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Function, functional conservation and interactions of the membrane-bound endo-1,4-beta-glucanases orthologous… Maloney, Victoria Jane 2010

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FUNCTION, FUNCTIONAL CONSERVATION AND INTERACTIONS OF THE MEMBRANE-BOUND ENDO-1,4-BETA-GLUCANASES ORTHOLOGOUS TO KORRIGAN  by VICTORIA JANE MALONEY B.Sc., The Kings University College, 1999 B.Sc. (Hon.), The University of British Columbia, 2003  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Forestry)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  April 2010  © Victoria Jane Maloney, 2010  Abstract Plant endoglucanses (E.C. 3.2.1.4) encompass multi-gene families across several plant clades, all belonging to the glycosyl hydrolase 9 (GH9) family.  One class of GH9  enzymes is unique in that all members possess sequences that encode an N-terminal membrane-anchoring domain. This class of enzymes, termed membrane-bound endo1,4-beta-glucanases, is the focus of this thesis. The most extensively studied enzyme was first discovered in Arabidopsis and was given the name KORRIGAN (KOR) because of the dwarfed phenotype and cellulose deficiency apparent in plants exhibiting KOR gene mutations. Research has principally focused on Arabidopsis and other nontree species and the possible role that the enzyme might play in primary cell wall development and cellulose synthesis. However, very little research with KOR has been conducted on trees and secondary cell wall development. Consequently, I investigated the effects of mis-regulating KOR in hybrid poplar and white spruce. I was able to demonstrate that the down-regulation of the hybrid poplar KOR gene increases the crystallinity of the secondary cell wall cellulose and affects the relationship between cellulose and the hemicellulose cell wall components. Concurrently, we were the first to isolate and characterize the KOR gene and suppress KOR gene activity in white spruce. Expression of white spruce KOR in Arabidopsis kor1-1 mutants demonstrated that the gene is able to rescue the mutant phenotype, providing evidence for functional equivalence. Additionally, suppression of the gene in white spruce reduced growth and cellulose content.  Since KOR has been demonstrated to be required in cells  undergoing cellulose synthesis, we investigated whether or not the KOR protein and the cellulose synthase complex (CSC) interact.  Although we were not able to provide  evidence for any KOR-protein interaction, we were able to disprove the hypothesis that KOR interacts with CesA7, a member of the secondary cell wall CSC. Collectively, the expression, functional characterization, and interaction data suggest that KOR does not function in direct contact with the CSC, but rather that it plays a role in the later stages of cell wall development, presumably in the relaxation of the stresses around the cellulose microfibril or in the separation of putative cellulose macrofibrils.  ii  Table of Contents Abstract ........................................................................................................................... ii Table of Contents ........................................................................................................... iii List of Tables ................................................................................................................. vii List of Figures ............................................................................................................... viii Acknowledgements.......................................................................................................... x Co-authorship Statement ................................................................................................ xi CHAPTER 1 Introduction ............................................................................................... 1 1.1 Overview ............................................................................................................... 2 1.2 Plant endo-1,4-β glucanases ................................................................................ 2 1.2.1 Activity ............................................................................................................ 2 1.2.2 Classes ........................................................................................................... 3 1.2.3 Physiological functions ................................................................................... 4 1.2.4 Membrane-bound endo-1,4-β-glucanases ...................................................... 4 1.2.5 Agrobacterium tumefaciens ............................................................................ 5 1.2.6 Tomato (Lycopersicon esculentum) ................................................................ 5 1.2.7 Arabidopsis thaliana ....................................................................................... 6 1.2.8 Oilseed rape (Brassica napus) ....................................................................... 8 1.2.9 Rice (Oryza) ................................................................................................... 9 1.2.10 Populus......................................................................................................... 9 1.3 Wood formation ....................................................................................................10 1.4 Cellulose biosynthesis ..........................................................................................11 1.4.1 Cellulose structure .........................................................................................12 1.4.2 Cellulose synthase complex ..........................................................................13 1.4.3 Cellulose synthase genes ..............................................................................13 1.4.4 Sucrose synthase ..........................................................................................14 1.4.5 Current cellulose biosynthetic model .............................................................15 1.5 Goals and hypotheses .........................................................................................15 1.5.1 Membrane-bound endo-1,4-β glucanase gene identification in hybrid poplar and white spruce ....................................................................................................15  iii  1.5.2 Functional characterization of a membrane-bound endo-1,4-β glucanase in secondary cell wall formation of hybrid poplar and white spruce ............................16 1.5.3 Determination of the degree of functional conservation between the white spruce and the Arabidopsis membrane-bound endo-1,4-β glucanases .................16 1.5.4 Determination of possible interactions between the Arabidopsis membranebound endo-1,4-β glucanase and the cellulose synthase complex or other proteins ...............................................................................................................................17 1.6 Model systems .....................................................................................................17 1.6.1 Populus .........................................................................................................17 1.6.2 Spruce ...........................................................................................................18 1.6.3 Arabidopsis thaliana ......................................................................................19 1.6.4 Saccharomyces cerevisiae ............................................................................19 1.7 References ...........................................................................................................21 CHAPTER 2 Characterization and varied expression of a membrane-bound endo-β1,4-glucanase in hybrid poplar .......................................................................................29 2.1 Introduction ..........................................................................................................30 2.2 Materials and methods .........................................................................................33 2.2.1 PaxgKOR isolation and construct development.............................................33 2.2.2 Generation of transgenic plants .....................................................................34 2.2.3 Genomic DNA extraction and purification ......................................................34 2.2.4 RNA extraction and transcript abundance .....................................................35 2.2.5 Analysis of wood composition........................................................................35 2.2.6 Antibody labelling ..........................................................................................36 2.2.7 Cellulose characterization..............................................................................37 2.3 Results .................................................................................................................37 2.3.1 Generation of transgenic hybrid poplar plants ...............................................37 2.3.2 Carbohydrate and lignin analyses .................................................................38 2.3.3 Cellulose characterization..............................................................................40 2.4 Discussion ............................................................................................................41 2.4.1 Previously proposed roles for KORRIGAN do not appear to hold true in poplar ...............................................................................................................................42  iv  2.4.2 Possible role for PaxgKOR in the relaxation of the stresses around the cellulose microfibril or in the separation of the cellulose macrofibril .......................43 2.4.3 Disrupting cellulose biosynthesis depletes soluble sucrose content ..............46 2.5 References ...........................................................................................................58 CHAPTER 3 Functional conservation of KORRIGAN, a putative membrane-bound endo-1,4-β-glucanase required for cellulose biosynthesis in vascular plants ................63 3.1 Introduction ..........................................................................................................64 3.2 Materials and methods .........................................................................................67 3.2.1 PgKOR isolation and construct development ................................................67 3.2.2 Plant strains and growth conditions ...............................................................68 3.2.3 Plant transformation ......................................................................................68 3.2.4 Genomic DNA extraction and screening ........................................................69 3.2.5 RNA extraction and realtime PCR .................................................................69 3.2.6 Structural carbohydrate analyses ..................................................................70 3.2.7 Cellulose characterization..............................................................................71 3.2.8 Cross sectional staining and microscopy .......................................................71 3.3 Results .................................................................................................................72 3.3.1 Molecular cloning and sequence analyses of PgKOR ...................................72 3.3.2 Complementation of Arabidopsis kor1-1 mutant ............................................73 3.3.3 Endogenous PgKOR expression ...................................................................73 3.3.4 RNAi suppression of endogenous PgKOR expression ..................................74 3.4 Discussion ............................................................................................................75 3.4.1 Molecular characterization of PgKOR ............................................................75 3.4.2 Endogenous PgKOR expression ...................................................................75 3.4.3 RNAi suppression of endogenous PgKOR expression ..................................76 3.4.4 Significance of the evolutionary conservation of KORRIGAN ........................77 3.5 References ...........................................................................................................91 CHAPTER 4 Investigation of KORRIGAN-protein interactions .....................................98 4.1 Introduction ..........................................................................................................99 4.2 Materials and methods .......................................................................................101 4.2.1 BRET construct development ......................................................................101 4.2.2 Plant strains and growth conditions .............................................................102 v  4.2.3 Plant transformations, screening and crossing ............................................102 4.2.4 RNA extraction and realtime PCR ...............................................................104 4.2.5 Toluidine blue staining and microscopy .......................................................104 4.2.6 BRET Assay ................................................................................................105 4.2.7 Bait construction and confirmation...............................................................105 4.2.8 Pilot screen and cDNA library screen ..........................................................106 4.3 Results ...............................................................................................................107 4.3.1 Complementation of Arabidopsis mutants with BRET constructs ................107 4.3.2 BRET assay.................................................................................................107 4.3.3 Yeast two hybrid assay ................................................................................108 4.4 Discussion ..........................................................................................................109 4.5 References .........................................................................................................117 CHAPTER 5 ................................................................................................................122 Thesis summary and future research ..........................................................................122 5.1 Thesis summary .................................................................................................123 5.2 Future research ..................................................................................................125 5.2.1 Detailed microscopy of KOR suppressed trees ...........................................126 5.2.2 High-resolution atomic force microscopy .....................................................126 5.2.3 Fluorescence microscopy ............................................................................127 5.3 Research significance ........................................................................................127 5.4 References .........................................................................................................128  vi  List of Tables Table 1.1. Summary of all known Arabidopsis KOR mutants ........................................20 Table 2.1. Structural cell wall carbohydrates and total lignin content of four-month old poplar trees . ..................................................................................................................48 Table 2.2. Microfibril angle and cell wall crystallinity of four-month old poplar trees .....49 Table 3.1. Structural carbohydrate analysis of wild-type, kor1-1 and complemented Arabidopsis plants indicating a recovering to wild type levels in the complemented plants .............................................................................................................................79 Table 3.2. Microfibril angle (MFA) and cell wall crystallinity as measured by x-ray diffraction of eight-week-old Arabidopsis stems .............................................................79 Table 3.3. Structural cell wall carbohydrates of wild-type and PgKOR-RNAi lines indicating a decrease of glucose levels in the PgKOR-RNAi lines .................................79 Table 3.4. Microfibril angle (MFA) and cell wall crystallinity, as measured by x-ray diffraction, of eighteen-month-old white spruce stems ...................................................80  vii  List of Figures Figure 2.1 Molecular characterization of KOR genes indicating similarities ..................50 Figure 2.2 Relative transcript abundances indicate decreased endogenous Pa×gKOR in RNAi lines and expression of AtKOR in the AtKOR over-expression lines of poplar .51 Figure 2.3 Height and caliper measurements of trees after four months of growth in the greenhouse indicates growth reduction in Pa×gKOR RNAi lines...................................52 Figure 2.4 Pa×gKOR RNAi trees display irx phenotype ................................................53 Figure 2.5 Decreased α-cellulose and holocellulose in Pa×gKOR RNAi lines of transgenic poplar ...........................................................................................................54 Figure 2.6 Histochemical staining of poplar cross-sections indicates the lower cellulose and slightly higher lignin cell wall chemistry associated with a down regulation of KOR in hybrid poplar ..................................................................................................................55 Figure 2.7 Xylan (LM10) and xyloglucan (anti-XG) immunolabelling of four-month-old poplar stem tissue indicates higher xylan concentrations in the Pa×gKOR RNAi ..........56 Figure 2.8 Soluble carbohydrates analyses of poplar leaf tissue indicates reduced sucrose and increased glucose and fructose in the Pa×gKOR RNAi lines 16 and 19, while AtKOR over-expression lines have no significant difference ...............................57 Figure 3.1 Molecular characterization of the KOR gene from a variety of plants indicates conserved features .........................................................................................82 Figure 3.2 Phylogenetic comparisons indicate that PgKOR is a conserved member of the KOR family...............................................................................................................83 Figure 3.3 Spruce KOR rescues the growth phenotype of the Arabidopsis kor1-1 mutant............................................................................................................................84 Figure 3.4 Quantitative real time PCR analyses indicates expression of PgKOR in kor11 mutant background. ....................................................................................................85 viii  Figure 3.5 Spruce KOR rescues the irregular xylem phenotype of the kor1-1 mutant ..86 Figure 3.6 Relative transcript abundance of endogenous PgKOR in five year old white spruce trees indicates highest expression in young tissues ..........................................87 Figure 3.7 Relative transcript abundance of endogenous PgKOR in eighteen-month old PgKOR-RNAi trees indicates substantial reduction from wild type levels ......................88 Figure 3.8 Impaired growth in PgKOR-RNAi white spruce trees ...................................89 Figure 3.9 Histochemical staining of eighteen-month old PgKOR RNAi white spruce cross-sections stained with either phloroglucinol for lignin or calcoflour white for cellulose .........................................................................................................................90 Figure 4.1 Four-week old wild type (WS), kor1-1, AtKOR, wild type (LER) and AtCesA7 Arabidopsis plants demonstrating the recovery from the mutant growth phenotype.. ..112 Figure 4.2 Four-week old stem cross-sections of wild type (WS), kor1-1, AtKOR, wild type (LER), irx3-1, and AtCesA7 complemented lines demonstrating the recovery from the mutant irx phenotype in the complemented lines ...................................................113 Figure 4.3 Relative transcript abundance of AtKOR or AtCesA7 in four-week old Arabidopsis plants .......................................................................................................114 Figure 4.4 Measurements of YFP, LUC, or BRET in wild type (WT), control (LY), AtKOR (KOR-LUC), AtCesA7 (CesA7-YFP), and AtKOR×AtCesA7 crossed (KOR×CesA7) lines indicates the presence of YFP fluorescence and LUC luminescence in the crossed lines, but no BRET................................................................................115 Figure 4.5 AtKOR bait is functionally expressed in NMY51 yeast. ..............................116  ix  Acknowledgements I sincerely thank my supervisor Dr. Shawn Mansfield and committee members Drs. A. Lacey Samuels and Geoffrey Wasteneys for their supervision, guidance and encouragement. I also thank the past and present members of the Mansfield Lab for contributing to the pleasant work environment and for providing technical and moral support. Special thanks are extended to my labmates Andrew (Rob) Robinson, Faride Unda, Lisa McDonnell and Thomas Canam for the pleasant company, research advice and wonderful shared experiences. I am also eternally grateful for my husband Phil; without his help this thesis would never have been completed. Lastly, I would like to thank my friends and family for providing an endless supply of support and encouragement.  x  Co-authorship Statement Chapter 3: Victoria Maloney was involved with performing research, data analysis and manuscript preparation. Shawn Mansfield was involved with identification and design of the research program and manuscript preparation. Chapter 3: Victoria Maloney was involved with performing research, data analysis and manuscript preparation. Shawn Mansfield and A. Lacey Samuels were involved with identification and design of the research program and manuscript preparation. Chapter 4: Victoria Maloney was involved with performing research, data analysis and manuscript preparation. Lisa McDonnel was involved with performing the research. Shawn Mansfield was involved with identification and design of the research program and manuscript preparation.  xi  CHAPTER 1  Introduction  1  1.1 Overview Understanding the biosynthesis of cellulose, the world’s most abundant polymer, has been an intriguing area of research for many years. In plants the production of cellulose is as essential as photosynthesis. The deposition of cellulose in the plant cell wall can affect many aspects of plant growth and development, including cell division and expansion, plant morphogenesis, and response to environmental cues.  Elucidating  even a small fraction of the biosynthetic mechanism of this polymer could be critical to future experiments aimed at increasing cellulose production in plants.  This thesis  examines one enzyme, an endoglucanse that has been indicated to influence cellulose synthesis, in an effort to further our knowledge of the cellulose biosynthetic mechanism. 1.2 Plant endo-1,4-β glucanases 1.2.1 Activity Conventionally cellulases are divided into two classes; the exoglucanases and the endoglucanases both which belong to the glycosyl hydrolase family (EC 3.2.1.x). Exoglucanases (EC 3.2.1.91) have been shown act at the ends of the cellulose chain by releasing cellobiose units and have an affinity to degrade crystalline cellulose in an orderly manner. Conversely, endoglucanases; (E.C. 3.2.1.4) have been shown to act more irregularly along the cellulose chain and are more active on amorphous cellulose. In bacteria and fungi many of the cellulases that do not form enzyme complexes contain a catalytic domain responsible for the hydrolysis reaction and a cellulose binding domain (CBD) that enables them to bind specifically to the substrate (Carrard et al., 2000).  Interestingly, many eukaryotic organisms, including plants, nematodes and  some insects, also produce endoglucanases.  In a report on the classification of  glycosyl hydrolases, Henrissat (1991) found that all endoglucanases present in plants belong to glycoside hydrolase family 9 (GH9). The reaction mechanism of the GH9 family is distinctive in that upon hydrolysis the hydroxide on the anomeric carbon is inverted to the α-position. Generally, plant endoglucanases lack a CBD, posses an Nterminal endoplasmic reticulum import sequence, and are secreted into the apoplast (Molhoj et al., 2002). endoglucanases  of  With the exception of a few putative CBD containing  unknown  function  (Trainotti  et  al.,  1999),  most  plant  endoglucanases are believed to depolymerize non-crystalline glucans, although these 2  proposed catalytic mechanisms have yet to be fully tested (Libertini et al., 2004; Master et al., 2004; Molhoj et al., 2001a; Ohmiya et al., 2003; Ohmiya et al., 2000). After extensive research, the exact in vivo substrate, manner of activity, or to what degree plant endoglucanses can modify cellulose or other β-1,4 glucans is still relatively unknown (Brummell et al., 1997a; Brummell et al., 1997b; del Campillo, 1999; Master et al., 2004; Molhoj et al., 2001a; Molhoj et al., 2002; Molhoj et al., 2001c; Nicol et al., 1998; Ohmiya et al., 2003; Ohmiya et al., 2000; Peng et al., 2002; Robert et al., 2005; Rudsander et al., 2003; Sato et al., 2001; Szyjanowicz et al., 2004; Takahashi et al., 2009).  1.2.2 Classes All carbohydrate active enzymes have been classified into a number of different families based on amino acid sequence similarities that were initially detected by hydrophobic cluster analysis (Henrissat et al., 1989). All endoglucanases belong to the glycosyl hydrolase (GH) family and were traditionally named by the first two letters of the organism of origin, followed by the main biochemical activity, the GH family number and a letter (Henrissat et al., 1998).  Recently a new classification scheme for plant  cellulases was proposed in which the name of the gene contains an indication of the genus or species, the designated GH family, a letter indicating the domain structure, and a number corresponding to the given isoenzyme (Urbanowicz et al., 2007). For example, the Arabidopsis genome contains approximately 25 genes that encode endoglucanases that belong to GH9 (Henrissat et al., 2001). According to Molhoj et al. (2002), who compared the relationship between the 25 Arabidopsis endoglucanases and their identified homologs in other plant species, these 25 genes separate into at least 9 different classes. Seven of the classes show that at least one Arabidopsis sequence matches up with one or more sequences from another species, suggesting that they represent conserved functional classes, whereas two classes contain only Arabidopsis sequences. The genes in these two classes may be specific to Arabidopsis or similar genes in other species are yet to be identified. Most of the genes organized into class III have a C-terminal extension that encodes a putative CBD.  