Molecular Analysis and Functional Elucidation of a Novel Plant O-Fucosyltransferase in Arabidopsis Thaliana and Brassica Napus

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MOLECULAR ANALYSIS AND FUNCTIONAL ELUCIDATION OF A NOVEL PLANT O-FUCOSYLTRANSFERASE IN ARABIDOPSIS THALIANA AND BRASSICA NAPUS

Jerlene Halliday

A Thesis Presented to The University of Guelph

In partial fulfilment of requirements for the degree of Doctor of Philosophy in Plant Agriculture

Guelph, Ontario, Canada

Abstract

Molecular Analysis and Functional Elucidation of a Novel Plant O-Fucosyltransferase in A. thaliana and B. napus

Jerlene Halliday Advisor:

University of Guelph, 2016 Dr. K. Peter Pauls

The addition of sugar to cell wall polysaccharides and proteins is mainly catalyzed by glycosyltransferases (GTs). Plant O-fucosyltransferases (O-FuTs) are a type of GTs that catalyze fucose transfer from the donor, guanosine diphosphate fucose (GDP-Fuc), to various acceptor molecules, including oligosaccharides, glycoproteins and glycolipids. Previous microarray analysis identified a group of genes that were highly expressed in embryogenic B. napus microspore culture cells in as early as 3 days after microspore embryogenesis induction. Of special interest was the Brassica transcript homologous to the Arabidopsis gene AT2G44500 since it was consistently upregulated (threefold increase) in embryogenic cells compared to pollen-like/non-responsive cells. This Arabidopsis gene contains a GDP-O-fucosyltransferase

(O-FuT) domain.

In the present study, the complete genomic and coding sequences of At2G44500 homologs were isolated and sequenced from B. napus cv. Topas. Sequence analyses of

At2G44500 and its B. napus homolog, BnAt2G (1) suggest that they encode type II-membrane proteins that contain conserved key amino acids essential for O-FuT catalytic activity.

BnAt2G(1)/At2G44500 are differentially expressed throughout plant development. In particular, At2G44500 was shown to be highly expressed in the shoot apical meristem (SAM)

ii and young vegetative tissues in A. thaliana. BnAt2G(1) was also shown to be expressed at higher levels in B. napus embryogenic microspore cultures as well as in vegetative tissues.

The putative transcription control elements identified in the in silico promoter analysis of

At2g44500/BnAt2G(1) suggest that their gene expression is controlled by various transcription factors that are involved in hormone-regulated stress responses. At2G44500 has also been shown to be co-expressed with genes that have known functions in cell wall biosynthesis, embryogenesis and signal transduction. The sub-cellular localization of a GFP::BnAt2G(1) fusion protein in root cells and leaf protoplasts suggest that these type-II membrane proteins are localized in the Golgi or Golgi-derived vesicles.

To elucidate the function of At2G44500/BnAt2G(1), knockdown T-DNA mutants and transgenic plants expressing RNAi silencing (pFGc 5941) and overexpressing constructs driven by a 35S promoter (pMDC32) were generated. The knockdown expression of

At2G44500/BnAt2G(1) led to an increased number of lateral roots and wider stem diameter whereas the overexpression of At2g44500/BnAt2G(1) led to plants with a decreased number of lateral roots and smaller stem diameter compared to wild type and knock down lines. Fourier-

Transformed Infra-Red (FT-IR) analyses of the mutant and transgenic lines revealed significant changes in their cell wall composition particularly in their methyl-esterified pectin levels. It is proposed that the manipulation of At2G44500/BnAt2G(1) expression has consequences on wall architecture by causing structural changes in the pectic network and thus lead to changes in the cell wall's biomechanical properties.

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List of Figures

Figure 1.1 Structural depiction of cell wall polysaccharides and glycoproteins in plants Figure 1.2 Fucosyltransferase (FuT) - catalyzed reactions Figure 1.3 Biosyntheses of xyloglucan (XyG) and N-linked glycoprotein by glycosyltransferases (GTs). Figure 2.1 Phylogenetic analyses between the members of the FuT family in Arabidopsis thaliana. Figure 2.2 Conserved amino acid regions and phylogenetic relationship of the members of the Arabidopsis O-FuT family. Figure 2.3 Expression profiles of O-FuT genes in Arabidopsis thaliana development Figure 2.4 Gene expression profiles of At2G44500 Figure 2.5 Gene network identification of At2G44500

Figure 3.1 Maps of vectors used in creating GFP::BnAt2G(1) expression constructs Figure 3.2 Amplification of B.napus genes homologous to At2G44500 Figure 3.3 DNA sequence alignments of BnAt2G(1) and BnAt2G(2). Figure 3.4 Southern Blot of BnAt2G gene copies from cv. Topas leaf genomic DNA (gDNA) Figure 3.5 Syntenic genes located on the partial segments of the A.thaliana and B. napus chromosomes Figure 3.6 BnAt2G(1) expression in vegetative tissues and microspore cultures. Figure 3.7 Protein Structure and Characteristics of Arabidopsis and B. napus O - FuTs Figure 3.8 Subcellular localization of GFP::BnAt2G(1) Figure 3.9 Brefeldin A (BFA) treatment of GFP::BnAt2G(1) expressing root cells and leaf protoplasts Figure 3.10 Regulatory elements found in the promoter regions of At2G44500 and BnAt2G(1)

Figure 4.1 Phenotyping methodology Figure 4.2 Identification and validation of At2G44500 TDNA insertion mutants. Figure 4.3 Colony PCR of E.coli clones harbouring the plasmid pFGC5941+RNAi insert Figure 4.4 BnAt2G(1) insert orientation verification Figure 4.5 Generation of RNAi::BnAt2G(1) in A. thaliana Figure 4.6 Multiple sequence alignment of the A. thaliana O-FuT family and the BnAt2G(1) gene insert sequence of the RNAi::BnAt2G(1) construct Figure 4.7 Generation of 35S::BnAt2G(1) Overexpression Lines. Figure 4.8 At2G44500 gene expression levels in mutant lines. Figure 4.9 Phenotypic traits measured for wt, At2G44500 mutant and transgenic lines Figure 4.10 Stem traits of wt, At2G44500 mutant mutant and transgenic Arabidopsis plants. iv

Figure 4.11 FT-IR spectra of wild type (wt), mutant lines and transgenic lines: g-20490, g- 74450, RNAi::BnAt2G(1) and 35S::BnAt2G(1) Figure 4.12 Principal Component Analysis (PCA) of FT-IR spectra in the cell wall polysaccharide fingerprint region Figure 4.13 Schematic diagram of pectin polysaccharide structure. Figure 4.14 Proposed mechanism that explains how BnAt2G(1)/At2G44500 could affect cell wall extensibility

List of Tables

Table 1.1 List of non-processive glycosyltransferases that are involved in cell wall polysaccharide biosynthesis from Arabidopsis thaliana. Table 1.2 Members of the O-Fucosyltransferase family in A. thaliana Table 3.1 Primers used to amplify BnAt2G(1) and BnAt2g(2) Table 3.2 At2G44500 and BnAt2G(1) Protein Topology Predictions Table 3.3 Cis acting elements identified from the At2g44500 and BnAt2G(1) promoter regions.

Table 4.1 Primers used to identify and genotype T-DNA insertion lines, generate and validate RNAi::BnAt2G(1) and 35S::BnAt2G(1) plants Table 4.2 ANOVA and Bonferroni-Holm analysis of Ct values of mutant lines compared tothe wild type control. Table 4.3 Phenotypic traits that were measured in mutant and wild type analyses. Table 4.4 FT-IR absorption of cell walls and their assignment to main cell wall components. Table 4.5 ANOVA and Bonferroni-Holm analysis of the 2nd stem internodes of mutant lines compared to the wild type control and to each other. Table 4.6 Summary of Results for Phenotypic Analyses Table 4.7 ANOVA and post-hoc analyses of individual wave numbers with designated CW component for wt and mutant lines. Table 4.8 Absorption values and the corresponding cell wall components Table 4.9 Summary of studies reporting the role pectin methylesterases (PMEs) in cell wall biomechanics

List of abbreviations

The following table describes the meaning of the abbreviations used throughout the thesis.

Abbreviation Meaning GTs glycosyltransferases CAZy Carbohydrate Active Enzyme database XyGs xyloglucans RG-I/II rhamnogalacturonan I/II AGPs arabinogalactan proteins HGs homogalacturonans FuTs Fucosyltransferases GDP-Fuc Guanosine Diphosphate Fucose O-FuTs O-fucosyltransferases GlcNAc N-acetylglucosamine Gal galactose AG arabinogalactan GFP Green Fluorescent Protein CLSC Cellulose Synthase – Like family C XTs xylosyltransferases ER endoplasmic reticulum MNS and MAN mannosidases GNT N-acetylglucosaminyltransferases ME microspore embryogenesis MDEs microspore-derived embryos HRGPs hydroxyproline-rich glycoproteins DUF246 Domain of Unknown Function-246 SAM shoot apical meristem RM the rib meristem GA Giberellic Acid ACC Ethylene analog IAA Indole-Acetic Acid (Auxin analog) JA (MJ) Jasmonic Acid (Methyl Jasmonate) BL Brassinolide LEA Late embryogenesis abundant RACK1A Receptor for Activated C Kinase AtFRB1 FRIABLE1 MSR1/2 mannan synthesis related genes pENTRY Gateway entry plasmid pDEST Gateway binary destination vector BODIPY-TR Golgi- marker ConA Concanavlin A (ER- marker) BFA Brefeldin A EST expressed sequence tag csr conserved sequence region vi

G-Mo.R-Se Gene Modelling Using RNA-Seq TM transmembrane FT-IR Fourier-Transformed Infrared GSPPs gene-specific primer pairs wt wild type

Oex overexpression BRAN number of branches SN seed number TSW total seed weight SILN silique numbers PCA principal component analysis PC 1/2 principal component 1/2 LRs Lateral roots

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Table of Contents

Chapter 1 ...... 1 Literature Review - Fucosytransferases ...... 1 1.1 Introduction ...... 1 1.2 Cell wall- related polysaccharides...... 2 1.3 Plant Fucosyltransferases ...... 6 1.3.1 GT-10: Plant α1,3/4 FuTs ...... 7 1.3.2 GT-37: Plant α1, 2-FuTs ...... 7 1.3.3 GT-65R: Plant O-Fucosyltransferase Proteins...... 9 1.4 Cell wall polysaccharide and glycoprotein biosynthesis – where does fucosylation fit in? 13 1.5 Microspore Embryogenesis ...... 16 1.5.1 Molecular and Genetic Mechanisms Underlying Microspore Embryogenesis ...... 17 1.6 The Cell Wall and Microspore Embryogenesis ...... 21 1.7 Practical Applications of FuT Reseach ...... 25 1.8 Hypothesis and Research Objectives ...... 26 Chapter 2 ...... 36 Bioinformatic Analyses of the Plant Fucosyltransferase Family and the In silico Characterization of a Putative O-Fucosyltransferase, At2G44500 ...... 36 Abstract ...... 36 2.1 Introduction ...... 36 2.2 Materials and Methods ...... 38 2.2.1 Sequence Information and Phylogenetic Analyses ...... 38 2.2.2 Gene Expression Profiles ...... 38 2.2.3 Genetic Network Analysis ...... 39 2.2.4 Protein Structure and Topology Prediction...... 40 2.3 Results ...... 40 2.3.1 Phylogenetic analysis of the AtFuT family ...... 40 2.3.2 Protein Sequence Analysis of the O-FuT family members ...... 44 2.3.3 Expression Profiles of the GT-65R/ O-FuT Gene Family and At2G44500 ...... 51 2.3.4 In silico Co-Expression and Network Profile of At2G44500 ...... 57 2.4 Discussion ...... 59

