucosyltransferases. Involved in Arabinogalactan-Protein Glycosylation

Transcription

1 Identification and Characterization of Galactosyltransferases and Fucosyltransferases Involved in Arabinogalactan-Protein Glycosylation 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 Yan Liang August Yan Liang. All Rights Reserved.

2 2 This dissertation titled Identification and Characterization of Galactosyltransferases and Fucosyltransferases Involved in Arabinogalactan-Protein Glycosylation by YAN LIANG 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 Howard Dewald Interim Dean, College of Arts and Sciences

3 3 ABSTRACT LIANG, YAN, Ph.D., August 2012, Molecular and Cellular Biology Identification and Characterization of Galactosyltransferases and Fucosyltransferases Involved in Arabinogalactan-Protein Glycosylation Director of Dissertation: (Allan M. Showalter) Arabinogalactan-proteins (AGPs) are highly glycosylated hydroxyproline-rich glycoproteins (HRGPs) that are frequently characterized by the presence of the repetitive dipeptide motifs [Ala-Hyp] or [Hyp-Ala]. Hydroxyproline (Hyp) residues in the dipeptide motifs are sites for the attachment of arabinogalactan (AG) sugar side chains. Fucose residues are found in some dicot AGPs, and AGP fucosylation is developmentally regulated. AGP galactosyltransferase (GalT) and fucosyltransferase (FUT) activities were investigated in three ways in this study. First, an in vitro AGP GalT assay was developed, which used permeabilized microsomal membranes from tobacco (Nicotiana tobacum) or Arabidopsis (Arabidopsis thaliana) suspension-cultured cells as the enzyme source and UDP-[ 14 C] Gal as the sugar donor. Two model AGP peptides, [Ala-Hyp] 7 or [AO] 7 and deglycosylated [Ala-Hyp] 51 or d[ao] 51, were used as substrate acceptors. Product analysis indicated that the [AO] 7 :GalT assay and the d[ao] 51 :GalT assay mainly detected GalT activities that added the first and the second Gal residues in the Hyp-AG side chain. Examination of the Hyp:GalT activity using various acceptor substrates, including two extensin sequences and a [AP] 7 peptide, indicated this activity was specific for Hyp dipeptide motifs in AGPs. The [AO] 7 :GalT and d[ao] 51 :GalT activities were localized to the endomembrane system of Arabidopsis suspension cultured cells following sucrose

4 4 density gradient centrifugation. Second, five candidate AGP GALT genes (GALT1: At1g26810; GALT3: At3g06440; GALT4: At1g27120; GALT5: At1g74800; GALT6: At5g62620) were expressed in Pichia pastoris and tobacco suspension-cultured cells and tested for AGP GalT activity using the [AO] 7 :GalT and d[ao] 51 :GalT assay systems. Preliminary results indicate that heterologously expressed GalT3, GalT5 and GalT6 have AGP GalT activities. Furthermore, GalT3 was shown to be Golgi localized in a tobacco leaf expression system. Third, functions of the FUT4 and FUT6 genes were investigated using Arabidopsis fut4, fut6 and fut4/fut6 mutant plants. Biochemical analysis indicated that FUT4 was required for fucosylation of leaf AGPs, while both FUT4 and FUT6 contributed to fucosylation of root AGPs. In addition, glycome profiling indicated that fucosylated AGPs may regulate intermolecular interactions between AGPs and other wall components. Finally, a model of HRGP biosynthesis is proposed which highlights the glycosyltransferases involved in this process. Approved: Allan M. Showalter Professor of Environmental and Plant Biology

5 5 ACKNOWLEDGMENTS First and foremost, I am thankful to my advisor, Dr. Allan Showalter, the one who provided guidance throughout my PhD study; the one who offered the research assistant fellowship to allow me to concentrate on my research; the one who encouraged and trusted me in trying out my own ideas in experiments. I especially thank Dr. Ahmed Faik, who shared his expertise in enzyme biochemistry, his ideas of experimental design and his lab equipment, which were indispensable for the progress of my project. I also thank Dr. Marcia Kieliszewski, who is a critical thinker in science and at the same time impressed me with the feeling that doing science is an enjoyable process. I am grateful to Dr. Sarah Wyatt, not only for the information she taught me, but also for her guidance and help in becoming socialized in the academic society. I thank Dr. Wen-liang Xu for his contribution to the RNA analysis and HPAEC analysis in the FUT project and Ms. Alexandra Venetos and Ms. Rebecca Vondrell for their help in phenotypic analysis of the fut mutants. Mr. Xiao Liu and Mr. Wuda Wang helped with the bacterium colony screening in the expression construct cloning of GALT genes. Dr. Wei Zeng tutored me in the techniques of microsomal protein extraction, making sucrose gradient density, HPAEC and MS analysis and Pichia protein expression. Dr. Li Tan provided the d[ao] 51 peptide substrate and helped to solve the technical problems with the RP-HPLC system. Ms. Debarati Basu helped with the GC/MS analysis and shared her experience in Pichia protein expression and activity tests. Dr. Michael Held helped to set up the tobacco leaf expression system and advised on the use of the confocal microscope. Dr. Betsy Briju, Ms. Laura Cristea and Mr. Vijayanand Nadella

6 6 provided suggestions in tobacco BY2 transformation. Mr. Yuning Chen and Mr. Dening Ye helped me with the liquid chromatography techniques. Dr. Glen Jackson and Dr. Hao Chen assisted with the ESI-MS/MS analysis. Dr. Jie Yang and Dr. Yizhu Zhang provided many helpful suggestions for both my research and my PhD study. The administrative and coordination work done by Ms. Connie Pollard, Ms. Martha Bishop and Dr. Melanie Schori was fantastic. Also, the previous and current members of the Showalter lab and Faik lab, Dr. Harjinder Sardar, Ms. Mohor Chatterjee, Mr. Nan Jiang, Mr. Brian Keppler, Mr. Richard Wiemel and Ms. Rebekah Whitley were such supportive and nice people to be around. Without the help and kindness from all the above people, my research would not have gone as smoothly and my study at OU would not have been so enjoyable. I thank the Department of Environmental and Plant Biology and the Molecular and Cellular Program for providing teaching assistant scholarships and funding for me to attend national meetings. Last but not least, I am indebted to my family and friends, who walked me through difficult times and provide me with endless love and support.

7 7 TABLE OF CONTENTS Page Abstract.3 Acknowledgments... 5 List of Tables 14 List of Figures 16 List of Abbreviations Chapter 1: Introduction Plant Cell Walls Biological and economic importance Structural model and compositions Biosynthesis of cell wall polysaccharides Hydroxyproline Rich-Glycoproteins (HRGPs) Classification Three major HRGP subfamilies: PRPs, EXTs and AGPs HRGP Glycosylation Arabinogalactan-Proteins (AGPs) Classification... 52

8 Post-translational modifications Biological functions Specific Aims of the Research Chapter 2: Identification and Characterization of in vitro Galactosyltransferase Activities Involved in Arabinogalactan-Protein Glycosylation in Tobacco and Arabidopsis Introduction Material and Methods Suspension culture of Arabidopsis cells Preparation of microsomal membranes from tobacco and Arabiodopsis suspension cultured cells Standard assay for galactosyltransferase (GalT) activities Analysis of the GalT assay products by reverse phase-high performance liquid chromatography (RP-HPLC) Monosaccharide composition analysis of the GalT assay product Hyp [ 14 C]Galactoside profile analysis Characterization of the Arabidopsis [AO] 7 :GalT activity Electrospray Ionization-Tandem Mass Spectrometry (ESI-MS/MS) of the [AO] 7 :GalT assay product... 73

9 Continuous sucrose gradient centrifugation Results Development of a GalT assay system with AGP-like peptides as acceptor substrates Characterization of the GalT assay products by Reverse-Phase High Performance Liquid Chromatography (RP-HPLC) analysis Extent of AGP peptide galactosylation Enzymatic characteristics of the [AO] 7 :GalT activity in Arabidopsis suspension-cultured cells Identification of the Arabidopsis [AO] 7 :GalT reaction product by Electrospray Ionization-Tandem Mass Spectrometry (ESI-MS/MS) analysis Subcellular localization of [AO] 7 :GalT and d[ao] 51 :GalT activities in Arabidopsis suspension cultured cells Discussion Chapter 3: Heterologous Expression of Putative AGP Galactosyltransferases in Pichia Pastoris and Tobacco BY2 Cells and Their Functional Characterization Introduction Material and Methods Obtaining full length cdna clones

10 Sequence analysis of the putative GalTs Expression construct cloning of GALT1, GALT3, GALT4, GALT5 and GALT6 for expression in Pichia Pichia transformation Expression construct cloning of GALT1, GALT3, GALT4, GALT5 and GALT6 for expression in tobacco BY2 suspension-cultured cells Agrobacterium transformation Transformation of tobacco BY2 suspension-cultured cells Isolation of microsomal membranes from Pichia Preparation of microsomal membranes from tobacco BY2 cell lines Western blotting analysis AGP GalT activity test Transient expression of GALT3-YELLOW FLUORESCENT PROTEIN (YFP) in tobacco leaves Results Comparison of the GALT sequences of the corresponding cdna clones and the GALT sequences from the Arabidopsis Information Resource (TAIR) database Expression construct cloning and yeast/tobacco BY2 cell transformation

11 Protein expression analyses and AGP GalT activity tests of the microsomal membranes isolated from Pichia strains transformed with the P AOX :6xHis-GALT genes Protein expression analyses and AGP GalT activity tests of the microsomal membranes isolated from tobacco BY2 cell lines transformed with the P hsp :6xHis-GALT genes Protein expression analyses and AGP GalT activity tests of the microsomal membranes isolated from tobacco BY2 cell lines transformed with the P 2x35S :6xHis-GALTs genes Subcellular localization of GalT3 in tobacco leaf epidermal cells Discussion Chapter 4: Biochemical and Physiological Characterization of fut4 and fut6 Mutants Defective in Arabinogalactan-Protein Fucosylation in Arabidopsis Introduction Materials and Methods Plant materials and growth conditions Mutant confirmation by PCR and RT-PCR Phenotypic analysis Eel lectin staining Monosaccharide composition analysis by GC-MS

12 Monosaccharide composition analysis by HPAEC Monoclonal antibodies Preparation of AIR (Alcohol Insoluble Residue) extracts and fractionation Total sugar estimation and ELISA Expression analysis of FUT4 and FUT6 genes based on microarray data from Genevestigator Results Isolation of T-DNA insertion lines of fut4 and fut6 and generation of the fut4/fut6 double mutant Phenotypic analysis of fut4, fut6 and fut4/fut6 mutants under physiological growth conditions Eel lectin shows a different staining pattern in roots of fut6 and fut4/fut6 mutant plants compared to fut4 and wild type plants fut4/fut6 mutants are defective in AGP fucosylation in roots and leaves Glycome profiling of root and leaf cell walls of wild type, fut4, fut6 and fut4/fut6 mutant plants Discussion Chapter 5: Conclusions

13 References Appendix A: Sequences and Locations of Primers Used in This Work Appendix B: Partial Sequence of the Final Expression Constructs Appendix C: Expanded List of All Plant Cell Wall Glycan-Directed Monoclonal Antibodies (MAbs) Used in This Study for Glycome Profiling

14 14 LIST OF TABLES Page Table 1.1 Polysaccharide synthases and GTs involved in plant cell wall biosynthesis from Arabidopsis thaliana and other plants. a Table 2.1 Incorporation of [ 14 C] radiolabel into the [AO] 7 acceptor substrate when various nucleotide sugars were used as the sugar donor Table 3.1 Structural features of the 20 putative Arabidopsis β-(1 3)-GalTs a Table 3.2 Expression constructs and transgenic Pichia or tobacco BY2 cell lines obtained in this research Table 3.3 Summary of the signal bands observed in microsomal membrane proteins from transgenic and control Pichia and tobacco BY2 cells in western blotting analysis Table 3.4 Results of the tests for AGP GalT activities in microsomal membranes from transgenic strains using the in vitro [AO] 7 :GalT and d[ao] 51 :GalT assay systems Table 3.5 AGP GalT activities of the upper membrane layer from wild type BY2 cells and BY2 lines transformed with the P hsp :6xHis-GALT or the P 2x35S :6xHis- GALT genes Table 4.1 Flowering time and silique numbers of wild type, fut4, fut6 and fut4/fut6 mutant plants grown in soil under physiological growth conditions Table 4.2 Neutral monosaccharide content of purified AGPs from roots and leaves of wild type, fut4, fut6 and fut4/fut6 mutant plants

15 15 Table 4.3 Expression analysis of the FUT4 and FUT6 genes in response to biotic and abiotic stimuli based on microarray data from Genevestigator ( 172 Table 5.1 GT activities involved in AGP glycosylation

16 16 LIST OF FIGURES Page Figure 1.1 Protein sequences of a classical AGP molecule, an EXT molecule and a hybrid PRP molecule identified in Arabidopsis Figure 1.2 A simplified HRGP model illustrating the Hyp contiguity hypothesis for a model AGP protein sequence and a model EXT protein sequence Figure 2.1 AG structure of an AGP molecule Figure 2.2 Total [ 14 C] radiolabel incorporation into the GalT reaction product in the absence or presence of [AO] 7 or d[ao] 51 acceptor substrate Figure 2.3 RP-HPLC fractionation of the [AO] 7 :GalT and d[ao] 51 :GalT reaction products on a PRP-1 reverse-phase column Figure 2.4 Monosaccharide analysis of the RP-HPLC purified tobacco d[ao] 51 :GalT reaction product (Panel A) and the Arabidopsis [AO] 7 :GalT reaction product (Panel B) Figure 2.5 Bio-gel P2 fractionation of the RP-HPLC purified GalT reaction product Figure 2.6 Bio-gel P2 fractionation of free Hyp amino acid (Panel A) or Hyp amino acid produced following base hydrolysis of the [AO] 7 acceptor substrate (Panel B) Figure 2.7 Biochemical characteristics of the Arabidopsis [AO] 7 :GalT activity Figure 2.8 Incorporation of [ 14 C] radiolabel into acceptor substrates containing various Hyp motifs Figure 2.9 ESI-MS/MS analysis of the Arabidopsis [AO] 7 :GalT reaction product

17 17 Figure 2.10 Fractionation of mixed-membranes of Arabidopsis on a sucrose gradient in the presence of 1 mm EDTA Figure 3.1 Schematic domain structure of the two classes of putative Arabidopsis β- (1 3)-GalTs Figure 3.2 PCR analysis of Pichia transformed with ppicza-6xhis-galt1, ppicza- 6xHis-GALT3, ppicza-6xhis-galt4, ppiczb-6xhis-galt5, ppiczb- 6xHis-GALT Figure 3.3 Verification of the constructs of pmdc30-6xhis-galt by diagnostic digestion with the AscI and SacI restriction enzymes Figure 3.4 PCR analysis of the Agrobacterium strains transformed with the pmdc30-6xhis-galt constructs Figure 3.5 PCR analysis of tobacco BY2 cell lines transformed with the hsp:6xhis-galt genes Figure 3.6 Verification of the constructs of pmdc32-6xhis-galts by diagnostic digestion and PCR Figure 3.7 PCR analysis of the Agrobacterium strains transformed with the pmdc32-6xhis-galt constructs Figure 3.8 PCR analysis of the tobacco BY2 cell lines transformed with the P 2x35S :6xHis- GALT genes Figure 3.9 Western blotting analysis of microsomal proteins isolated from Pichia transformants

18 18 Figure 3.10 RP-HPLC fractionation of the [AO] 7 substrate acceptor and the [AO] 7 :GalT reaction products on a PRP-1 reverse-phase column Figure 3.11 Western blotting analysis of microsomal proteins isolated from the tobacco BY2 lines transformed with the P hsp :6xHis-GALT genes and the wild type BY2 lines Figure 3.12 Western blotting analysis of proteins from microsomal membranes isolated from tobacco BY2 lines transformed with the P 2x35S :6xHis-GALT genes and wild type BY2 lines Figure 3.13 Subcellular localization of GalT3 in tobacco leaf epidermal cells Figure 4.1 Identification of T-DNA insertion lines of Arabidopsis fut4, fut6, and fut4/fut6 mutants by PCR Figure 4.2 RNA transcript levels of FUT4 and FUT6 genes in homozygous fut4, fut6, fut4/fut6 double mutants and wild type seedlings Figure 4.3 Phenotypic analysis of Arabidopsis fut4, fut6 and fut4/fut6 mutant plants compared to wild type plants under physiological growth conditions Figure 4.4 Germination rate of Arabidopsis fut4, fut6 and fut4/fut6 double mutant and wild type plants Figure 4.5 Root growth of Arabidopsis wild type, fut4, fut6 and fut4/fut6 mutant plants Figure 4.6 Eel lectin staining of roots from WT, fut4, fut6 and fut4/fut6 mutant plants. 158 Figure 4.7 Monosaccharide composition analysis of root AGPs from WT, fut4, fut6 and fut4/fut6 mutant plants by HPAEC

19 19 Figure 4.8 Glycome profiling of sequential cell wall extracts from roots of WT, fut4, fut6 and fut4/fut6 double mutant plants Figure 4.9 Glycome profiling of sequential cell wall extracts from leaves of WT, fut4, fut6 and fut4/fut6 double mutant plants Figure 5.1 A model for HRGP hydroxylation and Hyp glycosylation through the secretary pathway Figure 5.2 Predicted GT activities involved in the formation of a 15-sugar unit for AGP glycosylation

20 20 LIST OF ABBREVIATIONS [AO] 7 : [alanine-hydroxyproline] 7 AG: arabinogalactan AGP: arabinogalactan-protein AIR: alcohol insoluble residue Ala: alanine AOX: alcohol oxidase Ara: arabinose Arabidopsis: Arabidopsis thaliana AraT: arabinosyltransferase Arg: arginine Asn: asparagine Asp: aspartic acid CAZy: the Carbohydrate Active enzymes (CAZy) database CCRC: the Complex Carbohydrate Research Center CID: collision-induced dissociation Cys: cystine d[ao] 51 : deglycosylated [alanine-hydroxyproline] 51 dd[ao] 51 : double deglycosylated d[ao] 51 peptide DP: degree of polymerization EDTA: ethylenediaminetetraacetic acid ElISA: enzyme-linked immunosorbent assay ER: endoplasmic reticulum ESI-MS: Electrospray Ionization-Tandem Mass Spectrometry EST: expressed sequence tag EXT: extensin ExtP: extensin peptide FITC: fluorescein isothiocyanate FLA: fasciclin-like AGP

21 21 FUC XG: fucosylated xyloglucan Fuc: fucose FUT: fucosyltransferase G unit: guaiacyl unit of lignin monomer GABA: γ-aminobutyric acid GAGP: gum arabic glycoprotein Gal: galactose Gal: galactosyltransferase GalA: galacturonic acid Galectin domain: galactose-binding lectin domain GalNAcT: acetylgalactosaminyltransferases GAUT: galacturonosyltransferase GAX: glucuronoarabinoxylan GC-MS: gas chromatography-mass spectrometry GFP: green fluorescent protein Glc: glucose GlcA: glucuronic acid GlcA: glucuronyltransferase GlcT: glucosyltransferase Gln: glutamine Glu: glutamic acid Gly: glycine GPI: glycosylphosphatidylinositol GT: glycosyltransferase H unit: hydroxyphenyl unit of lignin monomer HF: hydrogen fluoride HG: homogalacturonan His: histidine HPAEC: High ph Anion Exchange Chromatography

22 22 HRGP: Hydroxyproline rich-glycoproteins Hyp: hydroxyproline Idt: isodityrosine Lys: lysine mab: monoclonal antibody Man: mannose MeClcA: O-methyl-GlcA N-glycan: N-linked oligosaccharide NMR: magnetic resonance NON-FUC XG: nonfucosylated xyloglucan P4H: proline 4-hydroxylase Pfam: protein family database PMSF: phenylmethylsulphonyl fluoride Pro: proline PRP: proline rich protein RG-I: rhamnogalacturonan I RG-II: rhamnogalacturonan I Rha: rhamnose RP-HPLC: reverse phase-high performance liquid chromatography S unit: syringyl unit of lignin monomer Ser: serine ST: sialyltransferase TFA: trifluoroacetic acid Thr: threonine Tyr: tyrosine Val: valine Xyl: xylose XylT: xylosyltransferase β-yariv reagent: (β-d-galactosyl) 3 or (β-d-glucosyl) 3 Yariv phenylglycosides

23 23 CHAPTER 1: INTRODUCTION 1.1. Plant Cell Walls Biological and economic importance Plant cell walls are a major feature of plant cells. Plant cell walls, with their rigid structure, define plant cell shapes and provide mechanical support for the plant body. Born at the cell plates during cell division, plant cell walls are involved in plant embryogenesis and organ formation. In differentiated cells, cell wall structures acquire heterogeneity at the tissue and individual cell levels, or even across a single cell wall to accommodate the diverse functions of plant cells (Knox, 2008; Burton et al., 2010). Lying as the outermost layer of plant cells, the plant cell wall is also an active player in plant response to biotic and aboitic factors in the environment (Vorwerk et al., 2004). Plant cell walls are not only of biological importance for plants; they are the major components of feedstock and used for the production of textile, constructional materials and paper in industry (Carpita and McCann, 2000). In agriculture, cell wall compositions directly affect food and forage properties such as digestibility and shelf life. Some essential food content, like dietary fiber, comes from the plant cell wall. Specific cell wall components, such as gum arabic and pectins, are widely-used food ingredients and serve as emulsifiers, stabilizers or carriers for flavoring. Furthermore, cell wall biomass is one of the potential sources for biofuel production to meet the urgent need of the world for sustainable energy.

24 24 Given the biological and economic importance of plant cell walls, cell wall biology is of particular interest in plant biology. Acquiring knowledge about cell wall structure, regulatory mechanisms, biosynthesis and degradation is essential in the attempt to manipulate plants for better agricultural and industrial uses Structural model and compositions The architecture of plant cell walls differ depending on plant species, developmental stages of a plant or even differentiated types of cells within a plant (Carpita and Gibeaut, 1993; Knox, 2008). All plant cells have primary cell walls, which are deposited at the cell plate and grow accordingly during cell expansion. Some specialized cell types (e.g. xylem cells) also build a secondary cell wall after cessation of cell growth, between the primary cell walls and the plasma membrane. The primary cell walls of flowering plants are classified into two types (Carpita and Gibeaut, 1993; Carpita and McCann, 2000). The type I cell wall is present in dicot plants and most of the monocot plants whereas the type II cell wall is found in commelinoid monocots. The type I cell walls are composed of cellulose microfibrils interconnected by the hemicellulose xyloglucan. This load-bearing framework is embedded in a network of pectin polymers, which constitute the wall matrix, controlling wall porosity and many other physiological properties. The type I cell walls have substantial amount of protein components playing structural roles in muro. In type II cell walls, glucuronoarabinoxylan (GAX) replaces xyloglucan as an intermolecular linker for cellulose microfibrils, with the exception of plants of the order Poales, for which mixed-linkage glucans serve as the major cross-linking hemicellulose. The type II cell wall is low in pectin polymers as well

25 25 as structural protein components but rich in phenolic substances. Compared to primary cell walls, secondary cell walls have higher content in cellulose and lower content in pectin and protein components (Albersheim et al., 2011). Secondary cell walls often feature the presence of lignin polymers, which enhance the mechanical strength of cell walls and serve as barriers against water diffusion and pathogen invasion. The cell wall model is a developing model with the incorporation of new concepts from the advances of researchers. For example, one of the major cell wall glycoproteins, extensins (EXTs), was originally considered to form an independent wall domain, functioning to lock microfibrils in position when cells cease in growth (Lamport, 1986; Carpita and Gibeaut, 1993). Recent findings showed that EXT molecules are capable of self-assembly and are likely to self-assemble into a scaffold which directs the assembly of newly synthesized wall materials at the cell plate during cytokinesis (Cannon et al., 2008; Lamport et al., 2011) Celluloses Cellulose is the principle structural component of plant cell walls. It composes 20-30% (w/w) of both the type I and type II cell walls and accounts for up to 50% (w/w) of secondary cell walls (Albersheim et al., 2011). Unlike most of the other cell wall polysaccharides, which have sugar backbones frequently substituted by monosaccharides or larger sugar side chains, cellulose molecules consist of unsubstituted β-d-(1 4)- glucan chains. Two to three dozen of the glucan chains align in parallel and assemble into cellulose microfibrils (Carpita, 2011). With the glucan chains bound tightly through hydrogen bonds (Nishiyama et al., 2002; Nishiyama et al., 2003), cellulose microfibrils

26 26 are present as a crystalline structure, endowing them with strength and stiffness. The length of glucan chains varies from several hundred to several thousand glucose (Glc) units (measuring up to a few micrometers in length) depending on wall type (Brett, 2000; Brown et al., 2005). Individual glucan chains are thought to begin and end at different places within the microfibril to constitute longer cellulose fibrils (Carpita and McCann, 2000) Xyloglucans Xyloglucans constitute 10-20% (w/w) of Type I cell walls (Fry, 1989) as the principle cross-linking molecules. Xyloglucan molecules have a β-d-(1 4)-glucan backbone, with sugar substitutions occurring at regular positions. Substitutions on Glc residues in xyloglucan are limited to a few types, each of which is given a one-letter code as an abbreviation. Whereas G denotes unsubstituted Glc residues; X denotes Glc residues substituted by a single α-d-xylose (Xyl) at the O-6 position; L and A denote O-6 of the Glc residues substituted by β-d-galactose (Gal) -(1 2)-α-D-Xyl and α-larabinose (Ara) -(1 2)-α-D-Xyl, respectively. If the Gal on the L chain is further substituted by an α-l-fucose (Fuc) residue, the chain is denoted as F (Fry et al., 1993). With these regular sugar substitutions, xyloglucan can be viewed as a polymer constructed of repeating oligosaccharide blocks. The predominant xyloglucan, the type I cell wall xyloglucan, is built up mainly of XXXG and XXFG blocks, although α-l-ara is added at some places along the glucan chain (Carpita and McCann, 2000). Other forms of xyloglucan exist. For solanaceous species and peppermint, xyloglucan is a mixture of AXGG, XAGG and AAGG subunits (York et al., 1996). In commelinoid monocots, the

27 small amount of xyloglucan present is modified by xylose residues at random places on the backbone (Kato et al., 1982) Xylans Xylans refer to a family of polysaccharides having a β-d-(1 4)-xylan backbone in common (Scheller and Ulvskov, 2011). Modifications of the Xyl residues mainly occur at C-2 and C-3 positions with the attachment of α-ara residues, α-glucuronic acid (GlcA) or O-methyl-α-GlcA (MeGlcA) residues. Different species of xylan molecules vary in the types of substitutions and are present in different plant species and tissues. Secondary cell walls of dicot plants are composed of up to 35% (w/w) glucuronoxylans, which have little or no Ara but have approximately 10% to 20% (w/w) of the Xyl residues substituted with MeGlcA residues (Zeng et al., 2008). In commelinoid monocots, GAX serves as the principle hemicellulose, making up to 20% (w/w) of the cell wall. GAX is substituted with α-ara at the C-3 and, less frequently, single GlcA at the C-2 of the xylosyl units (Carpita and Gibeaut, 1993). The percentage of substituted Xyl residues ranges from 10% to 90% in GAX molecules. The higher degree of GAX substitution prevents bonding between GAX to itself and to cellulose, and so favors the need of dividing and elongating cells (Carpita and Gibeaut, 1993). GAX of commelinoid plants have ferulic acid esters formed at O-5 of some of the Ara residues. Feruloylated GAX may form intra- and intermolecular linkages within the GAX group or to lignin molecules, which is predicted to contribute to the recalcitrant nature of cell walls (Scheller and Ulvskov, 2011).

28 Mannans and glucomannans Mannans and glucomannans are β-(1 4)-linked polysaccharides containing mannose (Man) in their backbones (Scheller and Ulvskov, 2011). Mannans and galactomannans have backbones composed entirely of Man residues. Mannans have little if any substitutions on the backbone while galactomannans are substituted by α-1,6- linked Gal to various extents (Reid et al., 1995). The backbones of glucomannans and galactoglucomannans contain Glc and Man residues connected in a nonrepeating pattern (Scheller and Ulvskov, 2011). D-Gal is bonded as single-unit side chains to the glucomannan chain through α-(1 6)-linkages (Willfor et al., 2003). Mannans and galactomannans are well known to be prevalent in the endosperm of legume seeds. Galactoglucomannans are major components of the secondary cell walls in gymnosperms (Ebringerová et al., 2005). In other cell types of higher plants, this group of molecules is present in low abundance but may play essential roles (Goubet et al., 2003) Mixed-linkage glucans Mixed-linkage glucans mainly consist of β-(1 4)-linked glucotriose and glucotetraose units interspersed by single β-(1 3)-linkages, although oligomers of five or more contiguous β-(1 4)-linked Glc units also occur (Carpita and McCann, 2000). Mixed-linkage glucans are found throughout the order of Poales but not in dicot plants. In Poales, mixed-linkage glucan content is developmentally regulated and plays roles in cell expansion and cellulose cross-linking (Scheller and Ulvskov, 2011).

29 Pectins Pectins are a family of cell wall polysaccharides rich in galacturonic acid (GalA). GalA residues are linked at O-1 and O-4 positions and comprise approximately 70% of the pectin composition (Mohnen, 2008). The three major components, homogalacturonan (HG), rhamnogalacturonan I (RG-I) and rhamnogalacturonan II (RG-II), compose around 65%, 20%, and 10% of the pectin polymers, respectively. HG is an unbranched α-(1 4)- linked GalA chain. HG is partially methylesterified at the C-6 position and may be acetylated at O-2 or O-3. RG-II has a HG backbone substituted by four types of oligosaccharide and polysaccharide side chains. The four types of RG-II side chains in total are comprised of 12 types of sugars in over 20 different linkages. The highly complex structure of RG-II side chains is conserved across species, implying functional importance of these structures (Carpita and McCann, 2000). RG-II is found to form dimers via apiosyl residues in the side chains (O'Neill et al., 2004). RG-I contains a backbone of a repeating disaccharide unit [-α-d-gala-(1 2)-α-L-Rha-(1 4)-]. In contrast to RG-II, the side chains of RG-I exhibit a high degree of structural variation depending on cell types and developmental stages. Studies suggest that HG, RG-I and RG-II are linked via their backbones to form pectic polymers (Ishii and Matsunaga, 2001; Nakamura et al., 2002). Pectic polymers are cross-linked by RG-II dimerization into a macromolecular pectin network (Fleischer et al., 1999). The negatively charged HG domains interact with each other in the presence of Ca 2+ ions, forming the gel structure of pectin. Bridging between HGs through Ca 2+ is blocked by HG methylesterification, which can be reversed by pectin methylesterases in the cell wall.

30 Lignin Lignin is a complex aromatic biopolymer based on 4-hydroxyphenylpropanoids (Simmons et al., 2010). Three major lignin monomers are hydroxyphenyl unit (H units), guaiacyl unit (G units), and syringyl unit (S units), which are predominantly interconnected through ether linkages and carbon-carbon linkages (Albersheim et al., 2010). Lignins are deposited predominantly in secondary cell walls, reinforcing the mechanical properties of the wall and serving as a barrier to prevent water diffusion in vascular tissues (Vanholme et al., 2010). Biosynthesis of lignin is affected by biotic and abiotic stresses, indicating a role of lignin in plant defense and resistance to unfavorable conditions (Moura et al., 2010) Plant cell wall proteins Plant cell wall proteins refer to proteins located outside of the plasma membrane or in the wall as either bound components or free molecules. Based on genomic and proteomic data, it has been estimated that hundreds of cell wall proteins are present in organ and tissue specific patterns (Robertson et al., 1997). According to a recent survey, 281 plant cell wall proteins (including predicted ones) are grouped into polysaccharide modifying proteins, oxido-reductases, structural proteins, signaling proteins, proteases, proteins with interacting domains, miscellaneous proteins, and proteins with unknown function (Jamet et al., 2006). Plant cell wall proteins play essential roles in the wall, including modifications of cell wall components, constitution of wall structure, signaling, interactions between the wall and plasma membrane, and responses to environmental

31 31 stimuli (Carpita and McCann, 2000). Although a few cell wall proteins have been studied extensively, e.g. expansins (Cosgrove, 2000) and EXTs (Lamport et al., 2011), the exact functions and regulatory mechanisms of most cell wall proteins remain to be confirmed and further explored Biosynthesis of cell wall polysaccharides The biosynthesis of plant cell walls involves enzymes to synthesize nucleotide sugars as building blocks, the polysaccharide synthases and glycosyltransferases (GTs) to assemble the polysaccharides as well as multiple modifying enzymes, such as glycohydrolases, to complete the products. Polysaccharide synthases catalyze the synthesis of the backbones of wall polysaccharides (Perrin et al., 2001). Backbones of polysaccharides usually consist of repeating units of a single type of monosaccharide. Accordingly, polysaccharide synthases are processive, i.e. the sugar-transfer product could be used continuously as the substrate for the same enzyme (delcardayre and Raines, 1994). GTs catalyze the formation of sugar side chains which are diverse in their composition and architecture (Perrin et al., 2001). GTs are non-processive. A basic difference between sugar synthesis and nucleic acid or protein synthesis is that sugar synthesis does not involve templates. The accurate assembly of the complex structures of polysaccharides relies on the specificity of GTs that recognize specific substrate structures and generate unique glycosyl linkages in products. Given that plant cell wall polymers consist of at least 14 different types of monosaccharides connected by over 100 distinct glycosyl linkages (Keegstra and Raikhel, 2001), there is no surprise that over 400 genes have been predicted as putative GTs in the Carbohydrate Active enzymes (CAZy)

32 32 database ( for the Arabidopsis (Arabidopsis thaliana) genome alone. The CAZy database classifies GTs into families on the basis of amino acid sequence similarities, which reflects the intrinsic structural and mechanistic features of the enzymes (Campbell et al., 1998; Coutinho et al., 2003). Polysaccharide synthases and GTs are usually membrane proteins and are present in low amounts in plant cells, making their purification notoriously difficult. However, substantial progress has been made in recent years in the identification of genes for the biosynthesis of cell wall polymers. A variety of strategies have been developed and are usually applied in combination, including homolog identification based on the polysaccharide synthases or the GTs known in other organisms; conventional biochemical purification methods coupled with proteomic methods; forward genetic and reverse genetic methods; and gene mining based on expression data of expressed sequence tags (ESTs) or microarray data. Table 1 summarizes the polysaccharide synthases and GTs identified unambiguously for the synthesis of specific wall polymers to date, with the activity tests and mutant phenotypes included if such information is available.

33 Table 1.1 Polysaccharide synthases and GTs involved in plant cell wall biosynthesis from Arabidopsis thaliana and other plants. a Species and Gene CAZy GT Family Activity Mutant (Mutagen) Mutant Wall Major Phenotype (s) Reference(s) AtCESA1 2 n.d. rsw1(ems) Reduced cellulose Swollen root, abnormal dark Antisense Slightly reduced cellulose in leaves (Beeckman et al., morphology, reduced growth of 2002; Reiter, 2002) hypocotyls and roots (rsw1-10) Stunted growth (Burn et al., 2002) AtCESA3 2 n.d. ixr1 (sd, EMS) Unaltered cellulose Semidominant resistance to isoxaben (Scheible et al., 2001; Cano- Delgado et al., 2003) cev1 (EMS) Reduced cellulose Stunted root growth, induction (Ellis et al., 2002) of defense responses eli1 (EMS) Reduced cellulose, ectopic lignin Stunted root growth, induction of defense responses (Cano-Delgado et al., 2003) Antisense Slightly reduced cellulose in leaves Stunted growth (Burn et al., 2002) AtCESA4 2 n.d. irx5 (Ds, EMS) Reduction of secondary cellulose Irregular xylem vessels (Taylor et al., 2003) 33

34 34 Table 1.1 (continued) AtCES6 2 n.d prc1 Reduced cellulose, Reduced growth of dark-grown (Reiter, 2002; (T-DNA) incomplete cell walls hypocotyls and roots Doblin et al., 2003) irx2 Unaltered cellulose Semidominant resistance to (Desprez et al., (sd, EMS) isoxaben 2002) AtCESA7 2 n.d. irx3 (EMS) Reduction of secondary cellulose fra5 Reduction of secondary (sd, EMS), fra5 cellulose (OX) Collapsed xylem, weak stem (Reiter, 2002; Doblin et al., 2003) Fragile fibers, weak stem, (Zhong et al., 2003) extremely thin fiber walls, collapsed xylem vessels AtCESA8 2 n.d. irx1 (EMS) Reduction of secondary cellulose fra6 (EMS) Reduction of secondary cellulose Weak stem, collapsed xylem (Reiter, 2002; vessels Doblin et al., 2003) Reduced fiber wall thickness (Zhong et al., 2003) AtGSL5 2 n.d. pmr4 (EMS, Lack of wound-induced Powdery mildew resistance, (Jacobs et al., 2003; /CalS12 RNAi) callose induction of defense response, Nishimura et al., (At4g03550) callose synthesis 2003) 34

35 35 Table 1.1 (continued) AtGSL6 2 n.d. Heterologous More callose accumulated n.d. (Hong et al., 2001) /CalS1 expression in at the cell plate (At1g05570) tobacco AtCSLC4 2 β-(1 4)-glucan Heterologous Production of soluble β n.d. (Cocuron et al., (At3g28180) synthase expression in glucan; long β-glucan 2007) Pichia produced in lines coexpressing AtCSLC4 and AtXT1 AtXT1 34 Xyloglucan Heterologous changed distribution of No morphological change, (Faik et al., 2002; (At3g62720) α-(1 6)-XylT expression in XyG and slight decrease of slightly changed distribution Cavalier and Pichia and XyG amount patterns of xyloglucan epitopes Keegstra, 2006; Drosophila Cavalier et al., xxt1 2008) (T-DNA) AtXT2 34 Xyloglucan Heterologous Absence of XyG in Aberrant root hairs and (Cavalier and (At4g02500) α-(1 6)-XylT. expression in xxt1/xxt2 changed mechanical properties Keegstra, 2006; Drosophila of cell wall in xxt1/xxt2 Cavalier et al., xxt2 2008) (T-DNA) 35

36 36 Table 1.1 (continued) AtMUR3 47 Xyloglucan mur3 (EMS) Absence of fucogalactosyl- Altered trichome papillae, (Madson et al., (At2g20370) β-(1 2)- GalT for the side-cain in xyloglucan reduced wall strength in 2003; Ryden et al., 3 rd Xyl in XXXG hypocotyls 2003) AtFUT1 37 Xyloglucan mur2 98% reduction of Altered trichome papillae (Perrin et al., 1999; (At2g03220) α-(1 2)- FUT (EMS) xyloglucan fucosylation, Vanzin et al., 2002; fut1 lower acetylation level on Perrin et al., 2003) (T-DNA) Gal over-expression increase in xyloglucan O- No visible phenotype (Perrin et al., 2003) acetylation PsFUT1 37 Xyloglucan antisense No change Wrinkled, collapsed root cell (Faik et al., 2000; (Q9M5Q1) α-(1 2)-FUT surface under electron Wen et al., 2008) microscope AtIRX9 43 Xylan backbone irx9 55% less xylose in stem; Irregular xylem; develop (Bauer et al., 2006; (At2g37090) synthase; microsome (T-DNA) decrease of xylan; decrease slower; dark green; narrower Brown et al., 2007; from irx9 is defect in in cellulose increase in the leaves; almost sterile Lee et al., 2007; Lee incorporating 14 C-Xyl proportion of xylan et al., 2012) to β-(1 4)-Xyl 6 branching by Me-GlcUA relative to GlcUA 36

37 37 Table 1.1 (continued) AtIRX14 43 Xylan backbone irx14 Decrease of Xyl in stem; Irregular xylem; stem thinner (Brown et al., 2007; (At4g36890) synthase; microsome (T-DNA) decrease of xylan; decrease and shorter; has less well- Keppler and from irx14 is defect in in cellulose; increase in the developed silique and fewer Showalter, 2010; incorporating 14 C-Xyl proportion of xylan seeds; drought-tolerant Lee et al., 2012) to β-(1 4)-Xyl 6 branching by Me-GlcUA phenotype relative to GlcUA AtIRX14L 43 n.d. irx14l No Xyl deduction in stem No visible phenotype for irx14l. (Keppler and (At5g67230) (T-DNA) irx14/irx14l double mutant has Showalter, 2010) severe growth defect, is dwarf; fail to produce inflorescence stem AtIRX10 47 Xylan backbone irx10 Decrease of Xyl in stem Minor irregular xylem (Brown et al., 2005; /GUT2 synthase (T-DNA) phenotype Brown et al., 2009; (At1g27440) Wu et al., 2009) 37

38 38 Table 1.1 (continued) AtIRX10L 47 Xylan backbone irx10l No visible phenotype for No visible phenotype for irx10l. (Brown et al., 2009; (At5g61840) synthase; microsome (T-DNA) irx14l. irx14/irx14l double irx10/irx10l double mutant is Wu et al., 2009) from irx10/irx10l is mutant showed large dwarf; develop slowly; dark defect in incorporating reduction of xylan in leaves; sterile; has decreased 14 C-Xyl to β-(1 4)- secondary cell wall and secondary cell wall formation Xyl 6 decrease in cellulose AtGUXT1 8 xylan MeGlcAT and guxt1 Reduced [Me]GlcA Stem strength is reduced and (Mortimer et al., (At3g18660) GlcAT (T-DNA) substitution of improved extractability of 2010) glucuronoxylan guxt1 and xylan from cell wall of AtGUXT2 (At4g33330) 8 xylan MeGlcAT and GlcAT guxt2 (T-DNA) guxt2 in single mutant; almost absence of substitution of xylan guxt1/guxt2 double mutant; though no growth defects found (Mortimer et al., 2010) backbone in guxt1/guxt2 double mutant TaXAT1 61 xylan RNAi RNAi line decreases α- No visible phenotype (Anders et al., 2012) α-(1 3)-AraT Heterologous (1 3) linked expression in arabinosyl substitution of Arabidopsis xylan; Arabidopsis transgenic line obtain arabinosylated xylan 38