Class IX  appears to be a unique class in that members do not contain a cleaved hydrophobic endoplasmic reticulum (ER) signal sequence and are not secreted directly into the 3  apoplast, but rather they contain an N-terminal membrane anchoring domain which acts as a start transfer sequence for ER targeting (Brummell et al., 1997b; Nicol et al., 1998). 1.2.3 Physiological functions Enzymes within the GH9 family have different subcellular locations which is suggestive of the number of functions that these enzymes might play with respect to plant physiology. Presumably, enzymes that are located at the plasma membrane act at the innermost layers of the cell wall and enzymes that are secreted act anywhere within the cell wall or at the outer layer (del Campillo, 1999; Rose et al., 2004). Previous reports suggest that plasma-membrane anchored endoglucanases function in the biosynthesis of the cell wall either by editing the cellulose microfibril or during the assembly of the cellulose-hemicellulose network (Brummell et al., 1997b; Molhoj et al., 2001a; Nicol et al., 1998; Sato et al., 2001; Zuo et al., 2000). Extracellular endoglucanases have specific roles in the metabolic breakdown of the cell wall and their activity can be simple and restricted to a small area or have a more substantial and destructive affect (del Campillo, 1999). 1.2.4 Membrane-bound endo-1,4-β-glucanases It is evident that endoglucanases comprise a diverse family of enzymes that participate in the breakdown of β-1,4 glucosidic linkages, however one group of membrane-bound endoglucanases has gained particular attention with regards to cellulose biosynthesis in plants. It is this group of enzymes that will be the focus of this thesis. A number of studies examining the roles of membrane-bound endoglucanases from a variety of species, including monocots and dicots, have been undertaken in an attempt to elucidate the role(s) these enzymes may play with respect to cell wall remodelling, and more generally the overall physiology of plants (Bhandari et al., 2006; Brummell et al., 1997a; Brummell et al., 1997b; Lane et al., 2001; Master et al., 2004; Molhoj et al., 2002; Molhoj et al., 2001c; Takahashi et al., 2009). Generally, the results from these studies indicate that plants possess one particular membrane-bound endoglucanase that appears to have similar functionality regardless of species. I will herein discuss the identification, expression analysis and the putative roles of this membrane-bound  4  endoglucanase from Agrobacterium tumefaciens, tomato (Lycopersicon esculentum), Arabidopsis thaliana, oilseed rape (Brassica napus), rice (Oryza), and Populus. 1.2.5 Agrobacterium tumefaciens While membrane-associated endoglucanase enzyme activity had previously been reported in bean (Koehler et al., 1976; Koehler and Lewis, 1976; Lewis and Koehler, 1979; Tucker et al., 1988) and pea (Bal et al., 1976), a membrane-bound endoglucanase in Agrobacterium tumefaciens was the first to be fully described (Matthysse et al., 1995a). The gene in Agrobacterium tumefaciens (CelC) is the last gene in the operon neighbouring the operon containing the cellulose synthase gene. The closest related gene to CelC is an endoglucanase gene involved in cellulose degradation from Cellulomonas uda with a 39% identity (Nakamura et al., 1986), and disruption of CelC by transposon insertion showed that CelC is indeed required for cellulose synthesis. The predicted protein contains 342 amino acids and hydrophobicity plotting of the predicted CelC protein revealed one possible membrane spanning domain. Although the function of the CelC membrane-anchored endoglucanase is still not known, it was proposed to participate in the transfer of lipid-linked glucan oligosaccharides to cellulose chains (Matthysse et al., 1995b; Matthysse et al., 1995c). 1.2.6 Tomato (Lycopersicon esculentum) Brummell et al. (1997b) described the complete full-length cDNA sequence of the first membrane-anchored endoglucanase from tomato (Cel3). The results revealed that the Cel3 protein was unlike other endoglucanases in that it lacked a typical cleavable signal peptide and contained three distinct domains: an N-terminal domain, a membranespanning domain, and a C-terminal catalytic domain. The predicted protein contained 617 amino acids and included seven potential sites for N-glycosylation. Expression analyses concluded that Cel3 mRNA accumulated in young vegetative tissue and during times of rapid expansion. Furthermore, analyses of enzyme activity indicated that the enzyme is active in both the Golgi and the plasma membrane suggesting that the enzyme resides in sites of or near cellulose biosynthesis.  5  1.2.7 Arabidopsis thaliana Nicol et al. (1998) reported a mutant of Arabidopsis, KORRIGAN (KOR), which was extremely dwarfed, defective in primary cell wall biosynthesis and had a T-DNA insertion in the promoter region approximately 200 base pairs upstream of the translation start codon of an endoglucanase gene. Similar to the previously described tomato endoglucanase, this endoglucanase (KOR) lacked the typical cleaved Nterminal signal peptide common in other plant endoglucanases. Instead, it displayed a predicted internal N-terminal domain which could act as an ER start transfer sequence to translocate the protein into the ER bi-layer.  From the ER the protein would  presumably be transferred by way of vesicle traffic through the Golgi apparatus to the plasma membrane. Its final destination, embedded in the plasma membrane, puts the KOR protein in the same membrane bi-layer where cellulose synthesis occurs (Delmer, 1999). Additional mutations in the Arabidopsis KOR gene (kor1-2) have been isolated and shown to cause the formation of aberrant cell plates, incomplete cell walls, and multinucleated cells, leading to abnormal seedling morphology (Zuo et al., 2000). Kor 1-2 was further shown to possess a T-DNA insertion which deleted a ~1-kb portion containing the entire KOR promoter and 5’UTR 22 base pairs upstream of the translation start site.  Expression of a KOR-GFP fusion construct in tobacco BY2 cells  suggested that KOR localizes to the cell plate while substitution mutations in the predicted polarized targeting signals (LL and YXXΦ) indicated the protein is also located at the plasma membrane and that these motifs are essential for cytokinesis. However, Molhoj et al. (2002) observed the accumulation of KOR in the phragmoplast adjacent to the cell plate, and accounted for these differences by hypothesizing that KOR cycles between the plasma membrane, or phragmoplast, and an intracellular compartment in dividing cells. Furthermore, using antibodies specific to a number of different pectic polysaccharides, it was demonstrated that KOR also plays a role in pectin metabolism and can induce changes in pectin composition of the primary cell walls (His et al., 2001). More importantly, morphological and chemical data on temperature sensitive, elongation deficient single base pair-substitution mutants of KOR (acw1, rsw2-1, rsw2-3 and rsw2-4) have pointed to KOR as having an important role in depositing cellulose in primary cell walls (Baskin et al., 1992; Lane et al., 2001; Peng et al., 2000; Sato et al., 2001). Cellulose content in these mutants has been shown to be reduced by as much 6  as 40% compared to wild type plants, which appeared to be compensated for by an increase in pectin content (up 162%) in the acw1 mutant (Sato et al., 2001). These data indicate the presence of a feedback mechanism controlling cell wall polysaccharide composition. Other effects of KOR mutations include delays in cell wall regeneration in mutant protoplasts (Sato et al., 2001), reduced cellulose crystallinity (Szyjanowicz et al., 2004), distorted flower morphology, and anther dehiscence impairment (Lane et al., 2001). Recently, a study characterizing the tumorous shoot development1 (tsd1) mutant of Arabidopsis (Frank et al., 2002) revealed that tsd1 is also a strong allele of the KOR gene (Krupkova and Schmuelling, 2009).  These  mutants were shown to develop short swollen hypocotyls, the cotyledons and root arrested early during development and undifferentiated callus formed instead of leaves (Krupkova and Schmuelling, 2009). Moreover, double mutants of cellulose synthase (rsw1) and KOR (rsw2) showed a further reduction in cellulose content, suggesting the need for both a glycosyl transferase and glycosyl hydrolase for successful cellulose biosynthesis in the primary cell walls of plants (Lane et al., 2001). In  Arabidopsis,  two  additional  genes  encoding  membrane-anchored  endoglucanases have also been characterized (Molhoj et al., 2001b). Promoter-GUS reporter assays of these genes, termed KOR2 and KOR3, suggest that there are different membrane-anchored endoglucanases that are expressed in different cell types. KOR2 is only active in cell types such as the floral organs and in trichomes, KOR3 is active in developing root hairs, whereas the original KOR gene is expressed throughout the entire plant (Molhoj et al., 2001b). In Populus trichocarpa five GH9 genes with a single transmembrane domain have been shown to be similar to the KOR genes from Arabidopsis (del Campillo, 1999; Molhoj et al., 2002) however, it is still not apparent whether orthologs of KOR2 and KOR3 occur in other plant species (Molhoj et al., 2002). While the close association of KOR with primary cell wall development has been widely discussed, it has also been reported that KOR may also play a role in secondary cell wall development (Szyjanowicz et al., 2004). Two irregular xylem mutations (irx2-1 and irx2-2) that are defective in secondary cell walls and display reduced cellulose synthesis in the xylem cells have been shown to be the result of two independent proline to leucine substitutions (Pro250Leu and Pro553Leu) in the KOR protein. 7  Examination of the 25 putative Arabidopsis endoglucanases indicates that both of these prolines are highly conserved. Furthermore, sequence analysis suggests that Pro553 lies in or near a potential catalytic site which could potentially affect the activity of the KOR protein (Szyjanowicz et al., 2004). The irx2 mutants have severe reductions in crystalline cellulose (30% of wild type) and concurrently displayed no change in the glucose of the non-cellulosic fractions, suggesting that cellulose synthesis in the mutants was specifically affected only in the secondary cell wall (Turner and Somerville, 1997). Unlike the kor1-1 mutant, the irx2 mutants did not show reduced hypocotyl growth in the dark, nor did it display the radial swelling phenotype typical of the acw1 and rsw2 mutants.  Other cellular abnormalities, such as abnormal cell shape and  incomplete cell plates, were also not observed in the irx2 mutants (Szyjanowicz et al., 2004).  Szyjanowicz et al. (2004) suggest that the exclusive secondary cell wall  cellulose reduction evident in the irx2 mutants might be due to a relatively weak mutation where there is still enough activity to produce the cellulose required by the primary cell walls. Furthermore, the stems of the stronger kor1-1 mutant, that was reported to be a primary cell wall mutant, also had severely collapsed xylem cells similar to the irx2 mutants (Szyjanowicz et al., 2004). For a summary of all Arabidopsis KOR mutants see Table 1.1.  1.2.8 Oilseed rape (Brassica napus) An investigation of membrane-anchored endoglucanases from Brassica napus showed that its Cel16 protein shares 94% identity to the KOR protein from Arabidopsis suggesting that the two genes are orthologous, performing the same function in the two organisms (Molhoj et al., 2001a; Molhoj et al., 2001c). Expression of a Cel16 promoterGUS reporter construct in Arabidopsis indicated that Cel16 is expressed the highest in the young root and main stem whereas lower levels of expression were found in the young stems, the elongation zone of the roots and the older root base. In situ Cel16 RT-PCR showed a similar pattern of transcript abundance in the oilseed rape suggesting that the expression of Cel16 is inversely correlated to elongation (Molhoj et al., 2001a). It was further deduced that Cel16 has a molecular mass of 69 kD and has the same predicted cytosolic charged N-terminus, transmembrane domain and periplasmic catalytic core as KOR. The functional analyses of a truncated version of 8  the Cel16 gene (missing residues 1 through 90, but containing the Cel16 catalytic domain) in Pichia pastoris revealed that this truncated version of Cel16 is highly N glycosylated as compared with the native protein. The truncated Cel16 appeared to be inhibited by EDTA, exhibited a strong dependence on calcium and showed substrate specificity for low substituted carboxymethyl-cellulose and amorphous cellulose (Molhoj et al., 2001c). 1.2.9 Rice (Oryza) The dwarf mutant glu was identified from screening a T-DNA tagged rice population and was found to be caused by a mutation in the OsGLU1 gene (Zhou et al., 2006). The mutated gene was further found to encode a membrane-bound endo-1,4-beta-Dglucanase and was assumed to be orthologous to the previously described plant membrane-bound endoglucanases because of its requirement for proper cell wall synthesis. It was shown that the mutation of the OsGLU1 leads to a reduction in cell elongation and cellulose content and an associated increase in pectin content, suggesting that OsGLU1 is involved in the modification of the components of the rice cell wall. RNAi and complementation studies provided further evidence that OsGLU1 plays an important role in cell wall synthesis and plant cell growth (Zhou et al., 2006). 1.2.10 Populus Interrogation of an EST database of 3000 cambial sequences assembled from hybrid aspen (Populus tremula × tremuloides) identified ten putative cellulose synthases and at least two family 9 glycosyl hydrolase genes that are upregulated during xylogenesis (Hertzberg et al., 2001; Sterky et al., 1998). The endoglucanase PttCel9A was further found to be up-regulated during secondary cell wall formation, which corresponded with a period of high cellulose synthesis (Rudsander et al., 2003). PttCel9A contains the three domains that are typical of the previously described membrane-bound endoglucanases and shares 82% identity with the Arabidopsis KOR gene. The catalytic domain of PttCel9A was purified and shown to be active against only low-substituted, soluble cellulosic polymers such as cellotetraose (Master et al., 2004). Additionally, the catalytic efficiency (kcat/Km) was only 0.04% of that of a GH9 enzyme, TfCel9A, from the soil bacterium Thermobifida fusca (Sakon et al., 1997). This low catalytic efficiency 9  along with the apparent narrow substrate range were attributed to the lack of aromatic amino acids in the substrate binding domain (Master et al., 2004).  More recently,  PttCel9A1 was shown to complement the kor1-1 mutant, indicating that it is indeed a KOR ortholog (Takahashi et al., 2009). Takahashi et al. (2009) also demonstrate that the over-expression of the PttCel9A1 gene in Arabidopsis leads to a decrease in cellulose crystallinity suggesting that the KOR protein works after the initial synthesis of cellulose. PtrKOR, a putative Arabidopsis KOR ortholog, was cloned from aspen (Populus tremuloides) xylem tissue and was shown to have significantly elevated expression on the upper side of the bent aspen stem in response to tension stress while expression was suppressed on the opposite side experiencing compression stress (Bhandari et al., 2006). It was also reported that the cellulose synthase genes from aspen which are closely associated with secondary cell wall development in xylem cells exhibited similar tension-stress response behaviour.  These results provided evidence that both the  secondary cell wall cellulose synthase proteins and PtrKOR are important for the production of the highly crystalline cellulose that is present in tension wood.  1.3 Wood formation Once plants are germinated, they must live their entire life in a single location. The lifespan of woody perennial plants can be many years and as a result they need to adapt distinctive traits to thrive in their environments. The growth habit of trees is unique when compared with other plants. The massive stature of most trees is the result of years of radial secondary growth. This secondary growth or wood formation is initiated at the vascular cambium, which consists of secondary meristematic stem cells that differentiate into bark and secondary phloem to the outside and wood (secondary xylem) to the inside of the cambium. In mature secondary xylem only the parenchyma cells are alive and they occur most abundantly in the rays that run radially through the wood of the tree, but also occur scattered axially throughout the xylem. Other common cells in wood include tracheids, vessel elements and fibres. It is the tracheids and vessel elements that are involved in the transport of xylem sap. As a general rule, gymnosperms have only tracheids, whereas angiosperms have both vessel elements and tracheids. 10  Xylem cells must go through several developmental stages including origin, enlargement, secondary wall thickening and lignification in order to achieve their final functional state. consisting  During elongation, the cells only possess thin primary cell walls,  pectin,  radially  orientated  cellulose  microfibrils  and  cross-linking  hemicelluloses. The primary wall then expands longitudinally and radially under the pressure of cell turgor until they reach their final size.  During this expansion the  microfibrils reorient and additional layers of microfibrils are laid onto the inside of the primary cell wall. Once primary cell growth is complete the primary cell wall is locked into its final shape and the secondary cell wall can begin forming. The secondary cell wall can be subdivided into distinct zones; namely the outer (S1), middle (S2) and inner (S3) sub-wall which are constructed from layers of microfibrils deposited in ordered orientations inside the primary cell wall along with hemicelluloses and lignin. Hemicellulose binds to cellulose, pectin and lignin to form a network of cross-linked fibres in the cell wall (for reviews see Cosgrove, 2001; Cosgrove, 2005). Depending on the function of the cell, different cell types undergo different degrees of cell wall thickening. Many factors contribute to total tree size including species, developmental and seasonal growth transitions, efficiency of photosynthesis, nutrient and water uptake and transport, and the ability to respond to biotic and abiotic stresses. These processes are controlled by many genetic and epigenetic factors that respond dynamically to environmental signals. Growth ultimately results in the structures that provide mechanical support, defense mechanisms and translocate water, solutes, and signaling molecules over extensive distances (for reviews see Chaffey et al., 2002; Plomion et al., 2001).  1.4 Cellulose biosynthesis The synthesis of cellulose, the most abundant polymer on earth, has been the subject of numerous research projects over the last few decades. In plants the production of cellulose is essential for numerous plant functions. Understanding the structure of cellulose and the possible players in its biosynthesis is essential.  11  1.4.1 Cellulose structure Cellulose consists of flat chains of 1,4-β-glucan that results from the polymerization of glucose from the precursor molecule UDP-glucose (Delmer and Amor, 1995).  The  ultrastructure of native cellulose (cellulose I) has been shown to possess two crystal phases: Iα and Iβ (Atalla and Vanderhart, 1984). The amounts of Iα and Iβ vary from the Iα-rich cell wall of algae and bacterial cellulose to the Iβ-rich cellulose found in cotton, wood, and ramie fibers (Atalla and Vanderhart, 1984; Kadla and Gilbert, 2000). The crystal and molecular structure of cellulose I has been examined using atomicresolution synchrotron and neutron diffraction data from cellulose isolated from algae and tunicin (cellulose Iβ). These structural data show that most native samples of cellulose also have varying degrees of amorphous cellulose, which is more reactive to chemical and enzymatic attack (Nishiyama et al., 2002; Nishiyama et al., 2003). Cellulose in the secondary cell wall of wood is organized in 15-60 nm diameter macrofibrils which are each composed of a number of 3 nm microfibrils (Altaner et al., 2006; Donaldson, 2007; Kennedy et al., 2007). A study on the direct visualization of maize stem pith demonstrated that a number of elementary fibrils are synthesized at the cellulose synthase complex and come together into much larger macrofibrils. These macrofibrils eventually split at the ends to form parallel microfibrils, which are then available to interact with other cell wall components such as hemicelluloses, pectin and lignin (Ding and Himmel, 2006). Approximately 36 β-glucan chains combine to form the microfibril, which is a strong, yet flexible, paracrystalline array of cellulose (Brown and Saxena, 2000; Delmer and Amor, 1995; Ding and Himmel, 2006; Levy et al., 2002). In the past, numerous models of cell wall structures have been proposed, but all show non-cellulosic polysaccharides associated with the surfaces of the cellulose microfibrils (Cosgrove, 2001). The exact determinant of cellulose microfibril orientation has been debated however the proper orientation of cellulose microfibrils, whether directed by microtubules or not, controls growth, anisotropy and ultimately the shape of the plant cells (Wasteneys, 2004). The strength of the cellulose microfibril is determined in part by the extent of crystallization between associated glucan chains. Crystallization of cellulose is influenced by inter- and intra-hydrogen and Van der Waals bonds that join the polymeric glucose chains. Generally, crystallization protects the glycoside bonds from enzymatic attack.  However, the highly crystalline areas are spaced within 12  amorphous or less crystalline areas that result in weaker zones or areas of dislocation in the microfibril (Levy et al., 2002). 1.4.2 Cellulose synthase complex Evidence suggests that the formation of cellulose in vascular plants involves an organized enzymatic unit known as the cellulose synthase complex (CSC).  The  ultrastructural morphology of this complex is known as a rosette because of its six-fold symmetrical arrangement of subunits. Presumably, these subunits each contain six separate catalytic molecules so that the microfibril that is exuded from the complex is composed of 36 individual glucan chains (Brown and Saxena, 2000; Delmer, 1999). However, the isolation of functional CSCs has proven difficult, and has limited our understanding of cellulose biosynthesis. 1.4.3 Cellulose synthase genes An additional challenge in understanding cellulose biosynthesis has been the identification of genes that encode components of these complexes. The gene for the first putative cellulose synthase (CesA) catalytic subunit (CelA) was cloned from trypsintreated membranes of the bacteria Acetobacter xylinum (Saxena et al., 1990). Analysis of additional cellulose synthase genes from A. xylinum and other known UDP-specific glycosyltransferases revealed conserved regions surrounding aspartate residues and a conserved downstream QXXRW amino acid motif that were suggested to be essential for the binding of UDP-glucose, the substrate for the CSC (Saxena and Brown, 1995). These discoveries led to the identification of cDNA clones representing putative CelA holologs from rice (Oryza sativa) and cotton (Gossypium hirsutum) (Pear et al., 1996). Further examination of the plant genes revealed the presence two internal insertions of sequence, one conserved and one hypervariable, which are not found in the bacterial gene sequences. The identification of the first plant CesA genes led to the identification of more putative CesA genes and gene families in a number of plants. Interestingly, at least three distinct CesA genes appear to be involved in primary cell wall synthesis, while an additional three CesA genes are involved in secondary cell wall biosynthesis (Delmer, 1999; Doblin et al., 2002; Joshi et al., 2004). These two different cell walls differ significantly in terms of degree of polymerization (DP), crystallininty, microfibril 13  orientation, and amount of cellulose they possess (Doblin et al., 2002; Kimura and Kondo, 2002; Samuga and Joshi, 2004). A mutation in any one of the CesA genes can result in altered cellulose biosynthesis, and consequently an alteration in cell wall ultrastructure and morphology (Scheible et al., 2001).  Predicted tertiary peptide  structures of these proteins provide evidence that the catalytic subunit has a number of transmembrane domains and a cytosolic globular region.  The globular cytosolic  component is the putative location of an active site involving UDP-glucose.  The  discovery of this cytosolic active site has led to the creation of additional models involving the enzymatic channelling of UDP-glucose to the CSC (Brown and Saxena, 2000; Brown et al., 1996; Delmer, 1999). 1.4.4 Sucrose synthase The cytosolic pool of UDP-glucose, the known substrate for the CSC, is an important determinant for the rate of cellulose synthesis.  This pool of UDP-glucose can be  directly or indirectly affected by a number of enzymes. One particular enzyme, sucrose synthase (SuSy; EC 2.4.1.13; sucrose + UDP ↔ UDP-glucose + fructose), has received the most attention with respect to cellulose biosynthesis. Research has suggested that there are two forms of this enzyme, a soluble cytosolic (S-SuSy) form and a plasma membrane-associated (P-SuSy) form (Amor et al., 1995; Carlson and Chourey, 1996a; Carlson and Chourey, 1996b; Hardin et al., 2004; Winter et al., 1997).  However,  research has not been able to demonstrate that these two enzymes are distinct gene products suggesting that they are more likely the result of post-translational modifications (Delmer, 1999; Duncan et al., 2006). Of the two forms of SuSy, P-SuSy has gained particular interest in regards to the production of cellulose in cells undergoing secondary cell wall synthesis.  Immunolocalization studies in cotton have  shown that the distribution of P-SuSy parallels that of the cellulose microfibrils (Robinson, 1996).  These results suggested that P-SuSy is localized close to, or  perhaps bound to, the CSC.  