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2.4.1 Phylogenetic and Protein Sequence Analyses ...... 59 2.4.2 Gene Expression Profiles ...... 61 2.4.3 Gene Network Analysis ...... 62 Chapter 3 ...... 64 Molecular and In Silico Characterization of At2G44500 and Its B.napus Homolog, BnAt2G(1) 64 Abstract ...... 64 3.1 Introduction ...... 64 3.2 Materials and Methods ...... 66 3.2.1 Plant Materials and Growth Conditions ...... 66 3.2.2 Microspore culture ...... 67 3.2.3 Homologous Gene Isolation ...... 68 3.2.4 Gene Cloning ...... 72 3.2.5 Agrobacterium and Arabidopsis Transformation ...... 75 3.2.6 In silico analysis ...... 77 3.2.7 Leaf Protoplast Isolation ...... 77 3.2.8 BODIPY-TR and Concanavlin A (ConA) Staining ...... 78 3.2.9 Brefeldin A (BFA) Treatments ...... 78 3.2.10 Microscopy ...... 78 3.3 Results ...... 79 3.3.1 Isolation of Brassica napus genes that are homologous to a putative Arabidopsis O- fucosyltransferase, At2G44500 ...... 79 3.3.2 Gene Copy Number ...... 91 3.3.3 At2G44500 locus is syntenic to regions located in B.napus chromosomes C04 and A05 92 3.3.4 BnAt2G(1) is overexpressed in embryogenic microspore cultures ...... 94 3.3.5 Functional POFUT motifs are conserved in At2G44500 paralogs and in its B. napus homologs ...... 97 3.3.6 Protein Structure Prediction and Topology...... 97 3.3.7 Subcellular Localization of BnAt2G(1) ...... 104 3.3.8 In silico Promoter Analysis of At2G44500 and BnAt2g(1) ...... 107 3.4 Discussion ...... 110 3.4.1 Homologous Gene Isolation ...... 110

3.4.2 Predicted Protein Topology and Subcellular Localization ...... 111 3.4.3 In silico Promoter Analysis ...... 114 3.4.4 Conclusions ...... 115 Chapter 4 ...... 116 Functional Characterization of At2G44500 and BnAt2G(1) ...... 116 Abstract ...... 116 4.1 Introduction ...... 116 4.2 Materials and Methods: ...... 118 4.2.1 Plant Material ...... 118 4.2.2 Genotyping of T-DNA Insertion Mutants...... 118 4.2.3 Generation of RNAi Construct ...... 118 4.2.4 Generation of 35S:: BnAt2G(1) Overexpression Construct ...... 119 4.2.5 Generation of Competent Agrobacterium ...... 119 4.2.6 EHA105 Transformation and Preparation for Arabidopsis Transformation ...... 120 4.2.7 Arabidopsis Transformation ...... 121 4.2.8 RNAExtraction and qRT-PCR ...... 121 4.2.9 Phenotyping ...... 122 4.2.10 Stem Stains...... 123 4.2.11 Fourier Transformed Infrared Spectroscopy ...... 124 4.2.12 Image Analysis...... 124 4.2.13 Statistical Analysis ...... 124 4.3 Results ...... 124 4.3.1 Confirmation and Genotyping of T-DNA Insertion in SALK Lines ...... 124 4.3.2 Generation of RNAi Lines ...... 127 4.3.3 Generation of 35S::BnAt2G(1) Lines ...... 135 4.3.4 Gene Expression Level of Mutants ...... 137 4.3.5 Phenotyping ...... 140 4.3.6 FTIR Results ...... 143 4.4 Discussion ...... 148 4.4.1 Lateral Roots and Stem Phenotypes ...... 149 4.4.2 FT-IR Results ...... 151 4.4.3 Role of Fucosylation in Pectin Biosynthesis and Cell Adhesion ...... 153 x

4.4.4 Proposed Role of At2G44500/BnAt2G(1) in Pectin Synthesis and Cell Adhesion 154 4.4.5 Study Limitations ...... 155 Chapter 5 ...... 169 General Summary and Future Directions ...... 169 References: ...... 171 Appendix 1 ...... 198 Appendix 2 ...... 199

1 Chapter 1

Literature Review - Fucosytransferases

1.1 Introduction

The addition of sugar moieties to cell wall polysaccharides and proteins is mainly catalyzed by glycosyltransferases (GTs). In plants, large numbers of distinct GTs are required because of the diversity of glycosidic linkage types that exist in cell wall polymers. Until recently, GTs were thought to have only limited effects on metabolism and development in plants. However, the identiﬁcation and functional characterization of the enormously diverse GT gene families has changed that view. GTs are classified into families on the basis of their amino acid sequence similarities in the Carbohydrate Active Enzyme database (CAZy - http://www.cazy.org/GlycosylTransferases.html) (Cantarel et al., 2009). The CAZy database contains proteins with demonstrated biochemical functions as well as orthologous, putative, GTs from other species. The rapid growth of this database reflects the current interest in discovering and functionally characterizing new GTs. To date, CAZy contains 97 GT families, numbered from

GT1 to GT97 and includes 186,016 GT's in total.

GTs may act in a processive or non-processive manner (Price, 2002). Processive glycosyltransferases (also referred to as glycan/polysaccharide synthases) transfer sugar moieties to the main linear polymeric backbone without releasing the acceptor substrate (Price, 2002). They are integral membrane proteins with multiple transmembrane domains (TMDs), localized in the plasma membrane. A classic example of this type of GT is cellulase synthase.

Non-processive GTs (commonly known as glycosyltransferases) add monosaccharides to specific positions of polysaccharide chains or glycoproteins and then move on to other acceptor sites. Unlike processive GTs/glycan synthases, non-processive GT's are localized in the secretory endomembrane system (ER, the Golgi apparatus and Golgi-derived vesicles) (Figure 1.1) and aid 1 in the synthesis and assembly of non-cellulosic matrix polysaccharides and the glycosylation of structural glycoproteins, such as extensins or arabinogalactan proteins (AGPs) (Scheible, 2004;

Driouich, 2012). These enzymes are classified as type II membrane proteins. Type II membrane proteins possess a hydrophobic transmembrane domain, which is a short amino-terminal domain that faces the cytosol, and a carboxy-terminal that contains the catalytic domain positioned within the lumen. Table 1.1 summarizes the known non-processive GT's that have been characterized, including their proposed roles in the synthesis of non-cellulosic cell wall matrix polysaccharides, such as: xyloglucans and rhamnogalacturonan I and II (RG-I and RG-II) and modification of matrix proteins, like arabinogalactan proteins (AGPs).

1.2 Cell wall- related polysaccharides

Cell walls are essential components that provide structural integrity and physical support for plants. They are also required for proper growth and development and play important roles in plant defences and responses to environmental stresses (Cosgrove, 2005; Keegstra, 2010).

Moreover, cell wall polysaccharides are critical materials for plant-based products, including: food, fuel and fibre (for textile, pulp and paper manufacturing). Cell walls are highly diverse and primarily (but not exclusively) composed of complex and extensive networks of polysaccharides

(90%) and glycoproteins (10%), whose syntheses are spatially and temporally regulated.

The types of polysaccharides present in cell walls vary, and are dependent on the following factors: plant species, cell type and location, developmental stage and history of responses to biotic and abiotic stresses. Figure 1.1 shows simplified structures of some of these cell wall polysaccharides. Cellulose is the major load-bearing polysaccharide in primary and secondary cell walls. It is organized into microfibrils and is synthesized by cellulose synthase in the plasma membrane (Scheibel, 2004). Hemicelluloses are often closely associated with cellulose

2 microfibrils and form networks with them. They are thought to have cross-linking functions in primary cell walls (Perrin, 2003). In dicots, the predominant hemicellulose is xyloglucan. Other types of hemicelluloses (such as arabinoxylans and mannans) are present in grass species (such as in wheat, maize and rice). Pectins are acidic polymers and are structurally complex and elaborate.

They have gel-like physicochemical properties and accumulate in the middle lamella between primary and secondary cell walls. The three main pectic polysaccharides are: homogalacturonan

(HG), rhamnogalacturonan I (RG-I) and rhamnogalacturonan II (RG-II) (Carpita, 1993; Mohnen,

2008; Scheller, 2010).

Cell wall polysaccharides and glycoproteins are abundantly expressed and are highly heterogeneous, with respect to their structures as well as in their carbohydrate and sugar compositions. Several approaches for studying cell-wall related genes have been utilized, including the analyses of different types of GTs that decorate protein and polysaccharide backbones with sugar moieties in the Golgi, before they are transported to the plasma membrane, apoplast or the cell wall.

(A)

Figure 1.1 Structural depictions of plant cell wall polysaccharides and glycoproteins. (A) Non-fucosylated polysaccharides such as cellulose, heteromannan, homogalacturonan and rhamnogalacturonan I (RG-I) (B) Fucosylated matrix polysaccharides such as xyloglucan and RG-II (C) Fucosylated glycoproteins. Arrows indicate the linkages formed by known fucosyltransferases (FuTs) and unidentified FuTs are represented by question marks. Asn: Asparagine; Ser: Serine; Thr: Threonine; Hyp: Hydroxyproline.

(B)

(C)

1.3 Plant Fucosyltransferases

Fucose is a deoxyhexose monosaccharide that is a common component of many N- and O-linked glycans and glycolipids of eukaryotic cells (Becker, 2003). Fucose frequently exists as a terminal modification of glycan and polysaccharide structures (Becker, 2003,

Figure 1.1). Fucosylation can confer unique functional properties to oligosaccharides and are often regulated during cell growth and differentiation. Important roles for fucosylated glycans and polysaccharides have been demonstrated in plants (Perrin, 1999; Faik, 2000;

Sarria, 2001; Vanzin, 2002; Strasser, 2004; Liang, 2013; Tryfona, 2014), several of which are reviewed here. However, because of the diversity of fucose-containing glycoconjugates and the difficulties inherent in studying the biological function of carbohydrates, it is likely that many additional functions for fucosylated glycans remain to be uncovered.