39 39 Table 1.1 (continued) TfManS 2 Mannan mannosylt over-expression Two-fold increase in No visible phenotype (Dhugga et al., (AAR23313) (putative mannan mannan 2004) synthase) EST identified TfGMGT 34 Galactomannan α- heterologous Higher substitution level of n.d. (Edwards et al., (CAB52246) (1 6)-GalT for the 3 rd expression in Gal for mannan 1999; Edwards et Man from nonreducing tobacco al., 2002; Reid et end al., 2003) AtFUT4 (At2g15390) 37 AGP α-(1 2)- FUT over-expression 33% increase in fucose, change in other MS in AIR extract from leaves No visible phenotype (Wu et al., 2010) AtFUT6 (At1g14080) 37 AGP α-(1 2)- FUT n.d. n.d. n.d. (Wu et al., 2010) XEG n.d. xeg113 Underarabinosylation of Elongated hypocotyls and (Gille et al., 2009) (At2g35610) (T-DNA) extensin longer petioles of the rosette leaves in the presence of xyloglucanase 39

40 40 Table 1.1 (continued) AtGAUT1 (At3g61130) 8 HG:α-(1 4)-GalAT heterologous expression in human cells n.d. n.d. (Sterling et al., 2006; Caffall et al., 2009) AtGAUT7 8 Form an active gaut7 No significant change No visible phenotype (Sterling et al., (At2g38650) complex with (T-DNA) 2006; Atmodjo et AtGAUT1, but no al., 2011) activity itself expressed in human cells RGXT1 77 RG-II: α-(1 3)-XylT rgx1 No significant difference No visible phenotype (Egelund et al., (At4g01770) (T-DNA) compare to WT 2006) RGXT2 77 RG-II: α-(1 3)-XylT rgx2 No significant difference No visible phenotype (Egelund et al., (At4g01750) (T-DNA) compare to WT 2006) a Data listed were modified from Scheible and Pauly (2004) and updated with recent findings. n.d., not determined, indicating the enzyme activities have not been proved by available in vitro GT assays. 40

41 41 Most cell wall polysaccharides are synthesized in the secretory pathway except for cellulose and callose which are assembled at the outer layer of the plasma membrane (Scheible and Pauly, 2004). Emerging evidence supports the idea that individual biosynthetic enzymes may organize into functional complexes for the biosynthesis of some cell wall polysaccharides. The cellulose synthase complex has been proven to be composed of three or more different subunits (Gardiner et al., 2003; Taylor et al., 2003; Wang et al., 2008). The formation of the xyloglucan backbone requires coordinated actions of β-(1 4)-glucosyltransferases (GlcTs) and α-(1 2)-xylosyltransferases (XylTs), very likely in a complex (Gordon and Maclachlan, 1989; Cocuron et al., 2007). Very recently, a protein complex with GAX synthesizing activity has been shown to contain three putative GTs in wheat (Zeng et al., 2010). In addition, the Arabidopsis galacturonosyltransferase 1 (GAUT1) functions in a protein complex with the homologous GAUT7 for pectin synthesis (Atmodjo et al., 2011). In contrast, backbone formation and substitutions with GlcA or MeGlcA residues for glucuronoxylan synthesis are apparently two independent processes (Mortimer et al., 2010). The study of cell wall biosynthesis is still at an early stage. The actual functions of the majority of putative GTs remain to be identified. Many other fundamental questions also remain open. How are the polysaccharides synthesized in the secretory pathway integrated into the cell wall? Do the modifications of wall polymers occur in the secretory pathway or at the cell surface? How are the cell wall biosynthetic enzymes regulated to construct heterologous wall structures in differentiated plant cells? What signals and how are wall modifications achieved in response to environmental stimuli? Answers to these

42 42 questions will not only unravel the mystery associated with the cell wall biosynthetic machinery, but will also provide a basis for attempts to manipulate cell wall structure for agricultural and industrial applications Hydroxyproline Rich-Glycoproteins (HRGPs) HRGPs are a family of plant cell wall proteins that are rich in hydroxyproline (Hyp) residues in the protein backbone and often undergo glycosylation modifications. In addition, the HRGP family is characterized by the presence of repetitive motifs in the protein backbone (Kieliszewski and Lamport, 1994) Classification Plant HRGPs can be subdivided into proline (Pro) rich proteins (PRPs), extensins (EXTs), arabinogalactan-proteins (AGPs), gum arabic glycoprotein (GAGP), solanaceous lectins and the hybrid and chimeric HRGPs subfamilies (Showalter, 1993; Kieliszewski and Lamport, 1994; Seifert and Roberts, 2007). HRGP subfamilies vary in the characteristic motifs of their protein backbones, glycosylation patterns and cross-linking properties. Hybrid HRGPs are composed of modules of different HRGP subfamilies, and chimeric HRGPs are defined as HRGPs containing one or more HRGP modules as well as non-hrgp protein sequences (Showalter et al., 2010). While GAGP and solanaceous lectins are restricted to certain plant species, EXTs, PRPs and AGPs are widely present in plants (Showalter, 1993). Protein sequences of a chimeric PRP molecule, a classical EXT molecule and a classical AGP molecule are shown in Fig Although HRGP subfamilies have different characteristic motifs, common rules for Pro hydroxylation and

43 43 Hyp glycosylation apply to the whole HRGP superfamily. Thus, HRGP subfamilies are better viewed as structurally and evolutionarily related glycoprotein groups, rather than discrete classes (Kieliszewski and Lamport, 1994). A classical AGP >At1g35230 (AGP5) MASKSVVVFL FLALVASSVV AQAPGPAPTI SPLPATPTPSQSPRATAPAPSPSANPPPSAPTTAPPVS QPPTESPPAPPTSTSPSGAPGTNVPSGEAGPAQSPLSGSPNAAAVSRVSLVGTFAGVAVIAA LLL An EXT >At1g21310 (EXT3) MASLVATLLVLTISLTFVSQSTANYFYSSPPPPVKHYTPP VKHYSPPPVYHSPPPPKKHYEYKSPPPP VKHYSPPPVYHSPPPPKKHYVYKSPPPP VKHYSPPPVYHSPPPPKKHYVYKSPPPP VKHYSPPPVYHSPPPPKKHYVYKSPPPP VKHYSPPPVYHSPPPPKKHYVYKSPPPP VKHYSPPPVYHSPPPPKKHYVYKSPPPP VKHYSPPPVYHSPPPPKKHYVYKSPPPP VKHYSPPPVYHSPPPPKKHYVYKSPPPP VKHYSPPPVYHSPPPPKKHYVYKSPPPP VKHYSPPPVYHSPPPPKKHYVYKSPPPP VKHYSPPPVYHSPPPPKKHYVYKSPPPP VKHYSPPPVYHSPPPPKKHYVYKSPPPP VKHYSPPPVYHSPPPPKEKYVYKSPPPP PVHHYSPPHHPYLYKSPPPPYHY A chimeric PRP >At3g62680 (PRP3) MAITRSSLAICLILSLVTITTADYYSPSSPPVYKSPEHKPTLPSPVYTPPVYKPTLSPPVYTKPTIPPPVYTP PVYKHTPSPPVYTKPTIPPPVYTPPVYKPTLSPPVYTKPTIPPPVYTPPVYKPTPVYT KPTIPPPVYTPPVYKPTPSPPVYKKSPSYSSPPPPYVPKPTYTPTTKPYVPEILKAVDGIILCKNGYETYPIL GAKIQIVCSDPASYGKSNTEVVIYSNPTDSKGYFHLSLTSIKDLAYCRVKLYLSPVETCKNPTNVNKGLTG VPLALYGYRFYPDKNLELFSVGPFYYTGPKAAPATPKY Figure 1.1 Protein sequences of a classical AGP molecule, an EXT molecule and a hybrid PRP molecule identified in Arabidopsis. Colored sequences at the N and C termini indicate predicted signal peptide (green) and GPI anchor (light blue) addition sequences if present. The sequence of AGP5 (Schultz et al., 2000; Showalter et al., 2010), a classical AGP, is mainly composed of Pro-dipeptide sequences (highlighted in yellow) but also contains a few Pro n motifs (highlighted in pink). The sequence of EXT3 (Hall and Cannon, 2002), representing a typical EXT sequence, contains the YVYK motif (highlighted in dark purple) and the Pro n motif

44 located in longer repetitive motifs (underlined). The PRP3 (Fowler et al., 1999) is a chimeric PRP with an N terminal PRP domain composed of PRP motifs (highlighted in red) located in longer repetitive motifs (underlined) and a C terminal region rich in Pro, Val, Lys and cysteine (Cys) residues Three major HRGP subfamilies: PRPs, EXTs and AGPs PRPs have repetitive structures with a high content of Pro and Hyp residues and are suggested to be involved in normal plant development as well as defense (Liu and Mehdy, 2007). A subclass of PRPs may function in plant nodule formation (Showalter, 1993). One of the common motifs of PRPs is Pro-Pro-Xaa-Yaa-lysine (Lys), where Xaa is valine (Val), histidine (His), threonine (Thr) or alanine (Ala) and Yaa is tyrosine (Tyr), Thr, glutamic acid (Glu) or Pro (Sommer-Knudsen et al., 1998). One or both of the two Pro residues may be modified into Hyp residues (Francisco and Tierney, 1990; Kieliszewski et al., 1992). Expanded versions or variations of the pentapeptide are also found, such as (Lys)-Pro-Pro-Xaa-Yaa-Lys-(Pro-Pro), Lys-Pro-Pro-Xaa-Lys-(Pro-Pro), where Xaa is normally glutamine (Gln), Val, Thr or Ala. Individual PRPs may have one or two such motifs or consist almost entirely of the repetitive motifs (Sommer-Knudsen et al., 1998). PRPs are normally lightly glycosylated and contain 2-27% (w/w) sugar (Shpak et al., 1999). Glycosylation of PRPs occurs mainly as arabinosylation of Hyp residues. Gal attachment or N-glycosylation are found in some PRPs (Sommer-Knudsen et al., 1998). However, two recent studies characterized chimeric PRPs from tobacco (Nicotiana alata) and Arabidopsis with a carbohydrate content of up to 75%, highlighting the structural diversity and complexity of HRGPs (Sommer-Knudsen et al., 1996; Hijazi et al., 2012).

45 45 EXTs are periodic amphiphiles composed of hydrophilic motifs of serine (Ser) - Hyp 4 and hydrophobic motifs of Tyr-Xaa-Tyr-Lys (where Xaa is Lys or Tyr) and/or Val- Tyr-Lys (Kieliszewski and Lamport, 1994; Cannon et al., 2008). Among HRGPs, EXTs are moderately glycosylated and have sugar components counting for ~50% of the molecular weight. Glycosylation of EXTs occurs on Ser-Hyp 4 motifs, with Ara oligosaccharides (commonly DP1 to DP4) attached to the Hyp residues and frequently, single Gal residues attached to the Ser residues (Kieliszewski et al., 1992; Kieliszewski and Lamport, 1994). Contiguous Hyp residues in Ser-Hyp 4 form a rigid poly-pro II structure, which is stabilized by the attached sugar side chains as well as by isodityrosine (Idt) formation between the two Tyr residues in the Tyr-Xaa-Tyr-Lys motif (Kieliszewski and Lamport, 1994; Velasquez et al., 2011). While Idt functions in intramolecular crosslinking (Epstein and Lamport, 1984), two EXT monomers are cross-linked by forming di- Idt (Held et al., 2004) or pulcherosine, i.e. cross-linking between two Idt residues or between one Idt and one Tyr residue from the Val-Tyr-Lys motif (Schnabelrauch et al., 1996). The discovery of EXT peroxidase (Everdeen et al., 1988), together with the visualization of the in vitro self-assembly of EXT monomers into dendritic structures (Cannon et al., 2008) provides evidence that soluble EXT monomers are likely to selfassemble and cross-link into an insoluble EXT network in vivo. Formation of the EXT network likely contributes to wall architecture and defense, consistent with previous studies which showed induced expression of EXTs by wounding and pathogen attack (Esquerre-Tugaye and Mazau, 1974; Esquerre-Tugaye and Lamport, 1979; Mouly et al., 1992; Merkouropoulos et al., 1999). An essential role of EXT in cell plate formation was

46 46 illustrated by an aberrant cross wall assembly in the Arabidopsis ext3 mutant and suggested that the EXT network may serve as a scaffold to template cell wall assembly through interactions with pectin polymers (Hall and Cannon, 2002; Cannon et al., 2008). Among HRGPs, AGPs are the least periodic but undergo glycosylation to the highest extent (Tan et al., 2003). In Arabidopsis, a recent bioinformatic study identified 85 putative AGPs, which are highly variable in sequence (Showalter et al., 2010). A common feature of AGP protein backbones is the biased amino acid compositions, which are rich in Pro, Ala, Ser, Thr and glycine (Gly) residues (Showalter, 1993; Nothnagel, 1997). For example, classical AGPs are considered to have Pro, Ala, Ser and Thr residues composing greater than 50% of their protein backbones (Showalter et al., 2010). Rather than the longer protein motifs present in PRPs or EXTs, dipeptide sequences, such as Ala-Hyp, Hyp-Ala, Ser-Hyp, Thr-Hyp, are frequently found in AGPs in clusters or sometimes in a more dispersed pattern (Gao et al., 1999; Showalter, 2001). The protein backbone typically counts for less than 10% of the molecular mass, whereas the majority of the AGP molecule is composed of carbohydrates (Nothnagel, 1997). O-glycosylation of Hyp residues with arabinogalactan (AG) polysaccharides is the dominant form of AGP glycosylation, though there may be arabino-oligosaccharides attached to Hyp residues, O-glycosylation of Ser or Thr residues, and N-glycosylation in some AGPs (Gao et al., 1999; Qin et al., 2001). Specific binding of AGPs to (β-d-galactosyl) 3 and (β-dglucosyl) 3 Yariv phenylglycosides (β-yariv reagent) is considered a characteristic feature of AGPs. This binding is likely to involve both the protein backbone and polysaccharide components (Nothnagel, 1997). AGPs are proposed to play a variety of roles in plant

47 growth and development (Seifert and Roberts, 2007; Ellis et al., 2010), which will be further discussed in Chapter HRGP Glycosylation Glycosylation is an important feature of HRGPs. Hyp arabinosylation has been suggested to stabilize the secondary structures of EXTs (Velasquez et al., 2011). For HRGPs with extensive glycosylation, like EXTs and AGPs, it is conceivable that glycosylation plays a crucial role in the molecular function by determining the interactive surface of the molecule (Kieliszewski, 2001). In addition, glycosylation has been suggested to facilitate the secretion of HRGPs into the plant cell wall (Xu et al., 2008). HRGP glycosylation mainly occurs through O-glycosylation of Hyp residues, which involves two basic steps: Pro hydroxylation by Pro-4-hydroxylases and subsequent Hyp glycosylation by GTs. In the protein backbones of HRGPs, some but not all Pro residues are converted to Hyp residues; the resulting Hyp residues may remain non-glycosylated, be glycosylated by arabino-oligosaccharides or be glycosylated by AG-polysaccharides (Kieliszewski, 2001). These selective processes of Pro hydroxylation and Hyp glycosylation have inspired the query for potential rules involved. Kieliszewski and colleagues first proposed rules for Pro hydroxylation and Hyp glycosylation based on protein sequence data and glycosylation patterns of isolated EXT and PRP molecules (Kieliszewski and Lamport, 1994). Hydroxylation of Pro residues is likely to be specified by amino acid sequences involving the Pro residue and its neighboring amino acids. While dipeptide Pro-Val is always hydroxylated, Lys-Pro, Tyr- Pro, and Phe-Pro are never hydroxylated. Gly-Pro may be or may not be hydroxylated.

48 48 Furthermore, the extent of Hyp glycosylation was predicted to be determined by the contiguity of Hyp residues, as summarized by the following Hyp contiguity hypothesis. While contiguous Hyp residues are the sites for glycosylation by arabinooligosaccharides, clustered, noncontiguous Hyp residues are the sites for glycosylation by AG polysaccharides (Kieliszewski and Lamport, 1994; Kieliszewski and Shpak, 2001). Contiguous Hyp residues can be symbolized as Hyp n, where n is between 2 and 5 in general. For example, the SerHyp 4 motif in EXTs represents a typical contiguous Hyp motif. The motif containing clustered, noncontiguous Hyp residues may be symbolized as [Hyp-Xaa-Hyp-Xaa] n, where Xaa represents one to a few amino acids and is most frequently Ser, Thr or Ala as in AGPs. A simplified model illustrating the Hyp contiguity hypothesis is shown in Fig. 1.2.

49 Figure 1.2 A simplified HRGP model illustrating the Hyp contiguity hypothesis for a model AGP protein sequence and a model EXT protein sequence. The Hyp contiguity hypothesis was proposed by Kieliszewski and colleagues (Kieliszewski and Lamport, 1994; Kieliszewski and Shpak, 2001). The AG structure is modified from Tan et al. (2004; 2010). The arabino-oligosaccharide structure is modified from Velasquez et al. (2011). Monosaccharide symbols used here are based on the Symbol and Text Nomenclature for Representation of Glycan Structure as proposed by the Consortium for Functional Glycomics ( 49

50 50 While structures of isolated EXTs and PRPs support the Hyp contiguity hypothesis, the link between AGP backbone sequences and the glycosylation pattern was obscure due to difficulties in purification of a single AGP molecule to homogeneity as well as in separation of the arabinosylated Hyp motifs and the AG-glycosylated Hyp motifs present in the same AGP molecule. To overcome these difficulties, a novel strategy was applied that involved the expression of synthetic genes encoding Hyp motifs in fusion with a green fluorescent protein (GFP) tag in tobacco (Nicotiana tabacum) BY2 cells and subsequent purification and analysis of the expressed glycoproteins. Information obtained from such a synthetic gene expression strategy indeed supports the Hyp contiguity hypothesis. For example, the expressed [Ser-Pro] 32 motif is post-translationally modified into [Ser-Hyp] 32 (representing a clustered, non-contiguous Hyp motif) and subsequently glycosylated by AG side chains (Shpak et al., 1999); while an expressed [Ser-Pro 4 ] 25 motif is modified into [Ser-Hyp 4 ] 25 (representing a contiguous Hyp motif), and glycosylated exclusively by arabino-oligosaccharide side chains (Shpak et al., 2001). The Hyp contiguity hypothesis also receives supports from the glycosylation pattern of a plant AGP, tomato LeAGP1, when expressed in BY2 cells (Zhao et al., 2002). Further investigation with the synthetic gene expression strategy indicates that the identities of Xaa residues in the [Hyp-Xaa-Hyp-Xaa] n motif influence the extent of Pro hydroxylation and Hyp glycosylation (Tan et al., 2003). The Hyp contiguity hypothesis is well supported; however, there are exceptional cases. For example, the gymnosperm PRP motif: Lys-Pro-Hyp-Val-Hyp-Val-Ile-Pro-Pro- Hyp-Val-Val-Lys-Pro-Hyp-Hyp-Val-Tyr contains clustered noncontiguous Hyp motifs

51 51 (underlined), that are non-glycosylated or arabinosylated instead of arabinogalactosylated (Kieliszewski et al., 1992; Kieliszewski et al., 1995; Tan et al., 2003). Refinement of the Hyp contiguity hypothesis, perhaps taking into account what is the minimum number of noncontiguous Hyp residues for arabinogalactosylation, may help to explain these exceptional cases. Interestingly, the same synthetic motif, [Ser-Hyp 4 ] 25 is similarly hydroxylated and glycosylated when expressed in tobacco and Arabidopsis suspension cultured cells, but obtains a different glycosylation pattern when expressed in Arabidopsis plants (Shpak et al., 2001; Estevez et al., 2006; Xu et al., 2008). This indicates that even though the same Hyp glycosylation code is shared between tobacco and Arabidopsis, the tissue or organ specific activities of P4H and GTs need to be taken into account when using the Hyp glycosylation code to predict the glycosylation pattern of HRGPs. The hydroxylation of Pro to 4-Hyp is catalyzed by the Pro-4-hydroxylase (P4H) activities. P4Hs identified in Chlamydomonas reinhardtii and Arabidopsis compose a multi-gene family (Yuasa et al., 2005; Keskiaho et al., 2007), in support of the previous speculation that sequence-specific P4Hs are involved in the hydroxylation of HRGPs (Kieliszewski and Lamport, 1994). AtP4H1 showed broader substrate specificity compared to AtP4H2 when expressed in insect cells. Although both AtP4H1 and AtP4H2 use EXT-like peptides as substrates, only AtP4H1 hydroxylates collagen-like peptides and hypoxia-inducible transcription factors from vertebrates (Hieta and Myllyharju, 2002; Tiainen et al., 2005). It would be interesting to compare the efficiencies of P4Hs in using EXT-like peptides versus AGP-like peptides as a substrate. In addition, the 13 P4Hs in

52 52 Arabidopsis showed distinct expression profiles based on in silica analysis (Velasquez et al., 2011). Much remains to be explored with respect to how the P4H family (i.e. 13 predicted members in Arabidopsis) coordinates hydroxylation within the much larger HPRG family (i.e. 166 predicted members in Arabidopsis, (Showalter et al., 2010)) in planta. Even less is known about the GTs involved in HRGP glycosylation. To date, one EXT ARABINOSYLTRANSFERASE (AraT) (Gille et al., 2009) and two AGP FUCOSYLTRANSFERASES 4 and 6 (FUT4 and FUT6) (Wu et al., 2010) are the only genes identified for glycosylation of HRGPs Arabinogalactan-Proteins (AGPs) Classification Based on their protein backbone structures, AGPs are classified into classical AGPs, AG peptides, hybrid AGPs and chimeric AGPs (Schultz et al., 2002). As discussed before (Chapter 1.2.2), the characteristic sequence of an AGP protein backbone is rich in Hyp, Ser, Thr, Ala and Gly amino acids, which are often present in repetitive SO, TO and AO motifs (Showalter, 1993). The backbone of classical AGPs has a domain mainly composed of AGP motifs, sandwiched by a signal peptide and often a site for GPI-anchor attachment located at the amino and carboxyl termini, respectively. The lengths of protein backbones of classical AGPs vary from 80 to hundreds of amino acids (Schultz et al., 2000). AG peptides are similar to classical AGPs but have an AGP domain shorter in length. The definitive length of the AGP domain for an AGP peptide is considered

53 53 differently by different researchers, ranging from below 20 amino acids to up to 90 amino acids (Schultz et al., 2004; Showalter et al., 2010). In contrast to classical AGPs and AG peptides, hybrid AGPs and chimeric AGPs contain non-agp domains in the core protein sequence. Hybrid AGPs have domains composed of motifs commonly found in other HRGPs, i.e. PRP motifs, EXT motifs, in addition to AGP motifs. If the additional structural domains are irrelevant to HRGPs, the AGPs are referred to as chimeric AGPs (Schultz et al., 2002). Structural domains found in chimeric AGPs include the fasciclin-like domain, Cys-rich domain, plastocyanin domain, etc. (Showalter, 2001; Showalter et al., 2010) Post-translational modifications AGPs undergo extensive post-translational modifications, including the removal of the N-terminal signal peptide, C-terminal processing involving the addition of the glycosylphosphatidylinositol (GPI) anchor, Pro hydroxylation to Hyp and glycosylation of the protein backbone. In addition, some AGPs may be subjected to degradation by proteases and glycosidases Removal of N-terminal signal peptides All of the cdnas isolated from confirmed AGPs encode N-terminal signal peptides (Nothnagel, 1997). The presence of an N-terminal signal peptide has been used as a prerequisite factor for the identification of putative AGPs by bioinformatic methods (Showalter et al., 2010). The N-terminal signal peptides direct the co-translational translocation of AGP core backbones into the endoplasmic reticulum (ER) lumen, an

54 54 entry point into the secretory pathway from ER to the Golgi apparatus and then to the cell surface. The signal peptides are cleaved in the ER lumen as evidenced by the absence of the signal sequences in mature AGPs or AG peptides (Mau et al., 1995; Schultz et al., 2004) Addition of C-terminal GPI anchors In eukaryotes, the GPI anchor provides an alternative way for the attachment of proteins to the plasma membrane other than via transmembrane domains. The GPI anchor has a core oligosaccharide structure, i.e., D-Man-α-(1 2)-D-Man-α-(1 6)-D-Man-α- (1 4)-D-GlcN-inositol. The C-terminus of a GPI-anchored protein is linked to the nonreducing end of the core oligosaccharide via ethanolamine phosphate. The inositol phospholipid moiety on the reducing end of the core oligosaccharide is embedded in the outer leaflet of the plasma membrane so as to anchor the attached protein to the cell surface (Oxley and Bacic, 1999). GPI-anchored proteins in plants present similar characteristics with respect to the sites for processing and addition of GPI anchors as in other eukaryotes (Takos et al., 2000). The amino acid residue at the site of GPI addition is designated ω, which is Ser, asparagine (Asn), Ala, Gly, aspartic acid (Asp), or Cys, whereas the ω+ 2 residue is generally Ala, Gly, Thr, or Ser. The ω+1 residue is less critical. A terminal 14 to 18 amino acid hydrophobic tail is often found following a four to eight amino acid spacer region, which often contains a basic residue [e. g. arginine (Arg) or Lys]. The amino acid sequence after the ω residue is cleaved before the preformed GPI anchor is transferred en

55 55 bloc to the ω residue on the backbone of a GPI-anchored protein by transamidase activity (Takos et al., 2000; Showalter, 2001). To date, AGPs with known GPI anchors include PcAGP1 from Pyrus communis (Youl et al., 1998), NaAGP1 from Nicotiana alata (Gilson et al., 2001), AtAGP10 from Arabidopsis (Schultz et al., 2000), LeAGP1 from Lycopersicon esculentum (Sun et al., 2004) and 12 AG-peptides from Arabidopsis (Schultz et al., 2004). Fifty six out of 85 AGPs, including those experimentally confirmed and those newly identified by a bioinformatic strategy, are predicted to be GPI-anchored in Arabidopsis (Showalter et al., 2010). Many potential functions of AGPs related to their GPI-anchored characteristics have been proposed. For example, the attachment of AGPs to the surface of the plasma membrane through GPI anchors may facilitate the interaction between AGPs and other membrane proteins (Kohorn, 2000). Another intriguing hypothesis is that phospholipases, which specifically cleave the linkage between AGPs and their GPI anchors, might control the release of AGPs to the cell wall where AGPs may function as structural components or as signal molecules (Borner et al., 2002). However, these functional speculations require experimental proof Glycosylation Classical AGPs may contain both contiguous Hyp motifs and clustered, noncontiguous Hyp motifs for glycosylation by arabino-oligosaccharides and AG polysaccharides, respectively (Gao et al., 1999; Seifert and Roberts, 2007). However, Hyp O-glycosylation with AG polysaccharide is the major defining structure for AGPs and will be the focus of discussion here. Besides Hyp O-glycosylation, single Gal

56 56 attached to Ser (Tsumuraya et al., 1984), unidentified O-glycosylation through Thr residues (Tsumuraya et al., 1987) and N-glycosylation on Asn-X-Ser/Thr motifs (Wang et al., 1993) are also reported for some AGPs. AG polysaccharides found in AGPs are type II AGs, which are characterized by a β-( 3)-galactan backbone branched with β-(1 6)-galactan side chains where oligosaccharides containing arabinosyl residues are attached. Except for the above key features, type II AGs were suggested to be highly heterogeneous. The polysaccharide size ranges from sugar residues (Gaspar et al., 2001; Showalter, 2001); the ratio of Gal: Ara varies among different AGP preparations; besides the Gal and Ara residues, AGPs from different species may contain different sugars, including rhamnose (Rha), GlcA, GalA, Fuc, Glc or monosaccharides modified by acetylation or O-methylation (Fincher et al., 1983; Serpe and Nothnagel, 1995; Goodrum et al., 2000; Hashimoto, 2000). The strategy of expressing synthetic genes encoding Hyp motifs fused to a GFP tag not only helped with deciphering the Hyp glycosylation code but also helped with the elucidation of the AG glycan structure. The AG polysaccharide attached to the [Ala-Pro] 51 -EGFP fusion protein expressed in tobacco BY2 cells was isolated and analyzed by nuclear magnetic resonance (NMR), which presented the first complete primary structure of a type II AG glycan (Tan et al., 2004) (Fig. 1.2). In a subsequent study, an interferon α2b- [Ser-Hyp] 20 fusion protein was expressed in tobacco BY2 cells. The AG polysaccharides isolated from the transgenically expressed peptides with sizes ranging from 14 to 22 residues were much smaller in size compared to naturally occurring AG polysaccharides (Tan et al., 2004; Tan et al., 2010). It is possible that the expression of the polypeptides

57 57 [Ala-Pro] 51 -EGFP and α2b-[ser-hyp] 20 driven by the 35S promoter may produce a large pool of substrates which exceeded the capacity of the endogenous GTs to perform complete glycosylation. Although varying in sizes, the AG polysaccharides from the [Ala-Pro] 51 -EGFP and α2b-[ser-hyp] 20 peptides are similar in structure to each other and to naturally occurring AG polysaccharides. Structural similarities in the AG polysaccharides from the [Ala-Pro] 51 -GFP and the interferon α2b-[ser-hyp] 20 fusion proteins and naturally occurring AG polysaccharides led to the suggestion that a ~15 sugar unit may constitute the fundamental building blocks for the assembly of type II AG (Tan et al., 2010). The 15 sugar unit has a β-(1 3) linked trigalactosyl main chain and bifurcated sidechains linked through Gal residues 6-linked to Gal-1 and Gal-2 of the main chain. The trigalactosyl main chains are repeatedly connected through β-(1 6) linkages whereas Gal side chains may extend with Ara oligosaccharides, Rha, GlcUA or other sugar residues depending on the plant species. The 15 sugar unit hypothesis is consistent with previous studies showing that AGPs are composed of repeated sugar blocks separated by periodate-sensitive residues (Churms et al., 1981; Fincher et al., 1983). AGPs in plants with differentiated tissues and organs tend to be more heterogeneous in nature. However, studies using the tobacco expression system may provide a simple solution to dissect the seemingly complicated AG structure. To date, little is known about the enzymatic machinery involved in AGP glycosylation, or more broadly, in HRGP glycosylation. In protein N-glycosylation, an oligosaccharide core structure is formed on a lipid intermediate before being transferred en bloc onto the protein backbone (Varki et al., 1999). If complex AG polysaccharides

58 58 are composed of units of simpler sugar building blocks (Churms et al., 1981; Fincher et al., 1983; Tan et al., 2010), it is reasonable to predict that the sugar building blocks are preformed before assembly into AG polysaccharides. Potential lipid intermediate formation in AG glycosylation similar to N-glycosylation is supported by the identification of a glycolipid containing [ 14 C]Gal in a pea membrane preparation incubated with UDP- [ 14 C]Gal (Hayashi and Maclachlan, 1984). However, the lack of detailed information of the glycolipid structure or any intermediate glycosylation products makes this idea speculative. Given the specificity of GTs (Scheible and Pauly, 2004), different GTs are likely to be involved in putting on specific sugars at specific positions of the AG side chain. Membrane preparation from rygrass (Lolium multiflorum) cell cultures showed galactosyltransferase (GalT) activity that transferred radiolabeled Gal to endogenous acceptors with type II AG structures (Mascara and Fincher, 1982; Schibeci et al., 1984). GalT activities involved in the formation of the β-(1 6)-galactosyl linkage preferably on β-(1 3)-galacto-oligomers were identified and characterized in a membrane preparation from radish roots (Raphanus sativus L.) (Kato et al., 2003). Suspension-cultured tobacco cells pulse-labeled with [ 14 C]Ara were shown to accumulate AGPs with [ 14 C]Ara in Golgi membranes (Kawasaki, 1987). Misawa et al. (1996) identified FUT activity that added Fuc to the L-Ara-α-(1,3)-D-Gal β-(1 6)-D-Gal-pyridylamino acceptor through an α-(1 2)-linkage and was distinct from FUT activity for xyloglucan fucosylation in the primary roots of radish. In addition, glucuronyltransferase (GlcAT) activities were also detected in radish roots using linear β-(1 3)-galactans or branched β-(1 6)-galactans as

59 59 the acceptors (Endo et al., 2004). These studies used endogenous acceptors or oligosaccharides mimicking partial sequences of the AG side chains as acceptors, preventing a precise determination of the positions of sugar addition by the AGP GT activities. However, valuable information on the characteristics of AGP GTs was ascertained, for example, the β-d-galt activities from both rygrass and radish roots and the α-l-fut activity from radish root all require Mn 2+ divalent ions but have slightly different ph optima, ranging from 6 to 7.5 (Mascara and Fincher, 1982; Misawa et al., 1996; Kato et al., 2003). Furthermore, subcellular localization data indicated that β-d- GalT, α-l-fut and GlcAT for AGP glycosylation all reside in the endomembrane system (Mascara and Fincher, 1982; Schibeci et al., 1984; Misawa et al., 1996; Kato et al., 2003; Endo et al., 2004). Recently, progress has been made in the identification of AGP GT genes with the help of bioinformatics strategies. Qu et al. (2008) identified 20 Arabidopsis homologues to the mammalian -(1 3)-GalT genes as candidate AGP GalTs in the CAZy GT 31 family. Although one of the twenty genes was proven to be a -(1 3)-GalT involved in protein N-glycosylation (Strasser et al., 2007), another candidate gene demonstrated activity to transfer Gal to an O-methylated Gal- -(1 3)- Gal disaccharide, an analogue to a partial structure of AGP side chains (Qu et al., 2008). Two Arabidopsis genes, AtFUT4 and AtFUT6, were identified from the CAZy GT37 and proven biochemically to encode AGP specific α-(1 2)-FUTs (Wu et al., 2010). In addition, Oka et al. (2010) utilized chemically synthesized AGP peptides conjugated to fluorescein isothiocyanate (FITC) tags as acceptors to study the GalT activity for adding Gal residues to the AGP protein backbones.

60 Biological functions Because AGPs are usually present in mixtures and are heterogeneous in nature, it is often not possible to isolate pure, individual molecules for functional elucidation. Exceptions include the purification of the tobacco transmitting tissue-specific protein (TTS) (Cheung et al., 1995) and the Zinnia elegans xylogen (Motose et al., 2004), which are highly enriched in specific plant organs or cell lines. The use of the β-yariv reagent and monoclonal antibodies (mab) against AGPs have greatly facilitated the investigations of AGPs (Nothnagel, 1997; Showalter, 2001). Besides being utilized as molecular probes for the identification and localization of AGPs, β-yariv reagent and mabs against AGP epitopes were applied to bind to and interfere with AGPs in living plants or cells, thus providing cues to deduce AGP function. However, both β-yariv and most AGP mabs are estimated to bind to different AGPs, and thus provide information on the distribution, expression and function of an AGP population rather than individual molecules (Ellis et al., 2010). In recent years, advances in molecular genetic techniques and the completion of genome sequencing of model plants, has allowed for the study of individual AGP molecules by generating and characterizing their genetic mutants. In addition, advances in bioinformatics (Schultz et al., 2002; Ma and Zhao, 2010; Showalter et al., 2010) and the accumulation of microarray data (Du et al., 2009; Bosch et al., 2011; Lin et al., 2011) provide new tools to guide the identification and functional studies of AGPs. With the above strategies, AGPs were demonstrated to be involved in various aspects of plant growth and development (Showalter, 2001; Seifert and Roberts, 2007;

61 61 Ellis et al., 2010). Treatment of the suspension-cultured cells with β-yariv reagent indicated that AGPs are involved in cell devision (Serpe and Nothnagel, 1994) and programmed cell death (Gao and Showalter, 1999). In embryogenesis, AGP epitopes serve as markers for differentiating cells (Pennell et al., 1991). The isolation of xylogen, a chimeric AGP with a nonspecific lipid-transfer domain, provided direct evidence that AGPs function to induce tracheary element differentiation (Motose et al., 2001, 2004). AGPs are involved in shoot and root growth, as indicated by shorter shoots and multifaceted defects in root growth when plant seedlings are grown in the presence of β- Yariv reagent (Willats and Knox, 1996; Ding and Zhu, 1997). Consistently, AGPs were shown to be over-represented in the root hair morphogenesis transcriptome (Jones et al., 2006). A null mutant of the Arabidopsis FASCICLIN-LIKE AGP (FLA) gene, fla1, was defective in shoot regeneration in tissue cultures (Johnson et al., 2011). In secondary cell wall biosynthesis, two Arabidopsis genes, FLA11 and FLA12, were suggested to modulate cellulose deposition and thus affect the mechanical properties of stems (MacMillan et al., 2010). AGPs also have established roles in plant reproduction. Different AGP molecules were suggested to function in female gametogenesis (Acosta- Garcia and Vielle-Calzada, 2004), pollen tube guidance (Clarke et al., 1979; Cheung et al., 1995), pollen incompatibility (Lind et al., 1994; Cheung et al., 1995; Wu et al., 2000; Lee et al., 2008) and pollen grain development (Levitin et al., 2008; Coimbra et al., 2009). In addition to the multifaceted roles of AGPs under physiological conditions, AGPs are implicated in plant resistance to stress, like salt stress (Shi et al., 2003; Lamport et al.,

62 ) and possibly play a role in plant-microbe interactions (Nam et al., 1999; Gaspar et al., 2004). Given the large number of AGP family members predicted by bioinformatics (e.g. 85 AGPs in Arabidopsis (Showalter et al., 2010) and 69 AGPs in rice (Ma and Zhao, 2010)), the precise function of the majority of AGP molecules remains unknown. The AGP family is likely to divide into subgroups for functional specialization. Classification of AGPs based on the protein backbone structure is not necessarily correlated to the functional specialization. For example, the relatively well-characterized Lys-rich AGP and the fasciclin-like AGP families are both composed of members with diverse expression patterns and functions (Shi et al., 2003; Acosta-Garcia and Vielle-Calzada, 2004; Gaspar et al., 2004; MacMillan et al., 2010). The molecular mechanism of AGP function remains elusive dispite knowledge of the protein backbone sequence, the addition of the GPI-anchor and the glycosylation patterns that have been suggested to play a role in the functionalities of AGPs (Ellis et al., 2010) Specific Aims of the Research The goal of this research is to extend our understanding of the AGP glycosylation machinery. The research focuses on the identification and characterization of the GalTs and the FUTs for AGP glycosylation with the following specific aims. Development of an AGP GalT assay system and characterization of AGP GalT activities in tobacco and Arabidopsis suspension-cultured cells To date, none of the GalT genes involved in AGP glycosylation have been conclusively identified. The first specific aim of this research is to develop an in vitro

63 63 assay to facilitate the specific detection of AGP GalT activity. The AGP GalT assay, once developed, will be applied to characterize AGP GalT activity in Arabidopsis suspensioncultured cells to provide insight to the AGP biosynthetic machinery. Heterologous expression and functional characterization of putative GalT genes for AGP galactosylation in Pichia pastoris and tobacco suspension cultured cells Using a bioinformatics method, six genes in CAZy GT31 were predicted to encode putative GalTs for AGP galactosylation (Qu et al., 2008; Egelund et al., 2011). Five of the six genes with cdna clones commercially available will be expressed heterologously in yeast and tobacco suspension-cultured cell lines with an expression tag for functional characterization. Subcellular localization of the GalT genes will be also studied using a transient expression system in tobacco leaves to support the functional characterization work. Characterization of the fut4 and fut6 mutant plants in Arabidopsis Arabidopsis FUT4 and FUT6 have been identified as AGP-specific FUTs using a biochemical approach (Wu et al., 2010). The functions of the FUT4 and FUT6 genes as well as the physiological roles of AGP fucosylation will be studied by characterizing the fut4, fut6 and the fut4/ fut6 mutant plants in Arabidopsis.