Furthermore, recent research on detergent-insoluble  rosettes isolated from the plasma membrane of bean epicotyls only showed cellulose synthesis activity in the presence of SuSy-like detergent-soluble granular particles that exhibited UDP-glucose binding activity. Immunogold labelling with anti-SuSy antibodies confirmed that the SuSy-like catalytic unit was incorporated into the rosette structure 14  (Fujii et al. 2010).  If SuSy is bound to the CSC it would then have the ability to  efficiently channel UDP-glucose directly to the CSC, preventing pooling of the substrate, and consequently its consumption in other biosynthetic processes.  Additionally,  glycosyltransferases, such as the CSC, are inhibited by UDP. Therefore, P-SuSy could potentially increase the rate of cellulose syntheses by converting the level of local concentrations of free UDP into the substrate for the CSC (Delmer and Amor, 1995; Haigler et al., 2001). 1.4.5 Current cellulose biosynthetic model The most recent cellulose biosynthetic models postulate that the entire process involves at least three steps: (1) P-SuSy directly channels the UDP-glucose substrate to cellulose synthesizing apparatus; (2) co-ordinately expressed multiple CESA, organized in the form of hexagonal rosettes, polymerize glucose monomers into glucan chains while recycling the freed UDP molecule back to SuSy, and (3) a membrane-associated endo-1,4-beta-glucanase (endoglucanase; EC 3.2.1.4), e.g. KOR, has been shown to be essential to the process, but its exact role is still unclear (Delmer and Haigler, 2002; Joshi et al., 2004; Molhoj et al., 2002). 1.5 Goals and hypotheses The main goal of my research was to study the effects of KOR on two of the most commercially important forest trees, hybrid poplar and white spruce.  In so doing I  hoped to help elucidate the function that this enzyme plays in regard to secondary cell wall biosynthesis. 1.5.1 Membrane-bound endo-1,4-β glucanase gene identification in hybrid poplar and white spruce The goal of these analyses was to identify the membrane-bound endo-1,4-β glucanase genes that are orthologous to the Arabidopsis KOR in both hybrid poplar (Populus alba×grandidentata) and white spruce (Picea glauca) and to examine their protein genomic sequences. The hypothesis was that both hybrid poplar and white spruce contain at least one gene that appears to be orthologous to the Arabidopsis gene with similar protein and genomic features. In particular, it was anticipated that all the genes 15  would have similar transmembrane domains, polarized targeting signals, predicted glycosylation sites, residues essential for catalytic activity, as well as the same amount of exons and introns in their genomic sequences. 1.5.2 Functional characterization of a membrane-bound endo-1,4-β glucanase in secondary cell wall formation of hybrid poplar and white spruce The goal of these experiments was to investigate the effects that the overexpression of the Arabidopsis KOR has on cellulose formation in hybrid poplar and to investigate the effects of down regulation of the endogenous gene on cellulose formation in hybrid poplar and white spruce. Analyses focussed on growth rate, phenotype, biochemistry and chemistry of tissues undergoing secondary cell wall development. The hypothesis was that varied expression of the membrane-bound endoglucanase would alter the amount and ultrastructure of the ensuing cellulose. Specifically, it was believed that any overexpression would cause an increase in a more highly crystalline cellulose whereas down regulation would severely disrupt cellulose production and produce a less crystalline cellulose polymer. 1.5.3 Determination of the degree of functional conservation between the white spruce and the Arabidopsis membrane-bound endo-1,4-β glucanases The goal of this experiment was to determine if the membrane-bound endo-1,4-β glucanase from white spruce performs the same function as the Arabidopsis KOR protein. Spatial expression levels of the endogenous white spruce gene were examined in an attempt to understand its physiological role in cell wall development. Additionally, the white spruce gene was transformed into the Arabidopsis kor1-1 mutant in order to determine if it is able to recover the phenotype.  The hypothesis was that the  endogenous white spruce gene would be expressed highest in tissues undergoing either primary or secondary cell wall development. Furthermore, it was predicted the white spruce gene would perform the same function as the Arabidopsis KOR and would be able to recover the kor1-1 phenotype.  16  1.5.4 Determination of possible interactions between the Arabidopsis membranebound endo-1,4-β glucanase and the cellulose synthase complex or other proteins The goal of these experiments was to determine if KOR interacts with the cellulose synthase complex (CSC) or with any other proteins.  Bioluminescence resonance  energy transfer (BRET) was used to determine if there is an interaction between the Arabidopsis KOR and AtCesA7, a member of the CSC, in planta. As a second line of evidence, a split-ubiquitin (DUALmembrane) yeast-based screening assay was used to determine if the Arabidopsis KOR and the CSC interact in the plasma membrane as well as to identify novel protein interactions. The hypothesis was that KOR does not directly interact with the CSC, but that it does interact with other cellulose modifying proteins involved in the later stages of cellulose deposition. 1.6 Model systems The research conducted in this thesis utilizes four different model systems to study the function of membrane-bound endoglucanases. Specifically, chapter two looks in depth into the function of the membrane-bound endoglucanase orthologous to KOR in hybrid poplar. Chapter three examines the KOR gene from white spruce and compares its functionality to that of the Arabidopsis KOR. Finally, chapter four examines possible protein interactions between KOR and the CSC using Arabidopsis and Saccharomyces cerevisiae. The following briefly describes the model systems and the advantages they provide to the current research environment. 1.6.1 Populus Together with Arabidopsis, Populus is found in the angiosperm Euroside I clade (Jansson and Douglas, 2007).  However, unlike Arabidopsis, Populus offers a new  model system to study secondary growth and wood formation that better represent the extent of plant biological processes.  In the last decade, numerous attributes have  allowed for the Populus species to emerge as the model tree for biological and genomic research. These attributes include Populus’ rapid growth rate (~30m3/ha/yr for hybrids), lower age of maturation, shorter rotation times compared to other hardwood species grown in similar environments, ease of vegetative propagation, and general amenability 17  to in vitro culture, regeneration and transformation (Cheng and Tuskan, 2009; Cisneros et al., 1996; Hinchee et al., 2009; Jansson and Douglas, 2007; Tuskan et al., 2006b). More importantly, Populus has a relatively small genome size when compared to other tree species (480 million bp) which makes it amenable to genome and genetic expression studies (Bradshaw et al., 2000; Jansson and Douglas, 2007; Wullschleger et al., 2002). Consequently, in 2006 black cottonwood (Populus trichocarpa) became the first tree species to have its genome completely sequenced (Tuskan et al., 2006a). In Canada, Populus is the most likely candidate to complement the hybrid cottonwood plantations that have been developed in extensive breeding programs in the United States and Europe, and the Eucalyptus plantations of the southern hemisphere (Park and Wilson, 2007). Significant Populus stands are found in every Canadian province, and Populus is estimated to represent over 50% of all hardwoods and approximately 11% of the entire Canadian timber resource (Peterson and Peterson, 1995). The most common Canadian Populus species include trembling aspen (Populus tremuloides), cottonwood (Populus deltoides), black cottonwood (Populus trichocarpa), balsam poplar (Populus balsamifera), narrowleaf cottonwood (Populus angustifolia), and largetooth aspen (Populus grandidentata). From the time of European settlement, these species, as well as other introduced Populus species, have been important for providing support and benefits to many different human activities (Park and Wilson, 2007; Peterson and Peterson, 1995; Richardson et al., 2007). 1.6.2 Spruce In contrast to Populus, spruce (Picea) does not have a rapid growth rate; however, it is the dominant species of the boreal forest and occur throughout Canada (Ohse et al., 2009). Five species occur in Canada: black (Picea mariana), red (Picea rubens), white (Picea glauca), Engelmann (Picea engelmannii) and Sitka (Picea sitchensis). Norway spruce (Picea abies) is also a valuable species in Northern Europe. In the Canadian forest industry, spruce is commonly harvested for both structural timber and pulp. Understanding and subsequently being able to control and/or predict intrinsic phenotypic fiber attributes will be paramount in generating and exploiting this high quality resource.  18  1.6.3 Arabidopsis thaliana Arabidopsis was first proposed to be used as a model organism in 1943 by Friedrich Laibach who in 1907 had already determined that Arabidopsis has only five chromosomes (Meyerowitz, 2001). In 2000, Arabidopsis was the first plant to have its genome completely sequenced (Arabidopsis Genome Initiative, 2000) and it was subsequently proven to have one of the smallest plant genomes at 157 million basepairs (Bennett et al., 2003). The small size of its genome makes Arabidopsis very useful for genetic mapping and sequencing. Many of its 27,000 genes and the 35,000 proteins they encode have been assigned functions (Bennett et al., 2003).  Other  factors that make Arabidopsis a useful model organism include the plant’s small size, rapid life cycle and ease of Agrobacterium-mediated gene transformation. 1.6.4 Saccharomyces cerevisiae The yeast Saccharomyces cerevisiae is an extremely important model organism in modern cell biology research. The availability of the complete genome sequence of S. cerevisiae as well as its high transformation efficiency has made the extensive genetic manipulation of this organism possible and new methods are constantly being developed in order to engineer the yeast for specific purposes (Ostergaard et al., 2000). The NMY51 yeast reporter strain is an optimized screening strain typically used for conventional yeast two-hybrid and DUALmembrane screens. Two growth markers and a lacZ reporter ensure the highest possible stringency in a library screen, resulting in fewer false positives (DualSystems Biotech).  19  Table 1.1. Summary of all known Arabidopsis KOR mutants Mutant Name kor1-1  kor1-2  acw1  Type of mutation T-DNA insertion in the promoter region approximately 200 base pairs upstream of the translation start site T-DNA insertion deleting a ~1-kb portion containing the entire KOR promoter and 5’UTR 22 base pairs upstream of the translation start site Single amino acid substitution in the coding region of KOR (Gly429 to Arg)  rsw2-1  Single amino acid substitution in the coding region of KOR (Gly429 to Arg)  rsw2-3  Single amino acid substitution in the coding region of KOR (Ser-183 to Asn)  rsw2-4  Single amino acid substitution in the coding region of KOR (Gly344 to Arg)  tsd1  Gly to Ala transition at the beginning of the second exon causing the amino acid change Gly -126 to Glu  irx2-1  Single amino acid substitution in the coding region of KOR (Pro-250 to Leu) Single amino acid substitution in the coding region of KOR (Pro-553 to Leu)  irx2-2  Phenotype Extremely dwarfed and defective in primary cell wall biosynthesis  Reference (Nicol et al., 1998)  Aberrant cell plates, incomplete cell walls, and multinucleated cells, leading to abnormal seedling morphology  (Zuo et al., 2000)  Temperature sensitive impairment of tissue elongation, reduced cellulose content with concurrent increase in pectin content Temperature sensitive cytokinesis and cell expansion abnormalities, reduced cellulose in roots and shoots Temperature sensitive cytokinesis and cell expansion abnormalities, reduced cellulose in roots and shoots Temperature sensitive cytokinesis and cell expansion abnormalities, reduced cellulose in roots and shoots Short swollen hypocotyls, the cotyledons and root arrest early during development and undifferentiated callus forms instead of leaves, abnormal hormonal response Cellulose deficiency in the secondary cell wall, reduced crystalline cellulose  (Sato et al., 2001)  Cellulose deficiency in the secondary cell wall, reduced crystalline cellulose  (Baskin et al., 1992; Lane et al., 2001; Peng et al., 2000) (Baskin et al., 1992; Lane et al., 2001; Peng et al., 2000) (Baskin et al., 1992; Lane et al., 2001; Peng et al., 2000) (Frank et al., 2002; Krupkova and Schmuelling, 2009) (Szyjanowicz et al., 2004; Turner and Somerville, 1997) (Szyjanowicz et al., 2004; Turner and Somerville, 1997)    20  1.7 References Altaner C, Apperley DC and Jarvis MC (2006) Spatial relationships between polymers in Sitka spruce: Proton spin-diffusion studies. 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Plant Molecular Biology 60:137-151. Zuo JR, Niu QW, Nishizawa N, Wu Y, Kost B and Chua NH (2000) KORRIGAN, an Arabidopsis endo-1,4-beta-glucanase, localizes to the cell plate by polarized targeting and is essential for cytokinesis. Plant Cell 12:1137-1152.  28  CHAPTER 2  Characterization and varied expression of a membrane-bound endo-β-1,4glucanase in hybrid poplar 1  1  A version of this chapter has been published. Maloney VJ and Mansfield SD (2010) Characterization and varied expression of a membrane-bound endo-b-1,4-glucanase in hybrid poplar. Plant Biotechnology Journal 8:294-307.  29  2.1 Introduction In plants, the process of cellulose biosynthesis is as fundamental and important as photosynthesis. The modulation of cellulose biosynthesis influences many aspects of plant growth and development, including cell division and expansion, plant morphogenesis, and response to environmental cues (Mellerowicz and Sundberg, 2008; Taylor, 2008). Despite the importance of cellulose, a complete and thorough understanding of the intricacies of its biosynthesis is still relatively unknown. However, recently there have been a number of cellulose deficient Arabidopsis thaliana mutants that have provided evidence for the mechanism of several genes outside the cellulose synthase complex that are required for complete and correct cellulose biosynthesis. One such gene, KORRIGAN (KOR), was originally isolated from the kor1-1 mutant which displayed pronounced architectural alterations in the primary cell wall when grown in the absence of light. KOR was subsequently proposed to play a role in cell wall loosening (Nicol et al., 1998). KOR has since been shown to be an endo-β-1,4glucanase (EGase) that is located primarily in the plasma membrane and presumably acts at the plasma membrane-cell wall interface because of its single N-terminal membrane spanning domain (Nicol et al., 1998; Zuo et al., 2000). Additional mutations in the KOR gene of Arabidopsis (kor1-2) have been isolated and shown to cause the formation of aberrant cell plates, incomplete cell walls, and multinucleated cells, leading to abnormal seedling morphology (Zuo et al., 2000). Kor 1-2 was further shown to possess a T-DNA insertion that deleted a ~1-kb portion containing the entire KOR promoter and 5′UTR 22 base pairs upstream of the translation start site.  Moreover,  double mutants of cellulose synthase (rsw1) and KOR (rsw2) resulted in further reductions in cellulose content, suggesting the need for both a glycosyl transferase and glycosyl hydrolase for successful cellulose biosynthesis and deposition in the primary cell walls of plants.  This result concurs with previous findings in Agrobacterium  (Matthysse et al., 1995), in which a membrane-anchored EGase was shown to be involved in the cleavage of lipid-linked oligosaccharides. Sato et al. (2001) suggested that the role of KOR in cell elongation and cellulose synthesis provides evidence that cell wall loosening occurs normally but that filling the voids created within the cellulose microfibrils, which aids in constraining anisotropic cell elongation, is impaired in the mutants.  Furthermore, it was demonstrated that KOR also influences pectin 30  metabolism and can induce changes in pectin composition in the primary cell wall (His et al., 2001).  More importantly, morphological and chemical data on temperature  sensitive, elongation deficient, single base pair-substitution mutants of KOR (acw1, rsw2-1, rsw2-3 and rsw2-4) have pointed to KOR as having an key role in primary cell wall cellulose deposition (Lane et al., 2001). Cellulose content in these mutants has been shown to be reduced by as much as 40% compared to wild type plants, and was compensated by an increase in pectin content (up 162%). This implies the presence of feedback mechanisms controlling cell wall polysaccharide depositions. Other effects of these mutations included delays in cell wall regeneration in mutant protoplasts, abnormal cytokinesis, distorted flower morphology, and anther dehiscence impairment. While the close association of KOR with primary cell wall development has been widely discussed, it has also been reported that KOR may also play a role in secondary cell wall development (Szyjanowicz et al., 2004). Two irregular xylem 2 mutations (irx21 and irx2-2) that are defective in secondary cell walls and display reduced cellulose synthesis in the xylem cells have been shown to be the result of two independent mutations (Pro250Leu and Pro553Leu substitutions) in the KOR protein.  The irx2  mutants have severe reductions in crystalline cellulose (30% of wild type) and concurrently display no increase in glucose in the non-cellulosic fractions, suggesting that cellulose synthesis in the irx2 mutants is specifically affected only in the secondary cell wall. Unlike the kor1-1 mutant, the irx2 mutants did not show reduced hypocotyl growth in the dark, nor did they display the radial swelling phenotype typical of the acw1 and rsw2 mutants.  Other cellular abnormalities, such as abnormal cell shape and  incomplete cell plates, were also not observed in the irx2 mutants. Conversely, kor1-1 stems that were reported to be primary cell wall mutants also have severely collapsed xylem cells similar to the irx2 mutants (Szyjanowicz et al., 2004). An investigation of membrane-anchored EGases from Brassica napus showed that its cel16 gene is orthologous to the KOR gene from Arabidopsis, performing the same function in the two organisms (Molhoj et al., 2001a; Molhoj et al., 2001c). Additionally the recombinant expression of the catalytic domain of the putative hybrid aspen KOR ortholog, PttCel9A (Master et al., 2004), and cel16 (Molhoj et al., 2001) shows  that  AtKOR  orthologs  have  substrate  specificity  for  low  substituted  carboxymethyl-cellulose. Genes encoding membrane-anchored EGases have further 31  been found in dicots such as tomato (Brummell et al., 1997), oilseed rape (Molhoj et al., 2001a), aspen (Bhandari et al., 2006; Master et al., 2004), melon (GenBank# AB271851), opium (GenBank# BF010444), clover (GenBank# AW692796), and cotton (GenBank# AF511408); in monocots such as orchid (Brummell et al., 1994) and rice (Zhou et al., 2006); and in the gymnosperm Pinus taeda (Molhoj et al., 2002). Two additional genes encoding membrane-anchored EGases from A. thaliana termed KOR2 and KOR3 have also been characterized (Molhoj et al., 2001b) which suggests that membrane-anchored EGases are expressed in different cell types. In Arabidopsis the KOR gene is expressed more often throughout the entire plant, while KOR2 and KOR3 are only active in more cell types such as the flowering organs and in the developing root hairs, respectively (Molhoj et al., 2001b). In Populus trichocarpa, five glycosyl transferase family 9 genes with a single transmembrane domain have been shown to be similar to the KOR genes from Arabidopsis (del Campillo, 1999; Molhoj et al., 2002).  However,  expression profiling on an EST database of 3000  cambial sequences assembled from hybrid aspen identified only two family 9 glycosyl hydrolase genes that are upregulated during xylogenesis (Hertzberg et al., 2001; Sterky et al., 1998). Recently Takahashi et al. (2009) demonstrated that only transcripts of PtCel9A1, which has been shown to be allelic to the membrane-bound EGase from P. tremula × tremuloides (PttCel9A; (Master et al., 2004; Rudsander et al., 2003) and P. tremuloides (PtrKOR; Bhandari et al. 2006), were abundant in wood-forming tissues. These genes were further found to be upregulated in tissues undergoing secondary cell wall formation, to contain the three domains that are typical of the previously described membrane-bound EGases and to share 82% identity with the Arabidopsis KOR gene (Bhandari et al., 2006; Rudsander et al., 2003; Takahashi et al., 2009). In the present study, we report on the mis-regulation of a poplar membranebound β-1,4-glucanase gene through RNAi-silencing and on the over-expression of AtKOR in transgenic lines of hybrid poplar trees (Populus alba × grandidentata; Pa×g). The results show that the transcription of the endogenous Pa×gKOR gene was partially suppressed in the RNAi transgenic plants, and there was a significant increase in transcript abundance of the foreign AtKOR gene in the transgenic lines. Using the RNAi lines, we demonstrate that altered expression of Pa×gKOR affected the amount and ultrastructure of the secondary cell wall cellulose produced by the plant, leading to 32  either moderate or severe deformities, while the AtKOR up-regulation was mainly limited to impacts on the ultrastructure of the cellulose produced by the trees. Together, using the results of these efforts, we discuss the possible role that KOR plays in the cellulose biosynthetic process in trees.  These cell wall architectural changes have  significant industrial implications because cellulose content, cellulose architecture and the interactions among cell wall biopolymers all influence lignocellulosic processing parameters. 2.2 Materials and methods 2.2.1 PaxgKOR isolation and construct development A full-length cDNA encoding the putative hybrid poplar KOR (Populus alba × grandidentata; Pa×gKOR) was obtained using primers designed from a membranebound endo β-1,4-glucanase from the Populus trichocarpa genome database (http://genome.jgi-psf.org/Poptr1_1/Poptr1_1.home .html) which had a greater than 97% similarity to Pa×g. oligonucleotides,  Once the full-length reading frame was elucidated, two Pa×gRNAiFW  (5′TTTCACAACCAGACCAGTATA3′)  and  Pa×gRNAiRV (5′CATGGCTCCAACAAGTGTATT3′) with the addition of either 5′ BamHI and 3′ ClaI (sense) or 5′ XhoI and 3′ KpnI (antisense) restrictions sites were used to amplify a 400 base pair fragment of the Pa×gKOR coding region from cDNA.  The  fragments were then digested with the appropriate restriction enzymes and ligated into the pKANNIBAL (Helliwell and Waterhouse, 2003) cloning vector.  Finally, the NotI  fragment from pKANNIBAL containing the hpRNA cassettes was subcloned into the binary vector pART27 (Gleave, 1992) and used for plant transformations.  The  Arabidopsis thaliana (AtKOR) gene sequence was determined from GenBank (Accession #AF073875) and two oligonucleotides, AtKORFw with a BamHI site (5′ACCTTGGATTCATTGTTGTTGTT3′)  and  AtKORRv  with  a  SacI  site  (5′CAGGAGCTCACAAGTCTAGCTTT3′), were used to clone the full open reading frame from wild type Columbia Arabidopsis cDNA. The appropriate fragment was then inserted into the pSM1 cloning vector (Canam et al., 2006) under the control of the enhanced tandem CaMV 35S (2×35S) constitutive promoter (Datla et al., 1993; Kay et al., 1987).  33  2.2.2  Generation of transgenic plants  Either the pART27-Pa×gKORRNAi or 2×35::AtKOR plasmid was transferred to Agrobacterium tumefaciens strain EHA105 by the freeze-thaw method. Transformation of hybrid poplar plant tissue was achieved using a standard leaf disk inoculation technique (Coleman et al., 2008a). Plants were confirmed as transgenic by screening genomic DNA with primers specific to either a 773bp fragment that is located just outside of the hairpin structure (Fw- 5′TCCCACAAAAATCTGAGCTTAA3′, RV- 5′TACCTTTTTAGAGACTCCAATC3′) in the RNAi lines or the same primers described above for the AtKOR lines. Plants were maintained in tissue culture with 16 h days until approximately 10 plants of equal size from each line could be propagated. Plants were then transferred to soil and grown under supplemental lights (@ 300 µmole/s/m2) on flood tables and watered with fertigated water daily in a greenhouse. After four months’ growth, five trees from each line were harvested and leaf, cambium, and xylem samples were collected.  2.2.3 Genomic DNA extraction and purification Genomic DNA was extracted from single leaves taken from tissue culture grown plants using a CTAB extraction method modified from Rogers and Bendich (1994). Single leaves were placed in microcentrifuge tubes and ground to a powder with liquid nitrogen. One mL of CTAB extraction buffer (2% [w/v] hexadecyltrimethylammonium bromide [CTAB; Sigma, St. Louis, MO, USA], 100 mM Tris- HCl, pH 8.0, 1.4M NaCl, 20 mM EDTA, 1% [w/v] polyvinylprylidone, and 0.2% [v/v] 2-mercaptoethanol) was added to each tube, and then incubated at 65°C for 60 min. The tube was then centrifuged in a microcentrifuge for 10 min followed by the addition of one volume of phenol:chloroform:isoamyl alcohol (Sigma, St. Louis, MO, USA) and re-centrifuged for 10 min. Genomic DNA was precipitated from the aqueous phase by addition of half volume of isopropanol alcohol and centrifuging for 5 min. One volume of ethanol was then added to the resulting pellet and the genomic DNA recovered by centrifugation. Finally, the DNA was re-suspended in 50 mL of TE buffer (10 mM Tris-HCl, pH 8.0, and 1 mM EDTA), quantified by A260, and stored at 4°C.  34  2.2.4 RNA extraction and transcript abundance Total RNA was isolated from approximately 500 mg of frozen ground cambial tissue using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. RNA yield was measured by absorption at 260nm, and 10 μg was treated with DNAase (Ambion TURBO DNA-free, Ambion Inc., Austin TX, USA). One μg of the resulting DNA-free RNA was evaluated on a 1% Tris-acetate EDTA agarose gel in order to determine quality. Equal quantities of RNA (1 μg) were used for the synthesis of cDNA with SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA) and (dT)16 primers, according to the manufacturer’s instructions. Samples were run in triplicate with Platinum SYBR Green qPCR Master mix (Invitrogen, Carlsbad, CA, USA) on an Mx3000p real-time PCR system (Stratagene, La Jolla, CA, USA). The primers used for the real-time PCR analysis of the Pa×gKOR RNAi lines were designed so that the reverse primer (5′GGATTGACAAGAACACCATAT3′) was located within the 3′ UTR of the Pa×gKOR gene and the forward primer (5′GCAGCAAAATCATCTTACCAA3′) was located immediately upstream of the KOR stop codon. The real-time PCR analyses of the AtKOR up-regulated lines were performed using the primers AtKORFw (5′TCCTTTGTTCCCTACTCCAC3′)and AtKORRv (5′AGACCGGCAACAGGATTCAA3′). Conditions for all PCR reactions were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 30 s, 55°C for 1 min, and 72°C for 30 s. Transcript abundances were determined based on changes in Ct values relative to translation initiation factor5A (Ralph  et  al.,  2006)  using  the  following  (5′GACGGTATTTTAGCTATGGAATTG3′)  and  primers:  TIF5AFw TIF5ARv  (5′CTGATAACACAAGTTCCCTGC3′), and compared with the relative transcript abundance of the wild-type trees.  