Fucosyltransferases (FuTs) are a type of non-processive glycosyltransferase that are widely expressed in eukaryotic organisms and catalyze fucose transfer from the donor,

Guanosine Diphosphate Fucose (GDP-Fuc), to various acceptor molecules, including oligosaccharides, glycoproteins and glycolipids (Oriol, 1999) (Figure 1.1). Generally, FuTs are divided into two super families, based on their protein sequence similarities and the type of linkage that they make with their target or acceptor substrate. The members of the first FuT superfamily are the α1, 2 and protein-O-fucosyltransferases (O-FuTs) which belong to the GT-37 and GT-65R families, respectively. The members of the second superfamily are the α1, 3/ 4 fucosyltransferases, which belong to the GT-10 family. Figure

1.2 depicts the basic reaction and the type of linkages that these FuTs make to their specific target substrates.

As seen in Table 1.1 several members of the GT-10 and GT-37 families had been recently characterized. However, little is known about plant GT-65R/O-FuTs even though they comprise a larger gene family than GT-10 and GT-37 (Figure 2.1). The following presents what is known about these plant FuTs, with particular emphasis on members of the

GT-65R family, which are putative plant O-Fucosyltransferases.

1.3.1 GT-10: Plant α1,3/4 FuTs

Three α1, 3/4-fucosyltransferases have been identified in Arabidopsis (Wilson,

2001). Members of this CAZy GT-10 family add fucose to the reducing terminal N- acetylglucosamine (GlcNAc) residues of N-glycans (Figures 1.1 C and 1.2 C) (Strasser,

2004). All N-linked carbohydrates are linked through N-Acetylglucosamine and the amino acid asparagine (N) as shown in figure 1.2.

The α1, 4 FuT activities is predominant in young leaf and root tissues (Lerouge,

1998). Moreover, high levels of α1, 4 FuT activity were also detected in tobacco seeds, male flowers, and during tobacco male gametophyte development (pollen maturation, pollen germination, and tube elongation) (Joly, 2002), suggesting that fucosylated N-linked glycoproteins may play important roles in plant reproductive development. Additionally, it has been suggested that plant α1, 4 fucosylation may be involved in cell-to-cell communication and/or recognition (Rayon, 1999). Interestingly, mammals lack this type of fucosylation and, therefore, this epitope is recognized by their immune systems as a foreign element and it triggers allergic reactions (Wilson, 1998; Hemmer, 2001).

1.3.2 GT-37: Plant α1, 2-FuTs

The members of the CAZy family GT-37 are α1,2 FuTs that are able to transfer fucose from Guanosine Diphosphate Fucose (GDP-Fuc) to cell wall related polysaccharides

7 such as in the galactose (Gal) residue at the reducing end of xyloglucan hemicellulose

(Figure 1.1 B) (Perrin, 1999; Faik 2000). This particular FuT was initially isolated and characterized in Arabidopsis (AtFUT1) (Sarria, 2001) and from pea epicotyls (PsFUT1)

(Faik, 2000). AtFUT1 and PsFUT1 were found to be Golgi-bound Type II membrane proteins that share 62.3% protein sequence identity with each other.

These enzymes have roles in xyloglucan biosynthesis (Perrin, 1999; Faik, 2000).

Knock-down (mur2) and knock-out (mur1/mur2) mutants of AtFUT1 showed a significant decreases (98%) in xyloglucan FuT activity and xyloglucans extracted from root and leaf cell walls had no detectable levels of fucose (Vanzin, 2002; Perrin, 2003). However, in spite of these biochemical defects, mur2 plants grew and developed normally with the exception of a morphological defect in trichome papillae formation (Vanzin, 2002). AtFut1 is expressed at similar levels in all plant organs, whereas, the other members of this family

(AtFut2 through AtFut10) displayed more complex expression patterns. Most of them failed to transfer fucose to xyloglucan (Sarria, 2001; Vanzin, 2002). These results imply that, although they are related to AtFut1 (by sequence similarity), AtFut2 through AtFut10 might be fucosylating xyloglucan in an organ- specific manner or that they are involved in the fucosylation of other cell wall-related polysaccharides.

Recently, Wu et al (2010) and Liang et al (2013) identified AtFut4 and AtFut6 genes as Arabinogalactan protein (AGP) -specific FUTs, based on their enzymatic activities when heterologously expressed in tobacco BY2 suspension-cultured cells. Knockdown fut4, fut6, and fut4/fut6 mutants where not phenotypically different from wild type plants under normal physiological conditions but they had shorter roots under salt stress (Liang,

2013; Tryfona, 2014). An AtFUT6-GFP chimeric protein was localized in the Golgi

8 apparatus, in agreement with evidence that indicates that much of the biosynthesis of the

AG side chains occurs within this organelle (Wu et al., 2010). Monosaccharide profiling of

AGPs extracted from Arabidopsis leaves showed a substantial reduction in fucose content in the fut4 but not in the fut6 mutant. Additionally, fucose was reduced in root AGP extracts and was absent in fut4/fut6 mutants. These data suggest that AtFUT4 is required for AGP fucosylation in leaves and is functionally redundant in root AGP fucosylation with

AtFUT6.

1.3.3 GT-65R: Plant O-Fucosyltransferase Proteins

O-fucosylation is a post-translational modification that occurs in target molecules such as cell wall components and glycoproteins (Figures 1.1). Most O-linked carbohydrates have covalent linkages with either the amino acids serine (S) or threonine (T) of a glycoprotein (Figure 1.3). The first O-fucose glycoprotein modification was discovered in human urinary plasminogen activator (Kentzer, 1990), followed by several serum glycoproteins involved in blood clot formation or dissolution (Bjoern, 1991; Nishimura,

1992). Recent studies have shown that O-fucose modifications also exist on the serine or threonine residues of the Epidermal Growth Factor (EGF)-like and Thrombospondin type repeats (TSR) (Wang, 1998; Haltiwanger, 2003) in mammalian cells. Currently, there are no known or isolated O-Fucosylated plant glycoproteins.

Table 1.2 summarizes the reported putative and experimentally determined functions, gene expression patterns, subcellular localizations and protein-protein interactions of members of the GT-65R/O-FuT family. Most members are localized in the

Golgi but were also observed to be present in the cell wall, plasma membrane, vacuole, chloroplast and the nucleus. Some members of this GT-65R/O-FuT family were also found

9 to physically interact with proteins involved in cell division (AT1G29200 and

AT5G35570) as well as in stress and hormone responses (AT1G76270 and AT2G44500).

(B)

(A) (B)

Figure 1.2 Fucosyltransferase (FuT) - catalyzed reactions. (A) Fucose is transferred from GDP-fucose and creates unique sugar linkages with specific acceptor substrates. (B) Addition of fucose to other nd carbohydrates such as in the 2 carbon(C) molecule of the galactose ring, thus establishing(D) an alpha 1, 2 fucose linkage. Fucose can also be N-linked or O-linked to glycoproteins (continued)

Figure 1.2 Fucosyltransferase (FuT) - catalyzed reactions. (C) In N-linked linkages, fucose is added to the N-Acetylglucosamine that is connected to an asparagine amino acid (N) residue of the protein molecule. (D) Fucose is directly added either the serine (S) or threonine (T) amino acid residues in an O-linked fucosylation of a glycoprotein.

1.4 Cell wall polysaccharide and glycoprotein biosynthesis – where does fucosylation fit in?

The synthesis and movement of cell wall polysaccharides have been tracked by using antibodies directed against specific sugar epitopes and by generating Green Fluorescent Protein

(GFP)-tagged GTs, followed by their localization with anti-GFP antibodies. Figure 1.3 depicts the biosynthesis of XyG, which is the most studied and abundant hemicellulose in the primary cell wall in dicots (Cosgrove, 2005). The sugar back bone of XyG is synthesized by proteins encoded by members of the Cellulose Synthase – Like family C (CLSC) genes. In Arabidopsis, the Golgi- localized, multi-spanning CLSC4 protein has its catalytic domain on the cytosolic side and uses

UDP-Glucose in the cytosol to synthesize the glucan backbone. The growing nascent glucan chain enters the Golgi lumen through a pore formed by the multi-transmembrane domains of CSLC

(Keegstra, 2010), where it is modified by different GTs to synthesize a mature XyG molecule. In

Arabidopsis, xylosyltransferases (AtXT1-5) catalyze the addition of xylose to the glucan chain

(Zabotina, 2012; Cavalier, 2008).

AtXT2 was shown to form a protein complex with CSLC4 (Chou, 2012) and AtXT1 was shown to be localized in the cis and medial cisternae of the Golgi (Chevalier, 2010). AtMUR3 was shown to be predominantly associated with the medial while AtFUT1 was mostly detected in the trans cisternae to add a terminal Fucose to the XyG molecule (Chevalier, 2010). Moreover, fucose- containing epitopes of XyG was found in the trans Golgi cisternae (Driouich, 2012;

Moore, 1991; Zhang, 1992).

As seen in Figure 1.1, N-linked glycoproteins and arabinogalactan Proteins (AGPs) also contain terminal fucose molecules. Nascent glycoproteins are synthesized in the ER but are transported to and modified in the Golgi. Modifying enzymes that act on N-linked glycoproteins such as mannosidases (MNS and MAN) and N-acetylglucosaminyltransferases (GNT1) function

13 in the cis and medial compartments of the Golgi (Figure 1.3) and the addition of fucose occurs predominantly in the trans-Golgi. This reaction is catalyzed by α1,3/4 FuTs (Rayon, 1997).

It appears that the initiation of cell wall polysaccharide synthesis and the addition of its sugar moieties occur in early Golgi compartments (cis and medial) while the addition of terminal fucose, occurs in the trans cisternae. In the case of glycoproteins, nascent molecules are synthesized in the ER but are modified in the Golgi. The addition of sugar moieties in glycoproteins is similar to the pattern seen for cell wall polysacccharides. However, there are other cell wall polysaccharides that contain terminal and non-terminal fucose linkages such as in

RG-II (Figure 1.1). Fucosyl residues were also shown to be linked to galacturonan of soybean pectin (Fransen et al., 2000). Fucose is also present in low-Mr glycosides found in plants (Guo,

2000). These glycosides are integral sugar moieties in saponins, which are glycosylated triterpenoids that have been characterized from Quillaja saponaria bark (Guo, 2000). This suggests that novel functions for FuT subfamilies in plants have yet to be elucidated.

Figure 1.3 Biosyntheses of xyloglucan (XyG) and N-linked glycoprotein by

glycosyltransferases (GTs). (left) The glucose (blue circle) backbone of XyG is synthesized by a member of the cellulose synthase-like family C, CSLC4. The growing glucan chain enters the Golgi lumen where xylosyltransferases (AtXT) catalyze the addition of xylose (red star) in the cis Golgi. Galactose (yellow circle) is then attached to the xylosyl residues by galactosyltransferases, AtXLT2 and AtMUR3 in the medial Golgi. Lastly, a terminal fucose is added to the galactosyl residue of XyG by an α-1,2 fucosyltransferase, AtFUT1. (right) N-linked glycoproteins are initially synthesized in the ER but are transported to and modified in the Golgi. Mannose (green circle) is added by mannosidases, MNS1/2, MANII/MANIIx in the cis and medial Golgi, respectively. Addition of N-acetylglucosamine (black square) is catalyzed by N- acetylglucosaminyltransferases, GNT1 and GNT2 in the cis and medial Golgi, respectively. Addition of xylose (red star) on mannose by a xylosyltransferase (XylT) occurs in the medial Golgi. Lastly, the addition of terminal galactose (yellow circle) by a galactosyltransferase and fucose (red triangle) by fucosyltransferase (FuT11/12 and FuT13) occur in the trans compartment of the Golgi.