64 64 CHAPTER 2: IDENTIFICATION AND CHARACTERIZATION OF IN VITRO GALACTOSYLTRANSFERASE ACTIVITIES INVOLVED IN ARABINOGALACTAN-PROTEIN GLYCOSYLATION IN TOBACCO AND ARABIDOPSIS This work has been published in the following manuscript. Liang Y, Faik A, Kieliszewski M, Tan L, Xu WL, Showalter AM (2010) Identification and characterization of in vitro galactosyltransferase activities involved in arabinogalactan-protein glycosylation in tobacco and Arabidopsis. Plant Physiol 154: Introduction Arabinogalactan-proteins (AGPs) are highly glycosylated hydroxyproline-rich glycoproteins (HRGPs) implicated in many physiological processes including plant somatic embryogenesis, programmed cell death, wound responses, and hormone signaling pathways (Seifert and Roberts, 2007; Ellis et al., 2010). AGPs are defined by the presence of arabinogalactan (AG) polysaccharides and reside mainly at the plasma membrane-cell wall interface and in plant exudates (Langan and Nothnagel, 1997; Oxley and Bacic, 1999; Sherrier et al., 1999; Svetek et al., 1999; Lamport et al., 2006; Seifert

65 65 and Roberts, 2007). These AG polysaccharides are added via O-glycosylation, which is widespread in plants and includes monogalactosylation of Ser residues and extensive modification of hydroxyproline (Hyp), which ranges from addition of oligoarabinosides to AG polysaccharide addition. The Hyp contiguity hypothesis predicts contiguous Hyp residues as sites of HRGP arabinosylation while clustered non-contiguous Hyp residues are sites of galactosylation that give rise to AG polysaccharides on AGPs (Kieliszewski and Lamport, 1994; Kieliszewski et al., 1995; Tan et al., 2003). Tests of the hypothesis in gum arabic glycoprotein (Goodrum et al., 2000) and tobacco (Nicotiana tobacum) (Shpak et al., 1999; Shpak et al., 2001; Zhao et al., 2002; Tan et al., 2003; Held et al., 2004) using naturally occurring AGPs and synthetic genes encoding only clustered noncontiguous Hyp or contiguous Hyp confirmed that AG polysaccharide was added only to clustered, non-contiguous Hyp, while arabinosylation (oligosaccharides composed of ~4 Ara residues) occurred on contiguous Hyp blocks. The structure of a well-characterized Hyp-AG isolated from tobacco was recently elucidated (Tan et al., 2004) (Fig. 2.1). Based on the structure of this AG polysaccharide and given the specificity of glycosyltransferases (GTs), there may be as many as fifteen transferase activities involved in the synthesis of this AG polysaccharide, namely one peptidyl Hyp- galactosyltransferase, one -(1,5)arabinosyltransferase, possibly four - (1,3)arabinosyltransferases, three -(1,3)galactosyltransferases, three - (1,6)galactosyltransferases that add the three branch sites on the AG core, two - (1,6)glucuronyltransferases, and one -(1,4)rhamnosyltransferase. In other species,

66 additional transferases are possible, as in Arabidopsis (Arabidopsis thaliana) where - (1,2)fucosyltransferase is involved in AGP glycosylation (van Hengel and Roberts, 2002). 66 Figure 2.1 AG structure of an AGP molecule. The protein backbone containing a clustered non-contiguous Hyp motif is shown. Although the two Hyp residues in the protein backbone are both glycosylated with AG when expressed in tobacco cells, only one AG side chain is shown here for simplicity. The focus of the present study is the GalT enzyme (shown in red) that adds the first Gal residue onto the AGP peptide backbone. Monosaccharide symbols used here are based on the Symbol and Text Nomenclature for Representation of Glycan Structure as proposed by the Consortium for Functional Glycomics ( Home.jsp). Modified from Tan et al Despite the fact that plant cell walls contain substantial amounts of AGPs, there is an embarrassing lack of knowledge on the enzymology of AGP (and HRGP) biosynthesis (Ellis et al., 2010). (Karr, 1972) partially characterized a microsomal fraction that arabinosylated extensin peptides and Bolwell (Bolwell, 1986) suggested a lipid-linked intermediate might be involved in HRGP glycosylation. In addition, galactosyltransferase

67 67 (GalT) activity associated with AGPs was identified in ryegrass (Mascara and Fincher, 1982) and pea (Hayashi and Maclachlan, 1984) membrane preparations; moreover, this activity in ryegrass was restricted to Golgi-derived membranes (Schibeci et al., 1984). Others identified an -fucosyltransferase activity (Misawa et al., 1996) and a - glucuronyltransferase activity (Endo et al., 2003) that might be involved in biosynthesis of radish root AGPs. The major reason for this lack of research progress is the difficulty in making appropriate acceptor substrates for these enzymes. Very recently, however, some progress on the enzymes responsible of AGP glycosylation was made. A bioinformatics study looking for Arabidopsis homologs to mammalian -(1,3)GalTs identified 20 putative Arabidopsis -(1,3)GalTs (Qu et al., 2008), one of which was previously identified as a -(1,3)GalT involved in the biosynthesis of protein-bound N-linked oligosaccharide (N-glycan) (Strasser et al., 2007). Additionally, Oka et al. (2010) utilized an in vitro assay system to detect and localize Hyp:GalT activity in the endoplasmic reticulum (ER) of Arabidopsis using a chemically synthesized AGP peptide and variants thereof conjugated to fluorescein isothiocyanate (FITC) via a γ-aminobutyric acid (GABA) as acceptor substrates. Finally, Wu et al. (2010) identified and characterized two α-(1,2)fucosyltransferases encoded by AtFUT4 and AtFUT6 in Arabidopsis that are specific to AGPs. With this as a background, we report here on the identification and characterization of in vitro GalT activities that are likely involved in the initial steps of AGP glycosylation in both tobacco and Arabidopsis. This work was achieved by developing an in vitro AGP GalT assay using synthetic and transgenically produced AGP peptides as acceptor

68 substrates, UDP-[ 14 C]Gal as the sugar donor, and permeablized microsomal membranes from tobacco BY2 and Arabidopsis suspension-cultured cells Material and Methods Suspension culture of Arabidopsis cells The tobacco (Nicotiana tabacum, BY2) and Arabidopsis suspension cultured cells (Arabidopsis thaliana, ecotype Columbia) were maintained in liquid NT-1 media (Arabidopsis Biological Resource Center, on a rotary shaker (120 rpm) at 24 C. NT-1 media (ph 5.8) contains 4.3 g/l Murashige and Skoog salts (Caisson), 30 g/l Suc, 180 mg/l KH 2 PO 4, 100 mg/l myo-inositol, 1mg/L thiamine HCl and 0.44 mg/l 2,4-dichlorophenoxacetic acid. Cells were subcultured weekly (1:10 v/v) into fresh culture media. The Arabidopsis cells were obtained from and established by Axelos et al. (1992) Preparation of microsomal membranes from tobacco and Arabiodopsis suspension cultured cells Microsomal membranes were prepared as described earlier (Zeng et al., 2008). The method was modified for the use of suspension cultured cells as the starting material. Briefly, 140 g of mid-log phase cells (7 day cultures) were harvested and thoroughly washed with double distilled water (ddh 2 O) at 4 C and resuspended in 70 ml extraction buffer (0.1 M Hepes KOH ph 7, 0.4 M sucrose, 0.1% BSA, 1 mm DTT, 5 mm MgCl 2, 5 mm MnCl 2, 1 mm phenylmethylsulphonyl fluoride (PMSF), 1 tablet of Roche complete

69 69 protease inhibitor cocktail). Cells were disrupted in a Brinkmann Homogenizer (Model: CPU11, Brinkmann Instruments Corp.) at speed 9 for 3 min (stopping for 30 s every 30 s during disruption). The homogenate was filtered through two layers of miracloth, and the filtrate was centrifuged at 3,000 x g for 20 min. The resulting supernatant was layered over a 1.8 M sucrose cushion buffer and centrifuged at 100,000 x g for 60 min. The uppermost layer was discarded without disturbing the membrane containing interphase layer. A discontinuous sucrose gradient was implemented by sequentially layering 1.1 M and 0.25 M sucrose solutions onto the interphase layer and centrifuging at 100,000 x g for 60 min. The microsomal membranes enriched at the 0.25/1.1 M sucrose interphase were collected and pelleted by another centrifugation at 100,000 x g for 30 min. The pellet was resuspended in 700 μl extraction buffer and stored at -80ºC until use Standard assay for galactosyltransferase (GalT) activities The standard GalT reaction mixture (75 μl) consisted of permeabilized microsomal membranes (~150 μg total protein), acceptor substrate peptide (50 μg) and UDP-[ 14 C]Gal (~3 μm, 90,000 cpm, 465 cpm/pmol, PerkinElmer Life Science Inc.). To achieve permeabilization, 50 μl of the microsome preparations were treated with 1% Triton X- 100 (15 min, 4ºC), followed by ultracentrifugation at 100,000 x g for 45 min. The pellet was resuspended in 50 μl extraction buffer (BSA eliminated). The [AO] 7 acceptor substrate was a chemically synthesized peptide (GenScript Corp.). The sequence of [AO] 7 was [Ala-Hyp-Ala-Hyp-Ala-Hyp-Ala-Hyp-Ala-Hyp-Ala-Hyp-Ala-Hyp]. The d[ao] 51 peptide was obtained from the [AO] 51 -EGFP fusion protein expressed in tobacco BY-2 cells as described previously (Tan et al., 2003). In the control reaction, no acceptor

70 70 substrate was included. Reactions were incubated at room temperature for 2 h and terminated by mixing with 400 μl anion-exchange resin (DOWEX 1X8-100 resin, Sigma-Aldrich) (1:1 v/v in ddh 2 O). The resin mixture was loaded on a Zeba spin column (Pierce) and centrifuged at 15,000 x g for 1 min. While the unreacted UDP-[ 14 C]Gal was retained by the ion-exchange resin beads, the flowthrough contained the incorporated [ 14 C] radiolabeled product and was analyzed with an LS6500 Multi-purpose Scintillation Counter (Beckman) Analysis of the GalT assay products by reverse phase-high performance liquid chromatography (RP-HPLC) The GalT assay product purified by the ion-exchange resin was loaded onto a polymeric reverse-phase column (PRP-1, 5 µm, mm, Hamilton) equilibrated with buffer A (2% acetonitrile 0.1% trifluoroacetic acid). Reaction products were eluted with isotonic buffer A for 10 min followed by a linear gradient of buffer B (100% acetonitrile with 0.1% trifluoroacetic acid) from 2 to 100% in 90 min at a flow rate of 0.5 ml/min. Chromatography was monitored by absorption at 220 nm. The eluate was collected in 0.5 ml fractions and counted for radioactivity in cpm Monosaccharide composition analysis of the GalT assay product The RP-HPLC fractions containing [ 14 C] radiolabeled peptides were collected and freeze dried. The dried powder was dissolved in ddh 2 O and subjected to total acid hydrolysis [2 M trifluoroacetic acid (TFA), 121 C, 60 min]. Excess TFA was removed by a freeze drying-dissolving (with ddh 2 O) cycle repeated three times. The product from

71 71 total acid hydrolysis was dissolved in deionized water and analyzed on a CarboPac PA10 column (4 x 250mm, Dionex) in a BioLC system using pulsed amperometric detection (ED50 electrochemical detector, Dionex). The column was equilibrated at a flow rate of 0.5 ml/min with 200 mm NaOH for 10 min, ddh 2 O for 10 min and 2.5 M NaOH for 30 min. The sample was eluted with 2.5 M NaOH at a flow rate of 0.5 ml/min. Each fraction (0.5 ml) was counted for radioactivity in cpm. Fucose, glucose, galactose, arabinose and xylose monosaccharide (Acros Organics) were used as standards Hyp [ 14 C]Galactoside profile analysis The RP-HPLC fractions containing [ 14 C] radiolabeled peptides were pooled and freeze dried. The dried powder was dissolved in ddh 2 O and analyzed by size exclusion chromatography before or after base hydrolysis (0.44 M NaOH, 18 h, 105 C). Size exclusion chromatography was performed with a Bio-gel P2 column (90 x 1.5 cm) under gravity with degassed ddh 2 O. Each fraction (2.3 ml) was counted for radioactivity in cpm. The base hydrolysate of [AO] 7 as well as pure Hyp amino acid (Sigma-Aldrich) was eluted on Bio-gel P2 under the same elution conditions as described above for comparison. Hyp fractions were analyzed using a colorimetric method described previously (Lamport and Miller, 1971). The column system was calibrated with high-m r dextran (V 0 ), galactose (V t ), xylo-oligosaccharides (DP2-6), and xyloglucanoligosaccharides (DP7-9) (Megazyme).

72 Characterization of the Arabidopsis [AO] 7 :GalT activity The standard GalT assay was modified for the enzymatic characterization tests using [AO] 7 peptide as the acceptor substrate. The assay product from each reaction was fractionated on RP-HPLC to specifically measure incorporated [ 14 C] radiolabel into acceptor substrates. To test the effect of ph, permeabilized microsomal membranes were dissolved in test buffers at a final concentration of 100 mm and used for the GalT assay. Test buffers included MES-KOH buffer at ph 4, 5, 6, and 7; HEPES-KOH buffer at ph 6, 7, and 8; Tris-HCl buffer at ph 8 and 9; CAPS-KOH buffer at ph 10. To test the effect of divalent ions, microsomal membranes were extracted with divalent ions eliminated from the extraction buffer. Permeabilized microsomal membranes were dissolved in extraction buffer with divalent ions eliminated and distributed into aliquots. MnCl 2, MgCl 2, CaCl 2, CuCl 2, NiCl 2 and ZnSO 4 at a final concentration of 5 mm was added when the GalT assays were performed. Deionized distilled H 2 O instead of divalent ions were added in the control reaction. To test the enzyme specificity for different acceptor substrates, the standard GalT assay was performed with 2 µg of various acceptor substrates. The [AO] 14, [AO] 7 and ExtP acceptor substrates were chemically synthesized peptides (GenScript Corp). The sequence of [AO] 14 is [Ala-Hyp-Ala-Hyp-Ala-Hyp-Ala-Hyp-Ala-Hyp-Ala-Hyp-Ala- Hyp-Ala-Hyp-Ala-Hyp-Ala-Hyp-Ala-Hyp-Ala-Hyp-Ala-Hyp-Ala-Hyp]. The sequence of [AP] 7 is [Ala-Pro-Ala-Pro-Ala-Pro-Ala-Pro-Ala-Pro-Ala-Pro-Ala-Pro]. The sequence of ExtP is [Ser-Hyp-Hyp-Hyp-Hyp-Tyr-Val-Tyr-Ser-Ser-Hyp-Hyp-Hyp-Hyp-Tyr]. The

73 73 dextp2 was isolated from tomato suspension cultured cells and deglycosylated as described previously (Smith et al., 1986). dextp2 has a highly periodic structure which is composed of di-and octapeptides, with sequences of [Tyr-Lys] and [Ser-Hyp-Hyp-Hyp- Hyp-Val-Tyr-Lys], respectively. To analyze the enzyme specificity for sugar donors, the standard activity assay was performed with [AO] 7 as the acceptor substrate and various [ 14 C]-labeled nucleotide sugars (90,000 cpm) as the sugar donor. The nucleotide sugars tested included UDP- [ 14 C]Glc (MP Biomedicals), UDP-[ 14 C]Xyl (PerkinElmer Life Sciences) and GDP- [ 14 C]Fuc (PerkinElmer Life Sciences) Electrospray Ionization-Tandem Mass Spectrometry (ESI-MS/MS) of the [AO] 7 :GalT assay product The GalT assay was conducted under standard assay conditions except that UDP- [ 14 C]Gal was replaced by non-radioactive (cold) UDP-Gal (12 µm). The cold GalT assay products were fractionated by RP-HPLC, and fractions were collected according to the retention time of the glycopeptide peak as determined by a preceding run with radiolabeled products. RP-HPLC purified products were lyophilized and dissolved in 50% methanol containing 0.1% acetic acid. The sample was introduced into the ESI source at 3 µl/min using a Cole-Parmer syringe pump. ESI-MS spectra were acquired on an Esquire 6000 Ion Trap analyzer (Bruker Daltonics) operated in positive ion mode with a capillary voltage of 4000 V, drying gas temperature of 300 C, drying gas flow rate of 5 L/min and a nebulizer pressure of 10 psi. Nitrogen was used as both the nebulizing gas and drying gas. Samples from the control or experimental assay product were subjected

74 74 to a full scan from m/z 600 to In MS/MS analysis, precursor ions with the target m/z values were isolated from the full scan spectrum and fragmented by collision-induced dissociation (CID) with helium as the collision gas and a collision voltage of 1 V. Target m/z values for precursor ions were set as 1470, 1632 and 1794, corresponding to the theoretical m/z values of protonated [AO] 7 with one, two and three Gal residues attached. Instrument control and data acquisition were performed with Esquire 5.0 software Continuous sucrose gradient centrifugation Linear 30-45% (w/v) sucrose gradients (20 ml) were poured over 4 ml cushions of 62% sucrose (w/v) in a centrifugation tube by Auto Densi-Flow (Labconco). All solutions are made up in 0.1 M Hepes KOH (ph 7) in the presence of 1 mm EDTA. Microsomal membranes from Arabidopsis suspension cultured cells (1 ml) were layered on top of the sucrose gradient. After centrifugation (100,000 x g, 16 h, 4 C, Beckman Coulter SW32 rotor), 20 equal fractions were collected from the top of the gradient downward with the electric gradient fractionator Auto Densi-Flow (Labconco). The cytochrome C reductase (an ER marker), inosine diphosphatase (a Golgi marker) and glycan synthase II (a plasma membrane marker) in each gradient fraction were measured according to procedures described previously (Shore and Maclachlan, 1975; Gibeaut and Carpita, 1990; Dhugga and Ray, 1994).

75 Results Development of a GalT assay system with AGP-like peptides as acceptor substrates An in vitro GalT assay system was developed with UDP-[ 14 C]Gal as the sugar donor and permeablized microsomal membranes from tobacco or Arabidopsis suspensioncultured cells as the enzyme resource. Two peptides with repetitive [AO] units, representing clustered noncontiguous Hyp motifs of AGPs, were used as acceptor substrates. One of the peptides, denoted as d[ao] 51, contained an [AO] motif repeated 51 times. The d[ao] 51 peptide was obtained from the [AO] 51 -EGFP fusion protein expressed in transgenic tobacco BY-2 cells; the EGFP tag was removed by trypsin digestion and the AG side chains were removed by deglycosylation with hydrogen fluoride (HF). The other peptide, namely [AO] 7, was chemically synthesized and contained seven [AO] repeats. A control reaction was set up with the peptide acceptors excluded. After the reaction, unreacted UDP-[ 14 C]Gal was removed with an ion-exchange resin column and radioactivity in the reaction solution was counted. As shown in Fig. 2.2, when tobacco permeablized microsomal membranes were used as the enzyme resource, radioactivity detected in the d[ao] 51 :GalT reactions was higher than the control reaction as well as the [AO] 7 :GalT reactions, which displayed a similar level of radioactivity as the control reactions. Compared to the tobacco reactions, the Arabidopsis reactions showed much lower background levels and high, roughly equivalent levels of incorporation into both d[ao] 51 and [AO] 7.

76 76 Figure 2.2 Total [ 14 C] radiolabel incorporation into the GalT reaction product in the absence or presence of [AO] 7 or d[ao] 51 acceptor substrate. Permeablized microsomal membranes from tobacco and Arabidopsis suspension cultured cells served as the enzyme source. Reactions were done in triplicate and mean values are presented Characterization of the GalT assay products by Reverse-Phase High Performance Liquid Chromatography (RP-HPLC) analysis To confirm that [ 14 C] radiolabel was transferred to the acceptors, reaction products were first fractionated by RP-HPLC. RP-HPLC fractionation of the tobacco and Arabidopsis control reaction products resolved a single radioactive peak with a low retention time (Peak I in Fig. 2.3 A and D). Peak I was also detected in the experimental reactions with [AO] 7 and d[ao] 51 acceptor substrates (Fig. 2.3 B, C, E and F). The identity of the components in this [ 14 C] labeled fraction is unknown. The small A 220nm peaks appearing at the corresponding retention time were solvent peaks and did not represent protein components. It is possible that this fraction is composed of oligosaccharides with [ 14 C]Gal incorporated into endogenous sugar acceptors during the

77 GalT assay. Alternatively, the fraction may represent free [ 14 C]Gal residues released during the assay by an endogenous galactosidase (Kato et al., 2003). 77 Figure 2.3 RP-HPLC fractionation of the [AO] 7 :GalT and d[ao] 51 :GalT reaction products on a PRP-1 reverse-phase column. Radioactive Peak II coeluted with the [AO] 7 or d[ao] 51 acceptor substrate and was used for subsequent product analysis and in monitoring enzyme activity. Another radioactive product peak co-eluting with the [AO] 7 and d[ao] 51 acceptor substrates appeared only in the experimental reactions (Peak II in Fig. 2.3.B, C, E and F). Control reactions, lacking the peptide acceptors, contained no such radioactive product peak. RP-HPLC fractionation provided evidence for incorporation of the [ 14 C] label from UDP-[ 14 C]Gal into the [AO] 7 and d[ao] 51 peptide acceptors in both the tobacco and

78 78 Arabidopsis assay systems, including the [AO] 7 :GalT reaction in tobacco, which showed no substantial increase in radioactivity in the crude product analysis (Fig. 2.2). To confirm that the [ 14 C] radiolabel remained associated with Gal, RP-HPLC fractions containing the [ 14 C] labeled GalT reaction product coeluting with the d[ao] 51 peptide acceptor in the tobacco assay were pooled and subjected to total acid hydrolysis, which degraded the product into free amino acids and monosaccharides. The resulting [ 14 C] labeled monosaccharide eluted as Gal following High ph Anion Exchange Chromatography (HPAEC) on a CarboPac PA-10 column (Dionex) (Fig. 2.4.A). Similarly, [ 14 C]-labeled monosaccharide released from the Arabidopsis [AO] 7 :GalT reaction product eluted as Gal (Fig. 2.4.B). Such analysis confirmed that [ 14 C]Gal was incorporated into the d[ao] 51 and [AO] 7 acceptors without any significant conversion into other monosaccharides.

79 79 Figure 2.4 Monosaccharide analysis of the RP-HPLC purified tobacco d[ao] 51 :GalT reaction product (Panel A) and the Arabidopsis [AO] 7 :GalT reaction product (Panel B). The [ 14 C]-labeled monosaccharides were analyzed by High-Performance Anion- Exchange Chromatography (HPAEC) on a CarboPac PA-10 column. Elution times of monosaccharide standards are as indicated with arrows at the top Extent of AGP peptide galactosylation In order to investigate how many Gal residues were incorporated into the reaction products, RP-HPLC fractions containing the [ 14 C] labeled GalT reaction products coeluting with the peptide acceptors were pooled and subjected to base hydrolysis. Base hydrolysis degrades peptide bonds but keeps Hyp-glycosidic bonds intact (Kieliszewski and Shpak, 2001). The molecular sizes of the resulting [ 14 C] labeled base hydrolysates were analyzed by gel filtration chromatography on a Bio-gel P2 column (Fig. 2.5). The

80 80 rationale here was the extent of Gal addition would be reflected in the sizes of the resulting Hyp-glycosides. As shown in Fig. 2.6.A and B, Hyp generated from base hydrolysis of the [AO] 7 peptide eluted as a degree of polymerization (DP) 3 size on the P2 column, which was identical to the elution profile of commercially available free Hyp amino acid. Before base hydrolysis treatment, intact [ 14 C] labeled d[ao] 51 and [AO] 7 peptides eluted at the void volume (V 0 ) of the P2 column, as shown in the upper portion of each panel in Fig When tobacco permeabilized microsomal membranes were used as the enzyme source, the base hydrolysate of the d[ao] 51 reaction product mainly eluted as DP5 (Fig. 2.5.A), while the major base hydrolysate released from [AO] 7 reaction product eluted as DP4 (Fig. 2.5.B). Given that Hyp eluted as DP3 (Fig. 2.6) under the same conditions, the results indicated that Hyp residues in [AO] 7 were glycosylated with one Gal residue, while Hyp residues in d[ao] 51 had glycosyl side chains corresponding to two Gal residues. Note that Hyp, but not Ala residues, are known sites of glycosylation for AGPs.

81 Figure 2.5 Bio-gel P2 fractionation of the RP-HPLC purified GalT reaction product. Elution profiles of the d[ao] 51 :GalT reaction product before and after base hydrolysis are shown for tobacco reactions (Panel A) and Arabidopsis reactions (Panel D); elution profiles of the [AO] 7 :GalT reaction product before and after base hydrolysis are shown for tobacco reactions (Panel B) and Arabidopsis reactions (Panel E); elution profiles of the dd[ao] 51 :GalT reaction product before and after base hydrolysis are shown for tobacco reactions (Panel C) and Arabidopsis reactions (Panel F); the elution position of free Hyp amino acid (corresponding to DP3) is shown with an arrow in each panel. The column was calibrated with high-m r dextran (V 0 ), galactose (V t ), xylo-oligosaccharides 81

82 82 with degree of polymerization (DP) 2 to 5 and xyloglucan-oligosaccharides (DP6-9); their elution positions are indicated with arrows at the top of the figure. Figure 2.6 Bio-gel P2 fractionation of free Hyp amino acid (Panel A) or Hyp amino acid produced following base hydrolysis of the [AO] 7 acceptor substrate (Panel B). The column was calibrated as described in Fig In the case of the d[ao] 51 acceptor, it was possible that two monosaccharide sugars were added to one Hyp residue in the GalT assay. However, it could not be excluded that one Gal residue might pre-exist on the Hyp residues of d[ao] 51 because of incomplete deglycosylation during the preparation of this acceptor substrate. To test this possibility, double deglycosylated d[ao] 51 (dd[ao] 51 ) was produced by subjecting d[ao] 51 to a second HF deglycosylation reaction and used as an acceptor in the GalT assay. The base hydrolysate generated from the tobacco dd[ao] 51 reaction products eluted as DP4 on the Bio-gel P2 column (Fig. 2.5.C), indicating not only that d[ao] 51 was incompletely

83 83 deglycoslyated, but also providing evidence for the presence of two GalT activities in tobacco microsomal membranes. One activity, a Hyp:GalT activity, transfers the initial Gal onto Hyp residues in dd[ao] 51 and [AO] 7, while the second activity, a likely Gal:GalT activity, transfers the next Gal residue onto galactosylated Hyp in the incompletely deglycosylated d[ao] 51. When Arabidopsis permeablized microsomal membranes were used as the enzyme source, a similar picture emerged. Here, the base hydrolysate from the [AO] 7 :GalT reaction product eluted as DP4 on the Bio-gel P2 column (Fig. 2.5.E). Moreover, the base hydrolysate of the dd[ao] 51 :GalT product eluted mainly as DP4 with a small amount of DP5, whereas the base hydrolysate of the d[ao] 51 :GalT product eluted with considerably more DP5 relative to DP4 (Fig. 2.5.D and F). The result indicated that Arabidopsis, like tobacco, contained two GalT activities; however, the relative activity of the putative Gal:GalT responsible for the DP5 peak was considerably greater in tobacco than in Arabidopsis Enzymatic characteristics of the [AO] 7 :GalT activity in Arabidopsis suspension-cultured cells Since Arabidopsis material will be a better source for identification of the AGP GalT enzyme(s) using proteomic strategies given its complete genome sequence, Arabidopsis [AO] 7 :GalT activity was further characterized as described below. With a total of 150 µg of microsomal proteins in the assay system, [AO] 7 :GalT activity approached saturation when 2 µg of [AO] 7 was included in the reaction mix (Fig. 2.7). With 2 µg [AO] 7 in the GalT assay system, incorporation of [ 14 C]Gal increased

84 84 proportionally with respect to the amount of microsomal protein up to 150 µg using an incubation time of 2 h (Fig. 2.7.B). The [AO] 7 :GalT activity had a ph optimum of 7 (Fig. 2.7.C) and a temperature optimum of 40 C (data not shown). The [AO] 7 :GalT activity increased approximately 4 fold in the presence of Mn 2+ but was unchanged with Mg 2+, in comparison to the control (Fig. 2.7.D). In addition, the presence of Ca 2+, Cu 2+, Zn 2+ and Ni 2+ divalent ions inhibited activity to different extents (Fig. 2.7.D).

85 85 Figure 2.7 Biochemical characteristics of the Arabidopsis [AO] 7 :GalT activity. Data are the average of triplicate assays. A. Relationship between [AO] 7 concentration and incorporation of [ 14 C]Gal into [AO] 7. B. Relationship between microsomal protein concentration and incorporation of [ 14 C]Gal into [AO] 7. C. Effect of ph on enzyme activity. D. Effect of different divalent ions (5 mm) on enzyme activity. The specificity of the Arabidopsis Hyp:GalT activity was investigated using various acceptor substrates (Fig. 2.8). Namely, [AO] 7, [AO] 14 and dd[ao] 51 were used as AGP peptides with repetitive [AO] units of various lengths. Incorporation of [ 14 C] radiolabel decreased with increasing lengths of these [AO] acceptor substrates. In contrast, [AP] 7 containing seven repetitive [Alanine-Proline] units did not serve as an acceptor substrate, indicating that peptidyl Hyp was required for galactosylation. In addition, two extensin sequences were used as potential acceptor substrates, a chemically synthesized extensin

86 86 peptide (ExtP) designed based on a repetitive sequence (i.e., SOOOOYVYSSOOOOY) found in several Arabidopsis extensins (e.g., At4g08410) and a deglycosylated tomato P2 extensin (dextp2) containing numerous SOOOOVYK and YK repeat units (Smith et al., 1986). Both of these extensin sequences, which contained contiguous peptidyl Hyp as well as other amino acid sequences, failed to act as acceptors, indicating this Hyp:GalT activity was specific for AGP sequences containing non-contiguous peptidyl Hyp. Figure 2.8 Incorporation of [ 14 C] radiolabel into acceptor substrates containing various Hyp motifs. [AO] 7, [AO] 14 and dd[ao] 51 contain seven, 14 and 51 [AO] units, respectively. A chemically synthesized extensin peptide (ExtP) and tomato deglycosylated extensin P2 (dextp2) contain repetitive SO 4 units. [AP] 7 contains seven [AP] units. Enzyme reactions were done in triplicate and mean values are presented. The requirement of a specific nucleotide sugar donor for [AO] 7 glycosylation was also tested. Arabidopsis microsomal membranes specifically incorporated [ 14 C]Gal, but not [ 14 C]glucose, [ 14 C]xylose or [ 14 C]fucose from their corresponding nucleotide sugars, into the [AO] 7 acceptor substrate (Table 2.1).

87 Table 2.1 Incorporation of [ 14 C] radiolabel into the [AO] 7 acceptor substrate when various nucleotide sugars were used as the sugar donor. 87 Sugar Donor [ 14 C] Incorporation (pmol/h/mg ) UDP-[ 14 C]Gal UDP-[ 14 C]Glc UDP-[ 14 C]Xyl GDP-[ 14 C]Fuc Identification of the Arabidopsis [AO] 7 :GalT reaction product by Electrospray Ionization-Tandem Mass Spectrometry (ESI-MS/MS) analysis When non-radioactive (cold) UDP-Gal was used in the GalT reactions as the sugar donor, the resulting RP-HPLC purified [AO] 7 :GalT reaction product was analyzed by ESI-MS/MS analysis. No product peak was visible in either the [AO] 7 :GalT product or the control product by ESI-MS under full scan mode (data not shown), due to a low signal to noise ratio. In MS/MS analysis, however, a mass/charge ratio (m/z) ion corresponding to the putative galactosylated [AO] 7 with one Gal attached was isolated in the [AO] 7 assay product (Fig. 2.9.A). The identity of the ion was confirmed by ion fragmentation using the collision-induced dissociation (CID) technique, which generated a series of peaks with their m/z matching the theoretical fragments from [AO] 7 with either a single Gal attached or not (Fig. 2.9.B). The fragmentation pattern of the m/z ion

88 also indicated that the attachment site of the Gal residue was at Hyp 12 or Hyp 14. Although an ion with a m/z of was isolated from the control assay product (Fig C), the fragmentation pattern of the m/z ion was not related to the [AO] 7 substrate or to galactosylated [AO] 7 (Fig. 2.9.D). Moreover, we were unable to detect or isolate ions corresponding to [AO] 7 peptide with more than one Gal attached from either the [AO] 7 assay product or control product. Figure 2.9 ESI-MS/MS analysis of the Arabidopsis [AO] 7 :GalT reaction product. The ion with m/z of corresponding to glycosylated [AO] 7 with one Gal attached was isolated from the [AO] 7 :GalT reaction product (Panel A). The fragments generated from ion following collision-induced dissociation (Panel B) were matched with the [AO] 7 fragments or galactosylated [AO] 7 fragments (b and y ions with asterisks) based on their calculated m/z. In ion , the Gal attachment site was deduced to be the Hyp residue at position 12 or position 14 (i.e., Hyp residues with asterisks in the

89 peptide). A peak with m/z of was isolated from the control reaction (Panel C). However, fragmentation of ion did not show peaks relevant to [AO] 7 fragments or the galactosylated [AO] 7 product (Panel D) Subcellular localization of [AO] 7 :GalT and d[ao] 51 :GalT activities in Arabidopsis suspension cultured cells A continuous sucrose density gradient was employed to fractionate microsomal membranes, followed by marker enzyme assays to identify subcellular membrane systems. Although various combinations of gradient conditions were used, including different sucrose gradients, with or without the addition of EDTA (1 mm or 5 mm) or Mg 2+ (1 mm or 5 mm), it was not possible to completely resolve ER membranes from Golgi membranes. The best separation of ER and Golgi membranes was achieved with a 30-45% (w/v) sucrose gradient in the presence of 1 mm EDTA (Fig A), in which both NADH cytochrome C reductase (an ER marker) and inosine diphosphatase (a Golgi marker) showed two activity peaks (Fig B). However, NADH cytochrome C reductase showed higher activity in the peak of lower density (fraction 2 to 5), while inosine diphosphatase had greater activity in the higher density peak (fraction 7 to 16). The plasma membrane exhibited a broad distribution with a single peak around fraction 13, as indicated by using glucan synthase II as a marker (Fig A). The gradient fractions were used for GalT assays. With both [AO] 7 and d[ao] 51 as acceptor substrates, two GalT activity peaks corresponding to the ER and Golgi marker enzyme activities were detected (Fig C and D).

90 Figure 2.10 Fractionation of mixed-membranes of Arabidopsis on a sucrose gradient in the presence of 1 mm EDTA. Each fraction was assayed for: A. density and glucan synthase II (a plasma membrane marker); B. activities of antimycin A-insensitive NADH cytochrome C reductase (an endoplasmic reticulum marker) and IDPase (a Golgi apparatus marker); C. [AO] 7 :GalT activity, and D. d[ao] 51 :GalT activity. 90

91 Discussion Considering GTs are linkage-specific (Keegstra and Raikhel, 2001), the complex AG structure implies the involvement of multiple GalTs in AG biosynthesis. Using chemically synthesized and transgenically produced AGP peptides as acceptor substrates and UDP-[ 14 C]Gal as the sugar donor, an in vitro GalT assay was developed to detect GalT activities involved in AGP glycosylation in tobacco and Arabidopsis. Product analysis by RP-HPLC and monosaccharide analysis showed that [ 14 C]Gal was incorporated into AGP peptides in the GalT assay (Figs. 2.3 and 2.4). In examining the extent of AGP peptide galactosylation in the [AO] 7, d[ao] 51 and dd[ao] 51 reaction products (Fig. 2.5), evidence for two distinct GalT activities was detected. The first activity, a Hyp:GalT activity, catalyzes the addition of Gal onto peptidyl Hyp residues, as demonstrated in reactions with [AO] 7 and dd[ao] 51 acceptor substrates. The second activity, a probable Gal:GalT activity, extends the sugar side chain with a second Gal as demonstrated in reactions with the d[ao] 51 acceptor substrate and was more pronounced in tobacco compared to Arabidopsis. The Arabidopsis Hyp:GalT activity observed here is similar to that reported by Oka et al. (2010) with respect to its properties (ph optimum, temperature optimum, requirement for Mn 2+, specificity for UDP-Gal) (Fig. 2.7, Table 2.1) and localization to the endomembrane system (Fig. 2.10). However, further analysis of the Hyp:GalT activity here revealed that it was specific for AGP sequences and not for other related protein sequences, including extensins with their characteristic SO 4 repeat units. This finding is consistent with the Hyp contiguity hypothesis which states that non-contiguous

92 92 Hyp residues are sites of arabinogalactan polysaccharide addition, whereas contiguous Hyp residues are sites for the addition of arabinose oligosaccharides (Kieliszewski and Lamport, 1994; Kieliszewski et al., 1995; Tan et al., 2003). In addition, ESI-MS/MS analysis of the [AO] 7 product demonstrated that only one galactose was added per peptide molecule to the C-terminal or pentultimate Hyp residue of this peptide. Whether this observation reflects the enzymatic mechanism associated with this Hyp:GalT activity or the in vitro nature of this work awaits further investigation. It should also be noted that Oka et al. used a different in vitro assay system involving a chemically synthesized AGP14 peptide and variants thereof conjugated to fluorescein isothiocyanate (FITC) via a γ-aminobutyric acid (GABA) linker as acceptor substrates. Regardless, such chemically synthesized peptides, whether it be [AO] 7 or AGP14, were only useful in demonstrating the addition of the first Gal residue to Hyp and provide no evidence for a second Gal:GalT activity, which could only be detected when deglycosylated AGP sequences were used as acceptor substrates. These observations also highlight the need to develop appropriate acceptor substrates to detect specific enzyme activities in the AG biosynthetic pathway as the small amount of product formed in these assays is apparently insufficient to serve as an effective substrate for the next glycosylation reaction. In ryegrass, AGP GalT activity was reported to incorporate [ 14 C]Gal into endogenous acceptor substrates, forming 66% ethanol insoluble products with (1,6)- linked galactosyl residues (Mascara and Fincher, 1982). AGP GalT activity was also detected in radish roots and catalyzed addition of Gal residues onto exogenous β-(1,3)- galactotriose acceptors through β-(1,6)-linkages (Kato et al., 2003). In these two studies,

93 93 AGP GalT activities were involved in elongation of AG side chains and so are likely to be different from the Hyp:GalT activity identified here. Indeed, the Hyp:GalT activity exhibited distinct features from the rygrass GalT and radish GalT activity. For example, Mg 2+ did not activate the Arabidopsis Hyp:GalT activity as it did for ryegrass GalT activities. The optimal temperature of Arabidopsis Hyp:GalT (40 C) is higher than that of radish GalT (30 C) and of ryegrass GalT (between 10 and 25 C). N-glycosylation and O-glycosylation are the two major types of protein glycosylation. The biosynthesis of N-glycans involves assembling an oligosaccharide precursor structure linked to lipid carriers. The oligosaccharide precursor is then transferred en block from lipid carriers to the protein N-glycosylation sites (Varki et al., 1999). In contrast, O-glycosylation is often viewed as the sequential addition of single sugar residues to the polypeptide, as exemplified by two well defined processes, O- mannosylation (Goto, 2007) and mucin type O-glycosylation (Hanisch, 2001). The identification of Hyp:GalT activity and likely Gal:GalT activity for the formation of a disaccharide galactosyl chain on Hyp as well as the lack of detection of en block transfer in this study is consistent with the notion that Gal residues are transferred sequentially to the protein backbone in AGP glycosylation. In addition, the recent identification and characterization of two AGP fucosyltransferases which have the ability to fucosylate AGPs lacking terminal fucose residues is also consistent with this notion of sequential sugar addition (Wu et al., 2010). Protein N-glycosylation is initiated in ER but completed in Golgi apparatus. Oka et al. (2010) proposed a hypothetical model for AG biosynthesis in which the first Gal

94 94 residue is added to peptidyl Hyp in an AGP backbone by GalT in the ER with further AG side chain elongation occurring in the Golgi apparatus. The subcellular localization of the Hyp:GalT and Gal:GalT activities here is consistent with this model in that these enzyme activities were localized in the endomembrane system (Fig. 2.10). Cloning and tagging of the Hyp:GalT and Gal:GalT enzymes in the future should allow for more precise subcellular localizations. Moreover, the subcellular localization of AGP GalT activities in rygrass (Mascara and Fincher, 1982; Schibeci et al., 1984) and radish root (Kato et al., 2003), AGP fucosyltransferase activity in radish root (Misawa et al., 1996), an AGP fucosyltransferase enzyme in tobacco leaves (Wu et al., 2010), and AGP arabinosyltransferase activity in tobacco cultured cells (Kawasaki, 1987) likewise are consistent with involvement of the endomembrane system, particularly the Golgi apparatus, in AG biosynthesis. Although a large number of GTs are predicted to be involved with the biosynthesis of plant cell wall components, only a few GTs are unambiguously identified and functionally characterized (Keegstra and Raikhel, 2001). One of the main obstacles for GT identification is the lack of specific enzyme activity assays. Here, an in vitro assay was developed which allows for the detection of two distinct AGP GalT activities in tobacco and Arabidopsis microsomal membranes. Clearly, this radioactive, in vitro assay to detect GalT activity using AGP peptide and glycopeptide acceptor substrates provides a valuable tool for the identification of AGP GalT proteins/genes and serves as an entry point for the elucidation of AG biosynthesis for AGPs. Finally, this assay can be readily

95 modified by using different AG acceptor substrates along with appropriate radiolabeled sugar nucleotides to detect other GT activities involved with AG biosynthesis. 95

96 96 CHAPTER 3: HETEROLOGOUS EXPRESSION OF PUTATIVE AGP GALACTOSYLTRANSFERASES IN PICHIA PASTORIS AND TOBACCO BY2 CELLS AND THEIR FUNCTIONAL CHARACTERIZATION 3.1. Introduction A coupling of bioinformatics prediction and experimental validation becomes a potent tool for the functional characterization of genes in the post-genomic era. The use of bioinformatics tools as a guide to identify cell wall biosynthetic enzymes has proven successful in many cases, i.e. the identification of Arabidopsis XYLOGLUCAN XylTs (XXT1 and XXT2) (Faik et al., 2002; Cavalier and Keegstra, 2006; Cavalier et al., 2008), AGP FUT4 and FUT6 (Sarria et al., 2001; Wu et al., 2010), XYLAN GlcATs (GUX1 and GUX2) (Mortimer et al., 2010), and XYLAN AraTs (XAT1 and XAT2) (Anders et al., 2012). In these studies, putative GTs were identified by searching for homologues of genes with known functions or based on the expression of genes with the event of cell wall polymer deposition. The functional confirmation of candidate GTs usually involves three strategies (Farrokhi et al., 2006): i) the gene is expressed heterologously, usually in fusion with a molecular tag, and the gene product isolated and tested for the proposed GT activity in vitro; ii) the loss-of-function mutants showing physiological or biochemical phenotypes provide evidence of the gene function in vivo; iii) function of genes may be demonstrated via various gain-of-function systems. In the type II AG polysaccharides of AGPs, Gal residues are mainly connected through β-(1 3)- and β-(1 6)-linkages. To identify candidate AGP β-(1 3)-GalTs, Qu et al. (2008) conducted a BLAST search of the Arabidopsis database with three reference

97 97 protein sequences from Homo sapiens, GalT1, GalT2 and GalT4. Human GalT1 and GalT2 both encode a UDP-galactose:β-N-acetylglucosamine β-(1 3)-GalT, while GalT4 encodes a UDP-galactose:β-N-acetyl-galactosamine β-(1 3)-GalT. This study identified 20 putative Arabidopsis β-(1 3)-GalTs, only one of which has a known function as a β- (1 3)-GalT for the biosynthesis of the Lewis a epitope of N-glycans; it was named Arabidopsis GalT1 (Strasser et al., 2007) (Table 3.1). All of the twenty genes belong to the CAZy GT31 family. The CAZy GT31 family contains other genes with known functions mainly as β-(1 3)-GTs, including β-(1 3)-GlcTs, β-(1 3)-GlcNAcTs, β- (1 3)-GalTs, β-(1 3)-GalNAcTs and one β-(1 4)-GalNAcT, for the protein glycosylation in mammals and bacteria (Qu et al., 2008; Egelund et al., 2011).