2.2.5 Analysis of wood composition Wood samples harvested from approximately 5 cm of the base of four-month-old greenhouse-grown trees were ground in a Wiley mill to pass a 0.4-mm screen (40 mesh) and Soxhlet extracted overnight in hot acetone to remove extractives. The extractive-free material was used for all further analyses. Lignin and carbohydrate content was determined with a modified Klason, in which extracted ground stem tissue (0.2 g) was treated with 3 mL of 72% H2SO4 and 35  stirred every 10 min for 2 h. Samples were then diluted with 112 mL DI water and autoclaved for 1 h at 121°C. The acid-insoluble lignin fraction was determined gravimetrically by filtration through a pre-weighed medium coarseness sintered-glass crucible,  while  the  acid-soluble  lignin  component  was  determined  spectrophotometrically by absorbance at 205 nm. Carbohydrate contents were determined by using anion exchange high-performance liquid chromatography (Dx-600; Dionex, Sunnyvale, CA, USA) equipped with an ion exchange PA1 (Dionex) column, a pulsed amperometric detector with a gold electrode, and a SpectraAS3500 auto injector (Spectra-Physics). For α-cellulose determination the extracted ground stem tissue was delignified with a modified version of Browning (1967) with two successive chlorite extractions by placing 200 mg of wood into 14 mL of buffer (60 mL of glacial acetic acid + 1.3 g NaOH/L) and 6 mL of 20% sodium chlorite solution (NaClO2) in a 50 mL Erlenmeyer flask, capped and gently shaken at 50°C for 14 h. The reaction solution was decanted and the wood washed with 25 mL of 1% glacial acetic acid. After the first extraction, the delignification procedure was repeated, and the wood was washed three times with 25 mL of 1% glacial acetic acid, followed by a 25 mL wash with acetone. The resulting holocellulose was permitted to dry in a 50°C oven overnight. α-cellulose content was then determined by extracting 50 mg of the oven dried holocellulose with 4 mL of 17.5% sodium hydroxide for 30 min at room temperature then adding 4 mL water and leaving it to react for another 29 min (Yokoyama et al., 2002). Soluble carbohydrates and starch content of leaf and xylem scrapings were determined as described in Coleman et al. (2006).  2.2.6 Antibody labelling Eight-month-old stem tissue from the Pa×gKOR RNAi lines was collected and immediately sectioned using a Leica CM1850 cryostat microtome, placed on a teflon coated multiwell slide (Cedarlane 63430-04) and then left to dry overnight. Sections were incubated in a blocking solution of 5% low fat dried milk (Carnation Co., Los Angeles, Cal., USA) in TBST (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween20). After the grids were blotted dry, they were incubated in either the rat monoclonal antibody LM10 for xylan (McCartney et al., 2005) or the rabbit polyclonal anti-XG for xyloglucan (Moore et al., 1986) diluted 1:20 in TBST for one hour. The grids were then 36  washed with TBST and a 1:100 dilution of the secondary antibody was added and incubated for 1 h. The grids were then washed again with TBST followed by nanopure water. All steps were carried out at room temperature. Imaging was conducted on a Zeiss Pascal Excite (Laser Scanning Confocal Microscope, Carl Zeiss, Toronto, Ont. Canada) with a two channel (two detector) scan head mounted on an upright AxioImager M1 microscope.  2.2.7 Cellulose characterization Microfibril angle estimates were generated by X-ray diffraction (Megraw et al., 1998). The 002 diffraction spectra from five individual four-month-old trees from each line were screened for T value distribution and symmetry on a Bruker D8 discover X-ray diffraction unit equipped with an area array detector (GADDS). Wide-angle diffraction was used in the transmission mode, and the measurements were performed with CuKα1 radiation (λ=1.54 Å). The X-ray source was fit with a 0.5-mm collimator, and the scattered photon was collected by a GADDS detector. Both the X-ray source and detector were set to theta=0°. The degree of cellulose crystallinity was determined from both stems as well as from isolated α-cellulose. Crystallinity estimates generated by X-ray diffraction were described by Mansfield et al. (1997) using the same X-ray parameters as for MFA determination with the exception of the source theta set at 17°. The molecular weight distribution of three samples from each Pa×gKOR RNAi line was obtained by Gel Permeation Chromatography (GPC) analyses of their tricarbanyl derivatives as described by Mansfield et al. (1997). 2.3 Results 2.3.1 Generation of transgenic hybrid poplar plants The membrane-bound β-1,4-glucanase isolated from Pa×g cDNA was shown to have a protein similarity of 82% when compared to the KOR protein from Arabidopsis (109 amino acid substututions) with a 77% identical transmembrane domain. Additionally, it was determined that Pa×gKOR is allelic to the two previously published KOR genes from Populus (PtrKOR AY535003 (Bhandari et al., 2006) and PttCel9A AY660967 (Takahashi et al., 2009) sharing 98% identity at the protein level with all proteins having 37  identical polarized targeting signals (Figure 2.1). Suppression of Pa×gKOR expression was achieved using a 400-bp hairpin RNAi construct designed from the putative fulllength cDNA-encoding Pa×gKOR. In contrast, KOR over-expression was achieved by using the ubiquitous 2×35S promoter fused to the full length open reading frame of the foreign AtKOR gene.  Agrobacterium-mediated transformation yielded numerous  independent transformants from each construct that were confirmed through genomic screening, from which seven Pa×gKOR RNAi lines and ten AtKOR lines were propagated and transferred to the greenhouse. Four lines from each construct were chosen for complete cell wall analyses based on preliminary expression data. A more detailed analysis of endogenous KOR and AtKOR expression was completed using real-time PCR, which revealed a substantial reduction in KOR transcript abundance in all four Pa×gKOR RNAi lines when compared with the wild-type trees, and a substantial increase in transcript levels of the foreign KOR gene in all four of the AtKOR lines when compared to the associated wild-type trees grown under the same conditions, as might be expected (Figure 2.2). In addition, measurements of height and diameter (measured as caliper at 10 cm above the root collar) of the four Pa×gKOR RNAi lines revealed a significant reduction in these growth parameters with the lowest two expressing lines having the largest reductions in diameter growth as well as leaf browning, reduced stem stability and a irregular xylem phenotype (Figure 2.3A and Figure 2.4). In contrast, only two of the AtKOR transgenic lines displayed a reduction in height and there was no significant change in diameter growth (Figure 2.3B). All trees were harvested after four months of growth and were used for all further analyses. 2.3.2 Carbohydrate and lignin analyses Structural carbohydrates and lignin composition were determined from extractive-free dried stem tissue of four-month-old trees to investigate the effects of KOR misregulation on carbohydrate-derived polymer synthesis. The total carbohydrates (arabinose, rhamnose, galactose, glucose, xylose, and mannose) and lignin (soluble and insoluble) composition, as a percentage of dry mass, of both wild-type and transgenic poplar lines is shown in Table 2.1. All of the Pa×gKOR RNAi lines, with the exception of line 7, show significant differences compared to the wild-type trees grown at the same time with respect to stem structural chemistry. When compared to the 38  wildtype, the transgenic trees show a 2% to 14% increase in arabinose, rhamnose, galactose, xylose and mannose, sugars that make up the hemicellulose matrix and pectic components of the cell wall, whereas there was an average decrease of 15% in glucose content, implying a decrease in cellulose content.  Conversely, total lignin  content was increased by an average of 6% in these same lines. In contrast to the Pa×gKOR RNAi transgenic trees, the AtKOR lines showed an average 7% decrease in xylose and minor changes in cell wall glucose content when compared to their associated wildtype samples.  The variations in cell wall composition are generally  consistent with the variations observed in the expression data. To confirm a modification in cell wall cellulose composition, as inferred by glucose quantification, in the Pa×gKOR RNAi trees, holocellulose and subsequently, αcellulose was isolated from the extractive free xylem tissue. These data concur with the glucose measurements derived from whole cell wall analysis, clearly demonstrating an overall decrease in cellulose in lines 2, 16 and 19 (Figure 2.5).  Additionally,  histochemical staining of stem cross section with calcofluor white (specific for cellulose) exhibited a considerable decrease in fluorescence in the Pa×gKOR RNAi lines, while the phenolic-specific (lignin) phloroglucinol stain showed only a slight increase in color intensity in the transgenic lines (Figure 2.6). These findings are consistent with the wet chemical analyses. Histochemical staining and imaging also revealed that the two most extremely KOR suppressed transgenic poplar lines display an irregular xylem phenotype, particularly in vessel elements that appear to be substantially deformed when compared to the wild-type trees (Figure 2.4). To further examine the change in hemicellulose content that was evident from the carbohydrate analyses of the Pa×gKOR RNAi lines, immuno-labelling for xylan and xyloglucan was performed on the two lines that had the lowest Pa×gKOR expression levels (16 and 19), as well as in the wild-type trees. LM10, a rat monoclonal antibody (McCartney et al., 2005), was used for xylan labelling, while anti-XG, a rabbit polyclonal antibody (Moore et al., 1986) was used for the xyloglucan labelling. LM10 has been shown to bind to only un- or low-substituted xylans, whereas the anti-XG is specific to xyloglucans. Immuno-labelled sections were imaged with a confocal microscope, and average pixel values were calculated for all sections using the entire image (0 being black and 255 being white). Based on histochemical quantification, both Pa×gKOR 39  RNAi lines 16 and 19 have more xylan than the wild-type trees (21.04) with average pixel values of 26.97 and 23.94, respectively.  Employing a similar technique and  common samples indicated that there is no quantifiable change in the amount of xyloglucan among samples (Figure 2.7).  These findings support the wet chemical  analyses and expression data, which suggested that the down-regulation of KOR influenced xylan synthesis. However, it permits the extension of these findings and suggests that the increased xylan, as determined by wet chemical composition analysis, is a function of perturbed xylan synthesis, and not xyloglucan synthesis. Total soluble carbohydrates (glucose, fructose and sucrose) and starch content of the transgenic tissues were also quantified from the upper leaves of four-month-old trees (Figure 2.8). The Pa×gKOR RNAi trees have reduced levels of sucrose and slightly more glucose and fructose when compared to the associated wild-type trees. In contrast, the AtKOR lines had similar sucrose composition compared to their associated wild-type tress, but had slightly less glucose and fructose. Leaf starch content was not significantly changed, whereas in the xylem scraping, it was significantly decreased in three of the four Pa×gKOR RNAi lines (data not shown). Soluble carbohydrate analyses of the xylem scrapings of the Pa×gKOR RNAi lines again revealed a slight reduction in sucrose, while glucose and fructose content was not significantly altered (data not shown). 2.3.3 Cellulose characterization Cellulose crystallinity and microfibril angle (MFA) measurements were determined using X-ray diffraction. MFA, the primary angle of the cellulose microfibrils with respect to the long axis of the growing cell, was on average 15% smaller in all of the Pa×gKOR RNAi transgenic lines, while the percent of crystalline cellulose concurrently increased an average of 17% when compared to the wildtype.  The extent of these cell wall  phenotypes within the Pa×gKOR RNAi lines again is largely consistent with the trends observed in the expression data.  In contrast, cellulose crystallinity decreased an  average 10% in the AtKOR lines (Table 2.2). The degree a polymerization of the isolated cellulose polymer in the Pa×gKOR RNAi lines was also determined and was shown not to be significantly altered (data not show).  40  2.4 Discussion KORRIGAN has been identified as an important player in the biosynthesis of cellulose in Arabidopsis and other non-woody species, but despite its importance, to date very little work has been conducted on membrane-bound EGases in trees. And, equally important, KOR has received very little attention with respect to the synthesis and deposition of the secondary cell wall. Cellulose, arguably the most abundant polymer on earth, is a very valuable biomaterial for humankind for textiles, pulp and paper production and more recently as an important material in the production of biofuel. Therefore, understanding the synthesis of cellulose in plant species that accumulate substantial biomass, such as the rapid growing Populus species, is very important both fundamentally and industrially. The aim of this study was to investigate changes in carbohydrate metabolism and secondary cell wall cellulose ultrastructure that occur as a result of the misregulation of the membrane-bound β-1,4-endoglucanase orthologous to KORRIGAN in Populus alba x grandidentata (Pa×gKOR). RNAi-suppression was used specifically to impair the expression of Pa×gKOR in transgenic hybrid poplar trees. This approach takes advantage of the endogenous RNA silencing pathways that are normally directed against invading viral nucleic acids (Brodersen and Voinnet, 2006). We found that down-regulation leads to moderate to severe defects in plant growth and significantly impacts the ultrastructure of the cellulose produced. Generally, the Pa×gKOR RNAi trees display an irregular xylem (irx) phenotype, commonly associated with KOR and other secondary cellulose-specific mutants in Arabidopsis.  The suppressed poplar  transgenic lines produced less, but more highly-crystalline cellulose, while the hemicellulose content and, more specifically, the xylose content, increased. In addition, the amount of available sucrose in the leaves and xylem decreased. Futhermore, the major angle that the cellulose microfibrils made with respect to the longitudinal cell axis (secondary cell wall microfibril angle) was significantly lower in all of the Pa×gKOR RNAi transgenic lines, while the degree of polymerization of the cellulose polymer was not affected. Conversely, the exogenous expression of AtKOR in poplar trees did not have a major affect on growth parameters (no irregular xylem evident), but it did impact the ultrastructure of the synthesized cellulose. The AtKOR lines have less crystalline cellulose and reduced secondary cell wall xylose content. 41  2.4.1 Previously proposed roles for KORRIGAN do not appear to hold true in poplar Past studies on the role of membrane-bound EGases in cellulose synthesis in other species have lead to three main hypotheses. The first, that KOR is required for the assembly of glucan chains in cellulose microfibrils, arose from studies that show that a truncated version of the Brassica ortholog, CEL16, without the N-terminal membrane anchor, hydrolyzed the artificial cellulose derivative carboxymethylcellulose (CMC) and amorphous cellulose.  Most importantly, unlike secreted EGases from plants, the  soluble KOR protein could not act on xyloglucans, but digested a variety of cellulose derivatives and colloidal Avicel (Molhoj et al., 2001c). Similarly, the Arabidopsis irx2 mutants had severe reductions in crystalline cellulose (30% of wild type) as measured by FT-IR analysis (Szyjanowicz et al., 2004). It was suggested that proteins intricately associated with cellulose deposition, such as the EGase KOR, play an editing role by partially removing defective complexes from their growing microfibrils. However, we report here that a targeted down-regulation of the Pa×gKOR gene in hybrid poplar actually leads to an increase in the crystallinity index of the secondary cell wall cellulose, whereas the expression of the exogenous AtKOR gene in poplar decreases cellulose crystallinity, as determined by X-ray diffraction (a more precise measure of crystallinity). Furthermore, the current evaluation focuses on the development of the secondary cell wall cellulose, while previous efforts have tended to focus on, and draw conclusions from, primary cell wall analysis. The second hypothesis implied that KOR regulates the degree of polymerization (DP) during or subsequent to microfibril assembly (Molhoj et al., 2002). Molhoj et al. (2001c) demonstrated that KOR could only digest specific carbohydrates and that no βglucan, xylan, or oligosaccharides shorter than cellopentose were hydrolyzed, suggesting that the natural substrate for KOR might be a longer chain oligosaccharide, such as cellulose. However, investigation into the molecular weight distribution of the cellulose produced in the Pa×gKOR down-regulated trees did not provide any evidence that the poplar KOR protein has such a function. KOR mis-regulation in poplar did not alter the average molecular weight of the cellulose polymer isolated from the transgenic and wild-type trees, nor did it alter the polydispersity of this macromolecule. These  42  findings suggest that KOR does not function as a developmental regulator of cellulose polymer length. Finally, studies have also suggested that KOR cleaves a precursor molecule in the β-1,4-glucan synthesis pathway. The first support for such a theory comes from a study conducted by Peng et al. (2001), who observed the deposition of non-crystalline glucans, typical of rsw1 and acw1 mutants, in response to treatment of cotton fibers with the experimental herbicide CGA325’615.  It was further determined that these  glucan polymers were linked to sitosterol accumulations, and consequently, sitosterol-βglucoside (SG) as a primer for chain elongation was hypothesized (Peng et al., 2002). Further evidence to support this hypothesis came from experiments showing that labelled SG added to isolated membrane fractions from fibres is incorporated into the cellulose polymer (Peng et al., 2002). As such, Peng et al. (2002) developed a model where a glucose residue from UDP-Glc is first transferred onto lipid β-sitosterol to form SG, which further acts as a primer for chain elongation catalyzed by the cellulose synthase complex (CesA) proteins. These lipid-linked oligosaccharides were proposed to be cleaved from the sitosterol primer by KOR and assist as a mechanism for cleavage of the SG primer from the β-1,4-glucan chain. However, studies in the Arabidopsis C-14 sterol reductase mutant FACKEL, show that this mutation had adverse effects on cellulose, but transcript levels of KOR were not affected (Schrick et al., 2004; Schrick et al., 2000).  Additionally, Robert et al. (2004) studied sterol  glucosides in the kor1-1 mutant using autoradiography and showed that equal amounts of sitosterol glucoside and sitosterol cellodextrin were apparent in the mutant and the wild-type plants. In the current study, we also screened metabolites from the misregulated KOR transgenic trees with gas chromatography mass spectrometry and could not discern any difference in sitosterol glucoside concentrations (data not shown). 2.4.2 Possible role for PaxgKOR in the relaxation of the stresses around the cellulose microfibril or in the separation of the cellulose macrofibril Despite the increasing amount of information that is available on KOR, its exact function during cellulose biosynthesis remains to be established. This is further complicated by the lack of information about the exact cellular location of the KOR protein. It has been suggested that KOR is located at the plasma membrane (Nicol et al., 1998; Peng et al., 43  2002), the cell plate (Zuo et al., 2000) and the phragmoplast (Molhoj et al., 2002). Furthermore, epitope-tagged KOR constructs have proven that there is little chance that KOR and CesA proteins interact, which suggests a less direct role for KOR in the synthesis of the cellulose microfibrils and perhaps indicates a role for KOR downstream in cell wall formation/cellulose deposition (Szyjanowicz et al., 2004). Atomic force microscopy has previously been used to visualize cellulose microfibrils (Micrasterias denticulate), where it was shown to possess regular periodicities and twisting within individual microfibrils (Hanley et al. 1997). It is conceivable that KOR plays a role in relaxing the inherent stresses that are introduced during synthesis, and ultimately causing the microfibril to twist. If KOR is not completely functional, the frequency of highly twisted regions within each cellulose microfibril may be altered, consequently preventing the proper movement of the cellulose synthase complex. Futhermore, it has been shown in numerous studies that microfibril angle increases with the speed of growth (reviewed in Donaldson, 2008). Herein, we demonstrate that a reduction in the expression of Pa×gKOR leads to a significant decrease in the amount of cellulose in the plant stem, and growth. Additionally, we show that MFA decreases in the RNAisuppressed lines.  Taken together, these findings support the concept that KOR  indirectly influences the rate of cellulose biosynthesis by potentially influencing the extent to which the growing microfibril is twisted. Moreover, the cellulose that was deposited was more crystalline, which could be indicative of a more closely packed, highly twisted microfibril caused by an increase in torsional stress. In contrast, the AtKOR over-expressing lines had cellulose that was less crystalline, which provides further evidence that KOR may play of role in relaxing the torsional stresses around the developing cellulose microfibril in hybrid poplar. An alternate cause for the changes in the cell wall crystallinity in the modified plants could be explained using a new model for the primary cell wall that was derived from direct visualization of maize stem pith (Ding and Himmel, 2006). The authors demonstrate that a number of elementary fibrils are synthesized at the cellulose synthase complex and come together into much larger macrofibrils. These macrofibrils eventually split at the ends to form parallel microfibrils, which are then available to interact with other cell wall components such as hemicelluloses, pectin and lignin. On the basis of these data, Ding and Himmel (2006) proposed a new molecular model 44  consisting of a 36-glucan-chain elementary fibril, in which the 36-glucan chains form both crystalline and sub-crystalline structures. In the past, numerous models of cell wall structures have been proposed, but all show non-cellulosic polysaccharides associated with the surfaces of the cellulose microfibrils (Cosgrove, 2001). In hardwoods, such as the hybrid poplar angiosperm employed herein, the main hemicellulose in the cell wall is glucuronoxylan. Hayashi  (1989) previously suggested that it may be possible for  hemicelluloses, such as glucuronoxylan, to become trapped in the microfibril as it forms, resulting in disordered regions. We demonstrated that the expression of AtKOR in hybrid poplar led to a decrease in xylose, whereas a reduction in Pa×gKOR activity led to an increase in xylose as well as some of the other hemicellulose-derived carbohydrates, whose main role is weaving the cellulose microfibril network together. These results provide evidence for a role for KOR in the splitting of the macrofibril into individual microfibrils, and any lack of function could prevent the macrofibril from being dispersed into the microfibrils.  Alternatively, the hemicellulose response may be a  consequence of the plants response to compensate for the altered available binding sites resulting from altered levels of twisting, as previously proposed. The combination of increased hemicellulose-derived carbohydrates, along with more crystalline cellulose, implies an inability of the hemicellulose moeities to interact with the cellulose as a result of a decrease in available microfibrils. Conversely, when KOR function is increased, as is the case with the AtKOR lines, the macrofibril undergoes increased splitting into more microfibrils offering sites for increased rates of cellulose-hemicellulose interaction. Furthermore, the cellulose synthase complex may be somewhat inhibited in movement within the cell wall if the macrofibril is not properly split, leading to impaired cellulose production.  Roberts et al. (1982) showed that preventing the proper formation of  cellulose in the alga Oocystis apiculata with calcofluor white leads to lens-shaped thickening of the cell wall, which might be suggestive that the cellulose synthase complex is not tracking properly in the plasma membrane. Additionally, it has been suggested that the propulsion of the cellulose synthase complex is through a polymerization-driven supramolecular motor that will function only if the proper formation of the cellulose microfibril occurs (Diotallevi and Mulder, 2007). Furthermore, a recent paper by Takahashi et al. (2009) demonstrates  that overexpressing  PttCel9A1, the putative AtKOR ortholog from hybrid aspen, causes a decrease in 45  cellulose crystallinity. These results, taken together with our current results, provide further evidence that KOR aids cellulose biosynthesis by modifying the ultrastructure of the cellulose microfibril. 