1.5 Microspore Embryogenesis

Isolated plant microspores, when stressed and cultured in vitro, can be switched from their normal gametophytic pathway towards embryonic development. This results in the formation of haploid embryos and ultimately doubled-haploid plants. This process is called microspore embryogenesis (ME), and it is widely used in plant breeding programs of some economically significant crop species to generate homozygous lines from heterozygous parents in a single generation. Also, microspore-derived embryos are similar to zygotic embryos in Brassica napus and Arabidopsis thaliana with respect to their morphologies, gene expression patterns and biochemical compositions (Ramesar-Fortner, 2011; Boutilier et al. 2002; Ilic-Grubor et al. 1998).

This similarity in developmental pathways, combined with the ability to obtain large and homogenous populations of microspores to produce embryos at diverse stages, has made microspore culture a valuable tool for studying a range of processes related to plant embryogenesis.

Brassica napus is one of the most commonly studied crops regarding the genetic and molecular control of microspore embryogenesis. Although the responsiveness to microspore embryogenesis induction within this species also varies, its popularity as a model organism is due to the speed, efficiency and synchronicity in producting embryogenic microspores and its similarity to the model plant species, Arabidopsis thaliana at the phylogenetic and genome sequence level. Although at present, not all crops of agronomic interest and market-value respond efficiently to microspore embryogenesis induction, the knowledge obtained from species that are responsive enough to be considered as model systems (canola/rapeseed, tobacco, wheat and barley) can help us understand the molecular mechanisms of this process and apply them to recalcitrant genotypes or species.

1.5.1 Molecular and Genetic Mechanisms Underlying Microspore Embryogenesis

Although the switch from microspore to embryo development varies among plant species, the underlying mechanisms that could explain developmental concepts such as competence and differentiation are most likely similar and could be used to understand this process. Most differential gene analyses using the microspore culture system have uncovered genes and mechanisms that are involved in two key stages of microspore embryogenesis. The first stage is the switch in the developmental program, where immature pollen grains are induced by a type of stress to reprogram their gametophytic development to embryo development. This switch would inevitably cause changes in the transcriptome, which would then lead to embryogenic cells to differentiate, undergo a series of cell divisions and would sequentially establish tissue and organ patterns that characterize a developing embryo and eventually a mature plant.

Analyses of the genetic factors underlying ME focus on the differential expression of genes and proteins between embryogenic (induced) and pollen-like (non-induced) cultures to

17 reveal aspects of the transcriptional switch between the microspore and embryo stages (Boutilier et al. 2006; Joosen et al. 2007; Malik et al. 2007). Different screening techniques such as subtractive hybridization (Boutilier, 2002); and differential display PCR (Reynolds, 1992; Custers et al. 2001) have been used to identify putative genes involved in early microspore embryogenesis. Transcriptome comparison between microspores and mature pollen in

Arabidopsis (Honys, 2004) and wheat (Tran et al. 2013) show that there is little overlap between these two transcriptomes. The microspore transcriptome is characterized by a higher proportion of transcripts encoding structural proteins, as well as proteins involved in translation and metabolism (Whittle et al. 2010) and has been shown to be more similar to those of other sporophytic stages of plant development (Joosen et al, 2007; Tran et al, 2013) and sporophytic tissues such as bicellular pollen (Honys, 2004) and ovules (Whittle et al. 2010).

The genes that are differentially expressed during the developmental transition and/or early embryogenic development are hypothesized to be involved in the switch between these two pathways and thus in the process of dedifferentiation and may even play a role in the course of embryogenesis. Genes such as FUSCA3; LEAF COTYLEDON1 (LEC1); LEC2; BABYBOOM

(Boutilier, 2002); WUSCHEL-Related Homeobox (WOX2 and WOX9); ABA Insensitive (ABI3) have been identified in these screens. Although ESTs for these genes were only detected after 7 days of culture, their expression could be detected by RT-PCR much earlier, at 1–2 days of culture, suggesting that embryo cell identity is established as early as the first few sporophytic cell divisions.

The utility of a subset of these genes as early markers for embryogenic growth in genotypes differing in their ability to form haploid embryos was also examined. Only the expression of one of the markers, LEC2, could distinguish between embryogenic and non-

18 embryogenic cultures at 3 days, but all of them distinguish the same cultures at 7 days.

BABYBOOM (BBM) is an AP2/ERF (Apeptala2/Ethylene Response Factor) transcription factor that is expressed in early androgenesis, zygotic embryogenesis as well as in microspores that were treated with heat stress to induce embryogenesis (Boutilier, 2002). Ectopic expression of this

BBM leads to spontaneous somatic embryogenesis and cotyledon-like structures on seedlings and also results in pleiotropic phenotypes such as neoplastic growth, hormone-free regeneration of explants and alterations in leaf and flower morphology (Boutilier, 2002). The expression pattern of BBM in developing seeds combined with the BBM overexpression phenotype suggests a role for this gene in promoting cell proliferation and morphogenesis during embryogenesis

(Boutilier, 2002). Ectopic expression of the LEC1and LEC2 transcription factors in seedlings is also sufficient to induce activation of embryo-expression programs, as well as the de novo induction of somatic embryo formation in Arabidopsis (Lotan et al. 1998; Stone et al. 2001).

In B.napus, the transcriptome and proteome analyses of 8-day old suspensor bearing microspore-derived embryos (MDEs) identified proteins that were upregulated in MDEs. These protein played general roles for protein synthesis, glycolysis, and ascorbate metabolism (Joosen et al 2007). One of the genes that were upregulated in this analysis was the BNM2 BURP domain protein, which was previously identified in two independent screens for embryo markers in MDE cultures (Boutilier et al. 2002; Tsuwamoto et al., 2006).

BURP domain proteins are an abundant class of secreted proteins of unknown function that are defined by their modular structure and conserved C-terminal BURP domain (Hattori et al.,

1998). BURP domain genes are expressed under a wide range of developmental and environmental conditions, which is reflected in the diverse processes in which they appear to participate, such as development (Batchelor et al, 2002; Wang et al, 2003) and stress response

(Shinozaki, 1993; Yu et al, 2004). Among the transcriptional regulators identified was the MADS- box transcription factor AGL53, which was also shown to be expressed in somatic embryo cultures (Lehti-Shiu et al, 2005).

Another effort in searching for early embryogenic genes led to a genome-wide analysis of candidate genes that are involved in the early stages of B.napus microspore embryogenesis (Chan,

2006). Since embryogenic cells can be segregated from those committed to pollen-like cells in the

B. napus microspore culture system (Pauls, 1998), a differential microarray experiment can then be used to characterize the relative expression levels of genes in these two types of cells. In this study, an Arabidopsis cDNA array was used to compare the transcriptomes of embryogenic cells with pollen-like microspores to identify genes that were differentially expressed during the early stages of microspore embryogenesis. The transcriptome profiles that resulted from this study revealed informative expression trends from clusters that represent genes that were (1) upregulated in microspore-derived pollen-like cells; (2) consistently upregulated in microspore- derived embryogenic cells and (3) down-regulated in embryogenic versus pollen-like cells (Chan,

2006). For example, the largest group of gene products that were significantly upregulated in the embryogenic cells encoded for proteins involved in processes such as: carbohydrate metabolism; lipid and amino acid biosynthesis; cytoskeletal and vesicular movement. This supports the notion that embryogenic cells are more metabolically active than those that are on a pollen maturation pathway. Also, a portion of these upregulated genes in embryogenic cells were transcription factors containing the Zinc-finger and the F-box motifs as well as proteins that are involved in cationic and macromolecular transport, cell cycle and stress response. These gene expression changes clearly indicate that the induction of embryogenesis in microspores evoke an integration of genetic and cellular responses and is characterized by changes in the cytosolic pH, cytoskeletal

20 reorganization, alteration in cellular metabolism and biosynthesis, entry into the cell division cycle and the expression of stress proteins, etc.

Intriguingly, a large number of the differentially expressed genes do not have a known molecular or biological function. One of the genes in this group was recently characterized and was found to be a bZIP a transcription factor gene, annotated as BnMicEmUP (Shahmir, 2014).

This gene was up-regulated ( >7-fold) in 3 day old embryogenic microspore cultures compared to the non-induced/pollen like cultures through qRT-PCR. Overexpression of BnMicEmUP in

Arabidopsis resulted in plants with significantly lower seed yield and its reduced expression resulted in transgenics that exceeded seed production compared to control plants. It is hypothesized that this putative transcription factor, play a role in an ABA-mediated transcriptional regulation.

Another gene that was significantly overexpressed in embryogenic cells is annotated as

At2G44500. This gene is highly expressed in cells located in the rib meristem of the shoot apical meristem and has a conserved GDP-O-Fucosyltransferase domain. This is gene is a member of the

GT-65R/O-FuT family in plants and is hypothesized to be involved in the biosynthesis of cell walls of microspore-derived embryos.

1.6 The Cell Wall and Microspore Embryogenesis

The dynamic changes in the formation and differentiation of cell walls reflect the dramatic cellular changes that occur during microspore embryogenesis. In spite of its importance in the formation and establishment of microspore-derived embryos, changes at the cell wall level are still poorly understood. These changes include the moderate growth of the innermost pecto-cellulosic wall and intine (Telmer, 1995; Schulze and Pauls, 2002; Solis, 2008),

21 an increase in pectin esterification (Barany, 2010) and the differential localization of arabinogalactan epitopes (El-Tantawy, 2013).

The cell walls of induced microspores and microspore-derived embryos have unique and intriguing features. For example, it was shown that embryogenic microspores exhibit abundant deposits of excreted cytoplasmic material (Corral-Martínez, 2013). These walls are also characterized by altered levels of certain cell wall components, such as xyloglucans, pectins

(Barany, 2010) and arabinogalactan proteins (El-Tantawy, 2013).

The distribution pattern of the major cell wall polymer, cellulose, has also been investigated. Schulze and Pauls et al (2002) observed that the first cell walls produced in Brassica napus embryogenic microspores are somatic-type and therefore, cellulose-rich. In olive, Solis et al. (2008) described the presence of cellulose in some embryogenic microspores, at both the inner cell walls and a thick peipheral layer located below the exine. In contrast,

Dubas et al. (2013) demonstrated that cellulose was absent from the first cell walls formed in very young B. napus embryogenic microspores, whereas older microspore-derived embryos presented abundant cellulose signal in their walls. In agreement with this observation, Parra-

Vega et al (2015) recently showed that B.napus microspore-derived embryos have callose-rich, cellulose-deficient subintinal layers and irregular and incomplete cell walls. They suggested that altered synthesis and/or deposition of cell wall components is a direct consequence of the exposure of microspores to the inductive treatment and that the formation of these atypical walls is one of the first signs of embryogenic commitment (Parra-Vega, 2015).