98 98 Table 3.1 Structural features of the 20 putative Arabidopsis β-(1 3)-GalTs a Name in this Gene Accession Clade b Length c (a.a.) Position of the GalT Position of the Galectin Subcellular Localization e Dissertation Domain d Domain r d GalT1 At1g Golgi GalT2 At4g Golgi GalT3 At3g Golgi GalT4 At1g Golgi GalT5 At1g Golgi GalT6 At5g Golgi - At1g Golgi - At1g Golgi - At2g Golgi - At4g Golgi - At1g Golgi - At1g Golgi - At1g Golgi - At1g Golgi - At2g Golgi - At4g Golgi - At5g Golgi - At2g Post-Golgi - At3g Post-Golgi - At1g Post-Golgi a The 20 putative Arabidopsis β-(1 3)-GalTs were identified by Qu et al. (2008). b The clade number is based on the phylogenetic grouping of Arabidopsis GT31 family members (Egelund et al., 2011). c Protein lengths (a.a.) are calculated based on the translated sequences of the open reading frame of the cdna clones for GalT1 to GalT6. For the other genes, the data for protein length (a.a.) is from the TAIR database (

99 d The positions of the GalT domain and the Galectin domain are predicted by the Pfam database ( e The subcellular localization information is from the study of Qu et al. (2008) based on data from the Golgi predictor program ( 99 On a basis of their phylogenetic relationship, genes in the CAZy GT31 family were classified into 11 clades (Egelund et al., 2011). The 20 candidate β-(1 3)-GalTs identified by Qu et al. (2008) are located in clade 7 and clade 10, two clades containing exclusively plant GTs. All of the twenty candidate β-(1 3)-GalTs contain a GalT domain (Pfam: PF01762) as identified by the Protein Family (Pfam) database ( which is not present in the remaining 13 Arabidopsis genes in CAZy GT31. Within the GalT domain, six conserved motifs are present, including a conserved hydrophobic region, an RxxxRxT/SW motif, an FxxG/A motif, a DXD motif, a GxxYxxS motif, an E/DDV plus GxW/C motif. The six conserved motifs are involved in sugar donor binding, sugar acceptor binding, catalysis, and may affect the specificities of the enzymes for different donor substrates (Qu et al., 2008; Egelund et al., 2011). The presence of these conserved motifs supports that the Arabidopsis accessions encode β- (1 3)-GTs because none of the six conserved motifs, with the exception of the DxD motif, occur in either β-(1 4)-GTs or α-(1 3)-GTs (Qu et al., 2008). Besides the conserved GalT domain, all of the twenty putative β-(1 3)-GalTs were predicted to be Gogi or post-golgi localized, with the majority of the genes containing a transmemberane (TM) domain close to the N terminus of the protein (Qu et al., 2008). Such predicted subcellular localization of the putative GalT genes is consistent with the

100 100 AGP GalT activities identified experimentally in various plant species (see Chapter ). The six putative Arabidopsis GalTs in GT31 clade 7 differ from those in clade 10 by the addition of a galectin domain (Pfam: 00337). A schematic summarizing the domain structure of the putative Arabidopsis β-(1 3)-GalTs in clade 7 and clade 10 is shown in Fig A galectin domain is defined by a conserved carbohydrate recognition domain that binds exclusively to β-galactosides (reviewed by Egelund et al. 2011). In Homo sapiens, polypeptide N-acetylgalactosaminyltransferases (GalNAcT) containing galectin domains catalyze the addition of the first GalNAc to initiate protein O- glycosylations. It has been hypothesized that the Arabidopsis GalTs in clade 7 may transfer the first Gal onto the HRGP backbone in a similar mechanism as human GalNAcTs (Qu et al., 2008; Egelund et al., 2011). To test this hypothesis, I plan to adopt the heterologous expression strategy to characterize the functions of five putative GalTs in the GT31 clade 7. For simplicity, the five putative GalTs are named GalT1, GalT3, GalT4, GalT5 and GalT6 in this research (Table 3.1). Five out of the six candidate GALT genes were chosen to study at the beginning because cdna clones of the five genes were available from public cdna libraries. Later, the cdna clone of GALT2 was obtained as a gift from Dr. Richard Strasser (Institute of Applied Genetics and Cell Biology, BOKU, University of Natural Resources and Applied Life Sciences) and the function of GALT2 is being characterized by Ms. Debarati Basu in our lab using a similar heterologous expression strategy as described below. Specifically, the five putative GalTs will be expressed with an N-terminal 6xHis tag in two heterologous expression systems,

101 101 including a methanotrophic yeast (Pichia pastoris) system and tobacco BY2 suspensioncultured cells. For Pichia expression, GALT genes with the N-terminal 6xHis tag are cloned into ppicza/b vector behind the alcohol oxidase promoter (P AOX ). After transformation, the P AOX :6xHis-GALTs are integrated into Pichia genome at the site of the genomic P AOX through homologous recombination. For tobacco expression, GALT genes with the N-terminal 6xHis tag will be cloned into expression vectors containing an inducible heat shock protein promoter (P hsp ) or a constitutive 2x35S promoter (P 2x35S ). The P hsp :6xHis-GALTs or P 2x35S :6xHis-GALTs are integrated into random positions of tobacco genome through an Agrobacterium mediated transformation. The expressed proteins will be analyzed for AGP GalT activities using the in vitro GalT assays developed in Chapter 2. In addition, subcellular localization of the putative GalTs is planned to be studied by transiently expressing tagged versions of these genes in tobacco leaves to corroborate functional speculations of the GalTs.

102 102 Figure 3.1 Schematic domain structure of the two classes of putative Arabidopsis β- (1 3)-GalTs. A. Classical β-(1 3)-GalTs as presented by Arabidopsis genes in GT31 clade 10. B. Galectin-containing domain β-(1 3)-GalTs as presented by the six Arabidopsis genes in GT31 clade 7. The figure is drawn to scale. The red triangle indicates the position of the DXD motif in each gene. TM, transmembrane region as predicted by the TMHMM server version 2.0 ( Galectin domain and GalT domain are predicted by Pfam ( database. GalT1 and GalT3 were predicted to contain no TM by TMHMM server but were predicted to be Golgi localized by Golgi predictor ( Material and Methods Obtaining full length cdna clones Full length cdna clones of RAFL09-24-L19 (At1g26810, GALT1), RAFL K06 (At3g06440, GALT3), RAFL19-55-I21 (Ag1g27120, GALT4), and RAFL09-69-H18 (At1g74800, GALT5) were ordered from the RIKEN Bioresource Center ( The full length cdna

103 clone of At5g62620 (GALT6, NCBI Accession BX831498) was ordered from the Arabidopsis thaliana GSLT EST libraries ( Sequence analysis of the putative GalTs Each of the cdna clone sequences was extracted from the RIKEN Bioresource Center ( for GALT1, GALT3, GALT4 and GalT5 with the accession numbers of pda09678, pda01627, pda11283, pda08691, respectively, and the Arabidopsis thaliana EST libraries ( for GALT6 with the accession number of BX The sequences from the cdna libraries were then compared to the predicted cdna sequences from the TAIR database ( by the BLAST2 Sequences program ( &BLAST_PROG_DEF=megaBlast&BLAST_SPEC=blast2seq). Translations of the cdnas were performed using the ExPASy Translate Tool ( ESTs for GALT4 were identified from a search of the following NCBI UniGene database ( Expression construct cloning of GALT1, GALT3, GALT4, GALT5 and GALT6 for expression in Pichia The N-terminal 6xHis-tag and restriction sites were incorporated into the putative GalT genes by PCR. The PCR product was first cloned into the pcr4-topo vector

104 104 (Invitrogen) and then subcloned into the Pichia expression vector ppicza/b (Invitrogen). Specifically, GALT1, GALT3 and GALT4 were subcloned from the pcr4-topo vector into the ppicza vector by a SacII and ApaI restriction digestion and ligation; GALT5 and GALT6 were subcloned from the pcr4-topo vector into the ppiczb vector by a SacII and XbaI restriction digestion and ligation. E.coli strains hosting the pcr4-galts were selected by kanamycin resistance and E.coli strains hosting ppicza/b-galts were selected by Zeocin resistance. Both the pcr4-galts and the ppicza/b-galts constructs were first examined by diagnostic digestion or PCR and then sequenced to insure that correct in-frame sequences were obtained for the N-terminal 6xHis-tag and the GALT coding regions. The primer sequences for GalT subcloning and sequencing are listed in Appendix A Table 1 and Fig. 1. Sequencing results for the final expression constructs were assembled into contigs (Appendix B Part I) using an open source software, Cap3 ( as described by Huang and Madan (1999) Pichia transformation The constructs of ppicza-galt1, ppicza-galt3, ppicza-galt4, ppiczb- GALT5 and ppiczb-galt6 were transformed individually into Pichia strain X-33 by electroporation using the Gene Pulser II system (Bio-Rad). Preparation of competent X- 33 cells and the electroporation were performed according to the manufacturer s instruction for the EasySelect Pichia Expression Kit (Invitrogen). The transformants were selected on the yeast peptone dextrose sorbitol (YPDS) medium plates containing 100mg/L Zeocin antibiotic. Positive clones were inoculated into the liquid yeast peptone

105 105 dextrose (YPD) medium containing 100mg/L Zeocin. Genomic DNA (gdna) from transgenic Pichia was isolated and verified by PCR analysis as described in Chapter Protocols for making the YPDS and YPD medium are described in the EasySelect Pichia Expression Kit (Invitrogen). The protocol for gdna isolation from Pichia was provided by Dr. Tomohiko Sugiyama (Department of Biological Sciences, Ohio University). Briefly, a 3 ml overnight Pichia culture was pelleted by repeating centrifugation (10,000 xg, 1 min) in 1.5 ml microcentrifuge tubes. The culture medium was discarded and 200 µl Blue Buffer (10 mm Tris-HCl buffer ph 8.0, containing 2% Triton X-100, 1% SDS, 100 mm NaCl, 1mM EDTA), 200 µl phenol/chloroform/isoamyl alcohol solution (125:24:1,v/v, Fisher), and 0.3 g glass beads ( µm, Sigma) were added to the cell pellet. The mixture was vortexed for 1 min to break the Pichia cells and then centrifuged at 10,000 x g for 2 min. gdna was recovered from the supernatant by ethanol precipitation Expression construct cloning of GALT1, GALT3, GALT4, GALT5 and GALT6 for expression in tobacco BY2 suspension-cultured cells The CACC Kozak sequence followed by a 6xHis tag was incorporated into the N- termini of the putative GalT genes by PCR. The PCR products were cloned into the pentr/d-topo Gateway vector by Topo cloning (Invitrogen). GALTs were then transferred from the pentr/d-topo vector digested with MluI to the pmdc30 or pmdc32 vectors by an LR Clonase (Invitrogen) reaction. The pmdc30 differs from the pmdc32 vector in that the former carries a heat shock promoter and the later carries a 2x35S promoter to drive the gene expression in plant cells. E.coli strains hosting the

106 106 pentr/d-galts, pmdc30-galts and pmdc32-galts were selected by kanamycin resistance. The pentr/d-galts, pmdc30-galts and pmdc32-galts and the ppicza/b-galts constructs were first examined by diagnostic digestion and PCR and then sequenced to make sure that correct in-fram sequences were obtained for the N- terminal 6xHis-tag and the GALT coding regions. The primer sequences for GalT subcloning and sequencing are listed in Appendix A Table 1 and Fig. 1. Sequencing results for the final expression constructs were assembled into contigs and shown in Appendix B Part II and III Agrobacterium transformation The constructs of the pmdc30-galts and the pmdc32-galts were transformed into the Agrobacterium strain EHA105 by electroporation using the Gene Pulser II system (Bio-Rad). The settings for electroporation with a 2 mm cuvette were: capacitance: 25 µf; voltage: 2.4 kv; resistance: 200 Ohm; pulse length: 5 msec. The transformed Agrobacterium strains were selected using LB medium containing 50 mg/l kanamycin and cultured at 28 C. The transformed colonies were confirmed by direct PCR of the Agrobacterium liquid cultures using GalT gene specific primers and the untransformed Agrobacterium strain as the control Transformation of tobacco BY2 suspension-cultured cells Tobacco (Nicotiana tabacum, BY2) suspension-cultured cells were maintained under the same conditions as described in Chapter

107 107 Overnight-grown Agrobacterium cultures were centrifuged at 3,000 xg for 5 min to remove the LB medium and washed twice with the NT-1 medium. The Agrobacterium pellet was then resuspended in 200 µl NT-1 medium and co-cultivated with 4-days-old tobacco BY2 cells in a petri plate covered with aluminum foil at 25 C with slow agitation at 40 rpm. After 3 days, the BY2 cells were transferred to a 50 ml conical tube and the co-cultivation medium was removed with a pipette. The BY2 cells were washed by 3 repeating cycles of adding 50 ml NT-1 medium to the tube, inverting the tube to resuspend the cells, letting the cells settle and removing the medium with a pipette. The cells were washed for a fourth time with NT-1 medium containing 400 mg/l timentin before being plated onto a NT-1 plate containing 400 mg/l timentin and 20 mg/l hygromycin B to form a thin layer of cells. Calli grown after three weeks were subcultured onto new plates containing 400 mg/l timentin and 20 mg/l hygromycin B. After three rounds of selection on the petri plates, the transgenic BY2 callus were transferred and maintained in liquid NT-1 medium containing 50 mg/l hygromicin B Isolation of microsomal membranes from Pichia Pichia strains transformed with the P AOX :6xHis-GalT genes or the empty ppiczb vector were induced for protein expression following the instructions in the EasySelect Pichia Expression Kit (Invitrogen). The Pichia culturing temperature was 28 C, and 0.5% methanol was applied to the cell culture media for the induction of protein expression. Pichia cells were harvested on the fifth day after induction by centrifugation at 3,000 xg for 10 min and preserved at -80C until use. Microsomal membranes were isolated from

108 108 Pichia strains with a method described previously (Tan et al., 2006) with some modifications. Briefly, 2 ml Pichia cells were mixed with 2 ml glass beads ( µm, Sigma) and 4 ml extraction buffer (0.1 M HEPES-KOH, ph 7, 0.4 M Suc, 0.1% BSA, 1 mm dithiothreitol, 5 mm MgCl 2, 5 mm MnCl 2, 1 mm phenylmethylsulfonylfluoride, 2.4 mg Roche complete protease inhibitor and 66 µl Protease Inhibitor Cocktail IV from the Research Product International Corp). The mixture was repeatedly vortexed for 30 s for 7 times with 30 s intervals on ice and then centrifuged at 3,000 x g for 15 min at 4 C. The supernatant was recovered and subjected to ultracentrifugation at 100,000 x g for 90 min. The resultig pellet was resuspended in 300 µl extraction buffer and preserved at -80C until use Preparation of microsomal membranes from tobacco BY2 cell lines The preparation of microsomal membranes from the transgenic tobacco BY2 cell lines followed the same protocol for the preparation of microsomal membranes from the wild type BY2 cell lines (Chapter 2.2.2) except for the following two modifications. First, the preparation was scaled down to allow for the processing of multiple tobacco cell lines. Five to 12 g of tobacco cells were used in each preparation with the volume of extraction buffer reduced accordingly. Second, the microsomal membranes enriched at the 0.25/1.1 M sucrose interface and at the 1.1/1.8 M sucrose interface were both collected and subjected to the final ultracentrifugation step. The pellets obtained were resuspended separately and named as the upper membrane layer and the lower membrane layer as discussed in Chapter The P hsp :6xHis-GalT transgenic BY2 lines were subjected to a

109 109 heat shock treatment before the isolation of microsomal membranes. For the heat shock treatment, 75 ml culture media of the transgenic BY2 line or control wild type line were transferred to a 1 L flask to form a thin layer of cells. The 1 L flask was incubated in a water bath at 40 C with agitation at 125 rpm for 2 h. These conditions were chosen based on conditions used in a previous study on heat shock induced proteins in the tobacco Wisconsin 38 cell line (Barnett et al., 1979) and a study which utilized the same heat shock promoter for the induction of protein expression in tobacco leaves (Schoffl et al., 1989) Western blotting analysis Microsomal membrane samples were quantified by the Bradford method using a Bio-Rad Protein Assay system before being subjected to western blotting analysis. Pichia microsomal membranes containing 30 µg protein and BY2 microsomal membranes containing either 5 µg or 2 µg protein were treated at 100 C for 10 min in the Laemmli Sample Buffer (Bio-Rad) supplemented with 5% β-mercaptoethanol and run on a 10% SDS-PAGE gel before being electro-blotted to a PVDF membrane. Ten microliters of the ten times diluted Precision Plus Protein Kaleidoscope standard (Bio-Rad) was used. Electrophoresis and transfer of proteins were carried out using the Mini-PROTEAN 3 Electrophoresis Cell and Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad). For immunoblotting, the membrane was treated with the blocking buffer (5% non-fat dry milk in PBS) overnight at 4 C and then incubated with the 6xHis Monoclonal Antibody (Clontech) for 1 h at room temperature. Membranes were then washed in the washing

110 110 buffer (5% non-fat dry milk in PBS containing 0.1% Tween 20) for 45 min at room temperature before incubation with Goat anti Mouse IgG (H&L) horseradish peroxidaseconjugated secondary antibodies (Genscript Corp) for 1 h at room temperature. The primary and secondary antibodies were diluted 10,000 and 20,000 times respectively in the washing buffer AGP GalT activity test The standard assay for GalT activity (Chapter 2.2.3) was used to detect AGP GalT activities in the transgenic Pichia or tobacco cell lines with the following modifications. Microsomal membranes containing 250 µg proteins from transgenic Pichia or tobacco cell lines were permeabilized and used as the enzyme source. [AO] 7 alone, d[ao] 51 alone, or [AO] 7 and d[ao] 51 together was used as the substrate acceptor in the GalT assay. Based on the experimental result that [AO] 7 :GalT activity approached saturation when 2 µg of [AO] 7 was included in the reaction mix (Chapter 2.3.4), 2 µg instead of 50 µg of each substrate acceptor was used in the AGP GalT assay Transient expression of GALT3-YELLOW FLUORESCENT PROTEIN (YFP) in tobacco leaves To construct the pvkh18en6-galt3 vector, a single nucleotide mutation from T to C at nucleotide position 1446 was introduced to the GALT3 coding region to eliminate an XbaI recognition site without affecting the amino acid codons. The mutagenesis was performed using the QuikChange II-E Site-Directed Mutagenesis Kit (Agilent Technology). The introduction of an XbaI restriction site to the 5 end, a SalI restriction

111 111 site to the 3 end of GALT3 coding region, and the removal of the stop codon of GALT3 were achieved through one PCR. The resulting PCR product was gel purified and then cloned into the pvkh18en6 vector by a cut and paste method using the XbaI and the SalI restriction sites. The construct of pvkh18en6-galt3 was verified by diagnostic digestion and PCR analysis and sequenced to insure an accurate sequence for the GALT3 coding region and that the YELLOW FLUORESCENT PROTEIN (YFP) gene at the C terminus of the GALT3 gene was in frame. Sequencing results for the final expression construct were assembled one contig and shown in Appendix B Part IV. The primer sequences for GALT subcloning and sequencing are listed in Appendix A Table 1 and Fig. 1. The pvkh18en6-galt3 construct was then transformed into Agrobacterium strain GV3101 as described in Transformed Agrobacterium was selected in LB medium containing 50 mg/l kanamycin, 50 mg/l rifampicin and 25 mg/l gentamicin and verified by PCR analysis using the GALT3 gene specific primers. The Agrobacterium strain with the expression construct for the fusion protein of α-2,6-sialyltranferase-green FLUORESCENT PROTEIN (ST-GFP) was provided by Dr. Michael Held (Department of Chemistry and Biochemistry, Ohio University). The Agrobacterium strains hosting the GALT3-YFP and ST-GFP constructs were co-infiltrated into tobacco leaves as described previously (Sparkes et al., 2006). After two days of infiltration, the epidermal cell layers were observed for fluorescence using a Zeiss LSM 510 laser-scanning confocal microscope. For co-localization of GFP and YFP in epidermal cells, a 488- and 514-nm argon ion laser was used. Fluorescence signals were separated using a dichromic beam splitter NFT; the emission signal was 458 nm for GFP and 514 nm for YFP. ZEN 2007

112 112 software was used to operate the system. Images of tobacco cells expressing GALT3-YFP or ST-GFP alone were taken to serve as a control to indicate that no cross-talk occurred between the two fluorochromes before image acquisition Results Comparison of the GALT sequences of the corresponding cdna clones and the GALT sequences from the Arabidopsis Information Resource (TAIR) database cdna sequences of the GALT1, GALT3, GALT4 and GALT5 genes were retrieved from the website of the RIKEN Bioresource Center ( which hosts the cdna library, and compared to the cdna sequences of GALT genes predicted from the Arabidopsis genome sequence retrieved from the TAIR database. The sequences for GALT3 and GALT5 in the cdna clones are identical to the corresponding cdna sequences from TAIR. For GALT1, a single nucleotide variation at position 1206 (relative to the first nucleotide of the start codon designated as position 1) is found between the sequences from the cdna library database and from the TAIR database without affecting the amino acid sequences encoded. A single nucleotide variation at nucleotide position 1379 for the GALT4 gene in the cdna database compared to the sequence from TAIR results in a change in the amino acid sequence at position 460 (relative to the first amino acid designated as position 1) from Val to Ala. Amino acid position 460 is located in the region between the Galectin domain and the GalT domain of the GalT4 protein (Table 3.1). The currently available overexpression sequence tags (ESTs) are not available to

113 113 cover nucleotide position 1379 in GALT4, making it difficult to determine the authentic gene sequence at this specific position. The GALT6 cdna clone was obtained from the Arabidopsis thaliana EST libraries ( however, the nucleotide sequence retrieved from this website with the accession number of BX is apparently wrong as it contains multiple stop codons in the translated sequence. Based on the above sequence analysis, cdna sequences from RIKEN Bioresource Center were utilized as the reference sequences for the GALT1, GALT3, GALT4 and GALT5 genes, while the predicted cdna sequence from the TAIR website was used as the reference sequence for the GALT6 gene for sequence analysis during expression construct cloning Expression construct cloning and yeast/tobacco BY2 cell transformation All of the five putative GALT genes, GALT1, GALT3, GALT4, GALT5 and GALT6, with a 5 sequence encoding an N terminal 6xHis tag, were subcloned into the ppicza/b vectors for their expression in Pichia under the control of the P AOX promoter. Also, all of the five GALT genes were subcloned into the pmdc30 and pmdc32 vectors for their expression in tobacco BY2 cells under the control of the P hsp promoter and the P 2x35S promote, respectively (Table 3.2).

114 114 Table 3.2 Expression constructs and transgenic Pichia or tobacco BY2 cell lines obtained in this research. Purpose of Expression Expression System Promoter Constructs Completed Number of Independent Transformation Events GalT activity test ppicza/b vector Alcohol P AOX :6xHis-GALT1 Eight events for each for Pichia oxidase P AOX :6xHis-GALT3 gene construct transformation and promoter P AOX :6xHis-GALT4 gene expression P AOX :6xHis-GALT5 P AOX :6xHis-GALT6 GalT activity test pmdc30 vector for Heat shock P hsp :6xHis-GALT1 One event for each of transformation and protein P hsp :6xHis-GALT3 the GALT1, GALT3 gene expression in promoter P hsp :6xHis-GALT4 and GALT6 gene tobacco BY2 P hsp :6xHis-GALT5 constructs suspension-cultured P hsp :6xHis-GALT6 cells GalT activity test pmdc32 vector for 2 x 35S P 2x35S :6xHis-GALT1 Five events for the transformation and P 2x35S :6xHis-GALT3 GALT5gene construct gene expression in P 2x35S :6xHis-GALT4 and two events for the tobacco BY2 P 2x35S :6xHis-GALT5 GALT6 gene construct suspension-cultured P 2x35S :6xHis-GALT6 cells Subcellular localization of GalT proteins pvkh18en6 vector for transformation and gene expression in tobacco leaf epidermis 35S P 35S :GALT3-YFP a N.A. a YFP, yellow fluorescent protein. N.A., not applicable.

115 115 The expression constructs of ppicza-6xhis-galt1/galt3/galt4 and ppiczb- 6xHis-GALT5/GALT6 were sequenced for full length coding regions of the 6xHis-GALTs and partial sequences of the promoter and terminator regions (Appendix B Part I) before transformation into Pichia competent cells. After transformation, the P AOX :6xHis-GALT genes integrate into the Pichia genome through homologous recombination at the locus close to the ALCOHOL OXIDASE 1 (AOX1) gene (Tschopp et al., 1987) (Fig. 3.2). Eight Pichia colonies representing eight independent transformation events for each GALT gene construct were selected by genotyping of the transgenic Pichia lines by PCR. The PCR analysis of one transformation event for each P AOX :6xHis-GALT transformation is shown in Fig. 3.2.

116 116 Figure 3.2 PCR analysis of Pichia transformed with ppicza-6xhis-galt1, ppicza-6xhis-galt3, ppicza-6xhis-galt4, ppiczb-6xhis-galt5, ppiczb- 6xHis-GALT6. A. Schemetic diagram of the genome sequence of the control Pichia strain tranformed with the empty ppicza/b vector. The Zeosin r gene is integrated into the control Pichia genome. B. Schemetic diagram of the genome sequence of Pichia strains transformed with the ppicza/b-6xhis-galt or ppicza-xt1 (XT1 stands for xyloglucan XylT1) constructs. The Zeosin r as well as the P AOX :6xHis-GALT or the P AOX :6xHis-XT1 genes are integrated into the Pichia genome for protein expression. C. Genotyping of the Pichia strains transformed with the P AOX :6xHis-GALT genes, the P AOX :6xHis-XT1 gene or the empty ppiczb vector by PCR using the AOX primer pair, the positions of which are shown in A with arrows. In addition to a common band for the AOX1 gene of approximately 2200 bp, each lane shows a gene specific band with a size of the coding region of each gene plus 325 bp from the ppicza vector or 323 bp from the ppiczb vector, i.e. GalT1: 2257 bp; GalT4: 2347 bp; GalT5: 2342 bp; GalT6: 2369 bp; XT: 1706bp; ppiczb, no additional band amplified. The sizes of the GalT3 specific band (2185 bp) and the AOX1 gene band (2200 bp) are too close to be discriminated. M, DNA size maker. The expression constructs of the pmdc30-6xhis-galts were verified by diagnostic digestion analysis (Fig. 3.3). The verified constructs were sequenced for the full length coding regions of the 6xHis-GALTs and partial sequences of the promoter and terminator

117 117 regions (Appendix B Part II) before transformation into Agrobacterium. The Agrobacterium strain carrying each of the expression constructs was selected by PCR (Fig. 3.4) and used for the transformation of tobacco BY2 cells. One transformation event in BY2 cells was obtained for the P hsp :6xHis-GALT1, P hsp :6xHis-GALT3 and P hs p:6xhis- GALT6 genes, respectively. Genotyping of the transgenic BY2 lines was performed by PCR with wild type BY2 as a control, the result of which is shown in Fig Figure 3.3 Verification of the constructs of pmdc30-6xhis-galt by diagnostic digestion with the AscI and SacI restriction enzymes. The estimated product sizes of each construct after AscI/SacI double digestion are as follows: GalT1: 9977 bp, 2020 bp; GalT3: 9985 bp, 1515 bp, 400 bp; GalT4: 9977 bp, 2112 bp; GalT5: 9977 bp, 1606 bp, 500 bp; GalT6: 9977 bp, 1019 bp, 802 bp, 300 bp. M, DNA size maker. Figure 3.4 PCR analysis of the Agrobacterium strains transformed with the pmdc30-6xhis-galt constructs. Gene specific primers were used to amplify the coding region of each GALT gene from the Agrobacterium transformants with the pmdc30-6xhis-galt constructs (T) or the

118 empty pmdc30 vector (NC) by PCR. The primer sequences and locations in the cdna coding region are shown in Appendix A Table 1 and Fig. 1. M, DNA size maker. 118 Figure 3.5 PCR analysis of tobacco BY2 cell lines transformed with the hsp:6xhis- GALT genes. Gene specific primers were used to amplify the coding region of each GALT gene from genomic DNA isolated from the BY2 tranformants with the P hsp :6xHis-GALT genes (T) or from a wild type BY2 cell line (NC) by PCR. The primer sequences and locations in the cdna coding region are shown in Appendix A Table 1 and Fig. 1. M, DNA size maker. The expression constructs of pmdc32-6xhis-galts were verified by diagnostic digestion and PCR analysis (Fig. 3.6). The verified constructs were sequenced for the full-length cdna coding region of the 6xHis-GALTs and partial sequences of the promoter and terminator regions (Appendix B Part III) before transformation into Agrobacterium. The Agrobacterium strain carrying each expression construct was selected by PCR (Fig. 3.7) and used for the transformation of tobacco BY2 cells. Five and two transformation events in BY2 cell lines were obtained for the P 2x35S :6xHis- GALT5 gene and the P 2x35S :6xHis-GALT6 gene, respectively. Genotyping of the transgenic BY2 cell lines was performed by PCR with wild type BY2 cells as a control, the result of which is shown in Fig. 3.8.

119 Figure 3.6 Verification of the constructs of pmdc32-6xhis-galts by diagnostic digestion and PCR. A. Diagnostic digestion of the pmdc32-galt constructs and the empty pmdc32 vector with the AscI and SacI restriction enzymes. The estimated product sizes of each construct after AscI/SacI double digestion are as follows: GalT1: 9985 bp, 2020 bp, 78 bp; GalT3: 9985 bp, 1507 bp, 441 bp, 78 bp; GalT4: 9977 bp, 2112 bp, 86 bp; GalT5: 9985 bp, 1598 bp, 509 bp, 78 bp; GalT6: 9985 bp, 1019 bp, 810 bp, 305 bp, 78 bp; pmdc32: 9985bp, 1767 bp. B. Gene specific primers were used to amplify the coding region of each GALT gene from the corresponding pmdc32-6xhis-galt constructs (T) and from the empty pmdc32 vector (NC) by PCR. The primer sequences and locations in the cdna coding region are shown in Appendix A Table 1 and Fig. 1. M, DNA size maker. 119

120 120 Figure 3.7 PCR analysis of the Agrobacterium strains transformed with the pmdc32-6xhis-galt constructs. Gene specific primers were used to amplify the coding region of each GALT gene from the Agrobacterium strains transformed with the corresponding pmdc32-6xhis-galt constructs (T) or with the empty pmdc32 vector (NC) by PCR. The primer sequences and locations in the cdna coding region are shown in Appendix A Table 1 and Fig. 1. M, DNA size maker. Figure 3.8 PCR analysis of the tobacco BY2 cell lines transformed with the P 2x35S :6xHis-GALT genes. Gene specific primers were used to amplify the coding region of GALT5 or GALT6 from the P 2x35S :6xHis-GALT5 transformants number 1 to number 5 (5-1 to 5-5), the P 2x35S :6xHis-GALT6 transformants number 1 and number 2 (6-1 and 6-2) and the wild type BY2 cell line by PCR. The primer sequences and locations in the cdna coding region are shown in Appendix A Table 1 and Fig. 1. M, DNA size maker Protein expression analyses and AGP GalT activity tests of the microsomal membranes isolated from Pichia strains transformed with the P AOX :6xHis-GALT genes Microsomal membranes were isolated from the transgenic Pichia strains carrying the P AOX :6xHis-GALT genes or the control Pichia strain transformed with the empty ppiczb vector and analyzed by western blotting analysis using an anti-6xhis antibody

121 121 (Fig. 3.9; Table 3.3). Double bands at approximately 75 kd, close to the predicted sizes of 6xHis-GalT proteins were detected in the microsomal membrane proteins of all the P AOX :6xHis-GALT transgenic Pichia strains analyzed so far, including the P AOX :6xHis- GALT3 transformants number 1, number 3, and number 4, the P AOX :6xHis-GALT4 transformants number 1 to number 5, the P AOX :6xHis-GALT5 transformants number 1 and number 2 and the P AOX :6xHis-GALT6 transformants number 1 to number 5. The bands close to 75 kd in the GALT transgenic strains likely indicated the expression of the 6xHis-GalT fusion proteins because in the control Pichia strain, bands at 75 kd, if any, are significantly weaker compared to the GalT transgenic strains. Given that the protein sequences of GalT3, GalT4, GalT5 and GalT6 all contain two N-glycosylation sites (data not shown), the 6xHis-GalT fusion proteins may undergo N-glycosylation and were thus present as double bands or smears. Double bands with the size of approximately 50 kd were present in most of the GALT transgenic strains as well as the control strain with a lower signal intensity. The 50 kd bands may due to partial degradation of the 6xHis- GalT fusion proteins. Although less likely, it cannot be excluded that the 75 kd and 50 kd bands represent non-specific binding of the anti-6xhis antibody to endogenous Pichia proteins whose expression was induced in the GalT transgenic lines. Western blotting analysis with GalT specific antibodies will allow more accurate detection of the expression of the GalT proteins.

122 Figure 3.9 Western blotting analysis of microsomal proteins isolated from Pichia transformants. Pichia strains analyzed include the P AOX :6xHis-GALT3 transformants number 1, 3 and 4 (3-1, 3-3, 3-4), the P AOX :6xHis-GALT4 transformants number 1 to 5 (4-1 to 4-5 ), the P AOX :6xHis-GALT5 transformants number 1 and 2 (5-1 and 5-2), the P AOX :6xHis-GALT6 transformants number 1 to 5 (6-1 to 6-5) and the control Pichia strain transformed with the empty ppiczb vector. The primary anti-6xhis antibody and the secondary goat anti mouse antibody were used at 1:10,000 and 1:20,000 dilutions, respectively. 122

123 Table 3.3 Summary of the signal bands observed in microsomal membrane proteins from transgenic and control Pichia and tobacco BY2 cells in western blotting analysis Fusion Observed Sizes of Protein Observed Sizes of Observed Sizes of Observed Sizes of Protein Observed Sizes of Proteins Bands in Microsomal Protein Bands in Protein Bands in Bands in Upper Layer Protein Bands in (Predicted Membranes from P AOX :6xHis- Upper Layer Lower Layer Membrane Proteins from Lower Layer Sizes) GALT Transformed Pichia Membrane Proteins Membrane Proteins P 2x35S :6xHis-GALT Membrane Proteins from P hsp :6xHis- from P hsp :6xHis- Transformed BY2 cells from P 2x35S :6xHis- GALT Transformed GALT Transformed GALT Transformed BY2 cells BY2 cells BY2 cells 6xHis- n.d. 1-1 showed bands at 1-1 showed bands at n.d. n.d. GalT1 approximately 75 kd, approximately 50 kd, (72 kd) 60 kd, 50 kd and 40 kd and below 30 kd below 30 kd 6xHis- 3-1, 3-2 and 3-3 showed two 3-1 showed signals for 3-1 showed bands at n.d. n.d. GalT3 bands at 70 to 80 kd and two almost the whole approximately 50 kd (71 kd) bands at 50 to 60 kd sample lane including and below 30 kd bands at 75 kd 123

124 124 Table 3.3 (continued) 6xHis- GalT4 (78 kd) 4-1, 4-2, 4-3, 4-4 and 4-5 showed two bands at 75 to 85 kd and two bands at 45 to 55 kd. The upper bands at approximately 85 kd is more intense in 4-2, 4-3, and 4-4 lines compared to showed bands at approximately 125 kd. n.d. n.d. n.d. n.d. 6xHis- 5-1 and 5-2 showed two bands at n.d. n.d. 5-1, 5-2, 5-3, 5-4 showed 5-1, 5-2, 5-3, 5-4 GalT5 75 to 85 kd and one band at faint bands at approximately showed bands at 50 kd (78 kd) approximately 50 kd 50 kd and strong signals and below 30 kd below 30 kd. 5-1 and 5-2 showed a smear at 75 to 120 kd. 5-3 and 5-4 showed a weaker smear at 75 to 85 kd 124

125 125 Table 3.3 (continued) 6xHis- 6-1 and 6-4 showed two bands at 6-1 showed signals for 6-1 showed bands at 6-1 showed a smear of 6-1, 6-2 showed bands GalT6 70 to 80 kd, one band at almost the whole approximately 50 kd, signals from 50 kd to above at 50 kd and below 30 (79 kd) approximately 60 kd and two sample lane including 40 kd and below 30 kd 250 kd and multiple bands kd bands at approximately 45 to 55 bands at 75 kd below 50 kd. 6-2 showed a kd. 6-2, 6-3 and 6-5 showed two faint smear at 75 kd, a band bands at 75 to 85 kd and two at 50 kd and below 30 kd. bands at 45 to 55 kd Negative Pichia transformed with empty WT showed signals for WT showed bands at WT showed a smear from 70 n.d. Controls ppiczb vector showed faint almost the whole approximately 150 kd, to 110 kd and bands below band at approximately 70 kd and sample lane including 50 kd and below 30 kd 30 kd. two faint bands at 45 to 50 kd bands at 75 kd n.d. not determined. 125

126 126 Microsomal membranes from transgenic Pichia strains were analyzed for AGP GalT activities with the in vitro AGP GalT assay system developed using Arabidopsis and tobacco microsomal membranes (Chapter 2.3). Based on the structural analysis of the Arabidopsis AGP GalT products, the assay using [AO] 7 as the substrate acceptor mainly detects the GalT activity ([AO] 7 :GalT activity) that transfers the first Gal to Hyp residues in the peptide backbone; while the assay using d[ao] 51 as the substrate acceptor mainly detects the GalT activity (d[ao] 51 :GalT activity) that adds a second Gal to the first Gal already attached to the peptide backbone (Chapter 2.4). The incorporation of [ 14 C]Gal from UDP-[ 14 C]Gal into the substrate acceptors is indicated by the coelution of the [ 14 C] radioactive peak and the elution peak of the [AO] 7 or the d[ao] 51 substrate acceptor upon HPLC fractionation of the assay product. Unfortunately, none of the transgenic Pichia strains analyzed displayed [AO] 7 :GalT or d[ao] 51 :GalT activity in their microsomal membranes given that no radioactive peak coeluting with the substrate acceptors upon HPLC fractionation of the reaction products was observed. A representative HPLC elution profile of the Pichia [AO] 7 :GalT assay product is shown in Fig B. The [AO] 7 :GalT and d[ao] 51 :GalT assay results for individual transgenic Pichia strains are summarized in Table 3.4.

127 Figure 3.10 RP-HPLC fractionation of the [AO] 7 substrate acceptor and the [AO] 7 :GalT reaction products on a PRP-1 reverse-phase column. A. Elution profile of the [AO] 7 substrate acceptor. B. Representative elution profile of the [AO] 7 :GalT reaction product using microsomal membranes from the Pichia strains tranformed with the P AOX :6xHis-GALT genes. C. Representative elution profile of the [AO] 7 :GalT reaction product using microsomal membranes from the tobacco BY2 lines transformed with the P hsp : 6xHis-GALT genes. The presence or absence of the Radioactive Peak II coeluting with the [AO] 7 acceptor substrate indicates the presence or absence of the [AO] 7 :GalT activities in the microsomal membranes. The [AO] 7 :GalT activity is quantified by combining the radioactive counts present in Radioactive Peak II. 127

128 128 Table 3.4 Results of the tests for AGP GalT activities in microsomal membranes from transgenic strains using the in vitro [AO] 7 :GalT and d[ao] 51 :GalT assay systems. Pichia Strain [AO] 7 :GalT Activity d[ao] 51 :GalT Activity Non GALT transformant None b None b P AOX :6xHis-GALT1-1 n.d. n.d. P AOX :6xHis-GALT1-2 n.d. n.d. P AOX :6xHis-GALT1-3 n.d. n.d. P AOX :6xHis-GALT1-4 n.d. n.d. P AOX :6xHis-GALT1-5 n.d. n.d. P AOX :6xHis-GALT3-1 None a n.d. P AOX :6xHis-GALT3-2 n.d. n.d. P AOX :6xHis-GALT3-3 None a n.d. P AOX :6xHis-GALT3-4 n.d. n.d. P AOX :6xHis-GALT3-5 n.d. n.d. P AOX :6xHis-GALT4-1 None a None a P AOX :6xHis-GALT4-2 None a None a P AOX :6xHis-GALT4-3 None b n.d. P AOX :6xHis-GALT4-4 None b n.d. P AOX :6xHis-GALT4-5 None b n.d. P AOX :6xHis-GALT5-1 None a n.d. P AOX :6xHis-GALT5-2 n.d. n.d. P AOX :6xHis-GALT5-3 None a None a P AOX :6xHis-GALT5-4 None a None a P AOX :6xHis-GALT5-5 None a None a P AOX :6xHis-GALT6-1 n.d. n.d. P AOX :6xHis-GALT6-2 None a n.d. P AOX :6xHis-GALT6-3 None a n.d. P AOX :6xHis-GALT6-4 n.d. n.d. P AOX :6xHis-GALT6-5 None a n.d. a Tests with one biological repeat. b Tests with two biological repeats. None, No detected [AO] 7 :GalT or d[ao] 51 :GalT activities, as indicated by no incorporation of the [ 14 C] radiolabel from UDP-[ 14 C]Gal into the [AO] 7 or the d[ao] 51

129 129 substrate acceptor on the HPLC fractionation of the GalT assay product. n.d., not determined Protein expression analyses and AGP GalT activity tests of the microsomal membranes isolated from tobacco BY2 cell lines transformed with the P hsp :6xHis- GALT genes The tobacco BY2 cell lines transformed with the P hsp :6xHis-GALT genes were subjected to a heat shock treatment before being analyzed for the 6xHis-GalT expression and the AGP GalT activities. Wild type BY2 cells were treated in the same manner as the control. Total microsomal membranes were isolated from the treated tobacco BY2 cell lines and fractionated on a discontinuous sucrose density gradient as described in Chapter Microsomal membranes located at the interface of the 0.25M and the 1.1M sucrose solutions were collected and named as the upper membrane layer while microsomal membranes located at the interface of the 1.1M and the 1.8M sucrose solution were collected and named as the lower membrane layer (Fig A). Western blotting analysis of the proteins in the upper membrane layer and the lower membrane layer using an anti 6xHis-tag antibody showed similar signal patterns for wild type BY2 cells and the P hsp :6xHis-GALT transformants (Fig B; Table 3.3). Specifically, protein bands with sizes less than 50 kd were detected in the lower membrane layer from both the wild type and the P hsp :6xHis-GALT transgenic BY2 lines. Multiple antibody reactive bands of different sizes, including 75 kd bands, were observed for the upper layer membrane proteins from both the wild type and the P hsp :6xHis-GALT transgenic BY2 lines.