2.4.3 Disrupting cellulose biosynthesis depletes soluble sucrose content Prior to cellulose synthesis, sucrose is delivered directly to sink cells via phloem transport, where sucrose synthase (SuSy: soluble or particulate) works in concert with the cellulose synthase complex to deliver UDP-glucose to the CesA complex (Delmer and Haigler, 2002). In the Pa×gKOR RNAi lines, reduced cellulose synthesis led to simultaneous reduction in the accumulation of sucrose in both the leaves and xylem. This reduction may be indicative of a feedback mechanism that prevents the unnecessary accumulation of sucrose in the cell. Additionally, the Pa×gKOR RNAi lines appeared to be under stress, as evidenced by their browning leaves (Figure 2.4). Microscopy of four-month-old stems revealed that the xylem in the Pa×gKOR RNAi lines was indeed slightly collapsed (Figure 2.4 & Figure 2.6). This collapsed xylem phenotype likely impedes the transport of water and ultimately imposes drought stresses, illustrated by the wilting observed in line 19 and 16. Furthermore, Coleman et al. (2008b) showed that the severe inhibition of cell wall lignification manifested by the RNAi suppression of the coumaroyl 3'-hydroxylase gene in hybrid poplar (Populus alba×grandidentata) produced trees with a collapsed xylem phenotype, resulting in compromised vascular integrity, and displayed reduced hydraulic conductivity and a greater susceptibility to wall failure and cavitation. Experiments in barley leaves have shown that both drought and osmotic stress resulted in a depletion of sucrose (Villadsen et al., 2005). This study showed that sucrose synthesis was not inhibited, but rather there was an increased influx into the hexose pools caused by an increase in sucrose hydrolysis. We also observed an increase of flux into the hexose pools in the Pa×gKOR RNAi lines, where increases in both glucose and fructose were evident (Figure 2.8A). Using a combination of techniques, we were able to demonstrate that the misregulation of the poplar KOR gene affects the ultrastructural properties of secondary cell wall cellulose and equally as important, the relationship between cellulose and the ancillary carbohydrates composing the hemicellulose cell wall components. Biochemical 46  characterization of the resultant cellulose produced by both the down- and up-regulated transgenic trees clearly demonstrates that Pa×gKOR plays a role in either in the relaxation of the stresses around the cellulose microfibril or in the separation of the cellulose macrofibril. Furthermore, a change in cellulose content and/or structure has effects upstream of cellulose biosynthesis causing changes in the soluble carbohydrate content in the leaf and xylem.  Further studies are necessary to determine the exact  cellular location of KOR in order to elucidate the precise role that it plays in the cellulose synthesis process. The changes manifested by altering cellulose synthesis will have major affects on the processing and quality of the cellulose arising from KOR misregulated trees, including cellulose as a feedstock for lignocellulose biofuels applications.  47  Table 2.1. Structural cell wall carbohydrates and total lignin content of four-month old poplar trees. (A) Wild-type and Pa×gKOR RNAi-suppressed lines of poplar (grown from November 9, 2007 to March 20, 2008), and (B) wild-type and AtKOR over-expression lines of transgenic poplar (grown from September 11, 2007 to January 16, 2008). Standard error of the mean in parentheses, n=5 biological replicates; values in bold have a t-test p-value of 0.05 or less relative to wild-type. Carbohydrates (% of dry mass) Line   Lignin (% of dry mass)  arabinose   Rhamnose   galactose   glucose   xylose   mannose       insoluble    soluble    total   (A)                                 WT   0.32 (0.02)   0.50 (0.01)   1.08 (0.08)   46.19 (0.60)   17.74 (0.32)   2.26 (0.16)      20.93 (0.45)   3.72 (0.30)   24.65 (0.60)   2   0.34 (0.01)   0.57 (0.01)   1.12 (0.06)   42.32 (0.95)   18.44 (0.35)   2.38 (0.09)      21.59 (0.93)   4.41 (0.35)   26.00 (1.07)   7   0.31 (0.01)   0.46 (0.04)   1.27 (0.08)   47.49 (0.99)   16.68 (0.47)   2.32 (0.08)      20.28 (0.63)   4.46 (0.33)   24.74 (0.89)   16   0.36 (0.01)   0.58 (0.05)   1.28 (0.08)   36.17 (0.90)   20.62 (0.27)   2.55 (0.17)      21.41 (0.40)   4.69 (0.30)   26.10 (0.50)   19   0.38 (0.02)   0.58 (0.04)   1.45 (0.23)   38.63 (1.44)   19.20 (0.49)   2.55 (0.07)       22.21 (0.76)   4.19 (0.52)   26.40 (0.36)   (B)                                 WT   0.26 (0.01)   0.25 (0.03)   0.82 (0.04)   47.23 (0.86)   18.65 (0.24)   1.99 (0.11)      19.48 (0.67)   3.47 (0.33)   22.95 (0.93)   3   0.23 (0.02)   0.15 (0.04)   0.89 (0.04   43.95 (1.44)   17.48 (0.63)   1.83 (0.11)      22.12 (1.42)   3.23 (0.25)   25.35 (1.62)   5   0.27 (0.01   0.18 (0.04)   1.03 (0.12)   46.18 (0.87)   17.19 (0.45)   1.66 (0.14)      21.06 (0.56)   3.06 (0.22)   24.12 (0.62)   9   0.21 (0.02)   0.14 (0.03)   0.78 (0.06)   43.50 (1.37)   17.45 (0.36)   1.77 (0.17)      22.24 (0.88)   3.46 (0.40)   25.71 (0.93)   16   0.22 (0.03)   0.14 (0.05)   0.91 (0.11)   46.72 (0.74)   17.41 (0.76)   1.86 (0.19)      20.09 (0.67)   3.40 (0.30)   23.49 (0.67)                                    48  Table 2.2. Microfibril angle and cell wall crystallinity of four-month old poplar trees. (A) Wild-type and Pa×gKOR KOR RNAi-suppressed transgenic lines of poplar (grown from November 9, 2007 to March 20, 2008) and (B) wild-type and AtKOR over-expression lines of transgenic poplar(grown from September 11, 2007 to January 16, 2008) Standard error of the mean in parentheses, n=5 biological replicates; values in bold have a t-test p-value of 0.05 or less relative to wild-type. %Crystalline   %Crystalline   MFA   (wood)   (α‐cellulose)   WT   18.42 (0.32)  46.20 (0.58)  76.80(1.77)  2   15.79 (0.22)  51.20 (2.35)  78.00(2.86)  7   16.47 (0.31)  52.40 (1.57)  79.60(3.24)  16   15.18 (0.27)  53.00 (2.77)  80.00(1.58)  19   15.25 (0.36)  67.40 (4.06)  79.25(3.35)  WT   19.47 (0.79)  54.40 (0.81)  ‐  3   17.52 (0.49)  49.75 (2.88)  ‐  5   18.51 (1.53)  49.75 (2.43)  ‐  9   17.52 (0.61)  48.75 (1.03)  ‐  16   17.65 (0.43)  47.60 (0.68)  ‐  Line  (A)   (B)   49  Figure 2.1 Molecular characterization of KOR genes indicating similarities. (A) Schematic map of the genomic structures of the AtKOR and Pa×gKOR gene. Triangles indicate the location and size of the introns. (B) Alignment of the protein sequences for AtKOR, Pa×gKOR, PttCel9A (AY660967) and PtrKOR (AY535003) (Red = identical, Orange = similar, Black = different). The conserved polarized targeting signals (PTS) and the transmembrane domain are indicated.  50  Figure 2.2 Relative transcript abundances indicate decreased endogenous Pa×gKOR in RNAi lines and expression of AtKOR in the AtKOR over-expression lines of poplar. (A) Pa×gKOR in wild-type and Pa×gKOR RNAi lines of poplar, and (B) AtKOR in wildtype and AtKOR over-expression lines of transgenic poplar. Transcript levels were determined based on changes in Ct values relative to translation initiation factor5A. Error bars = standard error of the mean, n=5 biological + 3 experimental replicates. Stars indicate a t-test p-value of 0.05 or less. 51  Figure 2.3 Height and caliper measurements of trees after four months of growth in the greenhouse indicates growth reduction in Pa×gKOR RNAi lines. (A) Pa×gKOR RNAi lines of poplar, and (B) AtKOR over-expression lines of transgenic poplar. Error bars = standard error of the mean, n=10 biological replicates. Stars indicate a t-test p-value of 0.05 or less. 52  Figure 2.4 Pa×gKOR RNAi trees display irx phenotype. Three-month-old greenhouse grown Pa×gKOR RNAi line 19 and wildtype trees. Inset shows leaf browning and irregular xylem in four-month old RNAi trees.  53  Figure 2.5 Decreased α-cellulose and holocellulose in Pa×gKOR RNAi lines of transgenic poplar. Percentage, based on dry weight cell wall material. Error bars = standard error of the mean, n=5 biological replicates. Stars indicate a t-test p-value of 0.05 or less.  54  A  B  C  D  Figure 2.6 Histochemical staining of poplar cross-sections indicates the lower cellulose (lower calcoflour florescence) and slightly higher lignin cell wall chemistry associated with a down regulation of KOR in hybrid poplar. Cross sections of four-month-old (A) WT and (B) Pa×gKOR RNAi line 19 poplar stems stained with calcoflour white for cellulose, and (C) WT and (D) Pa×gKOR RNAi line 19 poplar stem sections stained with phloroglucinol for lignin. Scale bars = 90µm.  55  Figure 2.7 Xylan (LM10) and xyloglucan (anti-XG) immunolabelling of four-month-old poplar stem tissue indicates higher xylan concentrations in the Pa×gKOR RNAi. Stars indicate a significant difference in average pixel count compared to the wild type as determined by a t-test p-value of 0.05 or less. 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Proceedings of the National Academy of Sciences of the United States of America 95:13330-13335. 61  Szyjanowicz PMJ, McKinnon I, Taylor NG, Gardiner J, Jarvis MC and Turner SR (2004) The irregular xylem 2 mutant is an allele of korrigan that affects the secondary cell wall of Arabidopsis thaliana. Plant Journal 37:730-740. Takahashi J, Rudsander UJ, Hedenstrom M, Banasiak A, Harholt J, Amelot N, Immerzeel P, Ryden P, Endo S, Ibatullin FM, Brumer H, del Campillo E, Master ER, Scheller HV, Sundberg B, Teeri TT and Mellerowicz EJ (2009) KORRIGAN1 and its aspen homolog PttCel9A1 decrease cellulose crystallinity in arabidopsis stems. Plant and Cell Physiology 50:1099-1115. Taylor NG (2008) Cellulose biosynthesis and deposition in higher plants. New Phytologist 178:239-252. Villadsen D, Rung JH and Nielsen TH (2005) Osmotic stress changes carbohydrate partitioning and fructose-2,6-bisphosphate metabolism in barley leaves. Functional Plant Biology 32:1033-1043. Yokoyama T, Kadla JF and Chang HM (2002) Microanalytical method for the characterization of fiber components and morphology of woody plants. Journal of Agricultural and Food Chemistry 50:1040-1044. Zhou HL, He SJ, Cao YR, Chen T, Du BX, Chu CC, Zhang JS and Chen SY (2006) OsGLU1, a putative membrane-bound endo-1,4-beta-D-glucanase from rice, affects plant internode elongation. Plant Molecular Biology 60:137-151. Zuo JR, Niu QW, Nishizawa N, Wu Y, Kost B and Chua NH (2000) KORRIGAN, an Arabidopsis endo-1,4-beta-glucanase, localizes to the cell plate by polarized targeting and is essential for cytokinesis. Plant Cell 12:1137-1152.  62  CHAPTER 3  Functional conservation of KORRIGAN, a putative membrane-bound endo-1,4-βglucanase required for cellulose biosynthesis in vascular plants2  2  A version of this chapter will be submitted for publication. Maloney VJ, Samuels AL and Mansfield SD (2010). Functional conservation of Korrigan, a putative membrane-bound endo-1,4-β-glucanase required for cellulose biosynthesis in vascular plants.  63  3.1 Introduction Plant evolution has been characterized by adaptive events, including the development of a stiff, upright stem that permitted the expansion of the growth habit of plants migrating from an aquatic to terrestrial ecosystem. Lignins, aromatic polymers derived from phenylpropanoid metabolism, participate in macromolecular cross linkages with cellulose and other cell wall components in order to reinforce the xylem of secondary cell walls. Until recently, lignification was assumed to be the key factor facilitating plants to evolve from water to land (Boyce et al., 2003; Kenrick and Crane, 1997; Peter and Neale, 2004), and it was believed that true lignins were absent in non vascular plants (Lewis, 1999; Peter and Neale, 2004). However, recently it was clearly demonstrated that the intertidal red algae, Calliarthron cheilosporioides, contains lignified secondary cell walls, and this paramount finding raises important questions about the evolution of cell wall architecture and the divergence of vascular and non-vascular plants (Martone et al., 2009). This finding suggests that other factors, in addition to structural lignin, were required in cell wall evolution to allow upright growth on land. Thus, the question remains: What were the key factors in the evolution of the cell wall of terrestrial plants? The arrangement of polysaccharides in vascular plant primary cell walls is important for their ability to undergo controlled growth, generating cell shape (Estevez et al., 2009; Siddhanta et al., 2009). The cellulose of non-vascular plants has a higher level of crystallinity and a relatively larger surface area than higher plant cellulose (Ek et al., 1998; Stromme et al., 2002). In addition, highly specialized proteins such as endotransglycosylases are present in higher plants which modify the complex  cross-linking  of  cellulose  with  hemicelluloses  during  growth  and  morphogenesis (Cosgrove, 2005). It is this cross-linking that permits the cell wall to be strong yet flexible, and creates cellulose microfibrils that are more disordered than those observed in algae. Some of these proteins act on the hemicellulose-cellulose network in the cell wall while other proteins act during cellulose deposition.  It is  possible that the evolution of the proteins necessary for the formation of the critical cellulose-hemicellulose network of the cell wall was a key element in allowing plants to move from aquatic to terrestrial environments. One particular enzyme, an endoglucanse, has recently been shown to be involved in cellulose synthesis as well as cellulose/hemicellulose interactions (Maloney 64  and Mansfield, 2010).  Endoglucanases (E.C.3.2.1.4) are members of the cellulase  superfamily, possess the capacity to hydrolyze the β-1,4 linkage forming cellulose chain, and have been shown to be more active on amorphous cellulose than on crystalline cellulose. In a report on the classification of glycosyl hydrolases, Henrissat (1991) found that all endoglucanases present in plants belong to glycoside hydrolase family 9 (GH9). The reaction mechanism of the GH9 family is distinctive in that upon hydrolysis the hydroxide on the anomeric carbon is inverted to the α-position. Generally, plant endoglucanases lack a CBD, posses an N-terminal endoplasmic reticulum import sequence, and are secreted into the apoplast (Molhoj et al., 2002). With the exception of a few putative CBD containing endoglucanases of unknown function (Trainotti et al., 1999), most plant endoglucanases are believed to depolymerize non-crystalline glucans; however, these proposed catalytic mechanisms have yet to be tested (Libertini et al., 2004; Master et al., 2004; Molhoj et al., 2001a; Ohmiya et al., 2003; Ohmiya et al., 2000). After extensive research, the exact in vivo substrate, manner of activity, or to what degree plant endoglucanses can modify cellulose or other β-1,4 glucans is still relatively unknown (Brummell et al., 1997a; Brummell et al., 1997b; del Campillo, 1999; Master et al., 2004; Molhoj et al., 2001a; Molhoj et al., 2002; Molhoj et al., 2001b; Nicol et al., 1998; Ohmiya et al., 2003; Ohmiya et al., 2000; Peng et al., 2002; Robert et al., 2005; Rudsander et al., 2003a; Sato et al., 2001; Szyjanowicz et al., 2004; Takahashi et al., 2009). The Arabidopsis genome contains approximately 25 genes that encode endoglucanases that belong to GH9. These 25 genes separate into at least 9 different classes according to Molhoj et al. (2002), who compared the relationship between the 25 endoglucanases in Arabidopsis to their identified homologs in other plant species. Class IX appears to be the most unique class in that members of this class do not contain an endoplasmic reticulum import sequence and are not secreted directly into the apoplast, but rather contain sequences that encode an N-terminal membrane anchoring domain (Brummell et al., 1997a; Nicol et al., 1998). A number of studies examining the roles of membrane-bound endoglucanases from a variety of species, including monocots and dicots, have been undertaken in an attempt to elucidate the role(s) these enzymes may play with respect to cell wall remodelling, and more generally the overall physiology of plants. Generally, the results from these studies 65  indicate that plants possess one particular membrane-bound endoglucanase that appears to have similar functionality regardless of species (Bhandari et al., 2006; Brummell et al., 1997a; Brummell et al., 1997b; Lane et al., 2001; Master et al., 2004; Molhoj et al., 2002; Molhoj et al., 2001b; Takahashi et al., 2009).  Furthermore,  evidence suggests that this endoglucanase may play a role in cell wall biosynthetic processes, presumably by editing cellulose synthesis or during the assembly of the cellulose-hemicellulose network (Brummell et al., 1997b; Maloney and Mansfield, 2010; Molhoj et al., 2001a; Nicol et al., 1998; Sato et al., 2001; Zuo et al., 2000). The membrane-bound endoglucanase from Arabidopsis that appears to play a direct role in cellulose biosynthesis was termed KORRIGAN (KOR), and was originally isolated in a mutant Arabidopsis thaliana plant (kor1-1) that showed pronounced architectural alterations in the primary cell wall when grown in the absence of light (Nicol et al., 1998). It was further shown that KOR is an endo-1,4-β-glucanase that is located primarily in the plasma membrane and presumably acts at the plasma membrane-cell wall interface because of its single N-terminal membrane spanning domain (Nicol et al., 1998; Zuo et al., 2000). Additional KOR mutations (kor1-2) have been isolated and shown to cause the formation of aberrant cell plates, incomplete cell walls, and multinucleated cells, leading to abnormal seedling morphology (Zuo et al., 2000).  These results concur with findings in tomato (Lycopersicon esculentum;  (Brummell et al., 1997a; Brummell et al., 1997b), oilseed rape (Brasica napus; (Molhoj et al., 2001a; Molhoj et al., 2001b), rice (Oryza; (Zhou et al., 2006), and Populus (Bhandari et al., 2006; Hertzberg et al., 2001; Master et al., 2004; Rudsander et al., 2003b; Sterky et al., 1998; Takahashi et al., 2009). Although it has been speculated that sequence similarities between AtKOR and an expressed sequence tag from Pinus indicate that KOR predates the split between angiosperms and gymnosperms (Molhoj et al., 2002), to date no characterization of a gymnosperm KOR has been undertaken. If there is functional conservation of these membrane-bound endoglucanases in the gymnosperms, it suggests that their role in vascular plant evolution goes back 300 mya, predating angiosperms that arose 130 mya. In this paper we report on the isolation and characterization of a membranebound endoglucanase gene from white spruce (Picea glauca; denoted as PgKOR). This tree species represents an organism that is important commercially, especially with 66  respect to global wood and fibre supply. Sequence alignments with other membranebound endoglucanase proteins and phylogenetic reconstruction analyses reveal that PgKOR contains conserved polarized targeting signals, as well as residues predicted to be essential for catalytic activity; however, the transmembrane domain varies between species. Expression of PgKOR in Arabidopsis kor1-1 mutants clearly demonstrates that this gene is able to recover the mutant phenotype, providing evidence for functional equivalence. Analyses of endogenous KOR expression in white spruce revealed the highest expression in young developing tissues, which corresponds with primary cell wall development.  Additionally, RNA interference (RNAi) of the endogenous gene  substantially reduced growth and cellulose content, but had no affect on cellulose ultrastructure. 3.2 Materials and methods 3.2.1 PgKOR isolation and construct development RNA was extracted from the green leader portion of Picea glauca 653 stem. A reverse primer (5'AAGGGACGTTTCAAGTATCA3') was designed based on a Picea glauca EST that showed a 73% similarity to the Arabidopsis KOR (AtKOR). The forward primer (5'CTGGTCTATTGGTTGGTCTTA3') was designed based on the aspen KOR sequence (PtrKOR) that has 83% similarity with the AtKOR sequence.  In order for any  amplification to occur, the forward primer had to be designed within the PtrKOR open reading frame (ORF), and we were therefore unable to amplify the entire PgKOR ORF. In order to obtain the rest of the ORF, 5′ RLM RACE (Ambion, Austin TX, USA) was employed to complete the 5′ sequence of the gene. Following sequence verification, oligonucleotides  (Fw  -5'CACCATGTATGGGCGAGATCCGTG3',  Rv-  5'TCAAGGATTCCAAGGGGCTG3') were created to generate a PCR product from cDNA for direct cloning into the pENTR/D-TOPO vector (Invitrogen, Carlsbad, CA, USA). The vector was then sequenced to verify the correct insertion of the PgKOR gene, and Gateway technology (Invitrogen) was used to insert the gene into the 35S::hRLUC::attR destination vector (Subramanian et al., 2004). The resulting binary plasmid, 35S::hRLUC::PgKOR, was transformed into Agrobacterium tumefaciens strain GV3101.  67  The PgKOR-RNAi construct was built using two oligonucleotides, PgRNAiFW (5′CCCAAAGAGGTAGATATAGTC3′)  and  PgRNAiRV  (5′AGCTGCATTTACCACATACTG3′) with the addition of either 5′ BamHI and 3′ ClaI (sense) or 5′ XhoI and 3′ KpnI (antisense) restrictions sites. These oligonucleotides were used to amplify a 400 base pair fragment of the PgKOR coding region from cDNA. The fragments were then digested with the appropriate restriction enzymes and ligated into the pKANNIBAL (Helliwell and Waterhouse, 2003) cloning vector. Finally, the NotI fragment from pKANNIBAL containing the hpRNA cassettes was sub-cloned into the binary vector pART27 (Gleave, 1992) and used for plant transformations. 3.2.2 Plant strains and growth conditions The T-DNA–mutagenized Arabidopsis thaliana line kor1-1 (Nicol et al., 1998) was acquired from the Arabidopsis Biological Resource Center (Columbus, OH). The TDNA insertion of kor1-1 carries a gene that results in the resistance to kanamycin. Seeds were sterilized as described by Clough and Bent (1998) and germinated at room temperature in a 16-h/8-h light/dark cycle on half MS medium (Murashige and Skoog, 1962) containing 50 mg/L kanamycin sulfate (Sigma, St. Louis, MO, USA) and no sugar. Plantlets were transferred to soil after the first four primary leaves had emerged, and the growth cycle was allowed to complete under the same conditions. The F1 progeny from the kor1-1 line were compared with the Wassilewskija (WS) ecotype, the background for kor1-1. White spruce somatic embryo tissue (Pg653) was acquired from Dr. Klimaszewska (CFS, Quebec, Canada). Embryos were matured according to Klimaszewska et al. (2001).  Plantlets were grown for eighteen months and then  destructively harvested. 3.2.3 Plant transformation kor1-1 plants were transformed by infiltration using Agrobacterium tumefaciens carrying the PgKOR construct. The harvested seeds were selected on MS medium containing 50mg/mL kanamycin and 85mg/mL PESTANAL (Glufosinate ammonium; Sigma). Plantlets resistant to both antibiotics were transferred to soil and placed in a growth chamber and grown under the same conditions described previously. Pg653 somatic 68  embryo tissue was transformed with the PgKOR-RNAi construct according to Klimaszewska et al. (2003). 3.2.4 Genomic DNA extraction and screening Genomic DNA was extracted from single leaves taken from young soil-grown Arabidopsis plants using a CTAB modified extraction method (Rogers and Bendich, 1994). Briefly, single leaves were placed in microcentrifuge tubes and ground to a powder with liquid nitrogen. One milliliter of CTAB extraction buffer (2% [w/v] hexadecyltrimethylammonium bromide [CTAB; Sigma], 100 mM Tris- HCl, pH 8.0, 1.4 M NaCl, 20 mM EDTA, 1% [w/v] polyvinylprylidone, and 0.2% [v/v] 2-mercaptoethanol) was added to each tube, and the tube was incubated at 65°C for 60 min. An equal volume of chloroform was added, and the tube was vortexed and then centrifuged in a microcentrifuge for 10 min. Genomic DNA was precipitated from the aqueous phase by addition of 1 volume of isopropanol, incubating at -20°C for 10 min, and centrifuging for 5 min. Genomic DNA was resuspended in RNase buffer (25 mM Tris-HCl, pH 7.5, 10 mM EDTA, and 100 mg/mL RNase A) and incubated at 37°C for 30 min. Two volumes of ethanol were added, and the genomic DNA was recovered by centrifugation. Finally, the DNA was re-suspended in 50 mL of EB buffer (10 mM Tris-HCl, pH 8.0, and 1 mM EDTA), quantified at A260, and stored at 4°C. PCR was performed on these plants to test for the presence of the T-DNA insert in both alleles with the primers RP (5′TGATGATGCTCTCTGATAAAG3′), LP (5′TGTGGTAGACTATCATTTTCA3′), and TAG7 (5′GGACTGACCACCCCAAGTGC3′) (Nicol et al., 1998) . The presence or absence of the PgKOR construct was determined with  the  primers  hLUC3′  Fw  (5′AAGAGCTTCGTGGAGAGAGTGCTGA3′)  and  PgKORgate Rv (5′TCAAGGATTCCAAGGGGCTG3′). 3.2.5 RNA extraction and realtime PCR Total RNA was extracted from approximately 500 mg of frozen ground eight-week-old Arabidopsis plants using TRIzol reagent (Sigma) according to the manufacturer’s instructions. Total RNA was isolated from white spruce tissues using a method developed in the Treenomix laboratory based on Wang et al. (2000) and Chang et al. (1993). RNA yield was measured by absorption at 260nm, and 10 μg was treated with DNAase (Ambion TURBO DNA-free).  One μg of the resulting DNA-free RNA was 69  evaluated on a 1% Tris-acetate EDTA agarose gel in order to determine quality. Equal quantities of RNA (1 μg) were used for the synthesis of cDNA with SuperScript II reverse transcriptase (Invitrogen) and (dT)16 primers, according to the manufacturer’s instructions. Samples were run in triplicate with Platinum SYBR Green qPCR Master mix (Invitrogen) on an Mx3000p real-time PCR system (Stratagene, La Jolla, CA, USA). The real-time PCR analysis of the Arabidopsis lines was performed using the primers PgKORRT  Fw  (5′TTGGTTGGTGGGTTTTATGAT3′)  (5′ACTTCGCACTGTACTCAATAA  3′)  (5′TCCCTACTCCACCACCTCCA′)  and  and  or  PgKORRT AtKORRT  AtKORRT  3′UTR  Rv Fw Rv  (5′ACCCCGTGTTTGTATCTAGTAA′), while the endogenous PgKOR expression in the wildtype  and  the  PgKORRNAiRTFw  PgKOR-RNAi  lines  was  performed  (5′TTGGTTGGTGGGTTTTATGAT3′)  and  using  the  primers  PgKORRNAiRTRv  (5′CAAACCTCGTCGATCCAATAT3′). Transcript abundances were determined based on changes in Ct values relative to elongation initiation factor5A (Yamada et al., 2003) for Arabidopsis and actin for white spruce using the following primers: AtEIF4AFw (5′ACTGACCTCTTAGCTCG3′), AtEIF5ARv (5′CAGATCGGCCACGTTC3′) (Gutierrez et al.,  2008),  PgActinFw  (5′ATCCTATTGAGCATGGTATTG3′)  PgActinRv  (5′AGCAGGCACATTAAAAGTTTC3′). Conditions for all PCR reactions were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 30 s, 55°C for 1 min, and 72°C for 30 s. 3.2.6 Structural carbohydrate analyses Ten-week old Arabidopsis stems and eighteen-month old greenhouse-grown white spruce stems were ground in a Wiley mill to pass a 0.4-mm screen (40 mesh) and Soxhlet extracted overnight in hot acetone to remove extractives. Lignin and carbohydrate content were determined with a modified Klason, in which extracted ground stem tissue (50 mg) was treated with 3 mL of 72% H2SO4 and stirred every 10 min for 2 h. Samples were then diluted with 112 mL deionized water and autoclaved for 1 h at 121°C. The acid-insoluble lignin fraction was determined gravimetrically by filtration through a pre-weighed medium coarseness sintered-glass crucible, while the acid-soluble lignin component was determined spectrophotometrically by absorbance at 205 nm. Carbohydrate contents were determined by using anion exchange high70  performance liquid chromatography (Dx-600; Dionex, Sunnyvale, CA, USA) equipped with an ion exchange PA1 (Dionex) column, a pulsed amperometric detector with a gold electrode, and a SpectraAS3500 auto injector (Spectra-Physics). 