Several lines of evidence suggest that cell wall matrix polysaccharides are involved in early development, specifically in cell fate determination and the establishment and maintenance of cell polarity (Berger, 1994; Dupree, 1996; McCabe, 1997; Pennell, 1998; Braam,

1999; Belanger, 2000). For example, proteins involved in secretion and cell wall matrix synthesis have been shown to be required for normal embryo development in Arabidopsis (Scheres,

1999; Vroemen et al., 1999). In the embryo, the positioning of the cell plate results in either symmetrical or asymmetrical cell divisions, with consequences for differentiation and development. Although a large number of cell wall- related embryo mutants have been studied at morphological and molecular levels, a unifying framework of molecular and cellular events responsible for the critical steps in embryogenesis has not been established.

The two best-studied cell-wall related genes required for embryo development in

Arabidopsis, GNOM and KNOLLE, are both associated with secretion and/or vesicular trafficking. GNOM encodes a membrane-associated guanine nucleotide exchange factor for ADP-

Ribosylation Factor G-protein. This G protein is involved in vesicular trafficking to the cell surface (Lukowitz, 1996; Shevell, 2000). Studies with GNOM mutants suggest that this protein is involved in the polar localization of auxin efflux carrier proteins (Steinmann et al., 1999) and the deposition of cell wall materials (Shevell et al., 2000). Polarization of the Arabidopsis zygote requires GNOM (Vroemen et al., 1996). KNOLLE is a putative t-SNARE and was shown to be cell plate specific (Lukowitz, 1996; Lauber, 1997). In other systems, t-SNARES associated with membranes have been shown to bind to v-SNARES on arriving vesicles, thus facilitating vesicle fusion during cytokinesis (Jantsch-Plunger, 1999).

Analyses of the protein compositions of the plant cell walls initially focused on hydroxyproline-rich glycoproteins (HRGP), following the discovery by Lamport (1974) that hydroxyproline was a major amino acid constituent in cell wall hydrolysates. HRGPs have subsequently been shown to fall into three classes: Proline-rich proteins (PRP), arabinogalactans

(AGPs), and extensins (Bhargava, 2010). The ROOT-SHOOT-HYPOCOTYL–DEFECTIVE (RSH)

23 gene encodes a HRGP that is a member of the larger extensin gene family and was shown to have a role in strengthening mature cell walls and in positioning the dividing line at cytokinesis (Hall,

2002). In Arabidopsis, there are 20 verified out of 63 potential or putative extensins (Cannon,

2008). Arabinogalactan proteins (AGPs) are characterized by arabinose-/galactose-rich side chains, which define their interactive molecular surface (Basu, 2013).

Analysis of AGP profiles in cell walls of Brassica napus microspore cultures show the varying temporal distribution of AGPs from mature pollen to microspore-derived embryos

(Testillano, 2013). It also revealed that a wide group of AGPs were induced and specifically localized in the cell walls of early embryos with just two or four cells, which were formed by the first embryogenic divisions of the microspore, during the switch from gametophytic to embryo development (Testillano, 2013). In microspore-derived embryos of Capsicum annuum, structural changes during the formation, growth and maturation of cell walls were also analyzed using antibodies specific to four major cell wall epitopes (JIM5 – low esterified pectins; JIM7 – high esterified pectins; anti-RGII – B-rhamnogalacturonan II (RGII) complexes and anti-XG – xyloglucans). The results from this study suggest that the differentiated wall of the mature pollen has high levels of de-esterified pectins, xyloglucans and RGII, while the newly formed walls of the proliferating cells of microspore-derived embryos, as well as in root meristematic cells, showed a high abundance of esterified pectins and low levels of xyloglucans and RGII (Barany,

2009). These findings also provide experimental support for the proposal that a high proportion of esterified pectins in walls could be a marker for cell proliferation events but also for an early marker of microspore embryogenesis (Testillano, 2013).

1.7 Practical Applications of FuT Reseach

Aside from their value in plant embryogenesis as well as in providing structural flexibility to the cell wall, the importance of fucosylated plant molecules is still being explored. Recent advances have shown that there are great practical applications in studying and understanding the mechanisms involved in fucosylation, particularly in human health. The ABO blood group antigens are fucosylated glycans. Fucosylated glycans have also been implicated in the pathogenesis of several human diseases. For example, an increased expression of fucosylated N- glycans had been found in cancerous cells (Yamashita, 1997; Orntoft, 1996) as well as in serum immunoglobins in both juvenile and adult rheumatoid arthritis patients (Flogel, 1998; Gornik,

1999). Thus, fucosylation can be used as an early diagnostic tool for cancer and the onset of rheumatoid arthritis. Plants are good potential hosts for the production of antibodies with the capacity to recruit immune cells and thereby trigger the death of cancerous cells. However, plant and mammalian N-glycans differ in their respective structures, particularly in their fucosylation and xylosylation patterns. The differences can be effectively overcome by controlling the glycosylation of glycan structures in the production host so that these antineoplastic/anti-cancer antibodies can be glycoengineered to produce non-fucosylated glycan structures in plant systems

(ref). Currently, a number of groups have generated monoclonal antibodies with a "humanized"

N-glycosylation pattern that lack immunogenic xylose and fucose epitopes in A.thaliana (Schahs,

2007) and N. benthamiana (Strasse, 2008) through RNAi-induced gene silencing. Promising plant-based expression technologies have also been developed to produce high yielding and good quality plant-based antineoplastic antibodies (Vezina, 2009; Schuster, 2007; Weise, 2007;

Koprivova, 2004)

The combined market for the three most successful antineoplastic antibodies

(Avastin®,Rituxan®, or Herceptin®) in 2007 was $6.8 billion and sales are projected to reach

$21.5 billion by 2016 (Komarova, 2012). The ability to generate transgenic plant lines that can produce humanized N-glycans together with plant-based expression systems that can produce high yielding and good quality plant-based antibodies, present an opportunity not only in anti-cancer antibody production but also the potential to develop novel antibody variants for other diseases

(such as rheumatoid arthritis). This also shows the value in continuing to understand the mechanisms of the biosynthesis, particularly of the glycosylation of N-linked glycoproteins as well as of biologically important molecules whose sugar moieties play significant roles in their structure and functions.

1.8 Hypothesis and Research Objectives

Previous microarray analysis identified genes with unknown functions that were highly expressed in embryogenic B. napus microspore culture cells, as early as 3 days after ME induction. Of special interest is the Brassica transcript homologous to the Arabidopsis gene

AT2G44500 since it was consistently upregulated (threefold increase) in embryogenic cells compared to pollen-like cells. This gene is highly expressed in cells located in the rib meristem of the shoot apical meristem and has a conserved GDP-O-Fucosyltransferase domain. There is a lack of information about the enzymes that catalyze the fucose transfer from the donor GDP-Fuc to various acceptor molecules in plants, even though fucosylated molecules are involved in a variety of biological processes such as tissue development and cell adhesion. The current study addresses this knowledge gap by studying the structure, expression patterns and developmental phenotypes controlled by a putative GDP-O-Fucosyltransferase (O-FuT) upregulated in B. napus microspore cultures.

Hypothesis: B. napus homologs of AT2G44500 are plant O-Fucosyltransferases (O-FuTs) that play important roles in plant development.

Objectives: The overall objective of the work is to elucidate and characterize the function of B. napus homologs of AT2G44500 by in silico analyses, cDNA analyses, mutant analyses and the creation of transgenics that overexpress or supress the expression of these genes. The specific goals of the work are to:

1. clone and sequence the genomic and cDNA homologs of AT2G44500 in B. napus;

2. analyze the obtained sequences using in silico methods to determine gene and protein

structures as well as their functional domains;

3. use bioinformatics approaches to establish synteny between Arabidopsis and the Brassica

members of the triangle of U (B. rapa, B. oleracea and B. napus);

4. measure transcript levels of these gene homologs in B. napus ME induced pollen like cells

and microspore-derived embryonic cells by real-time quantitative PCR using the primers

specific to the isolated B.napus sequence;

5. produce GFP::BnAt2G(1) lines to determine the sub-cellular localization of this particular

protein in A. thaliana;

6. generate mutant lines (over expression and knock down) in Arabidopsis and characterize

various growth phenotypes that may have been affected by the manipulation of this gene.

In the context of the B. napus microspore culture system, the expectation is that

information about the function of the specific O-FuT that is overexpressed in embryogenic

cells will lead to a better understanding about the cellular and molecular mechanisms that

are involved in the switch from the gametophytic to embryogenic development, especially

in the newly synthesized cell walls of microspore-derived embryos. This knowledge may

27 be used to improve the embryogenic response in microspore cultures of responsive crops/genotypes and may ultimately lead to the development of culture protocols for genotypes that are recalcitrant to ME induction.

Table 1.1 List of non-processive glycosyltransferases that are involved in cell wall polysaccharide biosynthesis from A. thaliana. nd: not determined; OE: Overexpression; XyG: Xyloglucan; AGP: Arabinogalactan Protein; Loc: Cellular localization

Gene Accession# Enzymatic Target CAZy Phenotypes of Loc References Name Activity Designation Mutants AtFuT1/ At2g03220 α1,2- XyG GT-37 - 98% reduction of Golgi Sarria, 2001; MUR2 Fucosytransf fucose in xyloglucan Vanzin, 2002 erase - increase in xyloglucan O- acetylation -altered trichome papillae in knock-out mutants

AtFuT3 At1g74420 α1,2- GT-37 OE mutants have nd Sarria, 2001 Fucosytransf stunted growth, curly erase leaves and unable to set seeds. AtFuT4 At2g15390 α1,2- AGP GT-37 - double mutant Golgi Sarria, 2001; Fucosytransf (fut4/fut6) show Wu, 2010; erase reduced root growth Liang, 2013; during salt stress Knoch, 2014 -functionally redundant with AtFuT6

AtFuT5 At2g15370 α1,2- AGP GT-37 OE mutants show 33% Golgi Sarria, 2001; Fucosytransf increase in fucose Showalter, erase levels 2010 AtFuT6 At1g14080 α1,2- AGP GT-37 - double mutant Golgi Sarria, 2001; Fucosytransf (fut4/fut6) show Wu, 2010; erase reduced root growth Liang, 2013; during salt stress Knoch, 2014 -functionally

redundant with AtFuT4 AtMUR3 At2g20370 beta-(1,2)- nd GT-47 - Reduced cell wall Golgi Chevalier, galactosyltra strength by 50% in 2010; Li, 2013 nsferase hypocotyls of knockout mur3 mutants. - lacks fucogalactosyl sidechain in Xyloglucan AtXT1 At3g62720 α1,6- XyG GT-34 -10% reduction in XyG Golgi Faik, 2002; Xylosyltransf -xxt1/xxt2 double Cavalier, 2006 erase mutants lack detecteble XyG levels AtXT2 At4g02500 α1,6- XyG GT-34 - 20% reduction in Golgi Egelund, 2006 Xylosyltransf XyG erase -xxt1/xxt2 double mutants lack detecteble XyG levels AtAXY4 Atlg70230 O- XyG - XyG is non acetylated Golgi Pena, 2004; Acetyltransfe gille, 2011 rase AtQUA1 At3g25140 α1,4- nd GT-8 -Dwarfed growth, nd Bouton, 2002 Galacturonos reduced cell adhesion yl - 25% reduction in cell transferase wall GalA content AtRGXT4 At4g01220 Xylosyltransf nd GT-77 - Defective root and Golgi Fangel, 2011; erase pollen tube growth Liu, 2011 AtARAD1 At2g35100 α1,5- Pectin GT-47 -Decrease in arabinose nd Harholt, 2012; Arabinosyltr content in RG-I Verhertbrugge ansferase n, 2013 AtXGD1 At5g33290 beta-(1,3)- nd GT-47 -mutants lack nd Jensen, 2008; xylosyltransf detectable Qin, 2013 erase xylogalacturonan in pectin fractions AtFRB1 At5g01100 Putative O- nd nd - mutant seedlings have Golgi Neumetzler, FuT fused hypocotyls, 2012 cotyledons or both.