130 Figure 3.11 Western blotting analysis of microsomal proteins isolated from the tobacco BY2 lines transformed with the P hsp :6xHis-GALT genes and the wild type BY2 lines. The transgenic and wild type BY2 cells were treated with a heat shock treatment at 40 C for 2 h before being used for microsome preparation. A. An image showing the separation of the upper membrane layer and the lower membrane layer of microsomal membranes by a discontinuous sucrose gradient. B. Western blotting analysis of the proteins in the two membrane layers as defined in A. U, upper membrane layer. L, lower membrane layer. The primary anti-6xhis antibody and the secondary goat anti mouse antibody were used at 1:10,000 and 1:20,000 dilutions, respectively. 130

131 131 The upper membrane layer from the P hsp :6xHis-GALT transgenic BY2 lines, were analyzed for AGP GalT activities using [AO] 7 as the substrate acceptor. The [AO] 7 :GalT assay products were fractionated by HPLC. A representative HPLC elution profile of the [AO] 7 :GalT assay product with upper layer membranes from transgenic BY2 cell lines is shown in Fig C. The total amount of radioactivity in the radioactive peak II coeluting with the substrate acceptor on HPLC was used as the measure for [AO] 7 :GalT activity. The BY2 cell lines transformed with the P hsp :6xHis-GALT3 and P hsp :6xHis- GALT6 genes showed higher [AO] 7 :GalT activities compared to the wild type BY2 lines after heat shock treatment (Table 3.5). In addition, higher [AO] 7 :GalT activities in the upper membrane layer was detected when the P hsp :6xHis-GALT3 transgenic cell line was subjected to heat shock treatment when compared to the same cell line cultured in normal conditions.

132 Table 3.5 AGP GalT activities of the upper membrane layer from wild type BY2 cells and BY2 lines transformed with the P hsp :6xHis-GALT or the P 2x35S :6xHis-GALT genes. Incorporation of the [ BY2 lines Heat Substrate shock treatment acceptors a radiolabel into substrate acceptors b (cpm) P hsp :6xHis-GALT1-1 Yes [AO] P hsp :6xHis-GALT3-1 Yes [AO] P hsp :6xHis-GALT3-1 No [AO] P hsp :6xHis-GALT6-1 Yes [AO] WT BY2 Yes [AO] P 2x35S :6xHis-GALT5-1 No [AO] 7 +d[ao] P 2x35S :6xHis-GALT5-2 No [AO] 7 +d[ao] P 2x35S :6xHis-GALT5-3 No [AO] 7 +d[ao] 51 n.d. P 2x35S :6xHis-GALT5-4 No [AO] 7 +d[ao] P 2x35S :6xHis-GALT6-1 No [AO] 7 +d[ao] P 2x35S :6xHis-GALT6-2 No [AO] 7 +d[ao] WT BY2 No [AO] 7 +d[ao] C] 132 a [AO] 7 alone or [AO] 7 together with d[ao] 51 was used as substrate acceptors in the in vitro GalT assays. Incorporation of the [ 14 C] radiolabel into substrate acceptors was measured by the amount of total radioactivity coeluting with the substrate acceptors on HPLC fractionation of the assay products. b Most of the test results have one biological repeat. The test results with two biological repeats were calculated for standard deviations shown as the number after the + symbol. n.d., not determined Protein expression analyses and AGP GalT activity tests of the microsomal membranes isolated from tobacco BY2 cell lines transformed with the P 2x35S :6xHis- GALTs genes In contrast to the P hsp :6xHis-GALTs genes whose expression requires the induction by a heat shock treatment, the expression of the P 2x35S :6xHis-GALTs genes is constitutive

133 133 in theory. Microsomal membranes were isolated from the BY2 cell lines transformed with the P 2x35S :6xHis-GALTs genes and fractionated into the upper and lower membrane layers as described in Chapter Western blotting analysis of the isolated proteins with an anti-6xhis antibody was performed. Microsomal membranes containing 2 µg proteins from the upper or lower membrane layer from the P 2x35S :6xHis-GALTs transgenic BY2 cells were loaded for each sample instead of 5 µg proteins as loaded for the analysis for the P hsp :6xHis-GALTs transgenic BY2 cells. Because of the lower sample loading, signals for non-specific antibody bindings were reduced in the western blotting analysis for the P 2x35S :6xHis-GALTs transformants (Fig. 3.12; Table 3.3) compared to previous analysis for the P hsp :6xHis-GALTs transformants (Fig. 3.11). For the lower layer membrane protein samples, only one band at around 50 kd was observed for all the samples. For the upper layer membrane protein samples, protein bands at the size of 75 kd or above 75 kd were present in both P 2x35S :6xHis-GALTs transformants and wild type samples. Proteins signals at around 75 kd or above 75 kd in the upper layer were stronger in the P 2x35S :6xHis-GALT5-1, GALT5-2, GALT6-1 lines, but weaker in the P 2x35S :6xHis-GALT5-3, GALT5-4, GALT6-2 lines compared to wild type.

134 134 Figure 3.12 Western blotting analysis of proteins from microsomal membranes isolated from tobacco BY2 lines transformed with the P 2x35S :6xHis-GALT genes and wild type BY2 lines. The upper layer membrane proteins (U) and the lower layer membrane proteins (L) are defined as in Fig A. The primary anti-6xhis antibody and the secondary goat antimouse antibody were used at 1:10,000 and 1:20,000 dilutions, respectively. AGP GalT activities were measured in the upper layer membranes from the P 2x35S :6xHis-GALT5 and the P 2x35S :6xHis-GALT6 transformants and the wild type BY2 cells (Table 3.5). Both the [AO] 7 and the d[ao] 51 substrate acceptors were included in the

135 135 same GalT activity assay. The assay product was fractionated by HPLC and the radioactive peak coeluting with the substrate acceptors was combined and tested for radioactivity. In the assays completed, P 2x35S :6xHis-GALT5-2, GALT6-1 and GALT6-2 showed slightly higher [ 14 C] radiolabel incorporation into the peptide substrate acceptors compared to wild type, while P 2x35S :6xHis-GALT5-1, GALT5-4 had lower [AO] 7 /d[ao] 51 :GalT activity compared to wild type (Fig C). Of note, the higher or lower [AO] 7 /d[ao] 51 :GalT activities do not correlate to the higher or lower signal intensities at around 75 kd in the western blotting analysis for upper membrane layer from the P 2x35S :6xHis-GALT transformants and the wild type line Subcellular localization of GalT3 in tobacco leaf epidermal cells To study the subcellular localization of GalT3, GalT3-yellow fluorescent protein (GalT3-YFP) fusion protein was constructed and expressed using a transient expression system in tobacco leaf epidermal cells (Sparkes et al., 2006). The expression of GalT3- YFP alone showed punctate structures typical of a Golgi-localized staining pattern (Fig. 3.13). In addition, the staining pattern of GalT3-YFP was distinct from the expression of an ER marker HDEL sequence fused with green fluorescent protein (HDEL-GFP) but overlapped with the expression pattern of a Golgi marker protein, α-2,6-sialyltransferase (Saint-Jore et al., 2002), fused with GFP (ST-GFP). Thus, the subcellular localization experiments indicate that GalT3 is Golgi-localized.

136 Figure 3.13 Subcellular localization of GalT3 in tobacco leaf epidermal cells. The expression of GalT3-YFP is shown as red dots and the expression of sialic acid transferase (ST, a Golgi marker)-gfp and HDEL (a ER Marker)-GFP fusion proteins is shown as green dots. The arrows indicate artificial signals. 136

137 Discussion For the purpose of protein expression for functional characterization, many heterologous expression systems or cell free expression systems are currently available. However, these expression systems all have advantages and disadvantages. There is no guarantee which system will be more preferable than another for the expression of a specific protein in its active form without experimental trials (Farrokhi et al., 2009). For this reason, two different heterologous expression systems were tested for expression of the five putative Arabidopsis GALTs, GALT1, GALT3, GALT4, GALT5 and GALT6. Foreign protein expression in a methanotrophic Pichia strain is driven by an alcohol oxidase promoter, P AOX. The commercially available Pichia strains have the advantages of high transformation efficiency and a short turnaround time for protein expression. Notably, Pichia has the basic cellular machinery for posttranslational modifications that are usually required for activity of an expressed protein of eukaryotic origin. Furthermore, Pichia strains are likely to have low background activity for expression of plant specific enzymes. Nonetheless, Pichia strains may lack some of the protein modification machineries or cofactors that are required for maintaining the activity of some plant proteins. The plant expression systems of tobacco BY2 suspension-cultured cells were utilized here to compensate for the deficiencies of the Pichia expression system. A major concern for the expression and activity test of the Arabidopsis GALT genes in tobacco cell cultures is the interference from endogenous GalT activities in tobacco, which were detected as discussed in Chapter 2. Thus, two types of promoters were used for the expression of the Arabidopsis GALT genes in tobacco. While the tandem 35S promoter

138 138 (P 2x35S ) is supposed to drive strong, constitutive expression of the gene downstream; the heat shock promoter (P hsp ) promotes gene expression specifically under heat shock conditions. Constructs for the expression of the five GALTs in both expression systems were successfully accomplished as shown by construct verification with PCR or diagnostic digestion analysis and sequence analysis. A sequence encoding a 6xHis tag was incorporated to the 5 end of the open reading frame of the GALT genes to facilitate downstream protein detection and purification procedures. Pichia transformants with each of the five 6xHis-GALTs were obtained successfully. Due to time limitations and technical difficulties, transformants of only some of the P hsp :6xHis-GALTs and P 2x35S :6xHis-GALTs in tobacco BY2 cells were obtained. A first screening for Pichia and BY2 transformants expressing active 6xHis-GalTs was performed by western blotting analysis and in vitro GalT assays using [AO] 7 and d[ao] 51 as substrate acceptors. It should be noted that most of the western blotting analysis and the GalT activity tests were conducted once or in duplicate, thus the data presented should be considered as preliminary and used as a guide for future investigations. Although western blotting showed that 6xHis-GalT3, 6xHis-GalT4, 6xHis-GalT5 and 6xHis-GalT6 are being expressed in Pichia cells, none of the Pichia transformants tested so far demonstrated the [AO] 7 :GalT activity or the d[ao] 51 :GalT activity. The protein expression analysis of the tobacco BY2 cells transformed with the P hsp :6xHis- GALT genes and the P 2x35S :6xHis-GALT genes is obscured by non-specific protein signals present in the wild type BY2 cells upon western blotting. However, in [AO] 7 :GalT

139 139 activity assays, the upper layer membranes from the P hsp :6xHis-GALT3 and the P hsp :6xHis-GALT6 transgenic BY2 lines showed higher GalT activity compared to the wild type BY2 line. Intriguingly, the upper membrane layer from the same P hsp :6xHis- GALT3 transgenic cell line showed higher [AO] 7 :GalT activity when the cells were treated with the heat shock treatment compared to the untreated cells, which suggests that the increase in the [AO] 7 :GalT activity is due to the expression of the 6xHis-GALT3 driven by the heat shock promoter. For the P 2x35S :6xHis-GALT transgenic cell lines, slightly higher GalT activity in assays using both [AO] 7 and d[ao] 51 as the substrate acceptors was detected in P 2x35S :6xHis-GALT5 transgenic line number 2, and the two P 2x35S :6xHis-GALT6 transgenic lines compared to wild type. The discrepancy between the yeast expression result and the BY2 expression result of 6xHis-GalTs may be attributable to many reasons. Firstly, expression analyses and GalT assays need to be repeated to confirm that higher GalT activity is present in the transgenic BY2 lines compared to wild type lines. In these tests, the [AO] 7 :GalT assay and the d[ao] 51 :GalT assay should be performed separately to discriminate the two GalT activities in the transgenic BY2 lines. Additionally, the d[ao] 51 :GalT assays for microsomal membranes from most of the transgenic Pichia strains have not been performed and need to be completed. Secondly, given that the differences in GalT activities between the transgenic BY2 cells and the wild type BY2 cells are subtle, the 6xHis-GalTs expressed in the P 2x35S :6xHis-GALT transgenic lines may need to be purified to show that the GalT activity is due to the expressed 6xHis-GalTs rather than endogenous GalTs in BY2 cells. The P hsp :6xHis-GALT transgenic lines have the

140 140 advantage that genetic variation is eliminated when GalT activity is tested for the same cell line under heat shock treatment versus no heat shock treatment. Nevertheless, the conditions for the heat shock treatment may be optimized for a more definitive result. Thirdly, parameters for Pichia protein expression may be optimized with respect to the temperature for Pichia culturing, the methanol concentration for the induction of protein expression, and the length of time for the induction of protein expression. The same methodologies used in this dissertation have been applied to express the 6xHis-GALT2 gene in Pichia, which showed detectable [AO] 7 :GalT activity in the microsomal extracts (unpublished data from Ms. Debarati Basu in the Showalter lab at Ohio University). Although the methodologies are proven to be valid for GALT2 expression, different genes may vary in their requirements for expression conditions. Fourthly, the western blotting analysis in this study utilized an anti 6xHis-tag antibody which detects non-specific signals with similar sizes to the estimated sizes for the 6xHis-GalT fusion proteins at a low level in the control Pichia lines and at a high level in the control BY2 lines. The generation and use of GalT specific antibodies may be more efficient for the identification of Pichia strains and BY2 lines with high expression levels of the GALT genes for the activity test. Fifthly, the GalT proteins may be inactive in Pichia for various reasons, such as low protein expression, incorrect folding or improper secondary modifications (Petersen et al., 2009). An example is the Arabidopsis xyloglucan XylT 2 (AtXT2), which showed xyloglucan XylT activity when expressed in Drosophila S2 (Schneider 2) cells but not in Pichia cells (Faik et al., 2002; Cavalier and Keegstra, 2006). Lastly, if a GT functions in an enzyme complex, it may not show activity when expressed

141 141 individually in heterologous systems. The last situation is exemplified by the recent discovery of the biological function of the Arabidopsis GALACTURONOSYLTRANSFERASE 7 (GAUT7), which did not show galacturonosyltransferase activity when expressed alone however is an essential component of the biosynthetic complex of HG (Atmodjo et al., 2011). Because microsomal proteins from the P hsp :6xHis-GALT3 transgenic line showed promising [AO] 7 :GalT activities, the subcellular localization of GalT3 in fusion with YFP was studied using a transient expression system in tobacco leaves. The subcellular localization result suggests that GalT3 is Golgi-localized, which is consistent with the expected localization of an AGP GalT.

142 142 CHAPTER 4: BIOCHEMICAL AND PHYSIOLOGICAL CHARACTERIZATION OF FUT4 AND FUT6 MUTANTS DEFECTIVE IN ARABINOGALACTAN- PROTEIN FUCOSYLATION IN ARABIDOPSIS This work has been submitted to the journal of Plant Physiology for review. Liang Y, Pattathil S, Xu W-L, Basu D, Venetos A, Faik A, Hahn MG, Showalter AM ( submitted) Biochemical and physiological characterization of fut4 and fut6 mutants defective in arabinogalactan-protein fucosylation in Arabidopsis Introduction Primary plant cell walls are a composite of complex carbohydrates and protein components. In dicots, cellulose microfibers cross-linked by xyloglucan hemicellulose constitute the load-bearing framework, which is embedded in a matrix of pectic polymers made of homogalacturonan (HG), rhamnogalacturonan I (RG-I), rhamnogalacturonan II (RG-II), and protein components (Carpita and Gibeaut, 1993). The hydroxyproline-rich glycoprotein (HRGP) family represents a major group of plant cell wall proteins. The three subfamilies of HRGPs, proline-rich proteins (PRPs), extensins (EXTs) and arabinogalactan-proteins (AGPs), share the common feature of having hydroxyproline (Hyp) residues in their protein backbones and undergo glycosylation to various extents

143 143 (Showalter, 1993; Shpak et al., 2001). PRPs are the least glycosylated and suggested to insert into and stabilize mature wall structure (Carpita and McCann, 2000). EXTs are moderately glycosylated, characterized by motifs for intra- and inter- molecular crosslinking, and are active players for wall self-assembly and plant defense (Memelink et al., 1993; Wei and Shirsat, 2006; Cannon et al., 2008; Lamport et al., 2011). AGPs, having the greatest number of family members and the highest level of glycosylation of all the HRGPs, are implicated in various aspects of plant growth and development (Nothnagel, 1997; Showalter, 2001; Seifert and Roberts, 2007; Ellis et al., 2010). As their name implies, AGPs are extensively glycosylated with type II arabinogalactan (AG) polysaccharides, which are mainly composed of galactose (Gal) and arabinose (Ara) residues, but may also contain other sugars, including rhamnose (Rha), glucuronic acid (GlcA), galacturonic acid (GalA), and fucose (Fuc) (Nothnagel, 1997; Tan et al., 2004; Ellis et al., 2010). Given that the sugar side chains typically account for > 90% of the molecular mass of AGPs, they are likely to define the interactive surface of the molecule and hence its function. Recently, 85 AGP genes were identified in Arabidopsis (Arabidopsis thaliana) using bioinformatics (Showalter et al., 2010). However, the precise functions and mechanisms of action of most AGPs remain elusive. To address the function and regulation of AGPs, it is necessary to understand the mechanism underlying AGP glycosylation. Although several glycosyltransferase activities were reported in in vitro assays for AGP glycosylation (Mascara and Fincher, 1982; Hayashi and Maclachlan, 1984; Schibeci et al., 1984; Misawa et al., 1996; Liang et al., 2010; Oka et al., 2010), there are to date, only

144 144 two Arabidopsis genes, FUCOSYLTRANSFERASE 4 and 6 (FUT4 and FUT6), identified to encode glycosyltransferases specific for AGPs (Wu et al., 2010). FUT4 and FUT6 belong to the Carbohydrate Active enzymes glycosyltransferase (GT) family 37 (CAZy GT37) ( the members of which are thought to encode α-1,2-futs (Sarria et al., 2001), based on the characterized function of FUT1, which specifically fucosylates xyloglucans in pea and Arabidopsis (Perrin et al., 1999; Faik et al., 2000). AGPs with terminal Fuc residues were mainly studied in cruciferous plants (Hashimoto, 2000). Monosaccharide composition analysis of AGPs from different organs of radish plants showed that AGP fucosylation was organ-specific and developmentally regulated (Tsumuraya et al., 1984; Tsumuraya et al., 1987; Tsumuraya et al., 1988). In Arabidopsis, Fuc was reported to be present in root AGPs (van Hengel and Roberts, 2002). Previously, we demonstrated that the Arabidopsis FUT4 and FUT6 proteins fucosylate AGPs in biochemical assays in vitro and in a tobacco BY-2 expression system, but no in planta data was provided (Wu et al., 2010). Although, van Hengel and Roberts (2002) have shown that reduction in Fuc in Arabidopsis roots affected their development, no direct link to the genes involved in AGPs fucosylation was demonstrated. Here we demonstrate that the FUT4 and FUT6 genes are responsible for AGP fucosylation in Arabidopsis plants. Specifically, we focus on the biochemical and physiological characterization of Arabidopsis fut4, fut6 and fut4/fut6 double mutants, to corroborate our previous findings and to obtain insight to the physiological functions of AGP fucosylation.

145 Materials and Methods Plant materials and growth conditions Arabidopsis (Arabidopsis thaliana) wild type and fut4, fut6 mutant plants were of the Columbia-0 ecotype. Seeds of T-DNA insertion lines of fut4 and fut6 were obtained from the Arabidopsis Biological Research Centre ( in Columbus, Ohio. Plants were grown in soil for mutant screening, seed harvesting and growth stage phenotypic analysis. For root harvesting, plants were grown hydroponically in water supplemented with Liquid All-Pro Plant Food (Dyna-gro). The Arabidopsis hydroponic growth system was as described previously (Gibeaut et al., 1997). For the phenotypic analysis of root growth, plants were grown on MS medium (Murashige and Skoog salts, Caisson) containing 0.5% sucrose and 1 g/l Phytogel. All plants were grown under long-day conditions (16 h of light /8 h of dark, 22 C, 55% humidity) in growth chambers or growth rooms Mutant confirmation by PCR and RT-PCR Genomic DNA isolation from fut4, fut6 and fut4/fut6 mutant leaves and subsequent PCR analysis was carried out using Extract-N-Amp Plant Kits (Sigma). The primer locations are indicated in Fig.1, and the corresponding primer sequences are listed in Appendix A Table 1. For sequencing purposes, PCR products were purified by gel extraction with QIAquick Gel Extraction Kit (Qiagen) and sequenced by the Ohio University Genomics Facility ( To analyze transcript levels of FUT4 and FUT6, total RNA was isolated from seedlings of wild type and mutant plants

146 15 DAG using the RNeasy Plant Mini Kit (Qiagen) and the RNase-Free DNase Set (Qiagen). Primer sequences for RT-PCR are listed in Appendix A Table 1 and Fig Phenotypic analysis A growth stage based phenotypic analysis method was adopted from Boyes et al. (2001). Plant growth parameters including rosette perimeters (at 29 DAG), plant height (at 43 DAG), branch numbers (at 43 DAG) and plant weight (at 49 DAG) were measured and compared among wild type, fut4, fut6 and fut4/fut6 double mutant plants (15 plants for each line). For the measurement of germination rate, mutant and wild type seeds (over 50 seeds for each line) were sown on the same MS plate. Germinated seeds were counted under the light microscope every 12 h after sowing the seeds. For root morphology analysis, mutant and wild type plants were grown on MS plates (over 24 plants for each line). The lengths of primary roots were recorded every 24 h from 3 DAG to 10 DAG, when the primary roots reach the edges of the petri plates. Statistical analysis was performed using the Two-Sample Independent t Test for continuous variables obtained from an open source software, OpenEpi ( Eel lectin staining Roots were harvested from two-week old wild type, fut4, fut6 and fut4/fut6 double mutant seedlings grown on MS plates. Freshly harvested roots were incubated in a solution of eel lectin conjugated to Texas-Red (EY Laboratories) dissolved in 20 µg ml -1 in 10 mm phosphate buffered saline (ph 7.3) for 3 h in the dark at room temperature.

147 147 After incubation, roots were rinsed with 10 mm phosphate buffered saline (ph 7.3) and observed under a Motic BA400 EPI-Fluorescence Upright Biological Microscope (Beith Dekel) using a Texas Red/Cy3.5 filter set. Images were captured by Motic Images Plus 2.0 software (Motic Instruments Inc.) Monosaccharide composition analysis by GC-MS AGPs were isolated from leaves and roots of wild type, fut4, fut6 and fut4/fut6 double mutant plants as reported previously (Schultz et al., 2000). AGP samples were quantified using a Yariv precipitation method (Xu et al., 2008) and gum arabic as standards. Samples were analyzed by both in-house GC-MS instruments at Ohio University and by analytical services at Complex Carbohydrate Research Center (CCRC) at the University of Georgia ( Briefly, 100 µg AGP samples were hydrolyzed in 2 M trifluoroacetic acid (2 h in sealed tube at 121 C), reduced with NaBD 4, and acetylated using acetic anhydride/trifluoroacetic acid. Inositol, 20 µg, was added as an internal standard to all samples. For the in-house GC-MS system, alditol acetates were analyzed on a Trace GC Ultra interfaced to a DSQII mass spectrometer (Thermo Scientific). Separation was performed on a 30 m FactorFOUR VF-23 ms capillary column (Varian). At the CCRC, alditol acetates were analyzed on a 7890A GC interfaced to a 5975C mass spectrometer (Agilent). Separation was performed on a 30 m Supelco 2330 capillary column (Sigma).

148 Monosaccharide composition analysis by HPAEC Root AGP samples (1 µg each) from wild type, fut4, fut6 and fut4/fut6 double mutant plants were hydrolyzed by total acid hydrolysis and fractionated on a CarboPac PA20 column (Dionex) as described previously (Liang et al., 2010). Fuc, Glc, Gal, Ara, and Xyl monosaccharides (Acros Organics) were used as standards and treated under the same conditions Monoclonal antibodies Monoclonal antibodies were obtained as hybridoma cell culture supernatants from laboratory stocks at the Complex Carbohydrate Research Center [available from CarboSource Services ( A detailed list of the antibodies used, grouped according to the polysaccharide primarily recognized by the antibodies (Pattathil et al. 2010), is provided in Appendix C, which also includes links to a database, WallMabDB ( containing more detailed information about each antibody Preparation of AIR (Alcohol Insoluble Residue) extracts and fractionation Plant tissues were isolated and ground to a fine powder using liquid nitrogen and a mortar and pestle. The powder was then suspended in 80% (v/v) ethanol, vortexed and centrifuged at 3000 g for 5 min. The residue was then suspended in absolute ethanol and centrifuged as above. Subsequently, the residue was suspended in chloroform:methanol (1:1 [v/v]) and stirred for one hour at room temperature. This suspension was centrifuged

149 149 again and the AIR extract was washed with acetone. All the above steps were repeated, and the final residue was air-dried at room temperature. AIR fractionation was done in order to obtain an overall picture of the glycan epitope composition of the walls and gain insight into how tightly these epitopes are bound into the wall matrix. For this purpose, AIR samples were sequentially extracted with 50 mm ammonium oxalate, ph 5.0, 50 mm sodium carbonate, ph 10 (containing 0.5% [w/v] sodium borohydride), 1 M KOH (containing 1% [w/v] sodium borohydride), and 4 M KOH (containing 1% [w/v] sodium borohydride). Each treatment was done for a period of 24 h, after which time the samples were centrifuged at 4000 g for 15 min and the supernatants decanted. The pellets were washed with water and centrifuged as before; the water wash was discarded and the pellet retained for the next extraction. Each wall extract was neutralized (if necessary), dialyzed extensively against water, and lyophilized for further analysis Total sugar estimation and ELISA Cell wall extracts were dissolved in de-ionized water (0.2 mg ml 1 ) and total sugar contents of cell wall extracts were estimated using the phenol-sulfuric acid method (DuBois et al., 1956; Masuko et al., 2005). All solubilized fractions were adjusted to an equal amount of total carbohydrate content prior ELISA assay. Cell wall extracts (60 μg sugar ml 1 ) were applied to the wells of 96-well ELISA plates (Costar 3598) at 50 μl per well and allowed to evaporate to dryness overnight at 37 C. A Biotek robotic system (Biotek) was used to perform fully automated ELISAs using a series of 152 monoclonal antibodies directed against diverse plant cell wall carbohydrate epitopes (Pattathil et al.,

150 ). ELISA data are presented as heatmaps (glycome profiles) in which the antibody order and groupings are based on a hierarchical clustering analysis that groups the antibodies based on similarities in their binding patterns to a panel of diverse plant glycans (Pattathil et al., 2010) Expression analysis of FUT4 and FUT6 genes based on microarray data from Genevestigator Expression data for the FUT4 and FUT6 genes were acquired from the Genevestigator web site ( using the perturbations condition search. Filter options were used to select for fold changes in expression levels (> 2 or < -2) and for P < Results Isolation of T-DNA insertion lines of fut4 and fut6 and generation of the fut4/fut6 double mutant Arabidopsis fut4 (SAIL_284_B05) and fut6 (SALK_099500) single mutants with T- DNA insertions in the exons of FUT4 or FUT6 genes were ordered from the Arabidopsis Biological Resource Center (ABRC, Columbus, OH) and used to obtain homozygous mutants. Genotyping of the homozygous mutant plants was performed by PCR analysis using wild type controls (Fig. 4.1). Flanking regions of left borders of the T-DNA insertions were amplified and sequenced with T-DNA left border (LB) primers and FUT4- or FUT6-specific right primers (RP). DNA sequencing showed that the insertion

151 151 sites were located at nucleotide position 1438 (relative to the first nucleotide of the start codon designated as position 1) for fut4 and nucleotide position 31 for fut6, which were consistent with the records of insertion sites of the mutant lines on the Salk Institute Genomic Analysis Laboratory web site (SIGnAL, To confirm that the flanking regions of the right borders of the T-DNA insertions were also restricted to the fut4 and fut6 genes, PCRs with T-DNA right border (RB) primers and FUT4- or FUT6-specific left primers (LP) were performed, but no amplifications were obtained. Instead, specific bands were amplified using primer pairs of LB and LP (data not shown). Sequencing results of the product amplified with LB and LP indicated that a second T-DNA insertion was located at nucleotide position 1477 in fut4, downstream and in the opposite direction of the insertion at position 1438 (Fig. 4.1). Similarly in fut6, sequencing showed that at least two T-DNA insertions, in opposite directions, interrupted FUT6 between nucleotide position -4 and position 31. The occurrence of such T-DNA insertions as tandem repeats is not infrequent (Ponce et al., 1998). Absence of the FUT4 and FUT6 transcripts in the corresponding single mutant lines was confirmed by RT-PCR (Fig. 4.2). However, it should be noted that the possibility that truncated versions of FUT4 and FUT6 transcripts are present cannot be excluded based on the locations of the primers used in these experiments (Appendix A Table 1 and Fig. 2).

152 152 Figure 4.1 Identification of T-DNA insertion lines of Arabidopsis fut4, fut6, and fut4/fut6 mutants by PCR. A, Schematic diagrams of mutant fut4 and fut6 genes with T-DNA insertions. Black box, grey box and white box represent exons, introns and untranslated regions, respectively. Both the fut4 (SAIL_284_B05) and fut6 (SALK_099500) lines were identified to contain double T-DNA insertions (black triangles), as discussed in the text. B, Genotyping of wild type (W), fut4 (4), fut6 (6) and fut4/fut6 (4/6) double mutant plants by PCR. Positions of the PCR primers are shown in A with arrows. The expected sizes amplified with primer pairs of F4LP and F4RP (1026 bp), LB3+F4RP (450 bp), F6LP and F6RP (450 bp), LBa1 and F6RP (808 bp) are indicated with arrows in B. The 2-Log DNA ladder (New England Biolabs) was used as a molecular weight marker (M).

153 153 Figure 4.2 RNA transcript levels of FUT4 and FUT6 genes in homozygous fut4, fut6, fut4/fut6 double mutants and wild type seedlings. RT-PCR of total RNAs isolated from Arabidopsis seedlings 15 days after germination (DAG) were performed using gene-specific primers (See Appendix A Table 1 and Fig. 2). ACTIN was used as a control for loading. Homozygous fut4 and fut6 single mutants were crossed, and the F1 generation was selfed before screening the F2 generation to obtain homozygous fut4/fut6 double mutants (Fig. 4.1). As expected, no FUT4 or FUT6 transcripts were observed in the double mutant line (Fig. 4.2) Phenotypic analysis of fut4, fut6 and fut4/fut6 mutants under physiological growth conditions Growth stage-based phenotypic analysis (Boyes et al., 2001) was performed to evaluate growth and reproduction of the fut4, fut6 and fut4/fut6 mutants compared to wild type Arabidopsis plants. However, no significant differences were observed among the fut4, fut6 and fut4/fut6 mutants and wild type plants in terms of rosette size, plant height, weight and stem branching numbers (Fig. 4.3). The fut4, fut6 and fut4/fut6 mutants did not show abnormalities in reproduction as indicated by similar flowering times, seed yields, and seed germination rates compared to wild type plants under physiological growth conditions (Table. 4.1; Fig. 4.4).

154 Figure 4.3 Phenotypic analysis of Arabidopsis fut4, fut6 and fut4/fut6 mutant plants compared to wild type plants under physiological growth conditions. A, Whole plant images of wild type, fut4, fut6 and fut4/fut6 mutant plants grown in soil at 32 DAG. B, The rosette perimeters (at 29 DAG), plant height (at 43 DAG), branch numbers (at 43 DAG) and plant weight (at 49 DAG) compared among wild type, fut4, fut6 and fut4/fut6 mutant plants. Data and error bars represent mean + SD (n = 15). No significant differences were identified between wild type and any of the mutant lines for the parameters measured, as indicated by P > 0.05 in student t tests for continuous variables. 154

155 Table 4.1 Flowering time and silique numbers of wild type, fut4, fut6 and fut4/fut6 mutant plants grown in soil under physiological growth conditions 155 Wild Type fut4 fut6 fut4/fut6 a Flowering Time (days + SD) b Number of Siliques (number per plant + SD) a Flowering time was recorded when the first flower buds were visible. b Number of siliques was counted at 49 DAG (n = 15). No significant differences were identified between wild type and any of the mutant lines for flowering time and number of siliques, as indicated by P > 0.05 in student t tests for continuous variables. Figure 4.4 Germination rate of Arabidopsis fut4, fut6 and fut4/fut6 double mutant and wild type plants. Plant seeds were sown on MS plates containing 0.5% sucrose. Over 50 seeds were analyzed for each line. To examine root growth of the mutant plants, fut4, fut6 and fut4/fut6 mutant and wild type plants were grown on media plates containing Murashige and Skoog (MS) salts supplemented with 0.5% sucrose. No significant differences in root morphology or root

156 156 growth rates were observed among wild type, fut4, fut6 and fut4/fut6 mutant plants (Fig. 4.5). In addition, transverse sections of roots from wild type and fut4/fut6 double mutant plants were stained with toluidine blue, observed under a light microscope, and found to be histologically identical (data not shown). Figure 4.5 Root growth of Arabidopsis wild type, fut4, fut6 and fut4/fut6 mutant plants. Plants were grown on MS plates containing 0.5% sucrose. A, Light microscope images of wild type, fut4, fut6 and fut4/fut6 roots at 15 DAG. Scale bars, 10 µm. B, Root length was measured daily as a function of time from 3-10 DAG. Values shown were averages of data from over 24 individual plants per line, with standard errors shown as error bars.

157 Eel lectin shows a different staining pattern in roots of fut6 and fut4/fut6 mutant plants compared to fut4 and wild type plants Screening for and characterization of Fuc residues on AGPs from cruciferous plants were performed using eel lectin (Hashimoto, 2000), a reagent that binds specifically to terminal α-l-fuc residues (Watkins and Morgan, 1952; Springer and Desai, 1971). van Hengel and Roberts (2002) showed that eel lectin recognizes α-l-fuc attached to α-l- Ara residues in Arabidopsis root AGPs, but not α-l-fuc-(1 2)-β-D-Gal in RG-I. The binding specificity of eel lectin is also different from the specificity of the monoclonal antibody, CCRC-M1, which recognizes the α-(1 2)-linked fucosyl epitope on xyloglucan (Puhlmann et al., 1994; van Hengel and Roberts, 2002). When whole roots of 14-day old Arabidopsis plants were subjected to staining with eel lectin conjugated to Texas-Red, the wild type and fut4 mutant plants showed a bright, patchy staining pattern on the root surface, which was absent in the fut6 and fut4/fut6 mutant roots (Fig. 4.6). No significant differences were observed for the leaves from wild type, fut4, fut6 and fut4/fut6 mutants stained with eel lectin conjugated to Texas-Red (data not shown).

158 158 Figure 4.6 Eel lectin staining of roots from WT, fut4, fut6 and fut4/fut6 mutant plants. Eel lectin conjugated to Texas-Red was used to stain the roots of Arabidopsis plants grown on MS media containing 0.5% sucrose for 14 days. The stained roots were observed under fluorescent light (FL) and white light (WL) as indicated. Scale bars, 10 µm fut4/fut6 mutants are defective in AGP fucosylation in roots and leaves Because FUT6 was mostly expressed in root while FUT4 showed the greatest expression in leaf but was also expressed in root (Sarria et al., 2001), leaf and root AGPs were purified using β-yariv reagent from wild type, fut4, fut6 and fut4/fut6 mutant plants. The purified root AGPs were acid hydrolyzed to monosaccharides and analyzed by High ph Anion Exchange Chromatography (HPAEC). As expected, HPAEC detected Ara and Gal as the two dominant peaks in all of the AGP samples. Glucose (Glc) residues were also detected in each root sample as a minor peak. Intriguingly, Fuc was detected in root AGP samples from wild type plants as well as the fut4 and fut6 plants but not in fut4/fut6 root AGPs (Fig. 4.7). To confirm this HPAEC analysis result, AGP samples from both roots and leaves were subjected to monosaccharide composition analysis using gas

159 159 chromatography-mass spectrometry (GC-MS). Consistent with the HPAEC result, the GC-MS data showed that AGPs from all samples were mainly composed of Gal and Ara residues, with a ratio of approximately 3:1 for root samples and approximately 2:1 for leaf samples in both wild type and mutant lines with subtle variations (Table 4.2.). Compared to wild type, both fut4 and fut6 showed a decrease of Fuc content by approximately 50% in root AGPs, while the fut4/fut6 double mutant contained no detectable Fuc in their root AGPs. Fuc was also not detected in AGPs from fut4/fut6 and fut4 mutant leaves. In contrast, the Fuc content in leaf AGPs from fut6 was increased by approximately 1.5 fold compared to wild type. Figure 4.7 Monosaccharide composition analysis of root AGPs from WT, fut4, fut6 and fut4/fut6 mutant plants by HPAEC. Peaks 1-3 are noise signals; peaks 4-7 represent Fuc, Ara, Gal and Glc respectively, as determined by retention times of a set of sugar standards including Fuc, Ara, Gal, Glc and xylose (Xyl). Representative profiles of two biological replicates of each sample were shown.

160 Table 4.2 Neutral monosaccharide content of purified AGPs from roots and leaves of wild type, fut4, fut6 and fut4/fut6 mutant plants. Root AGPs a Leaf AGPs a Wild Type fut4 fut6 fut4/fut6 Wild Type fut4 fut6 fut4/fut6 Ara Gal Fuc ND ND ND Glc ND ND ND ND a AGP samples were derivatized by the alditol acetate method and analyzed using GC-MS. The molar percentage of each sugar is presented. The values are averages of two biological replicates. The standard deviations are indicated. ND, not detected. 160

161 Glycome profiling of root and leaf cell walls of wild type, fut4, fut6 and fut4/fut6 mutant plants To examine alterations in the compositions and interactive properties of cell wall polysaccharides in fut4, fut6 and fut4/fut6 mutants compared to wild type plants, total cell walls from leaf and root materials were sequentially extracted and the solubilized glycans were subjected to glycome profiling. Glycome profiling provides both a qualitative and quantitative measure of cell wall glycan epitopes present in the walls using a highthroughput enzyme-linked immunosorbent assay (ELISA) method (Pattathil et al., 2012). Approximately, one hundred fifty monoclonal antibodies, which recognize 19 groups of glycan epitopes present in most major classes of cell wall glycans, were used in the ELISAs. The output of these ELISAs is typically shown as a heatmap, where the antibodies are grouped according to their reactivities with a diverse panel of plant cell wall glycans (Pattathil et al., 2010). Glycome profiles of four sequential extractions of cell wall materials from roots and leaves are shown in Fig. 4.8 and Fig. 4.9, respectively. The four sequential extractions included an oxalate extraction, followed by alkaline extractions, with progressively increasing strengths. Oxalate binds to and depletes Ca 2+ from cell walls, during and after which, the most loosely associated pectic polysaccharides as well as AGPs are solubilized (Gorshkova et al., 1996; Giunter et al., 2004). Alkali solutions of different strengths extract mainly hemicellulose (Fry, 1988), but also solubilize more tightly bound pectins and AGPs. For the wild type root sample, consistent with the extraction method, pectic and AG epitopes, including epitopes for RG-I/AG and AG-1, 2, 3, 4, showed the

162 162 strongest signals in the oxalate extract, while most epitopes for hemicelluloses, including epitopes for nonfucosylated xyloglucans (NON-FUC XG), fucosylated xyloglucans (FUC XG), and xylans (recognized by XYLAN-1, 2, 3, 4 groups of antibodies) were present in the 1 M KOH and 4 M KOH extracts (Fig. 4.8). It should be noted that fractionation by no means separates cell wall components into pure polymers (Fry, 1988) and that epitopes recognized by the same monoclonal antibodies may be present on different wall polymers (Pattathil et al., 2010). Furthermore, the different sub-fractions of wall glycans are bound into the wall matrix to different degrees. Thus, while the glycome profiles of wild type roots showed abundant pectic and AG epitopes in the oxalate extract, these epitopes were also observed in decreasing amounts in the carbonate, 1 M KOH, and 4 M KOH extracts (Fig. 4.8).