3.2.7 Cellulose characterization Microfibril angle estimates were generated by X-ray diffraction (Megraw et al., 1998). The 002 diffraction spectra from five individual eighteen-month-old white spruce trees from each of all the transgenic lines and ten-week-old Arabidopsis stems were screened for T value distribution and symmetry on a Bruker D8 discover X-ray diffraction unit equipped with an area array detector (GADDS). Wide-angle diffraction was used in the transmission mode, and the measurements were performed with CuKα1 radiation (λ=1.54 Å). The X-ray source was fit with a 0.5-mm collimator, and the scattered photon was collected by a GADDS detector. Both the X-ray source and detector were set to theta=0°. The degree of cellulose crystallinty was determined from white spruce and Arabidopsis stems as described by Mansfield et al. (1997) using the same X-ray parameters as for MFA determination with the exception of the source theta set at 17°. Significant differences were determined using student t-tests. 3.2.8 Cross sectional staining and microscopy Eighteen-month old white spruce stems were cut from the base into a one cm segment and submerged in dH2O at room temperature for a day. Samples were then radially cut into 20 μm cross sections using a Leica SM2000r hand sliding microtome (Leica Microsystems, Wetzlar, Germany) and again stored in dH2O until needed. Sections were either treated with 0.01% Calcofluor white in 1x PBS for 3 min, and washed 3 x 5 min in 1x PBS to remove excess stain (Falconer and Seagull, 1985) or with 10% phloroglucinal with the addition of concentrated HCl. Four-week-old Arabidopsis stems were also cut 1 cm above the base and further hand sectioned into approximately 500 nm thick cross sections. Sections were then stained in 0.05% toluidine blue stain for 5 min. Excess stain was washed away with dH2O. All sections were mounted onto glass slides and examined with a Leica DRM microscope (Leica Microsystems, Wetzlar, Germany) fitted with epifluorescence optics.  71  Photos were taken with a QICAM camera (QImaging, Surrey, Canada) and OpenLab software (PerkinElmer Inc., Waltham, USA). 3.3 Results 3.3.1 Molecular cloning and sequence analyses of PgKOR To identify and characterize the white spruce homolog of the Arabidopsis KORRIGAN gene (KOR), the initial sequence was obtained by screening a spruce EST library (http://www.treenomix.ca), from which a set of contigs was selected based on sequence similarity to the Arabiopsis KOR gene. A large portion of the gene was isolated with a forward primer designed from the already characterized Populus tremuloides KOR (PtrKOR; AY535003) and a reverse primer designed from the EST screen. In order to obtain the entire open reading frame, 5′ rapid amplification of cDNA ends (RACE) was employed.  The resulting gene, designated PgKOR (Picea glauca KORRIGAN),  encodes a putative 65 kD protein with 617 amino acids. A comparison of the AtKOR, PgKOR and PtKOR genomic sequence structure shows that each gene consists of the same number of exons and introns, but the sizes of the introns vary between species (Figure 3.1A).  A comparison of protein sequence with that of other putative KOR  homologs from other plant species demonstrates the strong overall similarity among these proteins (Figure 3.1B). Overall, PgKOR is most similar to the KOR from aspen (PtrKOR), sharing greater than 80% sequence identity and containing identical polarized targeting signals, predicted glycosylation sites, and residues essential for catalytic activity. However, the predicted transmembrane domains for all species vary substantially, with only 40% sequence similarity between PgKOR and AtKOR (Figure 3.1).  Additionally, in a phylogenetic comparison of PgKOR with all of the known  Arabidopsis glycosyl hydrolase family 9 proteins, PgKOR clusters the closest to KOR1/KOR2/KOR3, the class IX proteins that are known to contain sequences that encode an N-terminal membrane anchoring domain (Figure 3.2A). Furthermore, in a broad species-wide phylogenetic comparison PgKOR clusters with all the other known plant membrane-bound endoglucanses (Figure 3.2B).  72  3.3.2 Complementation of Arabidopsis kor1-1 mutant Having established similarities in the molecular organization of PgKOR and the KOR from Arabidopsis, we sought proof of conserved developmental function. Arabidopsis plants homozygous for the kor1-1 mutation (Nicol et al., 1998) were transformed with either the PgKOR gene or the endogenous AtKOR gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter.  Following the transgene insertion  event, PgKOR and AtKOR transformants were selected by screening for plants capable of growing on PESTANAL selection media. None of the transformants displayed the elongation deficient phenotype typical of the kor1-1 mutant. The T2 progeny, obtained after selfing, were again able to grow on PESTANAL selection media, and had a wildtype growth phenotype, as demonstrated in Figure 3.3A for an AtKOR line and two representative PgKOR lines called PgKOR4 and PgKOR5. Genomic DNA screening for the T-DNA insertion (Figure 3.3B) and real time quantitative PCR analyses for the presence of the endogenous AtKOR transcript (Figure 3.4A) confirmed that the representative lines were homozygous for the T-DNA insertion and had significantly lower native gene expression than the wild type. Furthermore, genomic DNA screening for the PgKOR gene construct (Figure 3.3C) and real time quantitative PCR analyses (Figure 3.4B) confirmed that the PgKOR transgene is indeed expressed in the homozygous mutant background. Toluidine blue staining of four-week old stem crosssections revealed that the irregular xylem common to the kor1-1 mutant was not seen in the transgenic lines and the wild-type xylem morphology phenotype indeed recovered (Figure 3.5).  Further evidence for complementation comes from the structural  carbohydrate analyses that show the near recovery of the structural glucose levels in all the lines when compared to the mutant and the wild-type plants (Table 3.1). X-ray diffraction data showed that the degree of cellulose crystallization did not differ between any of the lines, but that the microfibril angle (MFA) of the kor1-1 mutant was increased by 29% when compared to the wild-type. Furthermore, this increase was decreased considerably in the complemented lines (Table 3.2). 3.3.3 Endogenous PgKOR expression Using 5-year old white spruce trees, spatial endogenous PgKOR expression was measured from eight tissue types from four individual trees (Figure 3.6).  PgKOR 73  expression was highest in the young and young expanded needles that correspond to locations of increased primary growth.  Expression from samples taken from the  secondary growth of the stem was highest in the developing xylem. These results are consistent with previous findings in oil seed rape where a promoter-GUS fusion to an orthologous membrane-anchored endo-1,4-β-glucanase cDNA (Cel16), which exhibits 94% sequence similarity to the Arabidopsis KOR protein, demonstrated that Cel16 is expressed in all tissue types, but was highest in young tissues (Molhoj et al., 2001a). 3.3.4 RNAi suppression of endogenous PgKOR expression To further explore the PgKOR gene function, we characterized transgenic spruce trees that had a suppression of the native KOR gene. Suppression of PgKOR expression was achieved using a 400-bp hairpin RNAi construct designed from the putative full-length cDNA-encoding PgKOR.  Agrobacterium-mediated transformation of white spruce  somatic embryo tissue (Klimaszewska et al., 2003) yielded numerous independent transformants that were confirmed through genomic screening, from which seven PgKOR-RNAi lines were matured and transferred to the greenhouse. representative lines were chosen for growth and cell wall analyses.  Three  Endogenous  PgKOR expression was analysed by real-time PCR, which clearly showed a substantial reduction (down 87%) in transcript abundance in all three PgKOR-RNAi lines when compared with the wild-type trees grown under similar conditions (Figure 3.7). Student t-tests indicated that there was a significant reduction in average plant height (down 27%) and diameter (down 13%) of the three PgKOR-RNAi lines, which largely correlated with a reduction in gene expression (Figure 3.8A and B). All trees were harvested after eighteen months’ growth and were used for all further analyses. Histochemical staining of stem cross section with the cellulose stain, calcoflour white, or the lignin stain, phloroglucinol, did not reveal any obvious cell wall anomalies or any apparent gross changes in the cell wall chemistry of the PgKOR-RNAi lines (Figure 3.9). A detailed structural carbohydrate analysis of the tree cell walls revealed that the RNAi lines do indeed have a significant reduction in average glucose levels (down 19%), indicating a reduction in cellulose compared to the corresponding wild-type trees (Table 3.3). These findings are consistent with recent finding in poplar where KOR was RNAi-suppressed (Maloney and Mansfield, 2010). Additionally, X-ray diffraction was 74  used to characterize the cellulose produced by the spruce trees; however, only a slight reduction in MFA was evident, while there was no measurable change in cell wall crystallinity (Table 3.4). 3.4 Discussion 3.4.1 Molecular characterization of PgKOR Positive identification of the white spruce KOR gene was accomplished using both molecular and functional criteria. The comparison of genomic and coding sequences of KOR genes from a variety of different species clearly revealed sequence features common to all. These homologues are characterized by a region rich in hydrophobic amino acids located in the N-terminus indicative of a single transmembrane domain as well as the polarized targeting signals, glycosylation sites, and residues essential for catalytic activity predicted by Nicol et al. (1998). Most other plant endoglucanases do not contain this single transmembrane domain. These sequence analyses were the first indication that the endoglucanase gene isolated from white spruce (PgKOR) was a strong candidate for an Arabidopsis KOR gene ortholog. 3.4.2 Endogenous PgKOR expression To test the claim that the PgKOR gene is an ortholog of the Arabidopsis KOR gene, we sought proof that the two genes have similar expression profiles and, more importantly, that PgKOR expression is associated with cell wall development. Endogenous PgKOR expression was highest in the young and young expanded needles that correspond to locations of increased primary growth. In contrast, the transcript abundance of samples taken from the stem tissue was highest in the developing xylem. Expression analyses of a membrane-anchored endo-1,4-β-glucanase cDNA from oil seed rape (Cel16), which exhibits 94% sequence similarity to the Arabidopsis KOR protein, demonstrates that Cel16 is expressed in all tissue types, but it was highest in young tissues. Furthermore, a Cel16 promoter-GUS fusion construct closely paralleled the pattern of abundance of Cel16 mRNA transcript profiling (Molhoj et al., 2001a). Similarly, a recent study by Takahashi et al. (2009) analysed the activity of the Arabidopsis KOR promoter again using a β-glucuronidase (GUS)-promoter construct and showed high activity in young plants especially in actively expanding cells and vascular tissues, as well as 75  being consistently active in the stems undergoing secondary cell wall thickening. These data provide solid, yet indirect, evidence that KORRIGAN functions in similar locations in a variety of species, suggesting that the genes are functionally conserved. 3.4.3 RNAi suppression of endogenous PgKOR expression In coniferous trees, the water conducting cells of the xylem are generally tracheids, whereas in the xylem of flowering plants, vessel elements are the predominate water conducting cell type. In the Arabidospsis KORRIGAN knock-out mutants (Burn et al., 2002; Lane et al., 2001; Nicol et al., 1998; Szyjanowicz et al., 2004; Zuo et al., 2000) as well as KOR-RNAi hybrid poplar trees (Maloney and Mansfield, 2010) the altered phenotype is evident primarily in the vessel elements manifesting itself in an irregular xylem (irx) phenotype and disrupting water transport and ultimately growth.  In the  current study evaluating one and a half year old PgKOR-RNAi white spruce, histochemical staining and x-ray diffraction did not reveal any visible cell well anomalies. However, there was a substantial reduction in height and radial growth as well as in structural cell wall glucose content. The lack of an irregular xylem phenotype in the secondary growth of spruce may indicate that KOR function in the secondary cell wall is restricted in a cell specific manner to vessel elements, but not tracheids or fibers. Vessels expand more radially than tracheids, which suggest that KOR may work in the expansion zone of the secondary xylem during morphogenesis and therefore vessels are disproportionately affected. An alternative hypothesis is that the collapse of the xylem in plants displaying an irx phenotype only happens in cells that are involved in water transport and are subjected to negative hydraulic pressures (Turner and Somerville, 1997). Cells that have higher inherent lignin content, such as tracheids in spruce, have a greater resistance to negative hydraulic pressure which may help to explain why the tracheids did not collapse in the PgKOR-RNAi white spruce plants (Akiyama et al., 2005; Boyce et al., 2004). Previous reports on the function of KOR suggest that it functions in both the primary and secondary cell walls and affects cellulose biogenesis (Bhandari et al., 2006; Brummell et al., 1997a; Brummell et al., 1997b; His et al., 2001; Molhoj et al., 2001a; Molhoj et al., 2002; Nicol et al., 1998; Szyjanowicz et al., 2004; Takahashi et al., 76  2009; Turner and Somerville, 1997).  Additionally, we previously reported on the  possible role for KORRIGAN in xylan-cellulose crosslinkages, and we provided evidence that the lack of KORRIGAN function in hybrid poplar can manifest in an increase in cell wall xylan content (Maloney and Mansfield, 2010). Xylans are known to be the major cellulose-linking polysaccharide in the secondary cell walls of higher plants.  Furthermore, Carafa et al. (2005) used monoclonal antibodies to provide  evidence that xylans are not present in the cell walls of mosses. The occurrence of xylans in plants with tracheid cell types suggests that the appearance of these polysaccharides has been an important evolutionary event in the development of vascular and mechanical tissues. This also suggests that KOR may primarily play a role in xylan-cellulose interactions, and may represent a major evolutionary milestone in the establishment of higher plants in terrestrial environments. 3.4.4 Significance of the evolutionary conservation of KORRIGAN Extensive genome sequencing over the last few years has produced a large amount of data available for comparative genomics across different plant species. These data provide powerful insight into the genetic conservation and evolution of many different protein families. Complementation studies, like the one conducted in this paper, can provide experimental evidence of the functional conservation of genes across hugely varying plant genomes such as Arabidopsis and white spruce as demonstrated in this work. The evolution of large land plants necessitated the need for water conducting tissues in order to avoid excessive water stress. These water conducting tissues came in the form of a compression resistant cell wall skeleton that functions following programmed cell death of the protoplast. These evolutionary important features were accomplished early, with water conducting cells present in the fossil record 410 million years ago (Sperry, 2003). The tracheid cell type, which evolved first, is a narrow single cell with an intact axial cell wall. Vessels, in contrast, are water conducting tubes with either partially or completely open axial cell walls (Tyree and Zimmermann, 2002). Vessel elements evolved through a series of modifications to the tracheid developmental process (Boyce et al., 2003; Feild et al., 2002; Sperry, 2003), and in general provide greater xylem hydraulic capacity than tracheids (Tyree and 77  Zimmermann, 2002). The fossil records provide evidence for bryophytes being some of the earliest land plants, and the publishing of the draft genome sequence of the moss Physcomitrella patens provides extensive information into the bryophyte genome (Rensing et al., 2008).  An extensive interrogation of the recently published  Physcomitrella patens draft genome sequence with both the AtKOR and the PgKOR gene did not reveal any significant matches.  Additionally, as a second measure,  comparison of the Arabidopsis secondary cell wall cellulose synthase genes with the Physcomitrella database provide evidence that mosses lack true secondary cell wall specific cellulose synthase orthologues, which is consistent with previous reports (Popper, 2008; Roberts and Roberts, 2004).  However, the presence of conserved  regions from other cellulose synthase and cellulose synthase-like genes in mosses suggests that vascular plants and mosses diverged before the cellulose synthase genes became specialized for either primary or secondary cell wall development (Liepman et al., 2007; Popper, 2008; Roberts and Bushoven, 2007). While a previous study on the genes required for cellulose synthesis in Agrobacterium tumefaciens C58 showed that a membrane-anchored endoglucanase (CelC) was involved in the transfer of cellulose oligomers from a lipid carrier to the growing cellulose chain (Matthysse et al., 1995), this gene only shares an 11% identity to AtKOR.  Furthermore, a screen of the  Agrobacterium tumefaciens C58 genome database (Goodner et al., 2001; Wood et al., 2001) as well as the cellulose-producing Oomycetes Phytophthora spp genome database (Gajendran et al., 2006) did not reveal any significant matches to these membrane-bound endoglucanases.  These data and the current evidence for the  functional conservation of KOR in both tracheid and vessel-containing plants provide evidence for the evolution of KOR and the secondary cell wall specific cellulose synthases occurring sometime before the evolution of the tracheid cell type, but after the conquest of land by plants.  78  Table 3.1. Structural carbohydrate analysis of wild-type, kor1-1 and complemented Arabidopsis plants indicating a recovering to wild type levels in the complemented plants. Values represent means of pools of 20 individual plant stems. Carbohydrates (nmoles/mg) Line  fucose  arabinose  rhamnose  galactose  glucose  xylose  mannose  WT  5.01  69.14  46.93  64.89  1975.73  959.11  155.09  kor1-1  7.46  80.64  61.96  74.46  795.52  1004.71  152.65  At  6.42  72.71  54.07  67.42  1776.55  1020.28  166.17  Pg4  5.74  77.15  50.53  67.84  1644.26  907.51  149.58  Pg5  5.73  40.70  42.65  61.44  1828.78  1030.44  162.34  Table 3.2. Microfibril angle (MFA) and cell wall crystallinity as measured by x-ray diffraction of eight-week-old Arabidopsis stems. Standard error of the mean in parenthesis, n=10; values in bold have a p-value of 0.05 or less using Student t-test. Line  MFA  %Crystalline  WT  12.23 (0.36)  50.30 (1.23)  kor1-1  17.29 (1.04)  50.50 (1.27)  At  13.16 (0.80)  48.20 (2.34)  Pg4  14.10 (0.33)  49.80 (1.33)  Pg5  14.55 (0.57)  52.00 (1.64)  Table 3.3. Structural cell wall carbohydrates of wild-type and PgKOR-RNAi lines indicating a decrease of glucose levels in the PgKOR-RNAi lines. Standard error of the mean in parenthesis, n=7; values in bold have a p-value of 0.05 or less using Student ttest. Carbohydrates (nmoles/mg) Line  arabinose  rhamnose  galactose  glucose  xylose  mannose  WT  171.41 (4.72)  29.56 (2.89)  144.42 (6.59)  2500.49 (92.23)  663.37 (21.86)  573.36 (17.71)  2  157.25 (6.06)  20.56 (8.00)  141.69 (9.53)  2168.36 (131.00)  628.46 (20.20)  595.98 (20.29)  7  188.90 (12.66)  28.27 (6.59)  143.63 (5.96)  1751.13 (116.88)  591.42 (9.56)  614.37 (21.58)  10  186.73 (9.43)  29.13 (5.37)  135.39 (4.46)  2125.52 (98.16)  605.38 (23.34)  519.34 (24.82)  79  Table 3.4. Microfibril angle (MFA) and cell wall crystallinity, as measured by x-ray diffraction, of eighteen-month-old white spruce stems. Standard error of the mean in parenthesis, n=7; values in bold have a p-value of 0.05 or less using Student t-test.  Line  MFA  %Crystalline  WT  42.41 (0.42)  37.43 (1.00)  2  42.48 (0.53)  37.00 (1.41)  7  41.19 (0.47)  35.86 (1.68)  10  40.96 (0.14)  35.50 (2.53)  80  A AtKOR  79  74  117  102  171  ATG  TGA  PgKOR  1531  808  168  92  125  ATG  TGA  PtKOR  464  ATG  433  939  92  95  TGA  100bp  B PgKOR AtKOR PaxgKOR LeCel3 OsCel9A BnCel16 PtKOR  10 20 30 40 50 60 70 80 90 100 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| MYGRDPWGGPLEIN-ADSATDDDRSRNLQDFDRSALSR---QLDETQQSWLLGPGEQKKKK-YVDLGCIVCSRTVFKWIVISILLSGSIAGLVTLIVKLV MYGRDPWGGPLEINTADSATDDDRSRNLNDLDRAALSR---PLDETQQSWLLGPTEQKKKK-YVDLGCIIVSRKIFVWTVGTLVAAALLAGFITLIVKTV MYGRDPWGGPLEINAADSATDDDRSRNLNDLDRAALSR---PLDETQQSWLLGPAEQKKKKIYVDLGCIIVSRKICVWTVGSIVAAGLLVGLITLIVKTV MYGRDPWGGPLEIHTADSATDDDRSRNLQDFDRAAMSR---SLDETQQSWLLGPTEQKKKK-YVDLGCIIVSRKIFKWTVGCIIAAALLAGFITMIVKLV MYSANHWGGSFEIA-ADGAAEDDHSRNM-DLDRGALSARQHQLDETQQSWLLGPPEAKKKDKYVDLGCVVVKRKLLWWVLWTLLAAFILIGLPVIIAKSI MYGRDPWGGPLEIHATDSATDDDRSRNLNDIDRAALSR---PLDETQQSWLLGPTEQKKKK-YVDLGCIIVSRKIFVWTVGTIVAAALLAGFITLIVKTV MYGRDPWGGPLEINAADSATDDDRSRNLNDLDRAALSR---PLDETQQSWLLGPAEQKKKKIYVDLGCIIVSRKICVWTVGSIVAAGLLVGLITLIVKTV  PgKOR AtKOR PaxgKOR LeCel3 OsCel9A BnCel16 PtKOR  110 120 130 140 150 160 170 180 190 200 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| PRHNNSAPPPDSYTKALHKALMFFNAQRSGPLPKHNNVSWRGNSGMRDGLSDPGAAHASLVGGFYDAGDAIKFNFPGAFAMTMLSWSVIEYSAKYEAAGE PRHHPKTPPPDNYTIALHKALKFFNAQKSGKLPKHNNVSWRGNSGLQDGKGETGSFYKDLVGGYYDAGDAIKFNFPMAYAMTMLSWSVIEYSAKYEAAGE PRHHHSHAPADNYTLALHKALMFFNGQRSGKLPKHNNVSWRGSSCLSDGKGKQGSFYKDLVGGYYDAGDAIKFHFPASFSMTMLSWSVIEYNAKYEAAGE PRHKHHNPPPDNYTLALRKALMFFNAQKSGKLPKHNNVSWRGNSCLQDGKSDDSTMFKNLVGGYYDAGDAIKFNFPQSFALTMLSWSVIEYSAKYEAAGE PKKKPHAPPPDQYTDALHKALLFFNAQKSGRLPKNNGIKWRGNSGLSDG-SDLTDVKGGLVGGYYDAGDNIKFHFPLAFSMTMLSWSVIEYSAKYKAVGE PRHHRKTPPPDNYTIALHKALKFFNAQKSGKLPRHNNVSWRGNSGLQDGKGDSGSFYKDLVGGYYDAGDAIKFNFPMAYAMTMLSWSVIEYSAKYEAAGE PRHHHSHAPADNYTLALHKALMFFNGQRSGKLPKHNNVSWRGSSCLSDGKGKQGSFYKDLVGGYYDAGDAIKFHFPASFSMTMLSWSVIEYNAKYEAAGE  PgKOR AtKOR PaxgKOR LeCel3 OsCel9A BnCel16 PtKOR  210 220 230 240 250 260 270 280 290 300 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| LNHAKDIIKWGADYFLKTFNSSADTISLIHAQVGKGDTSTGPSNPNDHYCWMRPEDIDYPRPVYDCSS-CSDLAAEMAAALAAASIVFKDNKLYSQKLVK LTHVKELIKWGTDYFLKTFNSTADSIDDLVSQVGSGNTDDGNTDPNDHYCWMRPEDMDYKRPVTTCNGGCSDLAAEMAAALASASIVFKDNKEYSKKLVH LNHVKELIKWGADYFLKTFNSSADTIDRIVAQVGSGDTSGGSTTPNDHYCWMRPEDIDYDRPVTECSS-CSDLAAEMAAALASASIVFKDNKAYSQKLVH LAHVKDTIKWGTDYLLKTFNSSADTIDRIAAQVGKGDTTGGATDPNDHYCWVRPEDIDYARPVTECHG-CSDLAAEMAAALASASIVFKDNKAYSQKLVH YDHVRELIKWGTDYLLLTFNSSASTIDKVYSQVGIAKING--TQPDDHYCWNRPEDMAYPRPVQTAGS-APDLGGEMAAALAAASIVFRDNAAYSKKLVN LVHVKELIKWGTDYFLKTFNSTADSIDDLVSQVGSGNTDDGSTDPNDHYCWMRPEDMDYKRPVTTCNGGCSDLAAEMAAALASASIVFKDNREYSKKLVH LNHVKELIKWGADYFLKTFNSSADTIDRIVAQVGSGDTSGGSTTPNDHYCWMRPEDIDYDRPVTECSS-CSDLAAEMAAALASASIVFKDNKAYSQKLVH  PgKOR AtKOR PaxgKOR LeCel3 OsCel9A BnCel16 PtKOR  310 320 330 340 350 360 370 380 390 400 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| GATTLFKFSRT--QRGRYSPGGSDPSKFYNSTGYYDEYVWGGAWMYYATGNNSYLQLATTLGIAKHAGAFRGGQDYGVLSWDNKLAGAQVLLSRLRLFLS GAKVVYQFGRT--RRGRYSAGTAESSKFYNSSMYWDEFIWGGAWMYYATGNVTYLNLITQPTMAKHAGAFWGGPYYGVFSWDNKLAGAQLLLSRLRLFLS GAKTLFKFARD--QRGRYSAGGSEAATFYNSTSYWDEFIWGGAWLYYATGNNSYLQLATMPGLAKHAGAFWGGPDYGVLSWDNKLAGAQLLLSRLRLFLS GARTLFKFSRD--QRGRYSVG-NEAETFYNSTGYWDEFIWGAAWLYYATGNSSYLQLATTPGIAKHAGAFWGGPDYGVLSWDNKLTGAQVLLSRMRLFLS GAAAVYKFARSSGRRTPYSRGNQYIEYYYNSTSYWDEYMWSAAWMYYATGNNTYITFATDPRLPKNAKAFYSILDFSVFSWDNKLPGAELLLSRLRMFLN GAKTVYQFGRT--RRGRYSAGTAESAKFYNSSMYWDEFIWGGAWLYYATGNVTYLDLITKPTMAKHAGAFWGGPYYGVFSWDNKLAGAQLLLSRLRLFLS GAKTLFKFARD--QRGRYSAGGSEAATFYNSTSYWDEFIWGGAWLYYATGNNSYLQLATMPGLAKHAGAFWGGPDYGVLSWDNKLAGAQLLLSRLRLFLS  PgKOR AtKOR PaxgKOR LeCel3 OsCel9A BnCel16 PtKOR  410 420 430 440 450 460 470 480 490 500 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| PGYPYEDVLQSYHNQTNIVMCSYLPAFRSFNRTRGGLIQLNHGRPQPLQYVVNAAFLAAVYSDYLTAADIPGWNCGPTFFSTEALRNFARTQINYILGKN PGYPYEEILRTFHNQTSIVMCSYLPIFNKFNRTNGGLIELNHGAPQPLQYSVNAAFLATLYSDYLDAADTPGWYCGPNFYSTSVLRDFARSQIDYILGKN PGYPYEEILSTFHNQTSIIMCSYLPIFTKFNRTKGGLIELNHGRPQPLQYVVNAAFLATLFSDYLEAADTPGWYCGPNFYSTDVLRDFAKTQIDYILGKN PGYPYEEILRTFHNQTSIIMCSYLPIFTSFNRTKGGLIQLNHGRPQPLQYVVNAAFLATLFSDYLAAADTPGWYCGPNFYSTDVLRKFAETQIDYILGKN PGYPYEESLIGYHNTTSMNMCTYFPRFGAFNFTKGGLAQFNHGKGQPLQYTVANSFLAALYADYMESVNVPGWYCGPYFMTVDDLRSFARSQVNYILGDN PGYPYEEIVRTFHNQTSIVMCSYLPYFNKFNRTRGGLIELNHGDPQPLQYAANAAFLATLYSDYLDAADTPGWYCGPNFYSTNVLREFARTQIDYILGKN PGYPYEEILSTFHNQTSIIMCSYLPIFTKFNRTKGGLIELNHGRPQPLQYVVNAAFLATLFSDYLEAADTPGWYCGPNFYSTDVLRDFAKTQIDYILGKN  PgKOR AtKOR PaxgKOR LeCel3 OsCel9A BnCel16 PtKOR  510 520 530 540 550 560 570 580 590 600 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| PMKMSYLVGYGSHYPKHVHHRGASIPKNKIKYSCKGGWKWRDSPRPNPNVLVGAMVAGPDKRDMFHDVRTNYNYTEPTLAGNAGLVAALVSLS--GGDTG PRKMSYVVGFGTKYPRHVHHRGASIPKNKVKYNCKGGWKWRDSKKPNPNTIEGAMVAGPDKRDGYRDVRMNYNYTEPTLAGNAGLVAALVALS--GEEEA PRKMSYIVGFGNHYPKHLHHRGASIPKNKIRYNCKGGWKWRDTSKPNPNTLVGAMVAGPDRHDGFHDVRTNYNYTEPTIAGNAGLVAALVALS--GDKTT PRKMSYVVGFGNHYPKHVHHRGASIPKNKVKYNCKGGWKYRDSSKANPNTIVGAMVAGPDKHDGFRDVRSNYNYTEPTLAGNAGLVAALVALS--GDRDV PKKMSYVVGYGKKYPRRLHHRGASTPHNGIKYSCTGGYKWRDTKGADPNVLVGAMVGGPDKNDQFKDARLTYAQNEPTLVGNAGLVAALVALTNSGRGAG PRKMSYLVGFGTKYPKHVHHRGASIPKNKVKYNCKGGWKWRDSKKPNPNTIEGAMVAGPDKRDGFRDVRTNYNYTEPTLAGNAGLVAALVALS--GEEEA PRKMSYIVGFGNHYPKHLHHRGASIPKNKIRYNCKGGWKWRDTSKPNPNTLVGAMVAGPDRHDGFHDVRTNYNYTEPTIAGNAGLVAALVALS--GDKTT  PgKOR AtKOR PaxgKOR LeCel3 OsCel9A BnCel16 PtKOR  610 620 ....|....|....|....|....