- decreased abundance of galactose and arabinose contents in the Golgi which consequently led to the decrease levels of galactose in pectic cell wall fractions AtMSR1 At3g21190 Mannan Nd nd - single T-DNA Golgi Voxeur, 2012; synthase insertion in AtMSR1 Wang, 2012 related/ showed a 40% Putative O- decrease in mannose Fut levels AtMSR2 At5g61640 Mannan Nd nd - msr1/msr2 double Golgi Wang, 2012 synthase mutants showed more related/ Putative O- than 50% decrease in Fut mannose levels from extracted cell wall of stems.

Table 1.2 – Members of the O-Fucosyltransferase Proteins (POFUTs)/GT65R family in A. thaliana Reported References Subcellular Name Description Localization Reported Protein-Protein Interactions - Upregulated in pollen unknown Wang, 2008 germination and pollen tube AT1G11990 growth unknown - Upregulated in pollen unknown Wang, 2008 germination and pollen tube AT1G14020 growth unknown - Upregulated in pollen unknown Wang, 2008 germination and pollen tube AT1G20550 growth unknown - Upregulated in pollen unknown Wang, 2008 germination and pollen tube AT1G22460 growth unknown AT1G3210/SAMBA - Encodes a plant- Wang, 2008 - Upregulated in pollen specific negative regulator of the germination and pollen tube Anaphase Promoting Complex (APC/C) AT1G29200 growth unknown 15 - Downregulated when chloroplast unknown Dal Bosco, 2004 AT1G35510 ATP Synthase is inactivated unknown - Increased expression during unknown Ascencio, 2008 pathogen response and cell cycle AT1G38065 during gemnivirus infection unknown - Increased expression during unknown Ascencio, 2008 pathogen response and cell cycle AT1G38131 during gemnivirus infection unknown Golgi, Cell unknown Wang, 2013 AT1G51630 Mannan Synthesis Related2/MSR2 Wall - Increased expression during Ascencio, 2008; AT1G53770 pathogen response and cell cycle Vacuole unknown Jaquinod, 2007

during gemnivirus infection Voxeur, 2012; Sakamoto, 2013; - Identified as a putative RG-II Golgi, Nikolovski, 2012; AT1G62330 specific glycosyltransferase nucleus unknown Parsons, 2012 AT3G51920/CALMODULIN9/AtCML9 Nikolovski, 2012; Golgi, - Ca2+ Binding Protein; Mutations in Zhang, 2011; Plasma this gene alter plant responses to abiotic popescu, 2007; AT1G76270 Membrane stress and ABA. 16 Dunkley, 2006 - Downregulated when chloroplast unknown Dal Bosco, 2004 AT2G01480 ATP Synthase is inactivated Unknown - Highly expressed in the root unknown Zybailove, 2008; AT2G03280 quiscent centre Chloroplast Nakamura, 2006 - Upregulated in pollen unknown Wang, 2008; germination and pollen tube Dal Bosco, 2004 growth - Downregulated when chloroplast AT2G37980 ATP Synthase is inactivated - Upregulated in pollen Urano, 2015; germination and pollen tube Kundu, 2013; growth Boavida, 2009; - Increased expression during Wang, 2008; pathogen response and cell cycle Chan, 2006; during gemnivirus infection Goda, 2004; - Auxin and Brassinosteroid Navarro, 2004 Responsive Gene AT1G18080/ATARCA/AtRACK1A - - Upregulated in the innate immune Encodes a scaffold protein that plays response to flg22 regulatory roles in diverse signal - Upregulated in embryogenic transduction and stress response AT2G44500 microspore culutres in B.napus Unknown pathways. - Increased expression during unknown unknown Ascencio, 2008 AT3G02250 pathogen response and cell cycle

during gemnivirus infection EMBRYO SAC Parsons, 2012; DEVELOPMENT ARREST Chloroplast, Won, 2009; AT3G03810 30/EDA30 Golgi unknown Zybailov, 2008 - Upregulated in pollen unknown Wang, 2008 germination and pollen tube AT3G05320 growth AT3G07900 Chloroplast unknown Kleffmann, 2004 unknown Wang, 2013; - Mannan Synthesis Nikolivski, 2012; Related1/MSR1 Parsons, 2012; - Increased expression during Voxeur, 2012; Mitra, pathogen response and cell cycle 2009; Ascencio, during gemnivirus infection Plasma 2008; - Identified as a putative RG-II Membrane, AT3G21190 specific glycosyltransferase Golgi - Identified as a putative RG-II unknown Voxeur, 2012 AT3G26370 specific glycosyltransferase

AT3G30300 Golgi unknown Nikolovski, 2012 - Upregulated in pollen unknown Wang, 2008 germination and pollen tube AT3G54100 growth Golgi 4,7, unknown Nikolovski, 2012; Plasma Zhang, 2011; AT4G16650 Membrane 8 Dunkley, 2006 unknown Nikolovski, 2012; Parsons, 2012; AT4G17430 Golgi 4,5,7 Dunkley, 2006 AT4G24530 unknown - ROOTHAIR SPECIFIC 17/ unknown Wang, 2008 RHS17 AT4G38390 - Upregulated in pollen

germination and pollen tube growth AT5G01100 FRIABLE1/FRB1 Golgi Neumetzler, 2012 - Increased expression during Ascencio, 2012 pathogen response and cell cycle AT5G15740 during gemnivirus infection unknown AT1G02970/ATWEE1/WEE1 KINASE Cook, 2013; Parsons, HOMOLOG - Protein kinase that 2012; Zybailov, - Downregulated when chloroplast Chloroplast , negatively regulates the entry into 2008; Dal Basco, AT5G35570 ATP Synthase is inactivated Golgi mitosis 2004 AT5G50420 unknown unknown - Increased expression during unknown Ascencio, 2012 pathogen response and cell cycle AT5G63390 during gemnivirus infection 22 - Upregulated in pollen unknown Wang, 2008; germination and pollen tube Cytosol , Koroleva, 2005 AT5G64600 growth nucleus - Upregulated in pollen germination unknown Hummel, 2012; AT5G65470 and pollen tube growth Cytosol Wang, 2008

2 Chapter 2

Bioinformatic Analyses of the Plant Fucosyltransferase Family and the In silico Characterization of a Putative O-Fucosyltransferase, At2G44500

Abstract

Glycosyltransferases (GTs) have many functions in plants, but the majority are most likely involved in the biosynthesis of polysaccharides and glycoproteins in the plant cell wall. The fucosyltransferases (FuTs) are a gene family of GT's that encode for enzymes that create fucose

α1,2; α1,3/4 and O- linkages with a large variety of target molecules. Most FuTs function in the

Golgi, where synthesis of cell wall matrix polysaccharides and glycoproteins occur. Members of the α1,3/4-FuT family fucosylate N-linked glycoproteins while some members of α1,2 FuT family add fucose to cell wall components such as xyloglucan and arabinogalactan proteins. The A. thaliana O-FuT family contains 39 gene members. Bioinformatic analyses show that the cell wall- specific α1, 2 FuTs and the largely uncharacterized O-FuT family are closely related to each other.

It was also shown that the previously identified conserved peptide motifs (I, II and III) for O-FuTs are conserved in the gene members of the O-FuT family. Here, the current information about plant

FuTs in A. thaliana is presented with an emphasis on the O-FuT family, particularly in one gene member annotated as At2G44500.

2.1 Introduction

Fucosyltransferases (FuTs) are glycosyltransferase that catalyze the transfer of the sugar fucose from Guanosine Diphosphate Fucose (GDP-Fuc) to acceptor molecules, including: oligosaccharides, glycoproteins and glycolipids (Oriol, 1999). Generally, FuTs are divided into two superfamilies, based on their protein sequence similarities and the type of linkages they make

36 between fucose and the target or acceptor substrate (Figure 1.2). In Arabidopsis, there are three, ten and thirty-nine members of the GT-10 (α1, 3/4 FuTs), GT-37 (α1,2-FuTs) and GT-65 families

(O-FuTs), respectively.

Members of the GT-10 family function as α1,3/4-fucosyltransferase while the GT-37 family members were described to function as plant specific α1,2-fucosyltransferases in the

CAZy database. The remaining thirty-nine members were mostly uncharacterized genes, annotated with the Domain of Unknown Function-246 (DUF246), which was later merged into the

GDP-fucose protein O-fucosyltransferase (O-FuT) domain (Pfam identity number: PF10250)

(Hansen et al., 2012). Proteins with this O-FuT domain were predicted by Hansen et al. (2009) to be GT candidates that are distantly related to the CAZy family GT-65 (Coutinho et al., 2003).

Because these putative O-FuT proteins (POFUTs) were annotated on the basis of animal data

(Parsons, 2012) and O-FuT catalytic activity has yet to be shown in plants, it was proposed that this family should be named the the GT-65R family (Wang, 2013).

A previous microarray analysis led to the identification of a group of genes with unknown function that were highly expressed in embryogenic cells of B. napus microspore cultures, three days after induction (Chan, 2006). Of special interest is the homolog of the Arabidopsis gene

AT2G44500, which was consistently upregulated (threefold increase) in embryogenic cells compared to pollen-like cells. This gene is is highly expressed in young rosette leaves, the second internode of the stem as well as in meristematic cells. AT2G44500 is a member of the GT-65R plant O-FuT family. In the current study, bioinformatic and in silico expression analyses of the

GT-65R/O-FuT family (and in particular At2G44500), were performed to determine how members of this largely uncharacterized family function in plant development.