163 Figure 4.8 Glycome profiling of sequential cell wall extracts from roots of WT, fut4, fut6 and fut4/fut6 double mutant plants. The presence of cell wall glycan epitopes in each extract (as indicated at the bottom of each column) was determined by ELISAs using 152 glycan-directed monoclonal antibodies. The data are presented as heat maps, with bright yellow indicating strongest binding and black indicating no binding. The altered patterns of mab binding affinity in the mutant fractions compared to wild type fractions were highlighted in green (increased binding) or red (decreased binding) rectangles. 163

164 164 Figure 4.9 Glycome profiling of sequential cell wall extracts from leaves of WT, fut4, fut6 and fut4/fut6 double mutant plants. The presence of cell wall glycan epitopes in each extract was detected as described in Fig Glycome profiles of cell wall extracts from roots of fut4, fut6 and fut4/fut6 mutants were different to varying extents from the corresponding extracts prepared from wild type roots (Fig. 4.8). In the oxalate extract, epitopes for the HG BACKBONE and AG-3

165 165 groups of antibodies showed decreased abundance in the fut6 and fut4/fut6 lines, respectively. The carbonate extract of fut4 roots exhibited signals for hemicellulose and pectin backbone epitopes, i.e. NON-FUC XG, FUC XG, XYLAN-1, 3, HG BACKBONE and RG-I BACKBONE, which were largely absent in the same extract of wild type samples. The fut6 and fut4/fut6 lines showed decreased abundance for epitopes for NON- FUC XG and FUC XG in 1 M KOH and 4 M KOH extracts compared to wild type. Interestingly, in the fut6 and fut4/fut6 lines, epitopes for RG-I/AG and AG-4 had decreased signal intensities in the 1M KOH and 4M KOH extracts, but increased signal intensities in the carbonate extract. Similarly, in all three mutant lines, epitopes for XYLAN-3 showed reduced signal intensity in the 1 M KOH extract, but epitope signal intensity increased in the carbonate extract. Glycome profiles of leaves from fut4, fut6, fut4/fut6 and wild type plants showed similar patterns to one another in the carbonate and 4 M KOH extracts (Fig. 4.9). In the oxalate extract, increased signals for pectic AG epitopes (RG-I/AG) were observed in all three mutant lines and increased AG epitopes (AG-1, 2, 4) were observed in the fut4 and fut4/fut6 mutant lines compared to the wild type line. In addition, the 1 M KOH extract in the fut4/fut6 double mutant showed increased signals for multiple epitopes, including XYLAN-3, XYLAN-4, RG-I BACKBONE, LINSEED MUCILAGE RG-I, and RG-I/AG Discussion Over 1000 Arabidopsis genes have been predicted to encode genes involved in cell wall biosynthesis (Yong et al., 2005). However, to date, only about two dozen genes have been identified unambiguously as GTs or glycan synthases responsible for the

166 166 biosynthesis of specific cell wall molecules. Identification of cell wall biosynthetic enzymes through a biochemical purification route is often hampered by the low abundance of the enzymes or difficulties in maintaining enzymatic activities during the solubilization/purification process. Genetic strategies are also potentially hampered by functional redundancy of cell wall synthetic genes or the plasticity of cell wall structure, which is often insensitive to the disruption of single genes (Reiter et al., 1997). Alternatively, bioinformatics provides a means to identify candidate genes involved in the biosynthesis of particular wall molecules, although additional research is required to verify the function of the genes identified in this manner. Using a bioinformatic strategy, nine Arabidopsis genes, namely FUT2-FUT9 (Sarria et al., 2001), were identified as homologues to Arabidopsis FUT1, which has a defined function as an α-1,2-fut specific for xyloglucan fucosylation in all organs of Arabidopsis plants (Perrin et al., 1999; Faik et al., 2000). We previously demonstrated that the FUT4 and FUT6 genes are AGP FUTs by introducing the FUT4 and FUT6 genes into tobacco BY2 cells, which normally do not contain fucosylated AGPs, and showing that AGP FUT activities are acquired by the transgenic BY2 cells (Wu et al., 2010). In this report, fut4 and fut6 mutants as well as a fut4/fut6 double mutant were utilized to corroborate our previous biochemical findings using molecular genetics and to obtain insight to the physiological functions of FUT4 and FUT6 in Arabidopsis plants. T-DNA insertion sites in fut4 and fut6 mutants were identified and confirmed by PCR and sequencing. RT-PCR using primers across the T-DNA insertion sites showed the absence of FUT4 or/and FUT6 transcripts in the corresponding fut4, fut6 and fut4/fut6

167 167 mutants. Further RT-PCR analysis using primers located at the other regions of the FUT4 and FUT6 genes may be performed to determine whether the fut mutant lines are truely null mutants or instead contain truncated versions of FUT4 or FUT6. Expression analysis has shown that FUT6 is expressed nearly exclusively in the root with only trace expression in flowers, while FUT4 is expressed more ubiquitously in root, stem, leaf and flower and has the highest expression in leaf (Sarria et al., 2001). Monosaccharide composition analyses of the AGPs isolated from the fut mutants examined in this study (Table 4.2) are consistent with these previous gene expression studies. Specifically, Fuc residues are present in wild type root and leaf AGPs, but absent in root and leaf AGPs from the fut4/fut6 mutant. Moreover, the fut4 and fut6 single mutants had reduced Fuc content in their root AGPs. Fuc residues were absent in fut4 leaf AGPs, but present in fut6 leaf AGPs (Table 4.2). The fucosylation patterns of AGPs in wild type and mutant lines, together with the expression patterns of FUT4 and FUT6, clearly indicate that AGP fucosylation is attributable to both FUT4 and FUT6 genes in roots, but only to the FUT4 gene in leaves. It would be interesting to investigate the role of FUT4 in AGP fucosylation in stems and flowers, where the expression of FUT4 was also abundant at least at a transcriptional level (Sarria et al., 2001). The fact that FUT3, FUT5 and FUT7 to FUT10 were all expressed in leaves at different levels (Sarria et al., 2001), but did not compensate for the lost function of FUT4 in the fut4 mutant, indicates that these genes might not be involved in AGP fucosylation and instead may fucosylate other non-agp molecules. Alternatively, these genes may fucosylate AGPs, but only under certain nonphysiological conditions, which were not examined in this study. FUT4 and FUT6 were

168 168 shown to differentially fucosylate AGPs, i.e. adding Fuc residues to different sites on AG side chains (Wu et al., 2010). Given the extensive heterogeneity of polysaccharide structures present in AGP populations, some AG polysaccharides may lack Fuc, while others may contain Fuc added by one or both of the FUT enzymes. Moreover, FUT6 likely adds a terminal Fuc residue to AG polysaccharides that is particularly reactive with eel lectin, since the fut6 mutant roots contained Fuc in AGPs but were not stained by eel lectin (Fig. 4.6; Table 4.2). It is likely that epitopes recognized by eel lectin were synthesized by FUT6 and were abolished in fut6 roots, whereas FUT4 still functioned in AGP fucosylation, but added the Fuc at a different site compared to FUT6. AGPs are implicated in the regulation of root growth and root epidermal cell expansion, as treatment of Arabidopsis roots with β-yariv reagent, which specifically aggregates some AGPs, results in a reduction of root length and epidermal cell length, as well as produces a bulging phenotype of epidermal cells (Willats and Knox, 1996; Ding and Zhu, 1997). The Arabidopsis mur1 mutant exhibited root phenotypes of shortened root length, which were attributed to shortened, swollen root hair cells presented in periodic zones of cells, interspersed by zones containing cells of normal length (van Hengel and Roberts, 2002). In addition, the length of the tip of mur1 roots, measured from root cap to the first initiated root hair has a 40% reduction in length compared to wild type roots. The mur1 mutant was deficient in synthesizing GDP-L-Fuc (Bonin et al., 1997), the sugar donor substrate for the biosynthesis of Fuc containing polymers. Considering that AGPs from mur1 roots had a reduced Fuc content as compared to wild type root and that eel lectin treatment of Arabidopsis plants

169 169 phenocopies the mur1 mutant in root, van Hengel and Roberts (2002) postulated that under-fucosylated AGPs were the cause of the mur1 root phenotype. Consistent with the study of van Hengel and Roberts, Fuc residues were detected in AGPs from wild type Arabidopsis roots (Table 4.2). However, the fut4/fut6 double mutant, in which Fuc residues are completely eliminated from root AGPs, did not show changes in the root phenotype (Table 4.2; Fig. 4), indicating that the absence of Fuc residues in AGPs per se, does not result in an abnormality of root growth. Besides AGPs, common Fuc-containing cell wall polymers include xyloglucan, pectin polymers and N-glycans. Similar to the fut4/fut6 double mutant reported here, mutants deficient in the fucosylation of xyloglucan or N-glycan had normal root growth, indicating the fucosylation status of xyloglucan or N-glycan alone did not affect root morphogenesis (van Hengel and Roberts, 2002; Vanzin et al., 2002). Nonetheless, it cannot be excluded that the additive effects of underfucosylation of multiple cell wall polymers resulted in the defects in root growth observed in the mur1 mutant. Compared to wild type plants, fut4, fut6 and fut4/fut6 mutants showed distinct glycome profiles for root and leaf cell wall extracts (Fig. 4.8 and Fig. 4.9). Glycome profiling provides information about both the glycan epitope composition of cell walls, as well as how tightly those epitopes are bound into the wall matrix. To date, no antibody specifically directed at a fucosylated AGP epitope has been generated or characterized. The glycome profiles of the fut mutants examined here, not surprisingly, do not show the complete absence of a particular class of cell wall glycan epitopes. However, the glycome profiles of the fut mutants do show changes in the epitope compositions of individual

170 170 extracts, suggesting that the mutations have affected the extractability of the glycan epitopes. For example in the glycome profiles of cell wall extracts from roots (Fig. 4.8), many epitopes, including epitopes for NON-FUC XG, FUC XG, XYLAN-1, 3, HG BACKBONE and RG-I BACKBONE, showed increased signals in the carbonate extract from the fut4 mutant, indicating that some xyloglucan and pectin polymers become more extractable in the fut4 mutant root by carbonate. On the other hand, the coupling of a decrease of signals in the more alkaline fractions with an increase of signals in the less alkaline fractions for RG-I/AG, AG-4 and XYLAN-3 epitopes in the fut mutant root, indicates the corresponding polymers are more extractable in the mutants compared to wild type. In contrast, epitopes for NON-FUC XG and FUC XG showed decreased signals in the 1 M KOH and 4 M KOH extracts for fut6 and fut4/fut6 lines compared to wild type, indicating decreased extractability of some xyloglucan polymers in fut6 and fut4/fut6 roots. At a molecular level, AGPs have long been suggested to interact with other wall polymers or wall modifying enzymes to modulate cell wall architectures (Roy et al., 1998; Seifert and Roberts, 2007). Strong and specific binding between β-yariv reagent and some AGPs implies that such AGPs may interact with β-glycan in vivo (Seifert and Roberts, 2007). Interactions between AGPs and pectic polymers were often suggested based on co-purification of the two polymers (Carpita, 1989; Iraki et al., 1989; Immerzeel et al., 2006). Binding between AGPs and pectin fractions was also shown in an in vitro binding experiment (Baldwin et al., 1993). In the current study, the changes of the extractability of wall polymers as suggested by the variation of epitope abundance in different wall extracts, in essence, reflects alterations in the interactions among cell wall

171 171 polymers (i.e. AGPs, pectin, hemicellulose, and cellulose) in the fut4, fut6 and fut4/fut6 mutants. Thus, Fuc residues on AGPs might play a critical role in mediating such interactions among wall polymers. It is interesting that changes in cell wall structure in the fut mutants were not manifested as morphological phenotypes at the organismal level. Perhaps morphological phenotypes will be observed at the organismal or cell wall level with more rigorous examination e.g. structural examination by electron microscopy, or monitoring plant growth under a wide range of non-physiological conditions. As an initial attempt to look for potential roles of FUT4 and FUT6 under non-physiological conditions, expression profiles of the two genes were examined based on public microarray data, which show that the two genes are differentially affected by several treatments (Table 4.3). The microarray data, although requiring experimental verification, provide a guideline for the selection of growth conditions or treatments to further investigate the physiological roles of FUT4 and FUT6 in Arabidopsis.

172 Table 4.3 Expression analysis of the FUT4 and FUT6 genes in response to biotic and abiotic stimuli based on microarray data from Genevestigator ( FUT4 a FUT6 a Categories Stimulus Fold Change P Value Fold Change P Value Both FUT4 and FUT6 showed increase or decrease in expression for more than two folds under stimulus conditions Biotic P.syringae pv. maculicola (Col-0)/ mock treated leaf samples (Col-0) 22.1 < Stress hypoxia study 6 (Col-0)/untreated plant samples (Col-0) FUT4 but not FUT6 showed increase or decrease in expression for more than two folds under stimulus conditions Biotic P.syringae pv. syringae study 2 (Col-0)/ non-infected leaf samples (Col-0) Biotic P.parasitica (6h)/ non-infected root samples (Col-0) Biotic P.syringae pv. tomato study 12 (Col-0)/ untreated leaf tissue samples (Col-0) Biotic B.graminis (Col-0)/non-infected rosette leaf samples 3.33 < Biotic P.syringae pv.phaseolicola (24h)/mock inoculated leaf samples (24h) 2.73 < Biotic P.syringae pv.tomato study 3 (DC3000 hrc-)/mock inoculated leaf samples (24h) 2.39 < Chemical cycloheximide/mock treated seedlings Chemical syringolin study 3 (late)/ solvent treated leaf samples (Col-0; late) Chemical Chitin/mock treated seedlings 3.98 < Chemical ozone/air treated seedlings

173 173 Table 4.3 (continued) Chemical benzothiadiazole study 3 (Col-0)/ untreated (Col-0) plant samples Chemical AgNO3/mock treated seedlings Chemical phenathrene/ untreated Col plant samples Elicitor HrpZ (4h)/H2O treated leaf samples (4h) Elicitor FLG22 study 2 (1h)/ H2O treated Col-0 seedlings (1h) 5.73 < Elicitor FLG22 study 4 (Col-0)/ untreated leaf disc samples (Col-0) Elicitor GST-PP1(4h) 4.12 < Elicitor OGs (1h)/ H2O treated Col-0 seedlings (1h) Elicitor FLG22 (1h)/H2O treated leaf samples (1h) 3.98 < Elicitor FLG22 study 5 (Col-0)/ untreated leaf disc samples (Col-0) Hormone NAA + FLG22 (1h)/ untreated leaf disc samples (Col-0) Hormone salicylic acid study 4 (Col-0)/ mock treated seedlings Hormone IAA study 9 (Col-0)/ untreated seedling samples (Col-0) Nutrient N depletion (Col-0)/ seedlings grown under N-replete condition (Col-0) 2.65 < <0.001 Stress cold study 6 (Col-0)/ 20C/18C treated rosette samples (Col-0) Stress Osmotic (late)/untreated green tissue samples (late)

174 174 Table 4.3 (continued) FUT6 but not FUT4 showed increase or decrease in expression for more than two folds under stimulus conditions Hormone ABA study 6 (Col-0)/untreated plant samples (Col-0) Light light study 2/dark grown Col-0 seedlings Light light study 6 (Col-0)/dark grown Col-0 seedlings Nutrient nitrate starvation/untreated seedlings <0.001 Nutrient iron deficiency (LZ4)/mock treated root samples (LZ4) Stress salt study 2(late)/untreated root samples (late) Stress anoxia study 2/dark grown Col-0 seedling samples Stress osmotic study 2 (late)/untreated plant samples (Col-0) <0.001 Stress heat study 3/dark grown Col-0 seedling samples Stress heat/anoxia/dark grown Col-0 seedling samples a Expression data of FUT4 and FUT6 genes for Arabidopsis (Col-0 ecotype ) were shown. 174

175 175 In conclusion, the characterization of the fut4, fut6 and fut4/fut6 mutants reported here further supports our previous biochemical study of FUT4 and FUT6 (Wu et al., 2010). Furthermore, we show here that FUT4 fucosylates AGPs in leaves and that FUT4 and FUT6 fucosylate AGPs in roots of Arabidopsis. Non-fucosylated AGPs present in the mutants did not affect normal root growth, but nonetheless resulted in biochemical changes in overall cell wall structure in fut4, fut6 and fut4/fut6 mutants as indicated by glycome profiling. Based on this profiling work, it is likely that the Fuc residues present in AGPs function to regulate interactions between AGPs and other cell wall molecules and thus contribute to overall cell wall architecture. The current work also exemplifies the possibilities of refinement of cell wall structures by manipulation of a single or a few cell wall biosynthetic genes. Indeed, plants with such modified wall structures may display altered biomass recalcitrance and thus have the potential to enhance biofuel production.

176 176 CHAPTER 5: CONCLUSIONS AGPs represent a plant-cell-surface glycoprotein family which includes a large number of family members that play diverse and important roles in plant growth and development. As an initial attempt to decipher the glycosylation machinery involved in AGP biosynthesis, this dissertation research focused on the identification and characterization of the GalTs that incorporate the first one or two Gal residues and the FUTs that add the terminal Fuc residues in the AG polysaccharide side chains. An in vitro AGP GalT assay was developed, which used permeabilized microsomal membranes from either tobacco or Arabidopsis suspension-cultured cells as the enzyme source and UDP-[ 14 C]Gal as the sugar donor. When a chemically synthesized peptide, [AO] 7, was used as the substrate acceptor, the assay mainly detected a GalT activity that transferred the first Gal onto Hyp residues of the protein backbone. When a transgenically expressed and HF deglycosylated AGP polypeptide, d[ao] 51, was used as the substrate acceptor, the assay mainly detected the GalT activity that added the second Gal to Gal residues existing on the protein backbone (Fig. 2.5). The incorporation of [ 14 C]Gal into the peptide substrate acceptors was detected by RP-HPLC fractionation of the assay products after removal of unreacted UDP-[ 14 C]Gal by an ion-exchange column (Fig. 2.3). After RP-HPLC, the purified assay product was subjected to total acid hydrolysis coupled with an HPAEC analysis to determine the sugar transfer specificity (Fig. 2.4) and to base hydrolysis coupled with the Bio-gel P2 size exclusion chromatography to determine the extent of glycosylation in the GalT assay (Fig. 2.5). Moreover, when cold UDP-Gal was used in place of UDP-[ 14 C]Gal, the [AO] 7 :GalT

177 177 assay product was precisely identified by mass spectrometry analysis (Fig. 2.9). The sequences of the peptide acceptors mimic the Ala-Hyp dipeptide motifs in naturally occurring AGP molecules; thus the assay is most likely to detect AGP specific GalT activities. Indeed, the substrate specificity test showed that the enzyme activity only used [AO] n (where n=7, 14 or 51 as tested in the current research) as the substrate acceptors but not peptides representing an EXT protein backbone sequence (Fig. 2.8). This in vitro AGP GalT assay can be used to monitor AGP GalT activities during purification of the GalT enzyme, or alternatively, the assay can be (and was) utilized for the functional verification of candidate AGP GalT genes expressed in heterologous expression systems. The Hyp contiguity hypothesis predicted that clustered non-contiguous Hyp residues are sites for the attachment of AG polysaccharides and contiguous Hyp residues are sites of HRGP arabinosylation (Kieliszewski and Lamport, 1994; Kieliszewski et al., 1995). While the [AO] n peptides represented a clustered, non-contiguous Hyp motif, the EXT peptides represented a contiguous Hyp motif. The identification of the Hyp:GalT activity in Arabidopsis microsomal membranes specifically utilizing the [AO] n peptides but not the EXT peptides as the substrate acceptors provided further support for the Hyp contiguity hypothesis. The enzymatic characterization of the [AO] 7 :GalT and d[ao] 51 :GalT activities in Arabidopsis microsomal membranes provided insights into AGP GalT activities. Enzymatic characterization showed that the [AO] 7 :GalT activity significantly increased in the presence of Mn 2+ divalent ions (Fig. 2.7). The nucleotide sugar-dependent GTs adopt either a GT-A or a GT-B structural fold (Lairson et al., 2008). For the GTs of the

178 178 GT-A type, an essential divalent cation, coordinated by the DXD motif, facilitates departure of the nucleoside diphosphate leaving group by electrostatically stabilizing the developing negative charge. In contrast, GTs of the GT-B type use a metal ionindependent method to facilitate leaving group departure. Eukaryotic members possessing the GT-A fold are typically type II membrane proteins, i.e. having a short N- terminal cytoplasmic domain that is followed by transmembrane and stem regions leading to the globular catalytic region (Breton and Imberty, 1999). Thus, the enzyme responsible for the [AO] 7 :GalT activity is likely to be a type II membrane protein processing the GT-A fold in its catalytic domain. Previous studies suggested that AGP GalT activities for the extension of AG side chains are localized to the Golgi apparatus (Mascara and Fincher, 1982; Schibeci et al., 1984; Kato et al., 2003). Oka et al. (2010) proposed that the first GalT activity for AGP glycosylation, namely Hyp:GalT activity (an equivalent activity to the [AO] 7 :GalT activity), is located in the ER and the GalT activities after the initiation step are located in the Golgi. Using a similar sucrose density gradient strategy but different organelle markers as Oka et al. (2010), the ER marker and the Golgi marker could not be completely resolved on a sucrose density gradient in the current study (Fig. 2.10). Moreover, the [AO] 7 :GalT and d[ao] 51 :GalT activities have similar distribution profiles to each other and to both ER and Golgi markers on the sucrose density gradient. Results from sucrose density gradient experiments indicate that AGP GalT activities are located to the endomembrane system but are not sufficient to provide precise localization information of the enzymes. First, sucrose density gradients are limited in achieving complete separation of ER and Golgi organelles in Arabidopsis

179 179 and tobacco. Second, marker enzymes used to identify Golgi or ER fractions may not be representative of all the sub-compartments of ER and Golgi organelles. Using a bioinformatics strategy, 20 Arabidopsis genes were predicted to encodeβ- (1 3)-GalTs for AGP glycosylation. Six of the 20 genes were further assigned as candidate GalTs that function in the initiation of AGP glycosylation by transferring the first Gal residue to the protein backbone (Qu et al., 2008; Egelund et al., 2011). All of the six candidate GalTs were predicted to be Golgi-localized (Table 3.1) and to possess a GT-A fold (Coutinho et al., 2003), which is consistent with the proposed localization and conformation of the enzymes corresponding to the [AO] 7 :GalT activity. I have referred to the six candidate GalTs in this dissertation as GalT1 to GalT6 and initiated the functional characterization of these GalTs by expressing five of the six genes (GalT1, GalT3, GalT4, GalT5 and GalT6) in heterologous expression systems. Genetic constructs for the expression of all of the five genes in two expression systems were successfully built. The preliminary data indicated that all of the five genes were expressed in Pichia cells but none of the Pichia expressed proteins tested so far showed [AO] 7 :GalT activity or d[ao] 51 :GalT activity. However, GalT3 and GalT6, when expressed in tobacco BY2 suspension cultured cells under the control of a heat shock promoter, displayed higher [AO] 7 :GalT activity compared to the wild type BY2 cells after a heat shock treatment (Table 3.4). Some of the BY2 cell lines, transformed with GalT5 or GalT6 under the control of a 2x35S promoter, displayed higher GalT activity compared to wild type BY2 cells in GalT assays containing both the [AO] 7 and d[ao] 51 substrate acceptors. Nonetheless, the preliminary results of activity tests of heterologously expressed GalTs

180 180 require confirmation by further tests with optimized expression conditions. In addition, subcellular localization of GalT3 with a transient expression system confirmed the Golgi localization of GalT3 as predicted by bioinformatics methods (Table 3.1; Fig. 3.13). Future work needs to determine subcellular localization of all the candidate AGP GalTs as well as the substrate specificity of the GalTs, i.e. whether the GalTs are specific for the [AO] 7 or d[ao] 51 substrate acceptor, or utilize both of the substrate acceptors. The precise subcellular localization of AGP glycosylation will be determined only when the GalT localization data and the GalT specificity data are both available. To study the biological functions of the FUT4 and FUT6 genes previously characterized by a biochemical approach as AGP FUTs (Wu et al., 2010), fut4, fut6 and fut4/fut6 genetic mutants in Arabidopsis were obtained and characterized. Sugar composition analysis of AGPs from the fut mutant plants compared to wild type plants indicated that FUT4 was required for fucosylation of leaf AGPs while both FUT4 and FUT6 contributed to fucosylation of root AGPs (Table 4.2). Non-fucosylated AGPs present in the mutants did not affect the phenotype of Arabidopsis grown under physiological conditions, but nonetheless resulted in biochemical changes in overall cell wall structure in the fut4, fut6 and fut4/fut6 mutants as indicated by glycome profiling (Figs. 4.8 and 4.9). Based on this profiling work, I propose that the Fuc residues present in AGPs function to regulate interactions between AGPs and other cell wall molecules and thus contribute to overall cell wall architecture. Previous biochemical characterization of the heterologously expressed FUT4 and FUT6 proteins suggested that the two enzymes differentially fucosylate AGPs (Wu et al., 2010). In the current study,

181 181 fut4 mutant roots showed an eel lectin-staining pattern which was similar to wild type roots but this staining pattern was absent in fut6 and fut4/fut6 mutant roots. The cell wall fractions from the fut4 mutant roots also showed a distinct glycome profile compared to the glycome profiles of cell wall fractions from the fut6 and fut4/fut6 mutant roots. In addition, the expression of the FUT4 and FUT6 genes was differentially affected by nonphysiological treatments. Taken together, FUT4 and FUT6 are likely to catalyze the addition of Fuc residues to different sites on the AG side chains as suggested by Wu et al. (2010). Moreover, differentially fucosylated AGPs catalyzed by FUT4 and FUT6 may play different physiological roles in plants. Based on the concept of the Hyp contiguity hypothesis (Kieliszewski and Lamport, 1994; Kieliszewski and Shpak, 2001) and the GalT and FUT activities studied in this research, a model is proposed for HRGP hydroxylation and Hyp glycosylation as depicted in Fig In the model, the N-terminal signal peptides direct the cotranslational translocation of HRGP core backbones into the ER lumen. In the ER, approximately 65 % of the AGP molecules are predicted to be modified by the addition of a GPI anchor to the C terminus of the protein backbone and become attached to the ER membrane. The AGP molecules without a GPI anchor attachment site remain soluble in ER lumen. Prolyl residues in the AGP backbone sequence are recognized by membrane bound prolyl-4-hydroxylases (P4Hs) in the ER and modified into clustered, noncontiguous Hyp motifs which are recognized by AGP Hyp:GalTs located in the ER or Golgi to initiate AGP glycosylation. The AGP Hyp:GalTs are likely to contain a galectin domain, which is absent in the GTs for the extension of AG polysaccharides. The

182 182 structural model of AG sidechains on AGPs from tobacco BY2 cells indicates that a ~15 sugar unit constitutes the building block of AG polysaccharides. AGP glycosylation in Golgi may involve GTs for the formation of the ~15-sugar unit, which is repeatedly assembled to form AG polysaccharides. Given the specificity of GTs, at least eleven GT activities are required to synthesize the 15-sugar unit (Table 5.1 and Fig. 5.2). In species other than tobacco, additional transferases are possible, as in Arabidopsis, FUT4 and FUT6 add terminal Fuc residues to different positions of AG polysaccharides.

183 Figure 5.1 A model for HRGP hydroxylation and Hyp glycosylation through the secretary pathway. 183

184 Figure 5.1 (continued) The N-terminal signal peptides direct the co-translational translocation of HRGP core backbones into the ER lumen. In the ER, some of the AGP molecules are modified by the addition of a GPI-anchor to the C terminus and become attached to ER membrane. The other AGP molecules, EXT molecules and PRP molecules remain soluble in the ER lumen. Pro residues in the HRGP protein backbones are modified into Hyp residues by prolyl-4-hydroxylases (P4Hs) bound to the ER membrane. Contiguous Hyp motifs in AGP, EXT and PRP molecules are recognized and glycosylated by AraTs located in Golgi apparatus. Clustered, non-contiguous Hyp motifs in AGPs are recognized by β- (1,3)-GalTs with a galectin domain in addition to the GalT catalytic domain to initiate 184

185 glycosylation. The initiation of AG-type glycosylation may occur in the ER or Golgi while the extention of AG side chains mainly occurs in Golgi. AG polysaccharides may be considered as an assembly of ~15-sugar units. A set of eleven GTs may form an enzyme complex to construct AG polysaccharides by repeatedly forming and extending the 15-sugar units. The AG polysaccharides may be further decorated by GTs not included in the AGP GT complex, such as FUTs. Fully glycosylated AGP, EXT and PRP molecules are transported to the plant cell surface through the secretory pathway. At the plant cell surface, AGP molecules may attach to the plasma membrane through GPI anchors or be released as free molecules for AGPs without GPI anchors or after GPI anchors are processed by phospholipases. Membrane attached or free AGPs may interact with other cell wall polymers to affect the cell wall structure or perform other biological functions. The secreted EXT monomers self-assemble into insoluble networks by forming intra- and inter-molecular cross-links. The EXT network may serve as a scaffold for the deposition of newly-formed cell wall polymers. Glycosylated PRPs may form cross-links with EXT polymers to further lock the cell wall polymers in place. CW, cell wall. PM, plasma memberane. ER, endoplasmic rediculum. 185 Table 5.1 GT activities involved in AGP glycosylation. GT a GT activity Functions in the Formation of the 15 AG Structural Unit b # 1 peptidyl Hyp-β-GalT Initiation of the trigalactosyl main chain # 2 β-(1 3)-GalT Addition of the 2 nd Ga residue in the trigalactosyl main chain # 3 β-(1 3)-GalT Addition of the 3 nd Ga residue in the trigalactosyl main chain # 4 β-(1 6)-GalT Formation of the linkage between the trigalactosyl main chains # 5 β-(1 6)-GalT Addition of Gal residues to Gal-1of the main chain # 6 β-(1 6)-GalT Addition of Gal residues to Gal-2 of the main chain # 7 α-(1 3)-AraT Substitution of the sidechain GalT residue # 8 β-(1 6)-GlcAT Substitution of the sidechain GalT residue # 9 α-(1 3)-AraT Extension of the Ara side chain # 10 α-(1 5)-AraT Extension of the Ara side chain # 11 α-(1 4)-RhaT Substitution of the terminal GlcA residue FUT4 α-(1 2)-FUT Addition of terminal Fuc residues FUT6 α-(1 2)-FUT Addition of terminal Fuc residues at the sites different from FUT4 a GT#1 to #11 are predicted GT activities involved in the formation of the core 15-sugar unit in AG polysaccharides. FUT4 and FUT6 are two FUTs identified for AGP fucosylation in Arabidopsis. b Linkages in the AG side chain formed by the predicted GT activities were shown in Fig. 5.2.

186 186 Figure 5.2 Predicted GT activities involved in the formation of a 15-sugar unit for AGP glycosylation. The predicted GT activities (from GT#1 to GT#11) were listed in table 5.1. The sites of the addition of terminal Fuc residues catalyzed by the Arabidopsis FUT4 and FUT6 are hypothetical. The GTs are most likely to be type II membrane proteins, however, they may also adopt other strategies to be retained in the Golgi for their functionality. For example, GTs without a transmembrane domain may be anchored in the Golgi by associating with a GT which contains a transmembrane domain (Atmodjo et al., 2011). The GTs may form a complex for AGP glycosylation, thus individually expressed GalTs showed either no (as for GalT1, GalT3, GalT4, GalT5 and GalT6) or low GalT activity (as for GalT2) in the

187 187 Pichia expression system. In the tobacco BY2 expression system, heterologously expressed Arabidopsis GalTs may interact with tobacco GT partners and thus showed detectable GalT activities. However, FUT4 and FUT6 are apparently not essential components of the AGP GT complex because AGPs from the fut4/fut6 double mutant showed minimal sugar composition changes except for the absence of Fuc residues. Different types of AGPs, including AGPs with or without a GPI anchor and hybrid and chimeric AGPs with non-agp domains, may be modified through a common glycosylation pathway and be transported to the plant cell surface where AGPs interact with other cell wall polymers to affect the cell wall structure or perform other biological functions. In contrast to AGP molecules, Pro containing motifs in EXT and PRP molecules are modified into contiguous Hyp motifs by P4Hs in ER. In Golgi, the contiguous Hyp motifs are glycosylated with arabino-tetrasaccharide sidechains probably by an AraT complex, which involves one peptidyl Hyp-AraT, two β-(1 2)-AraTs and one α-(1 3)- AraT. EXT and PRP monomers are soluble in the ER and Golgi. After being transported to the plant cell surface, glycosylated EXT monomers self-assemble into insoluble networks by forming intra- and inter-molecular cross-links through the Tyr-Xaa-Tyr-Lys and Val-Tyr-Lys motifs. The EXT network may serve as a scaffold for the deposition of newly-formed cell wall polymers. Glycosylated PRPs also form cross-links with each other and with EXT polymers to further lock cell wall polymers in place. The functions of HRGP subfamilies are achieved by a combination of factors, including the various functional domains of the protein backbone, the glycosylation

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210 APPENDIX A: SEQUENCES AND LOCATIONS OF PRIMERS USED IN THIS WORK 210 Appendix A Table 1. Sequence and annealing temperatures of the primers used in this work Purpose Name Primer Sequences (5 3 ) Annealing Cloning of GALT1 to ppicza GalT1 yeast F CGCCGCGGATGCATCATCATCATCATCACA TGAAGAGATTTTATGGAGGGCTT 54 C GalT1 yeast R GGGCCCTTACCATTCGCGGCAGCA Cloning of GALT3 to ppicza GalT3 yeast F CGCCGCGG ATGCATCATCATCATCATCACATGAAGCAAT TCATGTCAG TGGT 54 C GalT3 yeast R GGGCCCTTATTCGCAGCAAATAGATTGGTTC Cloning of GALT4 to ppicza GalT4 yeast F CGCCGCGGATGCATCATCATCATCATCACAT GAAGAAGTCTAAACTCGATAATTC 54 C GalT4 yeast R GGGCCCTCATCTCATGTTGCAGCATTG Cloning of GALT5 to ppicza GalT5 yeast F CGCCGCGGATGCATCATCATCATCATCACAT GAAAAAACCCAAATTGTCG 54 C GalT5 yeast R TCTAGATCATCTCATGTTACAACACTC Cloning of GALT6 to ppicza GalT6 yeast F CGCCGCGGATGCATCATCATCATCATCACAT GAGGAAGCCCAAGTTGTCA 54 C GalT6 yeast R TCTAGATCATCTCATGTTGCAGCACTG Cloning of GALT1 to pmdc30 /pmdc32; verification of constructs or Agrobacterium or BY2 cells containing GALT1 GalT1BY2F2 galt1r by2 CACCATGCATCATCATCATCATCACAAGAGA TTTTATGGAGGGCTTC TTACCATTCGCGGCAGCAAA 54 C Cloning of GALT3 to pmdc30 /pmdc32; verification of constructs or Agrobacterium or BY2 cells containing GALT3 GalT3BY2F2 galt3r by2 CACCATGCATCATCATCATCATCACAAGCAA TTCATGTCAGTGGT TTATTCGCAGCAAATAGATTGGTTC 54 C Cloning of GALT4 to pmdc30 /pmdc32; verification of constructs or Agrobacterium or BY2 cells containing GALT4 GalT4BY2F2 galt4r by2 CACCATGCATCATCATCATCATCACAAGAAG TCTAAACTCGATAATTC TCATCTCATGTTGCAGCATTG 54 C Cloning of GALT5 to pmdc30 /pmdc32; verification of constructs or Agrobacterium or BY2 cells containing GALT5 GalT5BY2F2 galt5r by2 CACCATGCATCATCATCATCATCACAAAAAA CCCAAATTGTCG TCATCTCATGTTACAACACTCAG 54 C

211 211 Appendix A Table 1 (continued) Cloning of GALT6 to pmdc30 /pmdc32; verification of constructs or Agrobacterium or BY2 cells containing GALT6 GalT6BY2F2 galt6r by2 CACCATGCATCATCATCATCATCACAGGAAG CCCAAGTTGTCA TCATCTCATGTTGCAGCACTG 54 C GALT1 construct sequencing GalT1site544 GTTCCTCCATCACAGTTATTGG 55 C GalT1site651R CACAATGATGGGTGGATCAG 55 C GalT1site220R CACCAGATATCGCTTCTTGAG 55 C GALT3 construct sequencing GalT3 site462 GCATTTGATAAGGATTTGTCTGG 55 C GalT3site622R GACGAGTTTCTCCTGATAATCC 55 C GALT4 construct sequencing GalT4 site586 GATCTCACATTACCGTCGTG 55 C GalT4site717R AGGCGGATCTTCACCATC 55 C GalT4site222R ATTAGCTTCCCTACTACCACC 55 C GALT5 construct sequencing GalT5 site395f GGTCAGTGGAGCTTCACAAG 55 C GalT5 site586 CCGTGTGGTTTGACATTGG 55 C GalT5site726R ATCCTCTCCTTCAACAGTCTTC 55 C GalT5site222R GGGCTCTTGCTCGTTATTG 55 C GALT6 construct sequencing GalT6 site592 GGTCACATATTACAGTGGTTGG 55 C GalT6site722R TTCAAACCCTGAAGCTCCAAC 55 C GalT6site227R CTCTCTTGTAACTCTCTCTGAC 55 C GalT6site855R TTCACATCTTTGTGCTGAGCC 55 C GalT6site1939F GCATAGAGAATTACTTGACGGCG 55 C pmdc30/pmdc32 vector sequencing pmdc45paci CCGGCAACAGGATTCAATCTTAAG 55 C GALT3 site 1446 T to C t1446c GATGAGCTCCTATCAAGCCTAGAAGAAAGA CCGTC 55 C t1446cantisense GACGGTCTTTCTTCTAGGCTTGATAGGAGCT CATC Cloning of GALT3 to pvkh18en6 GalT3-C-F CAGGACGTCTAGATGAAGCAATTCATGTCA GTGGTGAGATTC 62 C GalT3-C-R CATGACCGTCGACTTTTCGCAGCAAATAGAT TGGTTCTC

212 212 Appendix A Table 1 (Continued) pvkh18en6-galt3 sequencing MHP-23 GAACTTCAGGGTCAGCTTGCC 58.5 C pvkh18en6-galt3 sequencing MHP-22 AGTCCGCCCTGAGCAAAG 57.8 C Absence of insertion of fut4 F4SLLP CCATGTAGTTACATTCCCAACCG 54 C F4SLRP1 CCACGTCGATGGAGCCTTGTTT Presence of insertion 1 in fut4 LB3 TAGCATCTGAATTTCATAACCAATCTCGATA CAC 49 C F4SLRP2 GAACATGTTTTCAGAGCGAGC Presence of insertion 2 in fut4 LB3 TAGCATCTGAATTTCATAACCAATCTCGATA CAC 54 C F4SLLP CCATGTAGTTACATTCCCAACCG Absence of insertion of fut6 F6LP2 CACATCTTTCAGATCTCCAGCG 55 C F6RP CTTTCTTGTAAGCATCCGTGC Presence of insertion 1 in fut6 LBa1 TGGTTCACGTAGTGGGCCATCG 54 C F6RP CTTTCTTGTAAGCATCCGTGC Presence of insertion 2 in fut6 LBa1 TGGTTCACGTAGTGGGCCATCG 54 C F6LP1 TGTTTTGCTAGCAACGAAAGC RT -PCR for fut4 46RTL1 TGGAGGATTAAAGCCATGGTTAC 52 C FUT4RP1 AATGAAAAAGGAAGATATATAACA RT -PCR for fut6 FUT6brtlp TCCAGCGAAGTTTTCAGAGC 52 C Fut6brtrp CGCAACCCACACAATGTATC

213 213 Appendix A Figure 1. Schematic diagrams showing the positions of primers used for expression cloning and verification of transgenic bacteria, Agrobacterium or tobacco BY2 cells.