|.... G--IDKNTMFSAVPPMFPTPPPPPAPWNP TGKIDKNTIFSAVPPLFPTPPPPPAPWKP G--IDKNTIFSAVPPMFPTPPPPPAPWKP G--IDKNTLFSAVPPMFPTPPPPPAPWKP VTAVDKNTMFSAVPPMFPATPPPPSKWKP SGTIDKNTIFSAVPPLFPTPPPPPAPWKP G--IDKNTIFSAVPPMFPTPPPPPAPWKP  81  Figure 3.1 Molecular characterization of the KOR gene from a variety of plants indicates conserved features. (A) Schematic map of the genomic structures of the AtKOR, PgKOR and PtKOR gene. Triangles indicate the location and size of the introns. (B) Alignment of seven different plant membrane-bound β-1,4 endoglucanase protein sequences. X = Polarized targeting signals, X = predicted transmembrane domain, X = predicted glycosylation sites, X = residues essential for catalytic activity. PgKOR= Picea glauca, AtKOR = Arabidopsis thaliana, Pa×gKOR = Populus alba × grandidentata, LeCel3 = Lycopersicon esculentum, OsCel9A = Oryza sativa, BnCel16 = Brassica napus, and PtKOR = Populus trichocarpa.  82  A  B  ATGH9B11  ATGH9A2 KOR2 100 100  100  100  ATGH9B6  61  ATGH9B14  Medicago truncatula ABD32918  99 99  90 100  100  79 67  ATGH9A3 KOR3  Pyrus communis BAC22690  66  Gossypium hirsutum AAQ08018  Class IX  54 47  Populus trichocarpa  42 100  ATGH9B5  28  ATGH9B7 ATGH9C3  Vitis vinifera XP002269783  Proteins  ATGH9A1 KOR1  100 100  Solanum lycopersicum  83  PgKOR *  Saccharum hybrid CBB36505  Sorghum bicolour XP002465324  56  ATGH9A2 KOR2 100  Zea mays ACN28577  Zea mays NP001147537 99  ATGH9A4 98  Phyllostachys edulis ACZ82300  Oryza sativa NP001050004 100  ATGH9B4 Cel5 100  Hordeum vulgare BAA94257  100  ATGH9B3 Cel3  100  Triticum_aestivum AAM13693  83  ATCEL2 Cel2  ATGH9B15  100  100  ATGH9B13  50  50  94  ATGH9B1 Cel1  100  54  99  Colocasia esculenta ABP96983  ATGH9B16 pseudo  ATGH9B18  76  99  ATGH9B17  100  43  100  ATGH9C2  Populus tremula x Populus tremuloides_AAT75041 PaxgKOR GU324115  100  Populus tremuloides  Cucumis melo 32  ATGH9B8 ATGH9C1  Ricinus communis XM002519348  Pinus taeda ABR15471 100  ATGH9B12  100  ATGH9B10 ATGH9B9 80  PgKOR * Picea sitchensis ABR16291  ATGH9A3 KOR3 ATGH9A1 KOR1  Figure 3.2 Phylogenetic comparisons indicate that PgKOR is a conserved member of the KOR family. PgKOR with (A) all of the known Arabidopsis glycosyl hydrolase family 9 proteins including the canonical AtKOR and (B) all the other known plant membranebound endoglucanses. GenBank accession numbers are given when available.  83  A  B  C  Figure 3.3 Spruce KOR rescues the growth phenotype of the Arabidopsis kor1-1 mutant. (A) Complementation of four-week-old kor1-1 Arabidopsis plants indicating the recovery from the mutant phenotype in transgenic lines carrying AtKOR, PgKOR4 and PgKOR5. (B) Genomic DNA screening indicating that the complemented lines AtKOR, PgKOR4, and PgKOR5 carry the homozygous T-DNA insertion of the mutant background. (C) Amplification of the transgene representing the presence of the foreign gene construct in the PgKOR4 and PgKOR5 genomic DNA.  84  A  B 110 35  100 90 80  25  Relative Expression  Relative Expression  30  20 15 10  70 60 50 40 30  5  20  0  10 Wt  kor1-1  AtKOR  Pg4  Pg5  0 WT  kor1-1  Pg4  Pg5  Figure 3.4 Quantitative real time PCR analyses indicates expression of PgKOR in kor11 mutant background. Relative transcript abundance of (A) the AtKOR 3′ UTR in wildtype, kor1-1 and complemented Arabidopsis lines, and (B) PgKOR in wild-type, kor1-1 and complemented Arabidopsis lines. Transcript levels were determined based on changes in Ct values relative to translation initiation factor5A. Error bars = standard error of the mean, n=6 (2 plant pools * 3 technical replicates).  85  A  B  C  D  E  Figure 3.5 Spruce KOR rescues the irregular xylem phenotype of the kor1-1 mutant. Four-week-old Arabidopsis inflorescence stem cross sections stained with toluidine blue to highlight secondary cell walls (blue). Wild-type (Wassilewskija ecotype) vascular bundles (A) contrast with irregular, collapsed xylem in kor1-1(B). Recovery of wild-type morphology in xylem is seen in transgenic lines carrying AtKOR (C), PgKOR4 (D) and PgKOR5 (E). Arrows indicate xylem elements. Scale bars = 15 µm.  86  35  Relative Expression  30 25 20 15 10 5 0 yn0  yne  on  ph  xy  bk  r  wr  Figure 3.6 Relative transcript abundance of endogenous PgKOR in five year old white spruce trees indicates highest expression in young tissues. Transcript levels were determined based on changes in Ct values relative to actin. Error bars = standard error of the mean, n=12 (four trees*three replicates). yn0= young needles, yne = expanded young needles, on= old needles, ph= phloem, xy = xylem, bk= bark, r= root and wr = woody root.  87  2.5  Relative Expression  2.0  1.5  1.0  0.5  0.0 WT  2  7  10  Figure 3.7 Relative transcript abundance of endogenous PgKOR in eighteen-month old PgKOR-RNAi trees indicates substantial reduction from wild type levels. Transcript levels were determined based on changes in Ct values relative to actin. Error bars = standard error of the mean, n=21 (seven trees*three replicates).  88  B 20  Height  18  Caliper 4  16  12 10 2  8 6 4 2 0  WT  2  7  10  Figure 3.8 Impaired growth in PgKOR-RNAi white spruce trees. (A) Eighteen-month old greenhouse grown white spruce trees and (B) Height and caliper measurements of eighteen-month old wildtype and PgKOR RNAi trees. 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Plant Molecular Biology 60:137-151. Zuo JR, Niu QW, Nishizawa N, Wu Y, Kost B and Chua NH (2000) KORRIGAN, an Arabidopsis endo-1,4-beta-glucanase, localizes to the cell plate by polarized targeting and is essential for cytokinesis. Plant Cell 12:1137-1152.  97  CHAPTER 4  Investigation of KORRIGAN-protein interactions3  3  A version of this chapter will be submitted for publication. Maloney, VJ, McDonnell, L, and Mansfield, SD. Investigation of KORRIGAN-protein interactions.  98  4.1 Introduction Evidence suggests that the formation of cellulose in higher plants involves organized protein complexes on the plasma membrane referred to as terminal (TC) or cellulose synthase complexes (CSC). The ultrastructure morphology of this complex is known as a rosette because of its six-fold symmetrical arrangement of subunits. Presumably, these subunits each contain six separate catalytic molecules, thus the microfibril that is exuded from the complex is composed of 36 individual glucan chains (Brown and Saxena, 2000; Delmer, 1999). The isolation of functional CSCs has proven difficult to date, and consequently has arguably been the greatest limiting factor in our understanding of cellulose biosynthesis. Initially, a major challenge that restricted the basic understanding of cellulose biosynthesis was the identification of genes that encode components of these complexes. However, this hurdle was surmounted with the discovery of UDP-specific glycosyltransferases displaying conserved regions surrounding aspartate residues and a conserved downstream QXXRW amino acid motif (Saxena and Brown, 1995) that ultimately led to the identification of several putative cellulose synthase genes (CesA) and gene families in a number of plants. Interestingly, at least three distinct CesA genes appear to be involved in primary cell wall synthesis, while an additional three CesA genes are involved in secondary cell wall biosynthesis (Delmer, 1999; Doblin et al., 2002; Joshi et al., 2004). Moreover, it has been predicted that at least three different CesA subunits are required for the assembly of the rosette and the cellulose microfibril (Gardiner et al., 2003; Joshi and Mansfield, 2007; Taylor et al., 2003). Predicted tertiary peptide structures of these genes provide evidence that the catalytic subunit has a number of transmembrane domains and a cytosolic globular region.  The globular cytosolic component is the putative location of an active site  involving UDP-glucose.  The discovery of this cytosolic active site has led to the  creation of additional models involving the enzymatic channelling of UDP-glucose to the CSC (Brown and Saxena, 2000; Brown et al., 1996; Delmer, 1999). Despite the recent advances, what is still lacking is a detailed understanding of how these cellulose synthase complexes and other proteins involved in cellulose synthesis utilize the chemical and genetic resources to synthesize the complex multifunctional plant cell walls.  This lack of understanding is in part due to limited knowledge about the  subcellular localizations of these proteins (Sarkar et al., 2009). 99  It has long been hypothesized that CSC movement is co-aligned with microtubules, and direct experimental evidence for the co-alignment hypothesis came from live cell imaging experiments in which both functional CesA subunits and cortical microtubules displayed corresponding distribution patterns (Gardiner et al., 2003; Paredez et al., 2006). In addition, research on the response of the microtubule network to the drug inhibitor of cellulose synthesis 2,6-dichlorobenzonitrile (DCB) showed that DCB treatments affected the transverse arrays of the cortical microtubules (Himmelspach et al., 2003).  Furthermore, in a screen for Arabidopsis mutants that  showed a hypersensitivity to the microtubule destabilizing drug oryzalin, a KORRIGAN (KOR) mutant was identified (Paredez et al., 2008). KOR, which has previously been shown to be involved in cellulose synthesis (Brummell et al., 1997a; Brummell et al., 1997b; del Campillo, 1999; Maloney and Mansfield, 2010; Master et al., 2004; Molhoj et al., 2001a; Molhoj et al., 2002; Molhoj et al., 2001b; Nicol et al., 1998; Ohmiya et al., 2003; Ohmiya et al., 2000; Peng et al., 2002; Robert et al., 2005; Rudsander et al., 2003; Sato et al., 2001; Szyjanowicz et al., 2004; Takahashi et al., 2009), has also been shown to be localized in cells undergoing active cellulose synthesis (Brummell et al., 1997b; Molhoj et al., 2001a; Takahashi et al., 2009), as well as to some degree with cortical microtubules (Szyjanowicz et al., 2004). Since KOR and the CSC appear to be closely associated and are required in the same cells for proper cellulose synthesis, it is important to determine whether or not they directly interact. A variety of analytical tools are available to detect and identify proteins that potentially interact, including the yeast two-hybrid systems and immunoprecipitation assays. However, these techniques fail to monitor protein-protein interactions in vivo in living plant cells. Bioluminescence resonance energy transfer (BRET) is a technique that has more recently been used extensively for the real-time in vivo study of proteinprotein interactions.  BRET is a form of energy transfer that occurs when two  compatible optical probes are brought into close molecular proximity (Angers et al., 2000). In order to probe for protein-protein interaction between two putative partner proteins, the proteins are linked to a blue light emitting luciferase (from the sea pansy Renilla; RLUC) and to a blue light absorbing yellow fluorescent protein (YFP). If the two hybrid proteins interact, the emission energy of the luciferase is transferred to the fluorescent protein, resulting in an easily detectable yellow-shift in the luminescence 100  spectrum (Angers et al., 2000; Xu et al., 1999).  BRET requires that the donor  fluorophore or bioluminescent protein and reporter fluorophore (longer wavelength acceptor) are less then 10 nm apart. Thus, energy transfer implies that these molecules are in close proximity, and therefore, the proteins of interest fused to the donor and acceptor interact directly or as part of a complex (Angers et al., 2000; Pfleger et al., 2006; Xu et al., 1999). In this study, we established a BRET system for two key proteins involved in plant secondary cell wall synthesis; KOR and a cellulose synthase subunit, CesA7. Using Arabidopsis as a model, we generated plants that co-expressed an N-terminal RLUC-AtKOR construct with an N-terminal YFP-AtCesA7 construct, and assessed the plants for in vivo energy transfer resulting from the oxidation of the coelenterazine substrate by the RLUC transferred to the YFP. As a secondary measure, we attempted to elucidate with which protein(s) AtKOR might interact using a yeast-two-hybrid (YTH) assay.  Acquiring information about interactions of integral membrane proteins has  posed a major challenge because of the hydrophobic nature of membrane-spanning domains. To overcome this hurdle with AtKOR, we used split-ubiquitin membrane YTH technology based on the cleavage of a transcription factor from the N terminus of membrane-inserted bait. 4.2 Materials and methods 4.2.1 BRET construct development The AtKOR BRET expression vector was produced by cloning the entire coding sequence of the Arabidopsis AtKOR gene from cDNA using the AtKORgate forward primer (5'CACCATGTACGGAAGAGATCCATGGGGAG3') and the AtKORgate reverse primer (5'AGGTTTCCATGGTGCTGGTGGAGGT3') under the control of the 35S promoter. Following sequence verification, the AtKOR coding sequence was cloned directly into the pENTR/D-TOPO vector (Invitrogen, Carlsbad, CA, USA). The vector was then sequenced to verify the correct insertion, and Gateway technology (Invitrogen, Carlsbad, CA) was employed to insert the gene into the pZP 35S::hRLUC::attR destination vector (Subramanian et al., 2004). To create the AtCesA7 BRET expression vector under the control of the native Arabidopsis CesA7 promoter (PA7) the 35S promoter from the pBin19 35S::YFP::attR (Subramanian et al., 2004) BRET expression 101  vector was replaced with PA7. PA7 was amplified from DNA using PCR primers A7PRFWClaI  (5'AGTCATCGATGCAGCAACAGCAGGAGAGGTACG3')  and  A7PRRVXmaJI (5'AGTCCCCGGGGCAGCAACAGCAGGAGAGGTACG3') to introduce a ClaI and XmaJI restriction site to the 5′ and 3′ terminus, respectively, and inserted into the TOPO Zero Blunt (Invitrogen) cloning vector. The promoter fragment was digested from the cloning vector with ClaI/XmaJI and ligated into the 35S::YFP::attR backbone which had the 35S promoter removed by digestion with SdaI/AvrII in order to create the PA7::YFP::attR destination vector.  The AtCesA7 coding fragment minus the start  codon was amplified from Arabidopsis cDNA with PCR primers A7FWCACC (5′CACCGAAGCTAGCGCCGGTCT3') (5′TCAGCAGTTGATGCCACACTTGGAA3′).  and  A7RV  Again, Gateway technology (Invitrogen)  was used to insert the gene into the PA7::YFP::attR destination vector. The resulting binary plasmids, 35S::hRLUC::AtKOR (ATKOR) and PA7::YFP::AtCesA7 (AtCesA7), were sequence-confirmed and transformed into Agrobacterium tumefaciens strain GV3101 (Hellens et al., 2000).     4.2.2 Plant strains and growth conditions The T-DNA–mutagenized Arabidopsis thaliana line kor1-1 (Nicol et al., 1998) and the single base-pair substitution mutant irx3-1 (Turner and Somerville, 1997) were acquired from the Arabidopsis Biological Resource Center (Columbus, OH).  Seeds were  sterilized as described by Clough and Bent (1998) and germinated at room temperature in a 16-h/8-h light/dark cycle on half MS medium (Murashige and Skoog, 1962) containing no sugar. Plantlets were transferred to soil after the first four primary leaves had emerged, and the growth cycle was allowed to complete under the same conditions. Plants were determined to be homozygous for the gene mutation using genomic DNA screening and sequencing. The F1 progeny from the kor1-1 line were compared with the Wassilewskija (WS) ecotype and the irx3-1 line were compared the Landsberg erecta (LER) ecotype, the respective backgrounds for the mutants. 4.2.3 Plant transformations, screening and crossing kor1-1 seedlings were transformed with the AtKOR construct and the irx3-1 seedlings were transformed with the AtCesA7 construct by infiltration using Agrobacterium 102  tumefaciens.  The plants were permitted to set seed, and then collected and  transformant seeds selected on MS medium containing either 50 mg/L kanamycin sulfate (Sigma, St. Louis, MO) and 85mg/mL PESTANAL (Glufosinate ammonium; Sigma, St. Louis, MO) for the 35S::hRLUC::AtKOR transfomants, or 75 mg/L kanamycin sulphate for the PA7::YFP::AtCesA7 transformants. Plantlets resistant to the antibiotics were then transferred to soil and placed in a growth chamber and grown under the previously described conditions. Transformed plants were confirmed by genomic DNA PCR  screening  using  gene  5'AAGAGCTTCGTGGAGAGAGTGCTGA3'  specific  oligonucleotides and  5'GCGGAATCTGCAGTGTTTAT3' for the 35S::hRLUC::AtKOR (5'ATCACATGGTCCTGCTGGAGTTCGT3')  and  (LUC3'FW AtKOR5'RV  lines and YFP3'FW A75'RV  (5'TCAGAGGCTTTGGCTCTTCATGGTTGTG3') for PA7::YFP::AtCesA7 lines).  The  CesA7 construct as well as the CesA7 complemented Arabidospis line were created by Lisa McDonnell in the Mansfield lab. Genomic DNA was extracted from single leaves taken from young soil-grown Arabidopsis plants using a CTAB modified extraction method (Rogers and Bendich, 1994). Briefly, single leaves were placed in microcentrifuge tubes and ground to a powder in liquid nitrogen. One milliliter of CTAB extraction buffer (2% [w/v] hexadecyltrimethylammonium bromide [CTAB; Sigma, St. Louis, MO], 100 mM Tris- HCl, pH 8.0, 1.4 M NaCl, 20 mM EDTA, 1% [w/v] polyvinylprylidone, and 0.2% [v/v] 2-mercaptoethanol) was added to each tube, and incubated at 65°C for 60 min. An equal volume of chloroform was added, and the tube was vortexed and then centrifuged in a microcentrifuge for 10 min. Genomic DNA was precipitated from the aqueous phase by addition of 1 volume of isopropanol, incubating at -20°C for 10 min, and centrifuging for 5 min. Genomic DNA was re-suspended in RNase buffer (25 mM Tris-HCl, pH 7.5, 10 mM EDTA, and 100 mg/mL RNase A) and incubated at 37°C for 30 min. Two volumes of ethanol were added, and the genomic DNA was recovered by centrifugation. Finally, the DNA was re-suspended in 50 mL of EB buffer (10 mM Tris-HCl, pH 8.0, and 1 mM EDTA), quantified at A260, and stored at 4°C. Following confirmation of transgenic plants, plants from each line were grown until early flowering (approximately three weeks). All flowering parts except ovaries were removed from the immature flowers on the AtCesA7 plants and then dabbed with 103  an anther of a mature flower from an AtKOR plant. Once as many crosses that could be made were completed, the meristem of all other buds was removed from the AtCesA7 plant to prevent further development. After silique maturation, the seeds were collected and were grown on the appropriate selection media. 4.2.4 RNA extraction and realtime PCR Total RNA was extracted from approximately 500 mg of frozen ground eight-week-old Arabidopsis plants using TRIzol reagent (Sigma, St. Louis, MO) according to the manufacturer’s instructions. RNA yield was measured by absorption at 260nm, and 10 μg was treated with DNAase (Ambion TURBO DNA-free). One μg of the resulting DNAfree RNA was evaluated on a 1% Tris-acetate EDTA agarose gel in order to determine quality. Equal quantities of RNA (1 μg) were used for the synthesis of cDNA with SuperScript II reverse transcriptase (Invitrogen) and (dT)16 primers, according to the manufacturer’s instructions. Samples were run in triplicate with Platinum SYBR Green qPCR Master mix (Invitrogen) on an Mx3000p real-time PCR system (Stratagene, La Jolla, CA). The real-time PCR analysis of the Arabidopsis lines was performed using the primers AtKORRTFW (5'GGGAATACTGATGATGGAAAT3') and AtKORRTRV (5'ATCCACCATTACAAGTAGTCA3') A7HVRIIFW  for  the  35S::hRLUC::AtKOR  (5'ACATGAATGGTGACGTAGCAGCCCTT3')  (5'ACCGCAGCTTATGACATGGATTGCCT3')  for  the  and  lines  and  A7HVRIIRV  PA7::YFP::AtCesA7  lines.  Transcript abundances were determined based on changes in Ct values relative to elongation  initiation  factor5A  (AtEIF5AFW  5′ACTGACCTCTTAGCTCG3′  and  AtEIF5ARV 5′CAGATCGGCCACGTTC3′) for the AtKOR line and ubiquitin (AtUBQ5FW 5'ACACCAAGCCGAAGAAGATCAAGCAC3'  and  AtUBQ5RV  5'AAATGACTCGCCATGAAAGTCCCAGC-3') for the AtCesA7 line (Gutierrez et al., 2008; Yamada et al., 2003) . Conditions for all PCR reactions were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 30 s, 55°C for 1 min, and 72°C for 30 s. 4.2.5 Toluidine blue staining and microscopy Four-week-old Arabidopsis stems were cut 1 cm above the root collar and further hand sectioned into approximately 500nm thick cross sections. Sections were then stained in 0.05% toluidine blue stain for 5 min. Excess stain was removed by washing with dH2O. 104  All sections were mounted onto glass slides and examined with a Leica DAR microscope (Leica Microsystems, Wetzlar, Germany) fitted with epifluorescence optics. Photos were taken with a QICAM camera (QImaging, Surrey, Canada) and OpenLab software (PerkinElmer Inc., Boston, MA, USA). 4.2.6 BRET assay Five-day-old Arabidopsis plantlets were placed in a 96 well Optiplate (PerkinElmer, Boston, MA) with 180 μl water. Immediately prior to taking plate readings 20 μl of 1 μM coolenterazine (Biotium Inc., Hayward, CA) was added to each well. Measurements of YFP and Luciferase (LUC) activity were recorded using a Wallac 1420 VICTOR3 (PerkinElmer, Boston, MA) plate reader equipped with a LUC emission filter (486/10), a YFP emission filter (530/10), and a YFP excitation filter (490).  BRET measurements  were recorded four times with a 30 sec delay in between the third and fourth measurement. LUC values were measured four times with a 2 min delay between the first and second measurement and a 1 min delay between the third and fourth measurements, while YFP values were recorded after a single excitation and then repeated. BRET calculations were performed according to Bacart et al. (2008). Briefly, the BRET ratio was calculated by dividing the light signal recorded at the acceptor wavelength (530) by the donor signal (486). To correct for background signal due to the overlap of donor emission at the acceptor wavelength, the BRET ratio was determined in parallel for plants expressing the donor alone (35S::hRLUC::AtKOR). The BRET value was calculated by subtracting this BRET background ratio from the BRET ratio obtained in plants co-expressing the two BRET partners. Results are expressed in milliBRET (mB) by multiplying by 1000.  The amount of the YFP acceptor was  determined in an independent reading by exciting all donor molecules and is expressed as total fluorescence minus background (WT values). The amount of the LUC donor was also determined in independent readings following the addition of the substrate and is expressed as total luminescence minus background (WT values). 4.2.7 Bait construction and confirmation Bait construction and confirmation were conducted according to the DUALmembrane starter kit N (Dualsystems Biotech, Schlieren, Switzerland).  AtKOR bait primers 105  (AtKORbait FW 5′ATTACAAGGCCATTACGGAAATGTACGGAAGAGATCCATG3′ and AtKORbait  RV  5′AACTGATTGGCCGAGGCGGCCTCAAGGTTTCCATGGTGCTG3′)  were used to amplify the entire coding sequence of the AtKOR gene from Arabidopsis cDNA and then subsequently cloned into the Sfi I restriction sites in the pBT3-N bait vector. The AtKOR bait construct was then confirmed by sequencing and transformed into the NMY51 yeast strain according to the manufacturer’s instructions. In order to assay the correct expression of the bait protein in the yeast the bait plasmid was cotransformed with the control plasmids pOst-NubI and pPR3-N. Co-expression of the bait with Ost1-NubI resulted in reconstitution of split-ubiquitin and the concurrent activation of the HIS3 and ADE reporter genes and thus growth on selection media. Growth of the yeast expressing the AtKOR bait and Ost-NubI indicated that the bait was functional in the DUALmembrane system. Yeast co-expressing the AtKORbait and the pPRN-3 did not grow on selective medium. 4.2.8 Pilot screen and cDNA library screen In order to simulate the conditions of the library screen the empty library vector pPR3-N was transformed into the AtKOR bait bearing yeast strain according to the manufacturer’s instruction.  To minimize inherent leaky HIS3 expression, the co-  transformed yeast was streaked out on minimal SD plates (−Trp/−Leu/−His/−Ade) containing increasing amounts of 3-amino-1,2,4-triazole (3-AT; Sigma, St. Louis, MO). 40mM of 3-AT was determined to be the amount that could abolish growth with the pPR3-N control prey.  An Arabidopsis NubGx cDNA library was purchased from  Dualsystems Biotech (Schlieren, Switzerland).  28 µg of the cDNA library was  transformed into the yeast reporter strain NMY51 expressing the AtKOR bait. Approximately 4 × 106 independent clones were screened for colonies that would grow on media lacking Trp, Ade, His, and Leu supplemented with 40 mM 3-AT. Colonies that grew on selection media were further screened using the HTX β-galactosidase assay kit (Dualsystems Biotech, Schlieren, Switzerland). Library plasmids were isolated from clones that appeared positive in both screens, amplified in Escherichia coli, and their sequences analyzed.  106  4.3 Results 4.3.1 Complementation of Arabidopsis mutants with BRET constructs Plants homozygous for the kor1-1 mutation (Nicol et al., 1998) were transformed with the AtKOR gene tagged with a Renilla luciferase (hRLUC) reporter gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter (AtKOR).  Plants  homozygous for the irx3-1 mutation (Turner and Somerville, 1997) were transformed with the AtCesA7 gene tagged with a yellow fluorescent protein (YFP) reporter gene under the control of the native CesA7 promoter (AtCesA7). Following the transgene insertion events, transformants were selected by screening for plants capable of growing on the appropriate selection media.  None of the AtKOR or AtCesA7  transformants displayed the elongation deficient phenotype typical of their mutant backgrounds (Figure 4.1). Additionally, toluidine blue staining of four-week old stem cross-sections revealed that neither the AtKOR nor the AtCesA7 transformants displayed the irregular xylem (irx) phenotype typical of their mutant backgrounds (Figure 4.2).  Real time quantitative PCR analyses further confirmed that the inserted  endogenous cis-genes were indeed expressed in the complemented lines (Figure 4.3A&B). Plants positive for the AtKOR-hRLUC tagged construct were crossed with plants positive for the AtCesA7-YFP tagged construct in order to create AtKOR×CesA7 plants. 4.3.2 BRET assay In order to determine if the AtKOR protein interacts with the AtCesA7 protein we conducted an in planta BRET assay using five-day old Arabidopsis plantlets. AtKOR×CesA7 plantlets along with wild-type, LUC-YFP positive controls, AtKOR, and AtCesA7 plantlets were assayed for BRET, LUC and YFP emission (Figure 4.4). AtKOR single transformants, as well as the AtKOR×CesA7 plantlets displayed significant increases (92 and 80 respectively) in average LUC emission when compared to the wild-type (0) and the non-LUC tagged CesA7 plants (4).  Additionally, the  AtCesA7 single transformants and the AtKOR×CesA7 plants had a significantly higher average YFP emission (1143 and 649 respectively) than the wild-type (0) and the nonYFP tagged AtKOR plants (224) (Figure 4.4A). However, the AtKOR×CesA7 plants did 107  not display BRET (Figure 4.4B), indicating that the two tagged constructs do not interact in planta. 4.3.3 Yeast two hybrid assay Since the BRET assay suggested that the AtKOR and AtCesA7 do not interact with each other, we sought evidence for potential interacting partner proteins of AtKOR that might be involved in cell wall synthesis. The AtKOR coding fragment was therefore inserted into the membrane-specific yeast bait expression vector pBT3-N.  