2.2 Materials and Methods

2.2.1 Sequence Information and Phylogenetic Analyses

Arabidopsis FuT nucleic and amino acid sequences and their corresponding AGI IDs were downloaded from the Arabidopsis Information Resource website (www.tair.com). Multiple sequence alignment was done using Clustal Omega and CLC Genomics. Phylogenetic trees were generated using CLC Genomics and FigTree.

2.2.2 Gene Expression Profiles

2.2.2.1 Generation of heat maps to uncover the expression of the gene members of GT-65R/O-

FuT family in meristematic, embryogenic and vegetative tissues

The developmental expression profile of the members of the GT-65R/O-FuT family was generated using the "Expression Browser" tool from the Bio-Analytical Resource database

(http://bar.utoronto.ca/affydb/cgi-bin/affy_db_exprss_browser_in.cgi) (Toughini, 2005). This data mining tool enables users to query up to 125 genes and generate their expression profiles based on the microarray data from the AtGenExpress project

(https://www.arabidopsis.org/portals/expression/microarray/ATGenExpress.jsp). For this particular analysis, the AGI ID’s of the 39 members of the GT-65R/O-FuT were inputted and the “Extended Tissue” series data set was queried. The following “research areas” were chosen to focus the search of expression profiles based on tissue type: “Shoot Apical Meristem and

Root Quiescent Centre”; “Embryogenesis” and “Developmental Baseline”. For the output parameters, the filter ‘Average of replicate treatments’ and “Cluster based on “research areas” were selected. The resulting output page produced tables of expression values for all the queried genes, which were also clustered based on their tissue expression specificity. This table was downloaded and the expression values were used to generate a heat map using Microsoft Excel.

2.2.2.2 Meta-analysis of At2G44500 gene expression in embryogenic and vegetative tissues

Data from Arabidopsis microarray experiments compiled in the Arabidopsis Electronic

Fluorescent Pictograph (eFP) browser from the University of Toronto

(http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi) were used to conduct a meta-analyses of

At2G44500 gene expression profile using the "developmental" series.

For the developmental analyses, the eFP browser was set to "Compare" mode. The

‘‘Compare’’ mode accepts two gene identifiers (primary and secondary) as input and compares the gene expression levels of the primary gene relative to the expression of the secondary

(control) gene in each tissue or treatment. Fold-change values are then calculated by the program which represent the expression level of the primary gene relative to the secondary

(control) gene (Winter et al., 2007). The primary gene used in the meta-analysis was the gene of interest, At2G44500 and its expression was compared to three housekeeping genes that are stably expressed and are used as internal references for normalization of gene expression data

(Chezchowski, 2005; Dekkers, 2012). These genes were: polyubiquitin (AtUBQ10); actin

(AtACT2); CREB binding protein (AtCBP20) (Chezchowski, 2005; Dekkers, 2012). The average fold change and standard deviation from these separate analyses in each of the series mentioned above (At2G44500 vs. AtUBQ10; At2G44500 vs. AtAct2 and At2G44500 vs.

AtCBP20) were calculated and were plotted using Microsoft Excel.

2.2.3 Genetic Network Analysis

Co-expression analysis was performed using standard settings of the ATTED-II

(http://atted.jp), geneCAT (http://genecat.mpg.de/5221/genecat.html) and geneMANIA

(http://geneMANIA.org) databases. These databases utilize current publicly available information

(RNA-seq, protein interactions, co-expression, etc) for Arabidopsis in order to elucidate gene

39 functional associations. The setting parameters to determine gene correlation were set to: MR <

16, Cor > 0.5 where the correlation (Cor) is the direct “correlation” and Mutual Rank (MR) was based on weighted Pearson’s correlation coefficients.

2.2.4 Protein Structure and Topology Prediction

The secondary and tertiary protein structures for both At2G44500 and BnAt2G(1) were predicted using Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index) and RaptorX

(http://raptorx.uchicago.edu/).

2.3 Results

2.3.1 Phylogenetic analysis of the AtFuT family

Thirty nine of the fifty-two Arabidopsis genes classified as fucosyltransferases contain a

GDP-O-FuT domain. There are only a few members of this family that have been functionally characterized (Wang, 2013; Neumetzler, 2012; Voxeur, 2012; Table 1.2). In spite of the conservation of key amino acids for enzymatic activity (Figure 2.2), members of the Arabidopsis

GT65R family show high protein sequence diversity and cluster into different clades/sub-groups

(Figure 2.1 A). The gene structure for each clade/sub-group is conserved but overall, the genomic structures of all these genes differ greatly between sub-groups and also from known mammalian

O-FuTs, which have a monoexonic coding sequence (Costache,1997) (Figure 2.1 B).

The consensus protein sequences that resulted from the alignment of all the members of the GT-37, GT-65R/O-FuT and GT-10 families were used for the phylogenetic analysis of the

FuT families (Figure 2.1 B). The analysis shows that the GT-37 and GT-65R families are more closely related with each other than the GT-10 family Figure 2.1 B). The consensus amino acid sequence of GT-10 matched with the crystal structure of a UDP-Glycosyltransferase, which is a member of the FuT-like family (template number: d2nzwa1). GT-37 matched with a nodulation

40 fucosyltransferase (nodZ) from Bradyrhizobium (template number: c2hlhA). NodZ is an alpha 1,6

FuT which adds fucose to the reducing N-acetylglucosamine residues of lipo-chitin oligosaccharide signal molecules (Brzezinski, 2007). AtO-FuT matched the crystal structure of

POFUT1, from Caenorhabditis elegans (template number:c3zy6A). In the Arabidopsis GT-10 family, the gene encoding alpha 1, 3 FuT has three exons and two introns, whereas genes encoding alpha 1,4 FuT have seven exons and six introns (Figure 2.1 B). The members of the GT-

37 family have a conserved gene structure, which is comprised of two exons and one intron. In contrast, members of the large O-FuT family have varying exon-intron organizations.

(A)

Figure 2.1 Phylogenetic relationships among the Fucosyltransferase (FuT) family in A. thaliana. The dendogram is based on protein sequences. (A) This glycosyltransferase (GT) family has three distinct groups - the GT-10/α1,3/4 FuTs (yellow box), GT-37/α1,2 FuTs (green box) and the large GT-65R/O-FuT subfamilies (blue box). The gene of interest, At2G44500 and its homolgs in B.napus (BnAt2G_1), B.rapa (Bra004841) and B.oleracea (Bol030094) form a sub-group (rectangle). Other members of the GT-65R/O- FuT family that have been previously characterized are in red.

(B)

Figure 2.1 (continued) (B) Phylogenetic relationships amongthe protein consensus sequences

for the three Fucosyltransferase (FuT) families in A, thaliana. The predicted protein structures

and gene models are indicated for each GT family. Black box: exons; line: non-coding regions.

2.3.2 Protein Sequence Analysis of the O-FuT family members

Multiple amino acid sequence alignments of the 39 members of the Arabidopsis GT65R family and the crystallized O-FuTs in C. elegans (Lira-Navarrete, 2011) and H. sapiens (Chen,

2012) show conserved amino acid residues within the previously identified conserved peptide motifs I, II and III for O-FuTs (Martinez-Duncker et al, 2003) (Figure 2.2). Amino acid residues in motif I, such as Histidine (H238), were shown to interact with the guanine moiety of GDP and

Arginine (R240) was associated with the enzyme's catalytic activity (Oriol, 1999; Martinez-

Duncker, 2003; Lira-Navarrete, 2011). Phenylalanine in positions (F261) and (F357) were also shown to bind GDP, while Asparagine (N43) is involved in fucose binding (Lira-Navarrete,

2011). In motifs II and III, amino residues D309 (II) and S355, T356 and F357 (III) were also shown to participate in GDP binding.

The amino acid N43, which is involved binding fucose, is conserved except in

AT3G03810 and AT3G30300 (Figure 2.2, arrowhead). Strict conservation of all amino acids involved in GDP binding was not observed, except in H238 and F357, in most of the GT65R/O-

FuT members. The HxR motif in conserved region I is conserved in most of the members of

GT65R/O-FuT, except in AT1G51630, AT3G21190, AT3G03810, AT3G30300 and AT4G17430.

AT3G21190 and AT1G51630 are also known as mannan synthesis- related genes (AtMSR1 and

AtMSR2), while AT3G03810 is known as the embryo development sac arrest 30 gene (EDA30).

Arginine (R240) (the essential amino acid for fucosyltransferase activity) is conserved, but the

H238 is replaced with Aspartic Acid (D) and Glutamine (Q), in AtMSR1/AtMSR2 and

AT4G17430, respectively but not in both EDA30 and AT3G30300.