214 Appendix A Figure 2 Schematic diagram showing the positions of primers used for RT-PCR analysis of fut4, fut4/fut6 and fut4/fut6 mutant plants. 214

215 215 APPENDIX B: PARTIAL SEQUENCE OF THE FINAL EXPRESSION CONSTRUCTS Part I. Sequencing results of the ppicza-6xhis-galt constructs. The expression constructs of ppicza-6xhis-galt1/galt3/galt4 and ppiczb-6xhis - GalT5/GalT6 were sequenced for full length coding regions of the 6xHis-GalT genes and partial sequences of the promoter and terminator regions. Color codes: blue: ppicza/b vector sequences; orange: SacII restriction site; yellow: ApaI restriction site; green: XbaI restriction site; purple: 6xHis tag; black: GalT coding region; red: nucleotide mutation without affecting the amino acid encoded. >ppicz-galt1 GATCAAAAAACAACTAATTATTCGAAACGAGGAATTCACGTGGCCCAGCCGGCCGTCTCG GATCGGTACCTCGAGCCGCGGATGCATCATCATCATCATCACATGAAGAGATTTTATGGA GGGCTTCTTGTTGTATCAATGTGTATGTTCTTGACGGTGTATAGATATGTAGACTTGAAC ACTCCTGTTGAAAAGCCTTATATTACTGCTGCTGCTTCTGTTGTTGTTACTCCTAACACC ACTCTTCCTATGGAATGGCTGCGGATTACTCTCCCTGACTTTATGAAAGAAGCGAGGAAT ACTCAAGAAGCGATATCTGGTGATGATATCGCTGTTGTCTCGGGTTTGTTTGTTGAGCAG AATGTGTCTAAAGAAGAGAGAGAGCCTTTGCTTACTTGGAACCGTTTGGAAAGTCTTGTT GATAATGCACAGAGTTTAGTTAATGGAGTTGATGCTATTAAGGAAGCTGGCATTGTTTGG GAGAGTCTTGTGTCTGCTGTTGAAGCTAAGAAACTAGTTGATGTTAATGAAAATCAGACG AGGAAAGGAAAAGAGGAGCTTTGTCCTCAGTTTCTAAGCAAAATGAATGCTACTGAAGCT GATGGGAGTAGTCTGAAGTTGCAAATTCCTTGTGGTTTGACTCAGGGTTCCTCCATCACA GTTATTGGCATCCCAGATGGTCTTGTTGGTAGTTTTCGGATTGATCTAACGGGACAACCG CTTCCAGGGGAGCCTGATCCACCCATCATTGTGCATTATAATGTTAGGCTTCTTGGTGAC AAATCGACGGAAGACCCTGTGATTGTTCAAAACAGCTGGACGGCATCTCAGGACTGGGGA GCTGAGGAACGCTGTCCAAAATTTGATCCTGATATGAATAAGAAAGTGGATGACTTGGAT GAATGCAACAAGATGGTTGGTGGAGAAATTAACCGAACTTCTTCAACTAGCTTGCAGTCC AACACTTCAAGGGGAGTTCCAGTAGCCAGGGAAGCATCTAAACATGAAAAATACTTTCCT TTCAAGCAGGGTTTCTTATCGGTTGCTACACTTAGGGTGGGAACAGAGGGAATGCAGATG ACAGTTGATGGGAAACATATAACTTCATTTGCTTTCCGCGATACACTGGAACCGTGGCTT GTTAGTGAAATACGGATTACAGGTGACTTTAGGTTAATATCCATTCTTGCCAGCGGTTTG CCCACATCAGAAGAATCAGAGCACGTTGTTGATCTAGAGGCACTAAAATCACCTACCCTT TCACCATTAAGGCCACTGGATCTCGTTATTGGTGTTTTCTCCACTGCGAACAATTTTAAA AGACGGATGGCTGTGAGGAGAACATGGATGCAGTATGATGATGTAAGATCTGGAAGAGTT GCAGTACGCTTTTTTGTTGGCCTTCACAAAAGTCCTCTTGTTAACTTGGAACTCTGGAAC GAGGCTCGGACTTACGGTGATGTTCAGCTAATGCCCTTTGTTGATTATTACAGTCTCATC AGTTGGAAAACACTAGCCATCTGCATCTTCGGGACAGAGGTTGACTCAGCCAAGTTCATC

216 216 ATGAAAACGGATGATGACGCCTTTGTTCGTGTAGATGAAGTGTTACTTTCTTTATCAATG ACCAACAACACTCGCGGGTTAATATACGGACTGATCAATTCCGACTCTCAACCTATTCGA AACCCTGATAGCAAATGGTACATCAGTTATGAGGAATGGCCTGAAGAGAAATATCCACCA TGGGCGCATGGTCCAGGCTACATTGTATCTCGTGACATAGCAGAATCGGTTGGTAAGCTT TTCAAAGAAGGAAACCTAAAGATGTTTAAGCTAGAAGATGTGGCAATGGGGATATGGATA GCTGAGCTGACAAAACATGGACTCGAGCCTCATTACGAAAACGATGGAAGGATCATTAGT GATGGATGCAAGGATGGTTATGTGGTTGCTCATTACCAAAGCCCTGCCGAAATGACTTGC CTTTGGCGTAAATACCAAGAAACCAAACGCTCTCTTTGCTGCCGCGAATGGTAAGGGCCC GAACAAAAACTCATCTCAGAAGAGGATCTGAATAGCGCCGTCGACCATCATCATCATCAT CATTGAGTTT >ppicza-galt3 AGATCAAAAAACAACTAATTATTCGAAACGAGGAATTCACGTGGCCCAGCCGGCCGTCTC GGATCGGTACCTCGAGCCGCGGATGCATCATCATCATCATCACATGAAGCAATTCATGTC AGTGGTGAGATTCAAATTTGGTTTCACTTCAGTCAGAATGAGGGATTGGTCGGTGGGAGT CTCCATTATGGTTCTTACATTGATCTTCATCATCCGTTATGAACAATCTGATCACACTCA CACTGTGGATGATTCTAGTATAGAAGGAGAGAGTGTTCATGAACCCGCAAAGAAGCCACA TTTTATGACTTTGGAAGATCTTGATTATCTATTTTCAAACAAGAGCTTTTTTGGAGAAGA AGAAGTGTCCAATGGAATGCTTGTATGGTCTCGAATGCGTCCATTTCTTGAAAGGCCAGA TGCTTTGCCAGAAACTGCTCAAGGGATAGAAGAAGCTACATTGGCAATGAAAGGTTTGGT TTTAGAAATCAATAGAGAGAAGAGAGCTTATTCTTCTGGTATGGTCTCTAAGGAAATTAG AAGAATCTGTCCGGATTTTGTCACTGCATTTGATAAGGATTTGTCTGGTTTAAGTCATGT ACTTCTTGAGCTTCCTTGTGGTTTAATTGAAGATTCTTCAATAACTTTGGTTGGTATTCC TGATGAACATTCTAGTAGCTTCCAGATTCAGCTCGTTGGCTCGGGATTATCAGGAGAAAC TCGTCGGCCAATAATCTTGCGTTACAATGTGAACTTTTCTAAACCATCGATAGTGCAAAA TACATGGACAGAGAAGCTAGGTTGGGGAAACGAAGAGCGATGCCAATATCATGGATCATT GAAAAATCATTTAGTTGATGAACTTCCTCTCTGCAACAAACAGACCGGTAGAATCATTTC GGAAAAGAGTTCCAACGATGATGCAACTATGGAACTTTCTCTTTCAAATGCTAATTTTCC ATTTCTCAAAGGGAGTCCCTTCACTGCCGCATTGTGGTTTGGCTTAGAAGGTTTTCATAT GACGATAAATGGGCGGCACGAGACTTCATTTGCTTACAGGGAGAAGCTCGAGCCATGGTT AGTCAGTGCAGTCAAAGTCTCAGGTGGTTTGAAAATTTTATCTGTCTTAGCCACAAGACT GCCCATTCCCGATGACCATGCATCTTTAATCATAGAAGAGAAACTTAAAGCTCCATCTCT TTCCGGGACAAGAATAGAACTATTGGTGGGTGTTTTCTCCACTGGAAATAATTTTAAGCG GCGTATGGCATTGAGAAGATCTTGGATGCAATACGAGGCAGTAAGATCTGGCAAAGTAGC TGTTCGATTTCTCATTGGCCTTCACACAAATGAAAAAGTCAATTTAGAGATGTGGAGAGA ATCTAAGGCATATGGAGACATTCAGTTTATGCCATTTGTTGACTACTATGGTTTACTTAG CTTGAAAACAGTTGCGCTTTGCATTCTCGGGACCAAAGTCATCCCAGCAAAATACATAAT GAAGACGGATGATGATGCGTTTGTACGGATTGATGAGCTCCTATCAAGTCTAGAAGAAAG

217 217 ACCGTCTAGTGCCCTTCTGTACGGTTTGATCTCATTTGATTCATCACCGGACCGTGAACA AGGCAGCAAATGGTTTATCCCTAAAGAGGAATGGCCTTTAGATTCATACCCTCCATGGGC ACATGGCCCTGGCTACATCATCTCTCATGATATAGCGAAATTTGTGGTGAAGGGTCACCG TCAAAGAGATCTTGGACTTTTCAAGCTGGAAGATGTGGCGATGGGGATATGGATTCAACA ATTCAACCAGACGATAAAAAGAGTGAAGTACATCAATGACAAAAGATTTCATAACAGTGA TTGTAAATCAAATTACATTCTTGTTCATTACCAAACTCCTAGACTAATTTTGTGTCTTTG GGAGAAGCTGCAAAAAGAGAACCAATCTATTTGCTGCGAATAAGGGCCCGAACAAAAACT CATCTCAGAAGAGGATCTGAATAGCGCCGTCGACCATCATCATCATCATCATTGAGTTT >ppicza-galt4 GATCAAAAAACAACTAATTATTCGAAACGAGGAATTCACGTGGCCCAGCCGGCCGTCTCG GATCGGTACCTCGAGCCGCGGATGCATCATCATCATCATCACATGAAGAAGTCTAAACTC GATAATTCTTCTTCACAGATTCGATTCGGGCTTGTTCAGTTCTTATTAGTTGTTCTGCTT TTTTACTTCCTCTGCATGAGCTTCGAGATCCCATTCATCTTCAGAACCGGGTCTGGGTCC GGGTCTGATGATGTTTCATCTTCTTCTTTTGCTGACGCATTACCGAGACCAATGGTTGTT GGTGGTGGTAGTAGGGAAGCTAATTGGGTTGTCGGAGAAGAAGAAGAAGCAGACCCACAT CGACATTTCAAGGATCCGGGTCGGGTACAGCTTCGGTTACCGGAGCGGAAAATGAGGGAA TTTAAGTCCGTCTCTGAGATTTTCGTCAACGAGAGCTTCTTCGACAATGGCGGATTCAGC GATGAATTCTCAATCTTTCACAAAACAGCGAAGCATGCGATTTCAATGGGTCGAAAAATG TGGGACGGACTCGATTCGGGTTTAATCAAACCCGATAAAGCTCCGGTTAAGACCCGGATT GAGAAATGTCCGGATATGGTTTCGGTTTCTGAGTCGGAGTTTGTGAACCGGAGTCGGATC TTGGTTTTGCCGTGTGGGTTAACGTTAGGATCTCACATTACCGTCGTGGCTACGCCGCAT TGGGCTCACGTTGAGAAAGATGGTGATAAGACGGCGATGGTGAGTCAGTTCATGATGGAG TTACAAGGATTAAAGGCGGTGGATGGTGAAGATCCGCCTCGGATACTTCATTTTAACCCG AGGATTAAAGGTGATTGGAGTGGAAGACCAGTGATTGAGCAAAACACTTGTTATCGAATG CAATGGGGCTCAGGTTTACGTTGTGATGGTCGTGAATCTAGTGATGATGAAGAATATGTT GATGGAGAGGTGAAATGTGAGAGGTGGAAGAGAGATGATGATGATGGTGGTAATAATGGT GATGATTTTGATGAATCAAAGAAGACATGGTGGTTGAATAGGTTGATGGGTCGGAGGAAG AAGATGATAACACATGATTGGGATTATCCTTTTGCTGAAGGGAAGCTTTTTGTTCTTACA CTTCGAGCTGGGATGGAAGGTTATCATATTAGTGTGAATGGAAGACATATCACATCTTTT CCTTATAGAACGGGGTTTGTTTTGGAGGATGCCACTGGATTAGCAGTCAAAGGAAACATT GATGTGCATTCTGTATATGCTGCCTCGTTACCTTCTACAAATCCTAGTTTTGCACCGCAG AAGCATCTCGAGATGCAAAGGATATGGAAAGCTCCTTCGTTACCTCAGAAGCCTGTAGAG TTGTTCATTGGAATTCTTTCTGCTGGTAATCATTTTGCAGAGAGAATGGCAGTGAGGAAG TCATGGATGCAGCAGAAGCTGGTCAGATCATCGAAAGTTGCTGCCCGGTTCTTTGTGGCA TTGCATGCAAGAAAGGAAGTCAATGTGGATTTAAAGAAAGAAGCTGAGTACTTTGGTGAT ATTGTCATAGTACCGTACATGGATCATTATGACCTTGTTGTGCTCAAGACAGTTGCCATC TGCGAATATGGGGTGAACACAGTGGCGGCAAAGTACGTTATGAAATGTGACGATGATACA TTTGTGCGTGTGGATGCTGTGATCCAGGAAGCAGAAAAGGTTAAGGGAAGAGAGAGCCTT

218 218 TATATTGGAAACATTAATTTTAACCATAAGCCATTGCGTACCGGGAAATGGGCTGTGACA TTCGAGGAATGGCCAGAAGAGTATTATCCTCCATATGCAAATGGTCCGGGTTACATCTTG TCATATGATGTAGCTAAGTTCATTGTCGATGATTTTGAACAAAAGCGATTAAGATTATTC AAGATGGAAGATGTGAGCATGGGAATGTGGGTGGAGAAGTTCAACGAGACTAGACCAGTG GCAGTGGTTCACAGCCTCAAGTTCTGTCAGTTTGGTTGCATAGAAGACTACTTCACCGCT CATTATCAGTCGCCTCGCCAGATGATTTGCATGTGGGATAAGCTGCAGAGACTCGGGAAG CCCCAATGCTGCAACATGAGATGAGGGCCCGAACAAAAACTCATCTCAGAAGAGGATCTG AATAGCGCCGTCGACCATCATCATCATCATCATTGAGTTTTAGCCTTAGACATGACTGTT CCTCAGTTCAAGTTGGGCACTTACGAGAAGACC >ppiczb-galt5 GATCAAAAAACAACTAATTATTCGAAACGAGGAATTCACGTGGCCCAGCCGGCCGTCTCG GATCGGTACCTCGAGCCGCGGATGCATCATCATCATCATCACATGAAAAAACCCAAATTA TCGAAAGTGGAAAAAATCGACAAGATTGATCTGTTCTCTTCACTATGGAAGCAGAGATCG GTTCGTGTAATAATGGCAATAGGGTTTCTCTATCTTGTAATTGTCTCAGTAGAGATACCT CTCGTTTTCAAATCCTGGTCCAGCAGCTCCGTGCCTCTTGATGCTCTTTCTCGACTCGAG AAGCTCAATAACGAGCAAGAGCCCCAAGTCGAGATTATCCCTAATCCTCCATTGGAGCCA GTTTCGTACCCGGTTTCGAACCCGACCATTGTTACCCGGACGGACCTTGTTCAGAACAAG GTCCGTGAACATCATCGGGGTGTTCTCTCAAGTTTGAGATTTGATTCGGAAACTTTCGAC CCGAGTAGTAAAGACGGGTCAGTGGAGCTTCACAAGTCAGCTAAGGAAGCTTGGCAGCTA GGTCGCAAGCTATGGAAGGAGCTTGAATCTGGAAGGCTTGAGAAACTAGTGGAGAAGCCA GAGAAGAACAAACCGGATTCATGTCCACATTCTGTTTCGCTAACCGGGTCTGAGTTTATG AACCGGGAGAACAAATTGATGGAGCTGCCGTGTGGTTTGACATTGGGTTCACACATAACT TTGGTGGGGAGGCCGAGGAAAGCTCATCCCAAGGAAGGAGATTGGTCTAAGTTGGTGTCT CAGTTTGTGATAGAGCTTCAAGGTTTGAAGACTGTTGAAGGAGAGGATCCTCCTAGGATT CTGCATTTCAATCCGAGGCTTAAGGGAGATTGGAGCAAAAAACCGGTGATTGAGCAGAAT AGTTGCTATAGGATGCAATGGGGACCTGCACAACGTTGCGAAGGATGGAAGTCAAGAGAT GATGAAGAGACTGTTGATAGTCATGTGAAGTGTGAAAAATGGATTCGTGATGATGACAAT TACTCAGAAGGGTCGAGGGCAAGATGGTGGTTGAATAGACTTATAGGAAGGAGGAAAAGG GTCAAAGTAGAATGGCCGTTTCCTTTTGTGGAAGAGAAGCTGTTCGTTCTAACTCTTAGC GCCGGTTTAGAGGGTTACCATATCAATGTTGATGGAAAGCATGTTACTTCTTTCCCTTAT CGCACTGGTTTCACCCTTGAGGATGCAACAGGGCTAACAGTAAACGGAGACATTGATGTC CATTCTGTTTTTGTTGCCTCTCTGCCAACATCACATCCTAGTTTTGCTCCCCAAAGGCAT CTCGAATTGTCAAAGAGATGGCAGGCTCCTGTAGTTCCCGATGGGCCTGTGGAGATCTTT ATAGGCATTCTTTCCGCAGGCAATCATTTCTCTGAGCGGATGGCTGTGAGGAAATCCTGG ATGCAGCATGTTCTTATTACATCTGCAAAAGTTGTTGCTCGTTTCTTTGTGGCGCTGCAT GGGAGGAAGGAGGTGAATGTGGAATTGAAGAAAGAAGCGGAGTATTTTGGGGACATTGTA CTTGTTCCTTACATGGATAGCTATGATCTTGTCGTGCTGAAAACTGTTGCCATATGTGAA CACGGAGCTCTTGCATTCTCTGCAAAGTACATAATGAAGTGTGACGATGATACATTTGTA

219 219 AAACTTGGCGCGGTGATCAATGAAGTGAAAAAAGTACCCGAAGGTAGAAGCCTGTACATT GGTAACATGAATTATTACCACAAACCTCTCCGTGGGGGTAAATGGGCAGTCACATATGAG GAATGGCCAGAGGAGGACTATCCGCCCTACGCAAATGGACCCGGATATGTTCTATCTTCT GACATTGCGCGCTTCATCGTGGACAAGTTTGAGAGACATAAATTACGGCTGTTCAAGATG GAGGACGTGAGTGTGGGAATGTGGGTTGAGCATTTCAAGAACACAACAAACCCAGTGGAT TACAGACACAGTCTGAGATTCTGCCAGTTTGGTTGTGTTGAGAACTACTACACAGCTCAT TACCAGTCGCCAAGACAGATGATATGCTTATGGGATAAGCTCTTAAGACAGAACAAGCCT GAGTGTTGTAACATGAGATGATCTAGAACAAAAACTCATCTCAGAAGAGGATCTGAATAG CGCCGTCGACCATCATCATCATCATCATTGAGTTTGTAGCCTTAGACATGACTGTTCCTC AGTTCAAG >ppiczb-galt6 ACGACACTTGAGAGATCAAAAAACAACTAATTATTCGAAACGAGGAATTCACGTGGCCCA GCCGGCCGTCTCGGATCGGTACCTCGAGCCGCGGATGCATCATCATCATCATCACATGAG GAAGCCCAAGTTGTCAAAACTCGAGAGGTTAGAGAAATTCGACATTTTCGTTTCTCTAAG CAAGCAGAGATCGGTTCAGATACTAATGGCGGTTGGGTTACTCTACATGCTTCTTATCAC ATTCGAAATCCCTTTCGTCTTCAAAACCGGGCTTAGTTCTTTATCTCAGGATCCGTTAAC CCGACCCGAGAAGCACAATAGTCAGAGAGAGTTACAAGAGAGACGAGCTCCGACTCGACC TTTAAAGAGTCTGCTTTACCAGGAATCACAATCGGAATCACCGGCTCAGGGTTTAAGAAG AAGGACTCGGATCCTTTCTAGTTTGAGATTCGACCCGGAAACGTTTAACCCGAGTAGCAA AGATGGGTCTGTGGAGCTTCATAAATCTGCTAAGGTAGCTTGGGAAGTTGGTCGAAAGAT ATGGGAAGAGCTTGAGTCTGGGAAAACGTTGAAAGCTTTGGAGAAGGAGAAGAAGAAGAA GATTGAGGAACATGGGACAAACTCGTGTTCTCTCTCTGTTTCCTTAACCGGGTCTGATCT TTTGAAACGTGGGAATATCATGGAGCTTCCATGTGGTTTAACTCTTGGGTCACATATTAC AGTGGTTGGGAAGCCACGAGCTGCTCATTCGGAGAAGGACCCTAAGATATCGATGTTAAA GGAAGGAGATGAAGCTGTGAAGGTTTCACAGTTTAAGTTGGAGCTTCAGGGTTTGAAAGC AGTGGAAGGAGAAGAGCCACCTCGGATACTCCACTTGAATCCAAGGCTTAAGGGTGATTG GAGTGGTAAGCCTGTGATTGAGCAGAACACTTGCTATAGAATGCAATGGGGCTCAGCACA AAGATGTGAAGGATGGAGATCTAGGGATGATGAAGAGACTGTTGATGGTCAGGTTAAGTG CGAGAAATGGGCTCGGGATGATAGCATTACATCTAAAGAAGAAGAGTCTAGCAAGGCGGC TTCATGGTGGCTTAGTCGATTAATAGGTCGGAGCAAGAAAGTAACTGTTGAATGGCCATT TCCATTCACAGTTGACAAGCTTTTCGTGCTTACTCTTAGTGCTGGATTGGAAGGCTACCA TGTTAGTGTCGATGGGAAGCATGTCACTTCCTTTCCATACCGAACTGGATTTACGCTTGA GGATGCTACTGGTCTAACCATTAACGGGGACATAGATGTTCACTCTGTTTTCGCTGGCTC TCTCCCAACCTCGCATCCTAGTTTTTCTCCTCAGAGGCATCTTGAGCTCTCGAGCAATTG GCAAGCCCCATCACTTCCTGATGAGCAAGTTGATATGTTCATTGGTATCCTTTCTGCTGG TAACCATTTTGCTGAGAGGATGGCTGTGAGGAGGTCGTGGATGCAACATAAACTCGTTAA ATCTTCCAAAGTAGTGGCTCGGTTCTTTGTTGCACTGCACTCAAGGAAAGAAGTAAATGT GGAGCTAAAGAAGGAAGCTGAATTCTTTGGGGACATAGTTATAGTCCCTTACATGGACAG

220 TTATGACCTTGTCGTCCTCAAAACCGTTGCAATTTGCGAGTACGGGGCTCATCAACTTGC AGCTAAATTCATCATGAAGTGTGATGACGATACATTTGTACAAGTGGATGCGGTTCTTAG TGAAGCAAAGAAAACACCCACAGATAGAAGTCTATACATTGGCAACATCAATTATTATCA CAAACCACTTCGCCAGGGTAAATGGTCTGTTACATATGAGGAATGGCCAGAGGAAGACTA TCCACCTTATGCTAATGGCCCCGGATACATATTATCAAACGATATATCTCGCTTTATCGT GAAAGAGTTTGAGAAACACAAATTAAGGATGTTCAAAATGGAAGATGTAAGCGTGGGAAT GTGGGTAGAACAATTCAACAATGGTACAAAACCGGTGGACTACATTCACAGCCTCAGGTT TTGTCAGTTTGGTTGCATAGAGAATTACTTGACGGCGCATTATCAGTCGCCGAGACAGAT GATTTGCTTGTGGGATAAGCTGGTGTTGACAGGCAAACCTCAGTGCTGCAACATGAGATG ATCTAGAACAAAAACTCATCTCAGAAGAGGATCTGAATAGCGCCGTCGACCATCATCATC ATCATCATTGAGTTTGTAGCCTTAGACATGACTGTTCCTCAGTTCAAGTTGGGCACTTAC GAGAAGACC 220

221 221 Part II. Sequencing results of the pmdc30-6xhis-galt constructs. The expression constructs of pmdc30-6xhis-galts were sequenced for full length coding regions of the 6xHis-GalT genes and partial sequences of the promoter and terminator regions. Color codes: blue: pmdc30 vector sequences; orange: the remaining pentr/d vector sequence after recombination; purple: 6xHis tag; black: GalT coding region without ATG. > pmdc30-6xhis-galt1 CAAGCTCAAGCTGCTCTAGCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATC GGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATT AAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGCCAA GCTTGCATGCCTGCAGGTCGACGGATCCCCCCTGGTAGACCAATCCTAACCAATGTCTGG TTAAGATGGTCCAATCCCGAAACTTCTAGTTGCGGTTCGAAGAAGTCCAGAATGTTTCTG AAAGTTTCAGAAAATTCTAGTTTTGAGATTTTCAGAAGTACGGCATGATGATGCATAACA AGGACTTTCTCGAAAGTACTATATTGCTCCTCTACATCATTTTAAATACCCCATGTGTCC TTTGAAGACACATCACAGAAAGAAGTGAAGGCATCGTTAGCAGTTTTGTAGATTCAACCT CAATTTGCAGAGTTACGTTCTAATATATTTACACAAGACTGGGGATCCTCTAGAGGATCC CCGGGTACCGGGCCCCCCCTCGAGGCGCGCCAAGCTATCAAACAAGTTTGTACAAAAAAG CAGGCTCCGCGGCCGCCCCCTTCACCATGCATCATCATCATCATCACAAGAGATTTTATG GAGGGCTTCTTGTTGTATCAATGTGTATGTTCTTGACGGTGTATAGATATGTAGACTTGA ACACTCCTGTTGAAAAGCCTTATATTACTGCTGCTGCTTCTGTTGTTGTTACTCCTAACA CCACTCTTCCTATGGAATGGCTGCGGATTACTCTCCCTGACTTTATGAAAGAAGCGAGGA ATACTCAAGAAGCGATATCTGGTGATGATATCGCTGTTGTCTCGGGTTTGTTTGTTGAGC AGAATGTGTCTAAAGAAGAGAGAGAGCCTTTGCTTACTTGGAACCGTTTGGAAAGTCTTG TTGATAATGCACAGAGTTTAGTTAATGGAGTTGATGCTATTAAGGAAGCTGGCATTGTTT GGGAGAGTCTTGTGTCTGCTGTTGAAGCTAAGAAACTAGTTGATGTTAATGAAAATCAGA CGAGGAAAGGAAAAGAGGAGCTTTGTCCTCAGTTTCTAAGCAAAATGAATGCTACTGAAG CTGATGGGAGTAGTCTGAAGTTGCAAATTCCTTGTGGTTTGACTCAGGGTTCCTCCATCA CAGTTATTGGCATCCCAGATGGTCTTGTTGGTAGTTTTCGGATTGATCTAACGGGACAAC CGCTTCCAGGGGAGCCTGATCCACCCATCATTGTGCATTATAATGTTAGGCTTCTTGGTG ACAAATCGACGGAAGACCCTGTGATTGTTCAAAACAGCTGGACGGCATCTCAGGACTGGG GAGCTGAGGAACGCTGTCCAAAATTTGATCCTGATATGAATAAGAAAGTGGATGACTTGG ATGAATGCAACAAGATGGTTGGTGGAGAAATTAACCGAACTTCTTCAACTAGCTTGCAGT CCAACACTTCAAGGGGAGTTCCAGTAGCCAGGGAAGCATCTAAACATGAAAAATACTTTC CTTTCAAGCAGGGTTTCTTATCGGTTGCTACACTTAGGGTGGGAACAGAGGGAATGCAGA TGACAGTTGATGGGAAACATATAACTTCATTTGCTTTCCGCGATACACTGGAACCGTGGC TTGTTAGTGAAATACGGATTACAGGTGACTTTAGGTTAATATCCATTCTTGCCAGCGGTT TGCCCACATCAGAAGAATCAGAGCACGTTGTTGATCTAGAGGCACTAAAATCACCTACCC TTTCACCATTAAGGCCACTGGATCTCGTTATTGGTGTTTTCTCCACTGCGAACAATTTTA

222 222 AAAGACGGATGGCTGTGAGGAGAACATGGATGCAGTATGATGATGTAAGATCTGGAAGAG TTGCAGTACGCTTTTTTGTTGGCCTTCACAAAAGTCCTCTTGTTAACTTGGAACTCTGGA ACGAGGCTCGGACTTACGGTGATGTTCAGCTAATGCCCTTTGTTGATTATTACAGTCTCA TCAGTTGGAAAACACTAGCCATCTGCATCTTCGGGACAGAGGTTGACTCAGCCAAGTTCA TCATGAAAACGGATGATGACGCCTTTGTTCGTGTAGATGAAGTGTTACTTTCTTTATCAA TGACCAACAACACTCGCGGGTTAATATACGGACTGATCAATTCCGACTCTCAACCTATTC GAAACCCTGATAGCAAATGGTACATCAGTTATGAGGAATGGCCTGAAGAGAAATATCCAC CATGGGCGCATGGTCCAGGCTACATTGTATCTCGTGACATAGCAGAATCGGTTGGTAAGC TTTTCAAAGAAGGAAACCTAAAGATGTTTAAGCTAGAAGATGTGGCAATGGGGATATGGA TAGCTGAGCTGACAAAACATGGACTCGAGCCTCATTACGAAAACGATGGAAGGATCATTA GTGATGGATGCAAGGATGGTTATGTGGTTGCTCATTACCAAAGCCCTGCCGAAATGACTT GCCTTTGGCGTAAATACCAAGAAACCAAACGCTCTCTTTGCTGCCGCGAATGGTAAAAGG GTGGGCGCGCCGACCCAGCTTTCTTGTACAAAGTGGTTCGATAATTCA > pmdc30-6xhis-galt3 TAATATATTTACACAAGACTGGGGATCCTCTAGAGGATCCCCCGGGTACCGGGCCCCCCC TCGAGGCGCGCCAAGCTATCAAACAAGTTTGTACAAAAAAGCAGGCTCCGCGGCCGCCCC CTTCACCATGCATCATCATCATCATCACAAGCAATTCATGTCAGTGGTGAGATTCAAATT TGGTTTCACTTCAGTCAGAATGAGGGATTGGTCGGTGGGAGTCTCCATTATGGTTCTTAC ATTGATCTTCATCATCCGTTATGAACAATCTGATCACACTCACACTGTGGATGATTCTAG TATAGAAGGAGAGAGTGTTCATGAACCCGCAAAGAAGCCACATTTTATGACTTTGGAAGA TCTTGATTATCTATTTTCAAACAAGAGCTTTTTTGGAGAAGAAGAAGTGTCCAATGGAAT GCTTGTATGGTCTCGAATGCGTCCATTTCTTGAAAGGCCAGATGCTTTGCCAGAAACTGC TCAAGGGATAGAAGAAGCTACATTGGCAATGAAAGGTTTGGTTTTAGAAATCAATAGAGA GAAGAGAGCTTATTCTTCTGGTATGGTCTCTAAGGAAATTAGAAGAATCTGTCCGGATTT TGTCACTGCATTTGATAAGGATTTGTCTGGTTTAAGTCATGTACTTCTTGAGCTTCCTTG TGGTTTAATTGAAGATTCTTCAATAACTTTGGTTGGTATTCCTGATGAACATTCTAGTAG CTTCCAGATTCAGCTCGTTGGCTCGGGATTATCAGGAGAAACTCGTCGGCCAATAATCTT GCGTTACAATGTGAACTTTTCTAAACCATCGATAGTGCAAAATACATGGACAGAGAAGCT AGGTTGGGGAAACGAAGAGCGATGCCAATATCATGGATCATTGAAAAATCATTTAGTTGA TGAACTTCCTCTCTGCAACAAACAGACCGGTAGAATCATTTCGGAAAAGAGTTCCAACGA TGATGCAACTATGGAACTTTCTCTTTCAAATGCTAATTTTCCATTTCTCAAAGGGAGTCC CTTCACTGCCGCATTGTGGTTTGGCTTAGAAGGTTTTCATATGACGATAAATGGGCGGCA CGAGACTTCATTTGCTTACAGGGAGAAGCTCGAGCCATGGTTAGTCAGTGCAGTCAAAGT CTCAGGTGGTTTGAAAATTTTATCTGTCTTAGCCACAAGACTGCCCATTCCCGATGACCA TGCATCTTTAATCATAGAAGAGAAACTTAAAGCTCCATCTCTTTCCGGGACAAGAATAGA ACTATTGGTGGGTGTTTTCTCCACTGGAAATAATTTTAAGCGGCGTATGGCATTGAGAAG ATCTTGGATGCAATACGAGGCAGTAAGATCTGGCAAAGTAGCTGTTCGATTTCTCATTGG CCTTCACACAAATGAAAAAGTCAATTTAGAGATGTGGAGAGAATCTAAGGCATATGGAGA

223 223 CATTCAGTTTATGCCATTTGTTGACTACTATGGTTTACTTAGCTTGAAAACAGTTGCGCT TTGCATTCTCGGGACCAAAGTCATCCCAGCAAAATACATAATGAAGACGGATGATGATGC GTTTGTACGGATTGATGAGCTCCTATCAAGTCTAGAAGAAAGACCGTCTAGTGCCCTTCT GTACGGTTTGATCTCATTTGATTCATCACCGGACCGTGAACAAGGCAGCAAATGGTTTAT CCCTAAAGAGGAATGGCCTTTAGATTCATACCCTCCATGGGCACATGGCCCTGGCTACAT CATCTCTCATGATATAGCGAAATTTGTGGTGAAGGGTCACCGTCAAAGAGATCTTGGACT TTTCAAGCTGGAAGATGTGGCGATGGGGATATGGATTCAACAATTCAACCAGACGATAAA AAGAGTGAAGTACATCAATGACAAAAGATTTCATAACAGTGATTGTAAATCAAATTACAT TCTTGTTCATTACCAAACTCCTAGACTAATTTTGTGTCTTTGGGAGAAGCTGCAAAAAGA GAACCAATCTATTTGCTGCGAATAAAAGGGTGGGCGCGCCGACCCAGCTTTCTTGTACAA AGTGGTTCGATAATTCTTAATTAACTAGTTCTAGAGCGGCCGCCACCGCGGTGGAGCTCG AATTCCCCGATCGTCAACA > pmdc30-6xhis-galt4 GAAGACACATCACAGAAAGAAGTGAAGGCATCGTTAGCAGTTTTGTAGATTCAACCTCAA TTTGCAGAGTTACGTTCTAATATATTTACACAAGACTGGGGATCCTCTAGAGGATCCCCG GGTACCGGGCCCCCCCTCGAGGCGCGCCAAGCTATCAAACAAGTTTGTACAAAAAAGCAG GCTCCGCGGCCGCCCCCTTCACCATGCATCATCATCATCATCACAAGAAGTCTAAACTCG ATAATTCTTCTTCACAGATTCGATTCGGGCTTGTTCAGTTCTTATTAGTTGTTCTGCTTT TTTACTTCCTCTGCATGAGCTTCGAGATCCCATTCATCTTCAGAACCGGGTCTGGGTCCG GGTCTGATGATGTTTCATCTTCTTCTTTTGCTGACGCATTACCGAGACCAATGGTTGTTG GTGGTGGTAGTAGGGAAGCTAATTGGGTTGTCGGAGAAGAAGAAGAAGCAGACCCACATC GACATTTCAAGGATCCGGGTCGGGTACAGCTTCGGTTACCGGAGCGGAAAATGAGGGAAT TTAAGTCCGTCTCTGAGATTTTCGTCAACGAGAGCTTCTTCGACAATGGCGGATTCAGCG ATGAATTCTCAATCTTTCACAAAACAGCGAAGCATGCGATTTCAATGGGTCGAAAAATGT GGGACGGACTCGATTCGGGTTTAATCAAACCCGATAAAGCTCCGGTTAAGACCCGGATTG AGAAATGTCCGGATATGGTTTCGGTTTCTGAGTCGGAGTTTGTGAACCGGAGTCGGATCT TGGTTTTGCCGTGTGGGTTAACGTTAGGATCTCACATTACCGTCGTGGCTACGCCGCATT GGGCTCACGTTGAGAAAGATGGTGATAAGACGGCGATGGTGAGTCAGTTCATGATGGAGT TACAAGGATTAAAGGCGGTGGATGGTGAAGATCCGCCTCGGATACTTCATTTTAACCCGA GGATTAAAGGTGATTGGAGTGGAAGACCAGTGATTGAGCAAAACACTTGTTATCGAATGC AATGGGGCTCAGGTTTACGTTGTGATGGTCGTGAATCTAGTGATGATGAAGAATATGTTG ATGGAGAGGTGAAATGTGAGAGGTGGAAGAGAGATGATGATGATGGTGGTAATAATGGTG ATGATTTTGATGAATCAAAGAAGACATGGTGGTTGAATAGGTTGATGGGTCGGAGGAAGA AGATGATAACACATGATTGGGATTATCCTTTTGCTGAAGGGAAGCTTTTTGTTCTTACAC TTCGAGCTGGGATGGAAGGTTATCATATTAGTGTGAATGGAAGACATATCACATCTTTTC CTTATAGAACGGGGTTTGTTTTGGAGGATGCCACTGGATTAGCAGTCAAAGGAAACATTG ATGTGCATTCTGTATATGCTGCCTCGTTACCTTCTACAAATCCTAGTTTTGCACCGCAGA AGCATCTCGAGATGCAAAGGATATGGAAAGCTCCTTCGTTACCTCAGAAGCCTGTAGAGT

224 224 TGTTCATTGGAATTCTTTCTGCTGGTAATCATTTTGCAGAGAGAATGGCAGTGAGGAAGT CATGGATGCAGCAGAAGCTGGTCAGATCATCGAAAGTTGCTGCCCGGTTCTTTGTGGCAT TGCATGCAAGAAAGGAAGTCAATGTGGATTTAAAGAAAGAAGCTGAGTACTTTGGTGATA TTGTCATAGTACCGTACATGGATCATTATGACCTTGTTGTGCTCAAGACAGTTGCCATCT GCGAATATGGGGTGAACACAGTGGCGGCAAAGTACGTTATGAAATGTGACGATGATACAT TTGTGCGTGTGGATGCTGTGATCCAGGAAGCAGAAAAGGTTAAGGGAAGAGAGAGCCTTT ATATTGGAAACATTAATTTTAACCATAAGCCATTGCGTACCGGGAAATGGGCTGTGACAT TCGAGGAATGGCCAGAAGAGTATTATCCTCCATATGCAAATGGTCCGGGTTACATCTTGT CATATGATGTAGCTAAGTTCATTGTCGATGATTTTGAACAAAAGCGATTAAGATTATTCA AGATGGAAGATGTGAGCATGGGAATGTGGGTGGAGAAGTTCAACGAGACTAGACCAGTGG CAGTGGTTCACAGCCTCAAGTTCTGTCAGTTTGGTTGCATAGAAGACTACTTCACCGCTC ATTATCAGTCGCCTCGCCAGATGATTTGCATGTGGGATAAGCTGCAGAGACTCGGGAAGC CCCAATGCTGCAACATGAGATGAAAGGGTGGGCGCGCCGACCCAGCTTTCTTGTACAAAG TGGTTCGATAATTCTTAATTAACTAGTTCTAGAGCGGCCGCCACCGCGGTGGAGCTCGAA TTCCCCGATCGTCAAACA > pmdc30-6xhis-galt5 TACATCATTTTAAATACCCCATGTGTCCTTNGAAGACACATCACAGAAAGAAGTGAAGGC ATCGTTAGCAGTTTTGTAGATTCAACCTCAATTTGCAGAGTTACGTTCTAATATATTTAC ACAAGACTGGGGATCCTCTAGAGGATCCCCGGGTACCGGGCCCCCCCTCGAGGCGCGCCA AGCTATCAAACAAGTTTGTACAAAAAAGCAGGCTCCGCGGCCGCCCCCTTCACCATGCAT CATCATCATCATCACAAAAAACCCAAATTGTCGAAAGTGGAAAAAATCGACAAGATTGAT CTGTTCTCTTCACTATGGAAGCAGAGATCGGTTCGTGTAATAATGGCAATAGGGTTTCTC TATCTTGTAATTGTCTCAGTAGAGATACCTCTCGTTTTCAAATCCTGGTCCAGCAGCTCC GTGCCTCTTGATGCTCTTTCTCGACTCGAGAAGCTCAATAACGAGCAAGAGCCCCAAGTC GAGATTATCCCTAATCCTCCATTGGAGCCAGTTTCGTACCCGGTTTCGAACCCGACCATT GTTACCCGGACGGACCTTGTTCAGAACAAGGTCCGTGAACATCATCGGGGTGTTCTCTCA AGTTTGAGATTTGATTCGGAAACTTTCGACCCGAGTAGTAAAGACGGGTCAGTGGAGCTT CACAAGTCAGCTAAGGAAGCTTGGCAGCTAGGTCGCAAGCTATGGAAGGAGCTTGAATCT GGAAGGCTTGAGAAACTAGTGGAGAAGCCAGAGAAGAACAAACCGGATTCATGTCCACAT TCTGTTTCGCTAACCGGGTCTGAGTTTATGAACCGGGAGAACAAATTGATGGAGCTGCCG TGTGGTTTGACATTGGGTTCACACATAACTTTGGTGGGGAGGCCGAGGAAAGCTCATCCC AAGGAAGGAGATTGGTCTAAGTTGGTGTCTCAGTTTGTGATAGAGCTTCAAGGTTTGAAG ACTGTTGAAGGAGAGGATCCTCCTAGGATTCTGCATTTCAATCCGAGGCTTAAGGGAGAT TGGAGCAAAAAACCGGTGATTGAGCAGAATAGTTGCTATAGGATGCAATGGGGACCTGCA CAACGTTGCGAAGGATGGAAGTCAAGAGATGATGAAGAGACTGTTGATAGTCATGTGAAG TGTGAAAAATGGATTCGTGATGATGACAATTACTCAGAAGGGTCGAGGGCAAGATGGTGG TTGAATAGACTTATAGGAAGGAGGAAAAGGGTCAAAGTAGAATGGCCGTTTCCTTTTGTG GAAGAGAAGCTGTTCGTTCTAACTCTTAGCGCCGGTTTAGAGGGTTACCATATCAATGTT