Proper  insertion of the bait protein into the yeast was determined using a functional assay (Figure 4.5). The functional assay uses a control prey construct, pOst-NubI, to express a fusion to the yeast resident ER protein Ost1 to the wild type Nub (N-terminus) portion of yeast ubiquitin. Growth on selection media when the bait is co-transformed with the control prey indicates that the Cub(C-terminus)-LexA-VP16 moiety attached to the bait construct was properly located on the cytosolic side of the membrane and resulted in the rapid reformation of ubiquitin from the split moieties. This reformation of the ubiquitin causes the ubiquitin specific proteases to cleave the LexA-VP16 transcription factor, which subsequently activates the yeast reporter genes. However, co-expression of the AtKOR bait protein with the NubG-nonsense peptide fused to the pPR3-N did not lead to split-ubiquitin formation, indicating that the AtKOR bait does not exhibit nonspecific background. In order to screen for possible KOR-protein interactions, optimal screening conditions were determined with a pilot screen. Once bait functionality and the proper screening conditions were determined, we screened the AtKOR bait against a NubG-fused Arabidopsis cDNA library. Approximately 300 white colonies grew on selection plates, however, they all were very tiny. After permitting the colonies to grow for two additional days, they were all streaked onto new selection plates and then subjected to the β-galactosidase assay. The β-galactosidase assay identified only three colonies with a strong protein-protein interaction. These colonies were then cultured in liquid media, after which their plasmids were extracted and transformed into E. coli. Eight E. coli colonies were picked from each transformation, and their plasmid screened for a library insert. Of the 24 colonies screened, only one contained a library plasmid of a significant size (approximately 1500bp).  Sequencing analyses of the plasmid  108  indicated that it was an ORF selection vector (pSOS) plasmid and thus a false positive. The Arabidopsis cDNA library was screened two more times with similar results. 4.4 Discussion There are several inconsistencies in the literature reporting on the localization of KOR. Some of the first studies provided evidence that the KOR protein co-purified with plasma membrane markers (Brummell et al., 1997b; Nicol et al., 1998). Conversely, in tobacco BY-2 cells Zuo et al. (2000) reported on a C-terminal KOR-GFP fusion construct where GFP signal accumulated in the intracellular organelles, interphase cells, and in the phragmoplast in dividing cells. However, the authors were never able to prove that the GFP-fusion construct was functional. A more recent study (Robert et al., 2005) with an N-terminal GFP-KOR fusion construct that was able to complement the kor1-1 mutant provided evidence for the localization of KOR in the Golgi apparatus, early endosomes, and the tonoplast of epidermal cells in the root meristem.  A  redistribution of GFP-KOR away from early endosomes to a homogeneous population of compartments which were concentrated close to the plasma membrane was observed after treatment with isoxaben and in cells at the later stages of cell elongation. A subpopulation of GFP-KOR-containing compartments followed trajectories along the plasma membrane, and this movement required intact microtubules (Robert et al., 2005).  Furthermore, a promoter-GUS fusion to a membrane-anchored endo-1,4-β-  glucanase cDNA from oil seed rape (Cel16), which exhibits 94% sequence similarity to the Arabidopsis KOR protein, demonstrated that Cel16 is expressed in all tissue types, but was highest in young tissues (Molhoj et al., 2001a). Similarly, a recent study by Takahashi et al. (2009) analysed the activity of a Arabidopsis KOR promoter-GUS construct, and showed high activity in young plants—especially in actively expanding cells and vascular tissues—as well as being consistently active in the stems undergoing secondary cell wall thickening. It has also been demonstrated that the KOR ortholog in hybrid aspen, PttCel9A, is up-regulated in the xylem (Hertzberg et al., 2001). Furthermore, co-expression of another Populus KOR ortholog with the three secondary cell wall CesA genes has also been reported (Bhandari et al., 2006). The localization of KOR at or near the plasma membrane and its requirement during periods of intensive cellulose synthesis implies that KOR interacts or is bound to 109  the CSC. However, a study on the irx2 mutant of KOR showed that KOR did not copurify with IRX3 (AtCesA7), but a very low signal was obtained using the anti-KOR antibody, which could have prevented the detection of smaller amounts of KOR coprecipitating with IRX3 (Szyjanowicz et al., 2004). Similarly, a study evaluating the organization of cellulose synthase complexes involved in primary cell wall synthesis in Arabidopsis showed that CesA3 and CesA6 do not co-immunoprecipitate with KOR (Desprez et al., 2007). Additionally, using a more direct and accurate method here we show that AtKOR and AtCesA7 do not interact with each other in planta.  Taken  together, it is apparent that KOR is essential for proper cellulose synthesis, but it is not an integral part of the cellulose synthase complex located at the plasma membrane. Sequence analyses of cellulose synthases have predicted that a large globular region is present in the cytosol and as a result, the cytosolic portion of the CSC is predicted to be much larger than what can be visualized on the cell surface (Saxena and Brown, 2005; Saxena and Brown, 2007). Since it has also been predicted that the putative CSC active site and consequently the site for potential protein-protein interactions is located in this globular region (Saxena and Brown, 2007), it is only logical that KOR, whose putative catalytic sites have an extracellular location (Nicol et al., 1998), would not directly interact with the CSC. Having established that KOR does not interact with the CSC but that it is highly expressed and involved in cellulose synthesis, we predicted that it may directly interact with some other key proteins involved in cell wall synthesis. However, after multiple screenings of an Arabidospis cDNA library for a potential AtKOR interactor, we were unable to detect any KOR-protein interactions on the cytosolic side of the plasma membrane. This may be a related to the hypothetical nature of the yet to be confirmed extracellular catalytic sites, but more likely indicates that KOR works alone and does not directly associate or form complexes with other proteins involved in cell wall synthesis. These findings are consistent with suggested roles for KOR downstream of cellulose synthesis (Maloney and Mansfield, 2010; Szyjanowicz et al., 2004; Takahashi et al., 2009), including severing microfibrils. These catalytic functions are consistent with the presence of secondary cell wall deposition at the corner of cells that can be observed in the KOR irx2 mutants (Taylor, 2008; Turner and Somerville, 1997). The corners of the cells represent the earliest sites of secondary cell wall deposition (Altamura et al., 110  2001), which further indicates that cellulose deposition starts but cannot be completed in the mutants. While we were not able to provide evidence for any direct KOR-protein interaction, we were able to provide evidence that disproves the hypothesis that KOR interacts with CesA7, a member of the secondary cell wall CSC.  However, additional  studies may be necessary to conclusively prove that the KOR protein is not involved in any other extracellular protein interactions.  111  WT-LER  irx3-1  AtCesA7  Figure 4.1 Four-week old wild type (WS), kor1-1, AtKOR, wild type (LER) and AtCesA7 Arabidopsis plants demonstrating the recovery from the mutant growth phenotype. AtKOR plants were transformed to express the AtKOR gene under the control of the 35S promoter in the kor1-1 mutant background while AtCesA7 lines were transformed to express the AtCesA7 gene under the control of the AtCesA7 promoter in the irx3 mutant background.  112  Figure 4.2 Four-week old stem cross-sections of wild type (WS), kor1-1, AtKOR, wild type (LER), irx3-1, and AtCesA7 complemented lines demonstrating the recovery from the mutant irx phenotype in the complemented lines. AtKOR plants were transformed to express the AtKOR gene under the control of the 35S promoter in the kor1-1 mutant background while AtCesA7 lines were transformed to express the AtCesA7 gene under the control of the AtCesA7 promoter in the irx3 mutant background. Arrows indicate xylem elements. 113  B  A 45  250  40  200  30  Relative Expression  Relative Expression  35  25 20 15 10 5  150  100  50  0  0 WS  kor1-1  AtKOR  LER  irx3-1  AtCesA7  Figure 4.3 Relative transcript abundance of AtKOR or AtCesA7 in four-week old Arabidopsis plants. (A) AtKOR in wild-type (WS), kor1-1 and the complemented AtKOR line, and (B) AtCesA7 in wild-type (LER), irx3-1 and complemented AtCesA7 lines. Transcript levels were determined based on changes in Ct values relative to (A) translation initiation factor5A or (B) ubiquitin. Error bars = standard error of the mean, n=6 (2 plant pools * 3 technical replicates).  114  B  A 1400  120  YFP  180  *  1200  160  *  800 60 600 40  *  400 20  200  Average milliBRET (mB)  80  LUC Luminescence  *  *  100  1000  YFP Fluorescence  200  *  LUC  140 120 100 80 60  *  *  * *  40 20  0  0 WT  LY  KOR-LUC  CesA7-YFP KORxCesA7  0 WT  LY  KOR-LUC  CesA7-YFP  KORxCesA7  Figure 4.4 Measurements of YFP, LUC, or BRET in wild type (WT), control (LY), AtKOR (KOR-LUC), AtCesA7 (CesA7-YFP), and AtKOR×AtCesA7 crossed (KOR×CesA7) lines indicates the presence of YFP fluorescence and LUC luminescence in the crossed lines, but no BRET. (A) Average YFP fluorescence and LUC luminescence. Stars indicate a significant difference from the wild type at a t-test value of p=0.05. (B) Average milliBRET. Stars indicate a significant difference from the LY positive control at a t-test value of p=0.05. Error bars are standard error of the mean WT,LY, KOR-LUC, and CesA7-YFP n=22, KOR×CesA7 n=43. BRET calculations were performed according to Bacart et al. (2008).  115  Figure 4.5 AtKOR bait is functionally expressed in NMY51 yeast. Transformation efficiency indicates growth on non-selective plates. Red colonies indicate that no protein interaction is taking place and the ADE2 gene is not transcribed and the adenine synthesis pathway is blocked. White colonies indicate that the AtKORbait and positive control prey are interacting and that the AtKOR bait is properly inserted in the yeast.  116  4.5 References Altamura MM, Possenti M, Matteucci A, Baima S, Ruberti I and Morelli G (2001) Development of the vascular system in the inflorescence stem of Arabidopsis. New Phytologist 151:381-389. Angers S, Salahpour A, Joly E, Hilairet S, Chelsky D, Dennis M and Bouvier M (2000) Detection of beta(2)-adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer (BRET). 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Saxena IM and Brown RM (2005) Cellulose biosynthesis: Current views and evolving concepts. Annals of Botany 96:9-21. Saxena IM and Brown RM, Jr. (2007) A perspective on the assembly of cellulosesynthesizing complexes: Possible role of KORRIGAN and microtubules in cellulose synthesis in plants. Cellulose: Molecular and Structural Biology:169181,363. Subramanian C, Xu Y, Johnson C and von Arnim A (2004) In vivo detection of proteinprotein interaction in plant cells using BRET. Methods in Molecular Biology 284:271-286. Szyjanowicz PMJ, McKinnon I, Taylor NG, Gardiner J, Jarvis MC and Turner SR (2004) The irregular xylem 2 mutant is an allele of korrigan that affects the secondary cell wall of Arabidopsis thaliana. Plant Journal 37:730-740. Takahashi J, Rudsander UJ, Hedenstrom M, Banasiak A, Harholt J, Amelot N, Immerzeel P, Ryden P, Endo S, Ibatullin FM, Brumer H, del Campillo E, Master ER, Scheller HV, Sundberg B, Teeri TT and Mellerowicz EJ (2009) KORRIGAN1 and its aspen homolog PttCel9A1 decrease cellulose crystallinity in Arabidopsis stems. Plant and Cell Physiology 50:1099-1115. Taylor NG (2008) Cellulose biosynthesis and deposition in higher plants. New Phytologist 178:239-252. Taylor NG, Howells RM, Huttly AK, Vickers K and Turner SR (2003) Interactions among three distinct CesA proteins essential for cellulose synthesis. Proceedings of the National Academy of Sciences of the United States of America 100:1450-1455. Turner SR and Somerville CR (1997) Collapsed xylem phenotype of Arabidopsis identifies mutants deficient in cellulose deposition in the secondary cell wall. Plant Cell 9:689-701. Xu Y, Piston DW and Johnson CH (1999) A bioluminescence resonance energy transfer (BRET) system: Application to interacting circadian clock proteins. Proceedings of the National Academy of Sciences of the United States of America 96:151-156. Yamada K, Lim J, Dale JM, Chen HM, Shinn P, Palm CJ, Southwick AM, Wu HC, Kim C, Nguyen M, Pham P, Cheuk R, Karlin-Newmann G, Liu SX, Lam B, Sakano H, Wu T, Yu GX, Miranda M, Quach HL, Tripp M, Chang CH, Lee JM, Toriumi M, Chan MMH, Tang CC, Onodera CS, Deng JM, Akiyama K, Ansari Y, Arakawa T, 120  Banh J, Banno F, Bowser L, Brooks S, Carninci P, Chao QM, Choy N, Enju A, Goldsmith AD, Gurjal M, Hansen NF, Hayashizaki Y, Johnson-Hopson C, Hsuan VW, Iida K, Karnes M, Khan S, Koesema E, Ishida J, Jiang PX, Jones T, Kawai J, Kamiya A, Meyers C, Nakajima M, Narusaka M, Seki M, Sakurai T, Satou M, Tamse R, Vaysberg M, Wallender EK, Wong C, Yamamura Y, Yuan SL, Shinozaki K, Davis RW, Theologis A and Ecker JR (2003) Empirical analysis of transcriptional activity in the Arabidopsis genome. Science 302:842-846. Zuo JR, Niu QW, Nishizawa N, Wu Y, Kost B and Chua NH (2000) KORRIGAN, an Arabidopsis endo-1,4-beta-glucanase, localizes to the cell plate by polarized targeting and is essential for cytokinesis. Plant Cell 12:1137-1152.  121  CHAPTER 5  Thesis summary and future research  122  5.1 Thesis summary A number of Arabidopsis mutants possessing modified xylem phenotypes (irx) have provided evidence for the requirement of a membrane-bound endoglucanase, termed KOR, for complete and correct cellulose biosynthesis (Lane et al., 2001; Nicol et al., 1998; Sato et al., 2001; Szyjanowicz et al., 2004; Zuo et al., 2000). Further research in other plant species evaluating orthologs of KOR have also concluded that KOR is integral in cellulose synthesis (del Campillo, 1999; Maloney and Mansfield, 2010; Master et al., 2004; Molhoj et al., 2001a; Molhoj et al., 2002; Molhoj et al., 2001b; Ohmiya et al., 2003; Ohmiya et al., 2000; Peng et al., 2002; Robert et al., 2005; Rudsander et al., 2003; Takahashi et al., 2009). Although the existing research clearly demonstrated that KOR is required in cellulose synthesis, its exact role was not thoroughly investigated. Furthermore, the majority of the said research focused on nonwoody species and on primary cell wall development. The goal of this thesis research was to study the effects of KOR on two of the most commercially important forest trees, hybrid poplar and white spruce, and more importantly how the mis-regulation of KOR genes manifests change in the secondary cell walls. The results of my efforts in hybrid poplar (Chapter 2) show that the mis-regulation of KOR does indeed affect the cellulose of the secondary cell wall. A down-regulation of KOR leads to moderate to severe defects in plant growth, an irregular xylem (irx) phenotype which is consistent with other species, and significantly impacts the ultrastructure of the cellulose synthesized. I was able to show for the first time that the RNAi-suppression of KOR led to the deposition of significantly reduced quantities of a more highly-crystalline cellulose, while the hemicellulose content, and more specifically, the xylose content increased. In addition, the amount of soluble sucrose in the leaves and xylem decreased. Conversely, the expression of the foreign exogeneous AtKOR gene in poplar did not significantly alter cell wall development or plant growth parameters, but it did impact the ultrastructure of the cellulose produced, generating trees with less crystalline cellulose and reduced xylose content. As previously mentioned, the endogenous KOR genes from a number of herbaceous plants including Arabidopsis, tomato and oilseed rape have been discovered and some have been extensively characterized (Brummell et al., 1997; Molhoj et al., 2001b; Nicol et al., 1998). However, to date no characterization of a 123  gymnosperm KOR has been undertaken, and it is not known whether the membranebound endoglucanases in these taxa, which evolved 300 mya, are orthologous to the angiosperms that diverged 130 mya.  To that end, I isolated and characterized a  membrane-bound endoglucanase gene from white spruce (Picea glauca; Chapter 3). This tree represents a species that is important both ecologically and commercially, especially with respect to global wood and fibre supply. Sequence alignments with other membrane-bound endoglucanase proteins and phylogenetic reconstruction analyses revealed that the white spruce KOR (PgKOR) contains conserved polarized targeting signals, as well as residues predicted to be essential for catalytic activity. Expression of PgKOR in Arabidopsis kor1-1 mutants clearly demonstrates that this gene is able to recover the mutant phenotype, providing evidence for functional equivalence. Analyses of endogenous KOR expression in white spruce revealed the highest expression in young developing tissues and tissues undergoing secondary cell wall development, which agrees with previous expression patterns in other species (Bhandari et al., 2006; Hertzberg et al., 2001; Molhoj et al., 2001a; Takahashi et al., 2009). Additionally, the suppression of the endogenous gene substantially reduced growth and cellulose content in spruce, but had no effect on cellulose ultrastructure. These results differ from my findings in hybrid poplar, where the suppression of the endogenous KOR gene manifested an irx phenotype, indicating that the suppression of KOR has a larger affect on vessels than on tracheids. The precise reasons for this are not known, but cells that have higher inherent lignin content, such as tracheids, have a greater resistance to negative hydraulic pressure, which might may help to explain why the tracheids did not collapse in the white spruce (Akiyama et al., 2005; Boyce et al., 2004). An extensive interrogation of the recently published Physcomitrella patens draft genome sequence (Rensing et al., 2008) with both the AtKOR and the PgKOR gene did not reveal any significant matches. These data and the evidence for the functional conservation of KOR in both tracheid and vessel-containing plants provide indirect proof that KOR evolved sometime before the evolution of the tracheid cell type, but after the conquest of land by plants.  124  To expand on the KOR expression analysis and gene discovery described in Chapters 2 and 3, where the requirement of KOR during periods of intensive cellulose synthesis in the primary and secondary cell wall was evident in trees, I sought evidence that KOR interacts or is bound to the cellulose synthesizing complex (CSC). Previous reports investigating the localization of KOR have been inconsistent (Brummell et al., 1997; Nicol et al., 1998; Robert et al., 2005; Zuo et al., 2000), but with the use of BRET technology, I was able to provide evidence that AtKOR does not interact with AtCesA7, a member of the secondary cell wall CSC. Furthermore, I was not able to provide evidence for any direct KOR-protein interaction using a membrane-specific yeast-two hybrid system. While this may be related to the hypothetical nature of the yet to be confirmed extracellular catalytic sites of KOR, it more likely indicates that KOR works alone and does not directly associate or form complexes with other proteins involved in cell wall synthesis.  These findings are consistent with suggested roles for KOR  downstream of cellulose synthesis (Maloney and Mansfield, 2010; Szyjanowicz et al., 2004; Takahashi et al., 2009), including cellulose editing or severing microfibrils. However, additional studies may be necessary to prove conclusively that the KOR protein is not involved in any other extracellular protein interactions. Collectively, the data clearly demonstrate that KOR is essential for both primary and secondary cell walls in forest trees. However, the data also suggest that KOR is not directly associated with the CSC, but that its role occurs in the later stages of cellulose deposition - likely representing a carbohydrate modifying enzyme. In summary, the research described represents the first time a mis-regulation of the KOR gene in forest trees has been conducted. In addition, this research provides powerful information with regards to evolution of plants that enabled habitation on land. 5.2 Future research The research described in this thesis has expanded the existing knowledge base related to the effects of KOR on plant cell wall synthesis, and more specifically the role of KOR in forest trees.  In addition, I have provided evidence that KOR-protein  interactions do not appear at the plasma membrane.  However, there are three key  research projects that could add to our understanding of the role KOR plays with respect to plant growth and development. 125  5.2.1 Detailed microscopy of KOR suppressed trees Chapters 2 and 3 of this thesis collectively showed that suppression of the endogenous KOR gene in hybrid poplar and white spruce confer slightly different phenotypes. While both trees exhibited reduced cellulose contents, only the hybrid poplar displayed an irx phenotype. This suggests, as previously discussed, that a cell specific role for KOR exists, where it affects vessel elements, but not tracheids. If we could determine whether a KOR down-regulation affects cell expansion or secondary wall deposition its cell specific role might be clarified. A detailed examination of the different secondary cell wall layers (S1, S2, S3) of white spruce tracheids might indicate whether or not KOR is acting only on the primary growth of the secondary vascular system. If this is the case it could be a possible explanation of why vessels are more vulnerable to perturbation due to their more rapid rates of expansion. Additionally, a comparison of the measurements of the radial diameter of the tracheids in the KOR suppressed white spruce to the wild-type could provide further evidence of whether or not KOR works in the expansion zone of the secondary vascular cambium. If radial growth is largely affected,  vessels,  which  expand  radially  more  than  tracheids,  would  be  disproportionately affected by a KOR downregulation. 5.2.2 High-resolution atomic force microscopy In Chapter 2 we report on the changes in the cell wall crystallinity and the amount of hemicelluloses in KOR mis-regulated hybrid poplar trees.  I hypothesize that the  phenotypes observed in the modified plants could be explained using a new model for the primary cell wall that was derived from direct visualization of maize stem pith (Ding and Himmel, 2006).  Using high-resolution atomic force microscopy, these authors  demonstrate that a number of elementary fibrils are synthesized at the cellulose synthase complex and come together into much larger macrofibrils. These macrofibrils eventually split at the ends to form parallel microfibrils, which are then available to interact with other cell wall components such as hemicelluloses, pectin and lignin. I believe that my results provide evidence for a role for KOR in the splitting of the "macrofibril" into individual microfibrils where any lack of function could prevent the macrofibril from being dispersed into the microfibrils. However, to prove this hypothesis  126  definitively it would be essential to examine the cellulose in the modified trees using high-resolution atomic force microscopy. 5.2.3 Fluorescence microscopy Chapter 4 of this thesis addressed possible KOR-protein interactions. While I was able to demonstrate that AtKOR does not physically interact with AtCesA7 I was not able to determine the distance between the two proteins.  Using the AtKOR×AtCesA7  Arabidopsis lines that we previously created we should be able to visualize the locations of the YFP and LUC tagged proteins using fluorescence microscopy.  Direct  visualization of the reporter proteins will not only allow us to see their relationship to each other, but also their cellular location. 5.3 Research significance In plants, the process of cellulose biosynthesis is as fundamental and important as photosynthesis. The modulation of cellulose biosynthesis influences many aspects of plant growth and development, including cell division and expansion, plant morphogenesis, and response to environmental cues.  Elucidating the biosynthetic  mechanism of this polymer is pivotal to future experiments aimed at augmenting cellulose production in plants.  If attempts to improve cellulose production in  commercially important plants are successful, they will deliver enormous benefits to global agriculture and forest products industries. In this thesis we have made significant increases in the general knowledge base in regards to cellulose and cell wall synthesis. It is my hope that this research will be expanded upon in order to further the attempts at elucidating the biosynthetic mechanism of this important polymer. .  127  5.4 References Akiyama T, Goto H, Nawawi DS, Syafii W, Matsumoto Y and Meshitsuka G (2005) Erythro/threo ratio of beta-O-4-structures as an important structural characteristic of lignin. Part 4: Variation in the erythro/threo ratio in softwood and hardwood lignins and its relation to syringyl/guaiacyl ratio. Holzforschung 59:276-281. 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Plant Physiology 125:131-134. del Campillo E (1999) Multiple Endo-1,4-beta-D-glucanase (cellulase) genes in Arabidopsis, in Current Topics in Developmental Biology, Vol 46 pp 39-+. Ding SY and Himmel ME (2006) The maize primary cell wall microfibril: A new model derived from direct visualization. Journal of Agricultural and Food Chemistry 54:597-606. Hayashi T (1989) Xyloglucans in the primary-cell wall. Annual Review of Plant Physiology and Plant Molecular Biology 40:139-168. Hertzberg M, Aspeborg H, Schrader J, Andersson A, Erlandsson R, Blomqvist K, Bhalerao R, Uhlen M, Teeri TT, Lundeberg J, Sundberg B, Nilsson P and Sandberg G (2001) A transcriptional roadmap to wood formation. Proceedings of the National Academy of Sciences of the United States of America 98:1473214737. 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