225 225 GATGGAAAGCATGTTACTTCTTTCCCTTATCGCACTGGTTTCACCCTTGAGGATGCAACA GGGCTAACAGTAAACGGAGACATTGATGTCCATTCTGTTTTTGTTGCCTCTCTGCCAACA TCACATCCTAGTTTTGCTCCCCAAAGGCATCTCGAATTGTCAAAGAGATGGCAGGCTCCT GTAGTTCCCGATGGGCCTGTGGAGATCTTTATAGGCATTCTTTCCGCAGGCAATCATTTC TCTGAGCGGATGGCTGTGAGGAAATCCTGGATGCAGCATGTTCTTATTACATCTGCAAAA GTTGTTGCTCGTTTCTTTGTGGCGCTGCATGGGAGGAAGGAGGTGAATGTGGAATTGAAG AAAGAAGCGGAGTATTTTGGGGACATTGTACTTGTTCCTTACATGGATAGCTATGATCTT GTCGTGCTGAAAACTGTTGCCATATGTGAACACGGAGCTCTTGCATTCTCTGCAAAGTAC ATAATGAAGTGTGACGATGATACATTTGTAAAACTTGGCGCGGTGATCAATGAAGTGAAA AAAGTACCCGAAGGTAGAAGCCTGTACATTGGTAACATGAATTATTACCACAAACCTCTC CGTGGGGGTAAATGGGCAGTCACATATGAGGAATGGCCAGAGGAGGACTATCCGCCCTAC GCAAATGGACCCGGATATGTTCTATCTTCTGACATTGCGCGCTTCATCGTGGACAAGTTT GAGAGACATAAATTACGGCTGTTCAAGATGGAGGACGTGAGTGTGGGAATGTGGGTTGAG CATTTCAAGAACACAACAAACCCAGTGGATTACAGACACAGTCTGAGATTCTGCCAGTTT GGTTGTGTTGAGAACTACTACACAGCTCATTACCAGTCGCCAAGACAGATGATATGCTTA TGGGATAAGCTCTTAAGACAGAACAAGCCTGAGTGTTGTAACATGAGATGAAAGGGTGGG CGCGCCGACCCAGCTTTCTTGTACAAAGTGGTTCGATAATTCTTAATTAACTAGTTCTAG AGCGGCCGCCACCGCGGTGGAGCTCGAATTCCCCGATCGTCAACA > pmdc30-6xhis-galt6 CCAAGCTCAAGCTGCTNTAGCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGAT CGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGAT TAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGCCA AGCTTGCATGCCTGCAGGTCGACGGATCCCCCCTGGTAGACCAATCCTAACCAATGTCTG GTTAAGATGGTCCAATCCCGAAACTTCTAGTTGCGGTTCGAAGAAGTCCAGAATGTTTCT GAAAGTTTCAGAAAATTCTAGTTTTGAGATTTTCAGAAGTACGGCATGATGATGCATAAC AAGGACTTTCTCGAAAGTACTATATTGCTCCTCTACATCATTTTAAATACCCCATGTGTC CTTTGAAGACACATCACAGAAAGAAGTGAAGGCATCGTTAGCAGTTTTGTAGATTCAACC TCAATTTGCAGAGTTACGTTCTAATATATTTACACAAGACTGGGGATCCTCTAGAGGATC CCCGGGTACCGGGCCCCCCCTCGAGGCGCGCCAAGCTATCAAACAAGTTTGTACAAAAAA GCAGGCTCCGCGGCCGCCCCCTTCACCATGCATCATCATCATCATCACAGGAAGCCCAAG TTGTCAAAACTCGAGAGGTTAGAGAAATTCGACATTTTCGTTTCTCTAAGCAAGCAGAGA TCGGTTCAGATACTAATGGCGGTTGGGTTACTCTACATGCTTCTTATCACATTCGAAATC CCTTTCGTCTTCAAAACCGGGCTTAGTTCTTTATCTCAGGATCCGTTAACCCGACCCGAG AAGCACAATAGTCAGAGAGAGTTACAAGAGAGACGAGCTCCGACTCGACCTTTAAAGAGT CTGCTTTACCAGGAATCACAATCGGAATCACCGGCTCAGGGTTTAAGAAGAAGGACTCGG ATCCTTTCTAGTTTGAGATTCGACCCGGAAACGTTTAACCCGAGTAGCAAAGATGGGTCT GTGGAGCTTCATAAATCTGCTAAGGTAGCTTGGGAAGTTGGTCGAAAGATATGGGAAGAG CTTGAGTCTGGGAAAACGTTGAAAGCTTTGGAGAAGGAGAAGAAGAAGAAGATTGAGGAA

226 CATGGGACAAACTCGTGTTCTCTCTCTGTTTCCTTAACCGGGTCTGATCTTTTGAAACGT GGGAATATCATGGAGCTTCCATGTGGTTTAACTCTTGGGTCACATATTACAGTGGTTGGG AAGCCACGAGCTGCTCATTCGGAGAAGGACCCTAAGATATCGATGTTAAAGGAAGGAGAT GAAGCTGTGAAGGTTTCACAGTTTAAGTTGGAGCTTCAGGGTTTGAAAGCAGTGGAAGGA GAAGAGCCACCTCGGATACTCCACTTGAATCCAAGGCTTAAGGGTGATTGGAGTGGTAAG CCTGTGATTGAGCAGAACACTTGCTATAGAATGCAATGGGGCTCAGCACAAAGATGTGAA GGATGGAGATCTAGGGATGATGAAGAGACTGTTGATGGTCAGGTTAAGTGCGAGAAATGG GCTCGGGATGATAGCATTACATCTAAAGAAGAAGAGTCTAGCAAGGCGGCTTCATGGTGG CTTAGTCGATTAATAGGTCGGAGCAAGAAAGTAACTGTTGAATGGCCATTTCCATTCACA GTTGACAAGCTTTTCGTGCTTACTCTTAGTGCTGGATTGGAAGGCTACCATGTTAGTGTC GATGGGAAGCATGTCACTTCCTTTCCATACCGAACTGGATTTACGCTTGAGGATGCTACT GGTCTAACCATTAACGGGGACATAGATGTTCACTCTGTTTTCGCTGGCTCTCTCCCAACC TCGCATCCTAGTTTTTCTCCTCAGAGGCATCTTGAGCTCTCGAGCAATTGGCAAGCCCCA TCACTTCCTGATGAGCAAGTTGATATGTTCATTGGTATCCTTTCTGCTGGTAACCATTTT GCTGAGAGGATGGCTGTGAGGAGGTCGTGGATGCAACATAAACTCGTTAAATCTTCCAAA GTAGTGGCTCGGTTCTTTGTTGCACTGCACTCAAGGAAAGAAGTAAATGTGGAGCTAAAG AAGGAAGCTGAATTCTTTGGGGACATAGTTATAGTCCCTTACATGGACAGTTATGACCTT GTCGTCCTCAAAACCGTTGCAATTTGCGAGTACGGGGCTCATCAACTTGCAGCTAAATTC ATCATGAAGTGTGATGACGATACATTTGTACAAGTGGATGCGGTTCTTAGTGAAGCAAAG AAAACACCCACAGATAGAAGTCTATACATTGGCAACATCAATTATTATCACAAACCACTT CGCCAGGGTAAATGGTCTGTTACATATGAGGAATGGCCAGAGGAAGACTATCCACCTTAT GCTAATGGCCCCGGATACATATTATCAAACGATATATCTCGCTTTATCGTGAAAGAGTTT GAGAAACACAAATTAAGGATGTTCAAAATGGAAGATGTAAGCGTGGGAATGTGGGTAGAA CAATTCAACAATGGTACAAAACCGGTGGACTACATTCACAGCCTCAGGTTTTGTCAGTTT GGTTGCATAGAGAATTACTTGACGGCGCATTATCAGTCGCCGAGACAGATGATTTGCTTG TGGGATAAGCTGGTGTTGACAGGCAAACCTCAGTGCTGCAACATGAGATGAAAGGGTGGG CGCGCCGACCCAGCTTTCTTGTACAAAGTGGTTCGATAATTCTTAATTAACTAGTTCTAG AGCGGCCGCCACCGCGGTGGAGCTCGAATTCCCCGATCGTCAA 226

227 227 Part III. Sequencing results of the pmdc32-6xhis-galt constructs. The expression constructs of pmdc32-6xhis-galts were sequenced for full length coding regions of the 6xHis-GalT genes and partial sequences of the promoter and terminator regions. Color codes: blue: pmdc32 vector sequences; orange: the remaining pentr/d vector sequence after recombination; purple: 6xHis tag; black: GalT coding region without ATG. >pmdc32-6xhis-galt1 AAAAGGAAGGTGGCTCCTACAAATGCCATCATTGCGATAAAGGAAAGGCCATCGTTGAAG ATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAA AAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATAACATGGTGGAGC ACGACACACTTGTCTACTCCAAAAATATCAAAGATACAGTCTCAGAAGACCAAAGGGCAA TTGAGACTTTTCAACAAAGGGTAATATCCGGAAACCTCCTCGGATTCCATTGCCCAGCTA TCTGTCACTTTATTGTGAAGATAGTGGAAAAGGAAGGTGGCTCCTACAAATGCCATCATT GCGATAAAGGAAAGGCCATCGTTGAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGAC CCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAG TGGATTGATGTGATATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGC AAGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGACCTCGACTCTAGAGG ATCCCCGGGTACCGGGCCCCCCCTCGAGGCGCGCCAAGCTATCAAACAAGTTTGTACAAA AAAGCAGGCTCCGCGGCCGCCCCCTTCACCATGCATCATCATCATCATCACAAGAGATTT TATGGAGGGCTTCTTGTTGTATCAATGTGTATGTTCTTGACGGTGTATAGATATGTAGAC TTGAACACTCCTGTTGAAAAGCCTTATATTACTGCTGCTGCTTCTGTTGTTGTTACTCCT AACACCACTCTTCCTATGGAATGGCTGCGGATTACTCTCCCTGACTTTATGAAAGAAGCG AGGAATACTCAAGAAGCGATATCTGGTGATGATATCGCTGTTGTCTCGGGTTTGTTTGTT GAGCAGAATGTGTCTAAAGAAGAGAGAGAGCCTTTGCTTACTTGGAACCGTTTGGAAAGT CTTGTTGATAATGCACAGAGTTTAGTTAATGGAGTTGATGCTATTAAGGAAGCTGGCATT GTTTGGGAGAGTCTTGTGTCTGCTGTTGAAGCTAAGAAACTAGTTGATGTTAATGAAAAT CAGACGAGGAAAGGAAAAGAGGAGCTTTGTCCTCAGTTTCTAAGCAAAATGAATGCTACT GAAGCTGATGGGAGTAGTCTGAAGTTGCAAATTCCTTGTGGTTTGACTCAGGGTTCCTCC ATCACAGTTATTGGCATCCCAGATGGTCTTGTTGGTAGTTTTCGGATTGATCTAACGGGA CAACCGCTTCCAGGGGAGCCTGATCCACCCATCATTGTGCATTATAATGTTAGGCTTCTT GGTGACAAATCGACGGAAGACCCTGTGATTGTTCAAAACAGCTGGACGGCATCTCAGGAC TGGGGAGCTGAGGAACGCTGTCCAAAATTTGATCCTGATATGAATAAGAAAGTGGATGAC TTGGATGAATGCAACAAGATGGTTGGTGGAGAAATTAACCGAACTTCTTCAACTAGCTTG CAGTCCAACACTTCAAGGGGAGTTCCAGTAGCCAGGGAAGCATCTAAACATGAAAAATAC TTTCCTTTCAAGCAGGGTTTCTTATCGGTTGCTACACTTAGGGTGGGAACAGAGGGAATG CAGATGACAGTTGATGGGAAACATATAACTTCATTTGCTTTCCGCGATACACTGGAACCG TGGCTTGTTAGTGAAATACGGATTACAGGTGACTTTAGGTTAATATCCATTCTTGCCAGC GGTTTGCCCACATCAGAAGAATCAGAGCACGTTGTTGATCTAGAGGCACTAAAATCACCT

228 228 ACCCTTTCACCATTAAGGCCACTGGATCTCGTTATTGGTGTTTTCTCCACTGCGAACAAT TTTAAAAGACGGATGGCTGTGAGGAGAACATGGATGCAGTATGATGATGTAAGATCTGGA AGAGTTGCAGTACGCTTTTTTGTTGGCCTTCACAAAAGTCCTCTTGTTAACTTGGAACTC TGGAACGAGGCTCGGACTTACGGTGATGTTCAGCTAATGCCCTTTGTTGATTATTACAGT CTCATCAGTTGGAAAACACTAGCCATCTGCATCTTCGGGACAGAGGTTGACTCAGCCAAG TTCATCATGAAAACGGATGATGACGCCTTTGTTCGTGTAGATGAAGTGTTACTTTCTTTA TCAATGACCAACAACACTCGCGGGTTAATATACGGACTGATCAATTCCGACTCTCAACCT ATTCGAAACCCTGATAGCAAATGGTACATCAGTTATGAGGAATGGCCTGAAGAGAAATAT CCACCATGGGCGCATGGTCCAGGCTACATTGTATCTCGTGACATAGCAGAATCGGTTGGT AAGCTTTTCAAAGAAGGAAACCTAAAGATGTTTAAGCTAGAAGATGTGGCAATGGGGATA TGGATAGCTGAGCTGACAAAACATGGACTCGAGCCTCATTACGAAAACGATGGAAGGATC ATTAGTGATGGATGCAAGGATGGTTATGTGGTTGCTCATTACCAAAGCCCTGCCGAAATG ACTTGCCTTTGGCGTAAATACCAAGAAACCAAACGCTCTCTTTGCTGCCGCGAATGGTAA AAGGGTGGGCGCGCCGACCCAGCTTTCTTGTACAAAGTGGTTCGATAATTCTTAATTAAC TAGTTCTAGAGCGGCCGCCACCGCGGTGGAGCTCGAATTTCCCCGATCGTTCAAACATTT GGCAATAAAGTTTCTTAAGATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATATAAT TTCTGTTGAATTACGTTAAGCATGTAATAATTAACATGTAATGCATGACGTTATTTATGA GATGGGTTTTTATGATTAGAGTCCCGCAATTATACNATTAATACGCGATAGAAAACAAAA TATAGCGCGCAAACTAGGATAACANG >pmdc32-6xhis-galt3 CTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGA AGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAG GGATGACGCACAATCCCACTATCCTTCGCAAGACCCTTCCTCTATATAAGGAAGTTCATT TCATTTGGAGAGGACCTCGACTCTAGAGGATCCCCGGGTACCGGGCCCCCCCTCGAGGCG CGCCAAGCTATCAAACAAGTTTGTACAAAAAAGCAGGCTCCGCGGCCGCCCCCTTCACCA TGCATCATCATCATCATCACAAGCAATTCATGTCAGTGGTGAGATTCAAATTTGGTTTCA CTTCAGTCAGAATGAGGGATTGGTCGGTGGGAGTCTCCATTATGGTTCTTACATTGATCT TCATCATCCGTTATGAACAATCTGATCACACTCACACTGTGGATGATTCTAGTATAGAAG GAGAGAGTGTTCATGAACCCGCAAAGAAGCCACATTTTATGACTTTGGAAGATCTTGATT ATCTATTTTCAAACAAGAGCTTTTTTGGAGAAGAAGAAGTGTCCAATGGAATGCTTGTAT GGTCTCGAATGCGTCCATTTCTTGAAAGGCCAGATGCTTTGCCAGAAACTGCTCAAGGGA TAGAAGAAGCTACATTGGCAATGAAAGGTTTGGTTTTAGAAATCAATAGAGAGAAGAGAG CTTATTCTTCTGGTATGGTCTCTAAGGAAATTAGAAGAATCTGTCCGGATTTTGTCACTG CATTTGATAAGGATTTGTCTGGTTTAAGTCATGTACTTCTTGAGCTTCCTTGTGGTTTAA TTGAAGATTCTTCAATAACTTTGGTTGGTATTCCTGATGAACATTCTAGTAGCTTCCAGA TTCAGCTCGTTGGCTCGGGATTATCAGGAGAAACTCGTCGGCCAATAATCTTGCGTTACA ATGTGAACTTTTCTAAACCATCGATAGTGCAAAATACATGGACAGAGAAGCTAGGTTGGG GAAACGAAGAGCGATGCCAATATCATGGATCATTGAAAAATCATTTAGTTGATGAACTTC

229 229 CTCTCTGCAACAAACAGACCGGTAGAATCATTTCGGAAAAGAGTTCCAACGATGATGCAA CTATGGAACTTTCTCTTTCAAATGCTAATTTTCCATTTCTCAAAGGGAGTCCCTTCACTG CCGCATTGTGGTTTGGCTTAGAAGGTTTTCATATGACGATAAATGGGCGGCACGAGACTT CATTTGCTTACAGGGAGAAGCTCGAGCCATGGTTAGTCAGTGCAGTCAAAGTCTCAGGTG GTTTGAAAATTTTATCTGTCTTAGCCACAAGACTGCCCATTCCCGATGACCATGCATCTT TAATCATAGAAGAGAAACTTAAAGCTCCATCTCTTTCCGGGACAAGAATAGAACTATTGG TGGGTGTTTTCTCCACTGGAAATAATTTTAAGCGGCGTATGGCATTGAGAAGATCTTGGA TGCAATACGAGGCAGTAAGATCTGGCAAAGTAGCTGTTCGATTTCTCATTGGCCTTCACA CAAATGAAAAAGTCAATTTAGAGATGTGGAGAGAATCTAAGGCATATGGAGACATTCAGT TTATGCCATTTGTTGACTACTATGGTTTACTTAGCTTGAAAACAGTTGCGCTTTGCATTC TCGGGACCAAAGTCATCCCAGCAAAATACATAATGAAGACGGATGATGATGCGTTTGTAC GGATTGATGAGCTCCTATCAAGTCTAGAAGAAAGACCGTCTAGTGCCCTTCTGTACGGTT TGATCTCATTTGATTCATCACCGGACCGTGAACAAGGCAGCAAATGGTTTATCCCTAAAG AGGAATGGCCTTTAGATTCATACCCTCCATGGGCACATGGCCCTGGCTACATCATCTCTC ATGATATAGCGAAATTTGTGGTGAAGGGTCACCGTCAAAGAGATCTTGGACTTTTCAAGC TGGAAGATGTGGCGATGGGGATATGGATTCAACAATTCAACCAGACGATAAAAAGAGTGA AGTACATCAATGACAAAAGATTTCATAACAGTGATTGTAAATCAAATTACATTCTTGTTC ATTACCAAACTCCTAGACTAATTTTGTGTCTTTGGGAGAAGCTGCAAAAAGAGAACCAAT CTATTTGCTGCGAATAAAAGGGTGGGCGCGCCGACCCAGCTTTCTTGTACAAAGTGGTTC GATAATTCTTAATTAACTAGTTCTAGAGCGGCCGCCACCGCGGTGGAGCTCGAACANG >pmdc32-6xhis-galt4 ACGTCTTCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATGACGCACAA TCCCACTATCCTTCGCAAGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGG ACCTCGACTCTAGAGGATCCCCGGGTACCGGGCCCCCCCTCGAGGCGCGCCAAGCTATCA AACAAGTTTGTACAAAAAAGCAGGCTCCGCGGCCGCCCCCTTCACCATGCATCATCATCA TCATCACAAGAAGTCTAAACTCGATAATTCTTCTTCACAGATTCGATTCGGGCTTGTTCA GTTCTTATTAGTTGTTCTGCTTTTTTACTTCCTCTGCATGAGCTTCGAGATCCCATTCAT CTTCAGAACCGGGTCTGGGTCCGGGTCTGATGATGTTTCATCTTCTTCTTTTGCTGACGC ATTACCGAGACCAATGGTTGTTGGTGGTGGTAGTAGGGAAGCTAATTGGGTTGTCGGAGA AGAAGAAGAAGCAGACCCACATCGACATTTCAAGGATCCGGGTCGGGTACAGCTTCGGTT ACCGGAGCGGAAAATGAGGGAATTTAAGTCCGTCTCTGAGATTTTCGTCAACGAGAGCTT CTTCGACAATGGCGGATTCAGCGATGAATTCTCAATCTTTCACAAAACAGCGAAGCATGC GATTTCAATGGGTCGAAAAATGTGGGACGGACTCGATTCGGGTTTAATCAAACCCGATAA AGCTCCGGTTAAGACCCGGATTGAGAAATGTCCGGATATGGTTTCGGTTTCTGAGTCGGA GTTTGTGAACCGGAGTCGGATCTTGGTTTTGCCGTGTGGGTTAACGTTAGGATCTCACAT TACCGTCGTGGCTACGCCGCATTGGGCTCACGTTGAGAAAGATGGTGATAAGACGGCGAT GGTGAGTCAGTTCATGATGGAGTTACAAGGATTAAAGGCGGTGGATGGTGAAGATCCGCC TCGGATACTTCATTTTAACCCGAGGATTAAAGGTGATTGGAGTGGAAGACCAGTGATTGA

230 230 GCAAAACACTTGTTATCGAATGCAATGGGGCTCAGGTTTACGTTGTGATGGTCGTGAATC TAGTGATGATGAAGAATATGTTGATGGAGAGGTGAAATGTGAGAGGTGGAAGAGAGATGA TGATGATGGTGGTAATAATGGTGATGATTTTGATGAATCAAAGAAGACATGGTGGTTGAA TAGGTTGATGGGTCGGAGGAAGAAGATGATAACACATGATTGGGATTATCCTTTTGCTGA AGGGAAGCTTTTTGTTCTTACACTTCGAGCTGGGATGGAAGGTTATCATATTAGTGTGAA TGGAAGACATATCACATCTTTTCCTTATAGAACGGGGTTTGTTTTGGAGGATGCCACTGG ATTAGCAGTCAAAGGAAACATTGATGTGCATTCTGTATATGCTGCCTCGTTACCTTCTAC AAATCCTAGTTTTGCACCGCAGAAGCATCTCGAGATGCAAAGGATATGGAAAGCTCCTTC GTTACCTCAGAAGCCTGTAGAGTTGTTCATTGGAATTCTTTCTGCTGGTAATCATTTTGC AGAGAGAATGGCAGTGAGGAAGTCATGGATGCAGCAGAAGCTGGTCAGATCATCGAAAGT TGCTGCCCGGTTCTTTGTGGCATTGCATGCAAGAAAGGAAGTCAATGTGGATTTAAAGAA AGAAGCTGAGTACTTTGGTGATATTGTCATAGTACCGTACATGGATCATTATGACCTTGT TGTGCTCAAGACAGTTGCCATCTGCGAATATGGGGTGAACACAGTGGCGGCAAAGTACGT TATGAAATGTGACGATGATACATTTGTGCGTGTGGATGCTGTGATCCAGGAAGCAGAAAA GGTTAAGGGAAGAGAGAGCCTTTATATTGGAAACATTAATTTTAACCATAAGCCATTGCG TACCGGGAAATGGGCTGTGACATTCGAGGAATGGCCAGAAGAGTATTATCCTCCATATGC AAATGGTCCGGGTTACATCTTGTCATATGATGTAGCTAAGTTCATTGTCGATGATTTTGA ACAAAAGCGATTAAGATTATTCAAGATGGAAGATGTGAGCATGGGAATGTGGGTGGAGAA GTTCAACGAGACTAGACCAGTGGCAGTGGTTCACAGCCTCAAGTTCTGTCAGTTTGGTTG CATAGAAGACTACTTCACCGCTCATTATCAGTCGCCTCGCCAGATGATTTGCATGTGGGA TAAGCTGCAGAGACTCGGGAAGCCCCAATGCTGCAACATGAGATGAAAGGGTGGGCGCGC CGACCCAGCTTTCTTGTACAAAGTGGTTCGATAATTCTTAATTAACTAGTTCTAGAGCGG CCGCCACCGCGGTGGAGCTCGAATTCCCCGATCGTCAACA >pmdc32-6xhis-galt5 AAGTTCATTTCATTTGGAGAGGACCTCGACTCTAGAGGATCCCCGGGTACCGGGCCCCCC CTCGAGGCGCGCCAAGCTATCAAACAAGTTTGTACAAAAAAGCAGGCTCCGCGGCCGCCC CCTTCACCATGCATCATCATCATCATCACAAAAAACCCAAATTGTCGAAAGTGGAAAAAA TCGACAAGATTGATCTGTTCTCTTCACTATGGAAGCAGAGATCGGTTCGTGTAATAATGG CAATAGGGTTTCTCTATCTTGTAATTGTCTCAGTAGAGATACCTCTCGTTTTCAAATCCT GGTCCAGCAGCTCCGTGCCTCTTGATGCTCTTTCTCGACTCGAGAAGCTCAATAACGAGC AAGAGCCCCAAGTCGAGATTATCCCTAATCCTCCATTGGAGCCAGTTTCGTACCCGGTTT CGAACCCGACCATTGTTACCCGGACGGACCTTGTTCAGAACAAGGTCCGTGAACATCATC GGGGTGTTCTCTCAAGTTTGAGATTTGATTCGGAAACTTTCGACCCGAGTAGTAAAGACG GGTCAGTGGAGCTTCACAAGTCAGCTAAGGAAGCTTGGCAGCTAGGTCGCAAGCTATGGA AGGAGCTTGAATCTGGAAGGCTTGAGAAACTAGTGGAGAAGCCAGAGAAGAACAAACCGG ATTCATGTCCACATTCTGTTTCGCTAACCGGGTCTGAGTTTATGAACCGGGAGAACAAAT TGATGGAGCTGCCGTGTGGTTTGACATTGGGTTCACACATAACTTTGGTGGGGAGGCCGA GGAAAGCTCATCCCAAGGAAGGAGATTGGTCTAAGTTGGTGTCTCAGTTTGTGATAGAGC

231 231 TTCAAGGTTTGAAGACTGTTGAAGGAGAGGATCCTCCTAGGATTCTGCATTTCAATCCGA GGCTTAAGGGAGATTGGAGCAAAAAACCGGTGATTGAGCAGAATAGTTGCTATAGGATGC AATGGGGACCTGCACAACGTTGCGAAGGATGGAAGTCAAGAGATGATGAAGAGACTGTTG ATAGTCATGTGAAGTGTGAAAAATGGATTCGTGATGATGACAATTACTCAGAAGGGTCGA GGGCAAGATGGTGGTTGAATAGACTTATAGGAAGGAGGAAAAGGGTCAAAGTAGAATGGC CGTTTCCTTTTGTGGAAGAGAAGCTGTTCGTTCTAACTCTTAGCGCCGGTTTAGAGGGTT ACCATATCAATGTTGATGGAAAGCATGTTACTTCTTTCCCTTATCGCACTGGTTTCACCC TTGAGGATGCAACAGGGCTAACAGTAAACGGAGACATTGATGTCCATTCTGTTTTTGTTG CCTCTCTGCCAACATCACATCCTAGTTTTGCTCCCCAAAGGCATCTCGAATTGTCAAAGA GATGGCAGGCTCCTGTAGTTCCCGATGGGCCTGTGGAGATCTTTATAGGCATTCTTTCCG CAGGCAATCATTTCTCTGAGCGGATGGCTGTGAGGAAATCCTGGATGCAGCATGTTCTTA TTACATCTGCAAAAGTTGTTGCTCGTTTCTTTGTGGCGCTGCATGGGAGGAAGGAGGTGA ATGTGGAATTGAAGAAAGAAGCGGAGTATTTTGGGGACATTGTACTTGTTCCTTACATGG ATAGCTATGATCTTGTCGTGCTGAAAACTGTTGCCATATGTGAACACGGAGCTCTTGCAT TCTCTGCAAAGTACATAATGAAGTGTGACGATGATACATTTGTAAAACTTGGCGCGGTGA TCAATGAAGTGAAAAAAGTACCCGAAGGTAGAAGCCTGTACATTGGTAACATGAATTATT ACCACAAACCTCTCCGTGGGGGTAAATGGGCAGTCACATATGAGGAATGGCCAGAGGAGG ACTATCCGCCCTACGCAAATGGACCCGGATATGTTCTATCTTCTGACATTGCGCGCTTCA TCGTGGACAAGTTTGAGAGACATAAATTACGGCTGTTCAAGATGGAGGACGTGAGTGTGG GAATGTGGGTTGAGCATTTCAAGAACACAACAAACCCAGTGGATTACAGACACAGTCTGA GATTCTGCCAGTTTGGTTGTGTTGAGAACTACTACACAGCTCATTACCAGTCGCCAAGAC AGATGATATGCTTATGGGATAAGCTCTTAAGACAGAACAAGCCTGAGTGTTGTAACATGA GATGAAAGGGTGGGCGCGCCGACCCAGCTTTCTTGTACAAAGTGGTTCGATAATTCTTAA TTAACTAGTTCTAGAGCGGCCGCCACCGCGGTGGAGCTCGAANTCCCCGATCGTCAAACA >pmdc32-6xhis-galt6 TGCCGACCNGTGGTCCCAAAAGATGGACCCCCCNNCCCACGAGGAGCATCGTGGAAAAAG NAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATATCTCCNCTGACGTA AGGGATGACGCACAATCCCACTATCNTTCGCAAGACCCTTCCTCTATATAAGGAAGTTCA TTTCATTTGGAGAGGACCTCGACTCTAGAGGATCCCCGGGTACCGGGCCCCCCCTCGAGG CGCGCCAAGCTATCAAACAAGTTTGTACAAAAAAGCAGGCTCCGCGGCCGCCCCCTTCAC CATGCATCATCATCATCATCACAGGAAGCCCAAGTTGTCAAAACTCGAGAGGTTAGAGAA ATTCGACATTTTCGTTTCTCTAAGCAAGCAGAGATCGGTTCAGATACTAATGGCGGTTGG GTTACTCTACATGCTTCTTATCACATTCGAAATCCCTTTCGTCTTCAAAACCGGGCTTAG TTCTTTATCTCAGGATCCGTTAACCCGACCCGAGAAGCACAATAGTCAGAGAGAGTTACA AGAGAGACGAGCTCCGACTCGACCTTTAAAGAGTCTGCTTTACCAGGAATCACAATCGGA ATCACCGGCTCAGGGTTTAAGAAGAAGGACTCGGATCCTTTCTAGTTTGAGATTCGACCC GGAAACGTTTAACCCGAGTAGCAAAGATGGGTCTGTGGAGCTTCATAAATCTGCTAAGGT AGCTTGGGAAGTTGGTCGAAAGATATGGGAAGAGCTTGAGTCTGGGAAAACGTTGAAAGC

232 TTTGGAGAAGGAGAAGAAGAAGAAGATTGAGGAACATGGGACAAACTCGTGTTCTCTCTC TGTTTCCTTAACCGGGTCTGATCTTTTGAAACGTGGGAATATCATGGAGCTTCCATGTGG TTTAACTCTTGGGTCACATATTACAGTGGTTGGGAAGCCACGAGCTGCTCATTCGGAGAA GGACCCTAAGATATCGATGTTAAAGGAAGGAGATGAAGCTGTGAAGGTTTCACAGTTTAA GTTGGAGCTTCAGGGTTTGAAAGCAGTGGAAGGAGAAGAGCCACCTCGGATACTCCACTT GAATCCAAGGCTTAAGGGTGATTGGAGTGGTAAGCCTGTGATTGAGCAGAACACTTGCTA TAGAATGCAATGGGGCTCAGCACAAAGATGTGAAGGATGGAGATCTAGGGATGATGAAGA GACTGTTGATGGTCAGGTTAAGTGCGAGAAATGGGCTCGGGATGATAGCATTACATCTAA AGAAGAAGAGTCTAGCAAGGCGGCTTCATGGTGGCTTAGTCGATTAATAGGTCGGAGCAA GAAAGTAACTGTTGAATGGCCATTTCCATTCACAGTTGACAAGCTTTTCGTGCTTACTCT TAGTGCTGGATTGGAAGGCTACCATGTTAGTGTCGATGGGAAGCATGTCACTTCCTTTCC ATACCGAACTGGATTTACGCTTGAGGATGCTACTGGTCTAACCATTAACGGGGACATAGA TGTTCACTCTGTTTTCGCTGGCTCTCTCCCAACCTCGCATCCTAGTTTTTCTCCTCAGAG GCATCTTGAGCTCTCGAGCAATTGGCAAGCCCCATCACTTCCTGATGAGCAAGTTGATAT GTTCATTGGTATCCTTTCTGCTGGTAACCATTTTGCTGAGAGGATGGCTGTGAGGAGGTC GTGGATGCAACATAAACTCGTTAAATCTTCCAAAGTAGTGGCTCGGTTCTTTGTTGCACT GCACTCAAGGAAAGAAGTAAATGTGGAGCTAAAGAAGGAAGCTGAATTCTTTGGGGACAT AGTTATAGTCCCTTACATGGACAGTTATGACCTTGTCGTCCTCAAAACCGTTGCAATTTG CGAGTACGGGGCTCATCAACTTGCAGCTAAATTCATCATGAAGTGTGATGACGATACATT TGTACAAGTGGATGCGGTTCTTAGTGAAGCAAAGAAAACACCCACAGATAGAAGTCTATA CATTGGCAACATCAATTATTATCACAAACCACTTCGCCAGGGTAAATGGTCTGTTACATA TGAGGAATGGCCAGAGGAAGACTATCCACCTTATGCTAATGGCCCCGGATACATATTATC AAACGATATATCTCGCTTTATCGTGAAAGAGTTTGAGAAACACAAATTAAGGATGTTCAA AATGGAAGATGTAAGCGTGGGAATGTGGGTAGAACAATTCAACAATGGTACAAAACCGGT GGACTACATTCACAGCCTCAGGTTTTGTCAGTTTGGTTGCATAGAGAATTACTTGACGGC GCATTATCAGTCGCCGAGACAGATGATTTGCTTGTGGGATAAGCTGGTGTTGACAGGCAA ACCTCAGTGCTGCAACATGAGATGAAAGGGTGGGCGCGCCGACCCAGCTTTCTTGTACAA AGTGGTTCGATAATTCTTAATTAACTAGTTCTAGAGCGGCCGCCACCGCGGTGGAGCTCG AACANG 232

233 233 Part IV. The sequencing result of the pvkh18en6-galt3-yfp construct The expression construct of pvkh18en6-6xhis-galt3-yfp were sequenced for full length coding region of the 6xHis-GalT3 gene and partial sequences of the vector sequence. Color codes: blue: pvkh18en6 vector sequences; black: GalT coding region without the stop codon TAA; red: single nucleotide mutation from T to C at nucleotide position 1446 for cloning. The amino acid encoded is not affected by the mutation. The YFP on the pvkh18en6 construct is in frame with the GalT3 gene. CAACAACATTACATTTTACATTCTACAACTACATCTAGATGAAGCAATTCATGTCAGTGG TGAGATTCAAATTTGGTTTCACTTCAGTCAGAATGAGGGATTGGTCGGTGGGAGTCTCCA TTATGGTTCTTACATTGATCTTCATCATCCGTTATGAACAATCTGATCACACTCACACTG TGGATGATTCTAGTATAGAAGGAGAGAGTGTTCATGAACCCGCAAAGAAGCCACATTTTA TGACTTTGGAAGATCTTGATTATCTATTTTCAAACAAGAGCTTTTTTGGAGAAGAAGAAG TGTCCAATGGAATGCTTGTATGGTCTCGAATGCGTCCATTTCTTGAAAGGCCAGATGCTT TGCCAGAAACTGCTCAAGGGATAGAAGAAGCTACATTGGCAATGAAAGGTTTGGTTTTAG AAATCAATAGAGAGAAGAGAGCTTATTCTTCTGGTATGGTCTCTAAGGAAATTAGAAGAA TCTGTCCGGATTTTGTCACTGCATTTGATAAGGATTTGTCTGGTTTAAGTCATGTACTTC TTGAGCTTCCTTGTGGTTTAATTGAAGATTCTTCAATAACTTTGGTTGGTATTCCTGATG AACATTCTAGTAGCTTCCAGATTCAGCTCGTTGGCTCGGGATTATCAGGAGAAACTCGTC GGCCAATAATCTTGCGTTACAATGTGAACTTTTCTAAACCATCGATAGTGCAAAATACAT GGACAGAGAAGCTAGGTTGGGGAAACGAAGAGCGATGCCAATATCATGGATCATTGAAAA ATCATTTAGTTGATGAACTTCCTCTCTGCAACAAACAGACCGGTAGAATCATTTCGGAAA AGAGTTCCAACGATGATGCAACTATGGAACTTTCTCTTTCAAATGCTAATTTTCCATTTC TCAAAGGGAGTCCCTTCACTGCCGCATTGTGGTTTGGCTTAGAAGGTTTTCATATGACGA TAAATGGGCGGCACGAGACTTCATTTGCTTACAGGGAGAAGCTCGAGCCATGGTTAGTCA GTGCAGTCAAAGTCTCAGGTGGTTTGAAAATTTTATCTGTCTTAGCCACAAGACTGCCCA TTCCCGATGACCATGCATCTTTAATCATAGAAGAGAAACTTAAAGCTCCATCTCTTTCCG GGACAAGAATAGAACTATTGGTGGGTGTTTTCTCCACTGGAAATAATTTTAAGCGGCGTA TGGCATTGAGAAGATCTTGGATGCAATACGAGGCAGTAAGATCTGGCAAAGTAGCTGTTC GATTTCTCATTGGCCTTCACACAAATGAAAAAGTCAATTTAGAGATGTGGAGAGAATCTA AGGCATATGGAGACATTCAGTTTATGCCATTTGTTGACTACTATGGTTTACTTAGCTTGA AAACAGTTGCGCTTTGCATTCTCGGGACCAAAGTCATCCCAGCAAAATACATAATGAAGA CGGATGATGATGCGTTTGTACGGATTGATGAGCTCCTATCAAGCTAGAAGAAAGACCGT CTAGTGCCCTTCTGTACGGTTTGATCTCATTTGATTCATCACCGGACCGTGAACAAGGCA GCAAATGGTTTATCCCTAAAGAGGAATGGCCTTTAGATTCATACCCTCCATGGGCACATG GCCCTGGCTACATCATCTCTCATGATATAGCGAAATTTGTGGTGAAGGGTCACCGTCAAA GAGATCTTGGACTTTTCAAGCTGGAAGATGTGGCGATGGGGATATGGATTCAACAATTCA ACCAGACGATAAAAAGAGTGAAGTACATCAATGACAAAAGATTTCATAACAGTGATTGTA

234 AATCAAATTACATTCTTGTTCATTACCAAACTCCTAGACTAATTTTGTGTCTTTGGGAGA AGCTGCAAAAAGAGAACCAATCTATTTGCTGCGAAAAG TCG ACT GTG AGC AAG GGC GAG GAG CTG TTC ACC GGG GTG GTG CCC ATC CTG GTC GAG CTG GAC GGC GAC GTA AAC GGC CAC AAG T 234

235 235 APPENDIX C: EXPANDED LIST OF ALL PLANT CELL WALL GLYCAN- DIRECTED MONOCLONAL ANTIBODIES (MABS) USED IN THIS STUDY FOR GLYCOME PROFILING The groupings of antibodies are based on a hierarchical clustering analysis of all mabs screened against a panel of plant polysaccharide preparations (Pattathil et al., 2010; 2012) that groups the mabs according to the polysaccharides that they predominantly recognize. The majority of listings link to the WallMabDB plant cell wall monoclonal antibody database ( that provides detailed descriptions of each mab, including immunogen, antibody isotype, epitope structure (to the extent known), supplier information, and related literature citations. Glycan Group Recognized Non-Fucosylated Xyloglucan mab Name CCRC-M54 CCRC-M48 CCRC-M49 CCRC-M96 CCRC-M50 CCRC-M51 CCRC-M53 CCRC-M100 CCRC-M103 CCRC-M58 CCRC-M86 CCRC-M55 CCRC-M52 CCRC-M99 CCRC-M95 CCRC-M101 CCRC-M104 CCRC-M89 CCRC-M93

236 236 CCRC-M87 CCRC-M88 CCRC-M57 CCRC-M90 Fucosylated Xyloglucan CCRC-M102 CCRC-M39 CCRC-M106 CCRC-M84 CCRC-M1 Xylan 1/XG CCRC-M111 CCRC-M108 CCRC-M109 Xylan 2 CCRC-M119 CCRC-M115 CCRC-M110 CCRC-M105 CCRC-M117 CCRC-M113 CCRC-M120 CCRC-M118 CCRC-M116 CCRC-M114 CCRC-M154 CCRC-M150 Xylan 3 CCRC-M160 CCRC-M137 CCRC-M152 CCRC-M149 CCRC-M144 CCRC-M146 CCRC-M145 CCRC-M155 Xylan 4 CCRC-M153 CCRC-M151 CCRC-M148 CCRC-M140 CCRC-M139 CCRC-M138 Seed Galactomannan CCRC-M75 CCRC-M70

237 237 Homogalacturonan Backbone Rhamnogalacturonan I Backbone Linseed Mucilage Rhamnogalacturonan I Physcomitrella Pectin Rhamnogalacturonan IA Rhamnogalacturonan IB Rhamnogalacturonan IC Rhamnogalacturonan I Arabinogalactan CCRC-M74 CCRC-M131 CCRC-M38 JIM5 JIM136 JIM7 CCRC-M34 CCRC-M69 CCRC-M35 CCRC-M36 CCRC-M14 CCRC-M129 CCRC-M72 CCRC-M40 CCRC-M98 CCRC-M94 CCRC-M5 CCRC-M2 CCRC-M23 CCRC-M17 CCRC-M19 CCRC-M18 CCRC-M56 CCRC-M16 JIM137 JIM101 CCRC-M61 CCRC-M30 CCRC-M60 CCRC-M41 CCRC-M80 CCRC-M79 CCRC-M44 CCRC-M33 CCRC-M32 CCRC-M13 CCRC-M42 CCRC-M24 CCRC-M12 CCRC-M7

238 238 Arabinogalactan 1 Arabinogalactan 2 Arabinogalactan 3 CCRC-M77 CCRC-M25 CCRC-M9 CCRC-M128 CCRC-M126 CCRC-M134 CCRC-M125 CCRC-M123 CCRC-M122 CCRC-M121 CCRC-M112 CCRC-M21 JIM131 CCRC-M22 JIM132 JIM1 CCRC-M15 CCRC-M8 MH4.3E5 JIM16 JIM93 JIM94 JIM11 MAC204 JIM20 JIM14 MAC207 JIM19 JIM12 CCRC-M133 CCRC-M107 JIM4 CCRC-M31 JIM17 CCRC-M26 JIM15 JIM8 CCRC-M85 CCRC-M81 MAC266 PN 16.4B4

239 239 Arabinogalactan 4 Unidentified JIM133 JIM13 CCRC-M92 CCRC-M91 CCRC-M78 MAC265 CCRC-M97

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