AT1G04910 83 NGGLNQQRSAICNAVLAA----RIMNATLVLPEL-DA-NSF------AT1G11990 180 NGGINQQRVAVCNIVVVA----RLLNAALVIPKF-ML-SDV------AT1G14020 87 NGGLNQMRSAICDMVTVA----RLLNLTLVVPEL-DK-TSF------AT1G14970 144 NGGLNQQRTSICNAVAVA----GYLNATLVIPNF-HY-HSI------AT1G17270 209 SGQMSNHLICLEKHMFFA----ALLDRVLVIPSSK-F-D------YQYD------AT1G20550 108 SGGLNQQRTGIVDAVVAA----RILNATLVIPKL-DQ-KSY------AT1G22460 158 NGGLNQMRAGICDMVAIA----RIINATLVVPEL-DK-RSF------AT1G29200 172 NGGLNQQRVAICNAVAVA----ALLNATLVLPRF-LY-SNV------AT1G35510 150 NGGLNQQRLSICDAVAVA----GLLNATLVIPIF-HL-NSV------AT1G38065 31 NGGLNQQRSAICNAVAVA----GLLNAVLVIPRF-EF-HAI------AT1G38131 138 NGGLNQQRSAICNAVAVA----GLLNAVLVIPRF-EF-HAI------AT1G51630 90 TNGPEYHVSQITDAVMVA----KHLGATLVLPDI-RG-S-K------AT1G52630 75 YGGLNQMRRDLCDGVGIA----RLLNATLVLPKF-EV-AAY------AT1G53770 204 SGQMSNHLICLEKHMFFA----ALLKRVLVIPSHR-F-D------YHYS------AT1G62330 204 NGGINQQRVAVCNIVVVA----RMLNATLVIPKF-MF-SDV------AT1G76270 105 SGGLNQQRTGIVDAVVAA----RILNATLVVPKL-DQ-KSY------AT2G01480 151 NGGLNQQRTSICNAVAVA----GYLNATLVIPNF-HY-HSI------AT2G03280 82 NGGLNQMRAAICDMVTVA----RLLNLTLVVPEL-DK-KSF------AT2G37980 167 NGGLNQMRTGICDMVAAA----KIMNATLVLPLL-DH-ESF------AT2G44500 167 SGGMNQQRNQIVDAVVIA----RILGASLVVPVL-QV-NVI------AT3G02250 100 NGGLNQMRAAICDMVTIA----RYMNVTLIVPEL-DK-TSF------AT3G03810 93 FGGFDKIRSSICDLVTIS----RLLNATLVIPEL-QE-SLRSKG------AT3G05320 64 VWGLNNQKIAFARACLTA----RMMNRTLLMPSL-SA-SLF------AT3G07900 180 SGGLNQQKIQIVDAVVIA----RILGAVLVVPIL-QI-NLI------AT3G21190 89 TNGPEYHISQITDAVMVA----KHLGATLVLPDI-RG-S-K------AT3G26370 152 EGGLNQQRIAICNAVAVA----KIMNATLILPVL-KQ-DQI------AT3G30300 98 QGGFHEIRNSIPDVVAVS----RLLNATLVIPEI-QS-TTSSKG------AT3G54100 171 NGGLNQMRTGICDMVAVA----KIMNATLVLPLL-DH-ESF------AT4G16650 115 SGGLNQQRTGITDAVVVA----RILNATLVVPEL-DH-HSY------AT4G17430 76 HSGFSNQLSEFKNALLMA----GILNRTLIIPPILDH-HAVALGSCPKFRVLSPSEIRIS AT4G24530 96 DGGLNQQRMGICDAVAVA----KIMNVTLVIPRL-EV-NTV------AT4G38390 116 SGGLNQQRTGIIDAVVAA----YILNATLVVPKL-DQ-KSY------AT5G01100 161 NGGLNQMRTGICDMVAIA----KIMNATLVLPFL-DH-SSF------AT5G15740 100 NGGLNQMRAAICDMVTVA----RYMNVTLIVPEL-DK-TSF------AT5G35570 200 NGGLNQMRFGICDMVAVA----KIMKATLVLPSL-DH-SSY------AT5G50420 211 SGQMSNHLICLEKHMFFA----ALLDRVLVIPSSK-F-D------YQYD------AT5G63390 157 SGGLNQQRNQIVDAVVIA----MILEAALVVPVL-QV-NRV------AT5G64600 110 NGGLNQMRTGIADIVAVA----HIMNATLVIPEL-DK-RSF------AT5G65470 86 DGGLNQQRMGICDAVAVA----KILNATLVIPYL-EV-NPV------POFUT1_C 31 MGRFGNQVDQFLGVLAFA----KALDRTLVLPNF-IEFKHP------POFUT2_H 39 PEGFNLRRDVYIRIASLLKTLLKTEEWVLVLPPW-GR-LYH------

Figure 2.2 Conserved amino acid regions among members of the Arabidopsis GT65R/ O-FuT family. Multiple sequence alignment of O-FuT proteins in A. thaliana, C. elegans and H. sapiens. This family is characterized by the presence of three conserved peptide regions (called motifs I, II, and III). Red asterisks signify the conserved motif essential for the catalytic activity of O-FuTs (HxR). Red triangles = GDP-Fucose binding site.

I AT1G04910 240 ------GHFMSIHLRFEM------DMLAFAGCFDIFN-PE AT1G11990 340 ------SYYLALHLRFEI------DMVAHSLCYFGGG-ET AT1G14020 242 ------GPFVALHLRYEM------DMLAFSGCTHGCT-EE AT1G14970 303 ------N-NAGKYVSVHLRFEE------DMVAFSCCVFDGG-DQ AT1G17270 392 ------GKNFISLHLRRHGF-LKFC------NA------AT1G20550 260 ------SKHFIALHLRFEP------DMLAFSGCYYGGG-EK AT1G22460 310 ------GLYIALHLRFEK------EMLAFSGCNHGLS-AS AT1G29200 337 EEALLGESMVKS----TVKGEE-EPLKYLALHLRFEE------DMVAYSLCDFGGG-EA AT1G35510 309 ------E-SGGKYVSVHLRFEM------DMVAFSCCEYDFG-QA AT1G38065 190 ------A-TGGKYVSVHLRFEE------DMVAFSCCLYEGG-RA AT1G38131 297 ------A-TGGKYVSVHLRFEE------DMVAFSCCLYEGG-RA AT1G51630 245 ------K-SGGRFIAIDLRVE------ILEKKNCHETGA--- AT1G52630 224 ------KPFLSLHLRFEP------DMVAYSQCEYPNLSPS AT1G53770 391 ------GKNYIALHFRRHGF-LKFWYDLFSTSLLHST----- AT1G62330 370 DPYLVGPKFASFILDKKAGPLH-KASKYLAVHLRFEI------DMVAHSLCYFGGG-DA AT1G76270 257 ------SKHFIALHLRYEP------DMLAFSGCYYGGG-DK AT2G01480 310 ------N-HGAKYVSVHLRFEE------DMVAFSCCIFDGG-NQ AT2G03280 264 ------GSFVALHLRYEM------DMLAFSGCTHGCT-DE AT2G37980 319 ------SEPFIALHLRYEK------DMLAFTGCSHNLT-AG AT2G44500 318 ------QGQYLSLHLRMEK------DVWVRTGCLPGLT-PE AT3G02250 256 ------GPFLVLHLRYEM------DMLAFSGCSHGCN-RY AT3G03810 256 ------GQPFLAYHPGLVR------EKLAYHGCAELFQ-DI AT3G05320 235 ------EILGPVPFVAVHMRIEI------DWMIH--CKKLEQ-RK AT3G07900 331 ------KGPYIALHLRMEK------DVWVRTGCLSGLS-SK AT3G21190 244 ------K-SGGRFIAVDLRID------ILEKKNCHTTGV--- AT3G26370 313 ------NPNPYMALHLRFEK------GMVGLSFCDFVGT-RE AT3G30300 261 ------GRPFIAYDPGMTR------EALAYHGCAELFQ-DV AT3G54100 323 ------TEPYIALHLRYEK------DMLAFTGCNHNLT-TE AT4G16650 269 ------AKRFIAVHLRFEP------DMLAFSGCDFGGG-EK AT4G17430 332 ------KAPFLCAQLRLLDGQFKNHR------ES------AT4G24530 264 SGTMDPT------DRINTIVKA-GAGKFAVLHLRFDK------DMAAHSGCDFEGG-KA AT4G38390 270 ------AKHFVALHLRFEP------DMLAFSGCYYGGG-QK AT5G01100 313 ------NEPYLALHLRYEK------DMLAFTGCNHSLS-NE AT5G15740 256 ------GPFLVLHLRYEM------DMLAFSGCSHGCN-PE AT5G35570 352 ------RGPYLALHLRYEK------DMLAFTGCSHSLT-AE AT5G50420 394 ------GKNFIALHFRRHGF-LKFC------NA------AT5G63390 308 ------EGPYIALHLRLEK------DVWVRTGCLTGLG-SE AT5G64600 261 ------AGRYIALHLRYEK------DMLAFTGCTYGLT-DA AT5G65470 255 QKNVDHLG-----DMTNPHRRQ-EPGKFAVLHLRFDK------DMAAHSACDFGGG-KA POFUT1_C 224 ------AKPFVAVHLRNDA------DWVRV--CEHIDT-TT POFUT2_H 269 ------A-LGGPYLGVHLRRKDF------IWGHRQDVPSL---- ***

II AT1G04910 299 ------PEEVGLIL-RAMRFDNSTRIYLAAGELFGGEQF-----MK------P AT1G11990 407 ------PEEAVLML-AALGFNRETRVFVAGANIYGGSKR-----LA------V AT1G14020 303 ------PEEVALVL-KALGFEKNTQIYIAAGEIYGSEHR-----LS------V AT1G14970 371 ------PLEVGLML-RGMGFNKSTYIFLAAGPIYSANRT-----MA------P AT1G17270 414 PSCFYPI-PQAADCIS-RMVERANAPVIYLSTDAAESETGL-----LQSLVVVDGKV-VP AT1G20550 320 ------PEEVGLML-RALGYGSDVHIYVASGEVYGGEKS-----LA------P AT1G22460 371 ------PKEVGILL-TALGYSSDTPVYIAAGEIYGGESR-----LA------D AT1G29200 424 ------PEEATLVL-AGLGFKRKTYIYLAGSQIYGGSSR-----ML------P AT1G35510 377 ------PLEVGMML-RGMGFNNSTLVYVAAGNIYKADKY-----MA------P AT1G38065 258 ------PLEVGMML-RGMGFDNNTSIYLASGRIYQPEKH-----LA------P AT1G38131 365 ------PLEVGMML-RGMGFDNNTSIYLASGRIYQPEKH-----LA------P AT1G51630 279 ------AQEIALFL-RKLGFDSDTTIYLTQPRW---ESS-----LN------I AT1G52630 284 ------PNETVLML-QSLNIPTSTNIYLAAGD---GLME-----ME------G AT1G53770 451 PSCFFPI-PQAASCIT-RLIEKVEAPVLYLSTDAAESETGL-----LQSLLILNGKT-VP AT1G62330 461 ------PEEAVLML-AGLGFSRKTRVFVAGANIYGGNKR-----LA------A AT1G76270 317 ------PEEVGLML-RALGYGSDVHIYVASGEVYGGEES-----LA------P AT2G01480 378 ------PLEVGLML-RGMGFNKSTYIFLASGEIYDANRT-----MA------P AT2G03280 325 ------PEEAVLVL-KALGFQKDTQIYIAAGEIFGGAKR-----LA------L AT2G37980 381 ------PREAAIFL-KAMGYPSSTTVYIVAGEIYGG-NS-----MD------A AT2G44500 382 ------ALEVTRLL-KALEAPKDARIYWAGGEPLGGKEV-----LE------P AT3G02250 317 ------PEETALTL-SALGIDRNVQIYIAAGEIYGGKRR-----LK------A AT3G03810 322 ------PEEVGILL-KALGYSQKAIIYLAGSEIFGGQRV-----LI------P AT3G05320 274 ------REILDRVG-NISGLKTPTVLYLAVADTLLEEKEEDSSVLS------G AT3G07900 395 ------AKEVTRLL-RALGAPRDARIYWAGGEPLGGKEA-----LK------P AT3G21190 278 ------AQEIALFL-RKLGFASDTTIYLTQPRW---DSS-----LN------I AT3G26370 381 ------PGEVAVIL-RAMGYPKETQIYVASGQVYGGQNR-----MA------P AT3G30300 327 ------PEEVGILL-RAYGYSWDTIIYVAGGEVFGGQRT-----LI------P AT3G54100 385 ------PREAAIFL-KAMGYPSSTTVYIVAGEIYGS-ES-----MD------A AT4G16650 329 ------PHEVGLML-RALGFTNDTYIYVASGEIYGGEKT-----LK------P AT4G17430 362 LEALSVKNPGLINVFVMTDLPEFNWTGTYL------GDLSK-----NSTNF----KLHFI AT4G24530 345 ------PEEIGLLL-SALGFSNNTRLYLASHQVYGGEAR-----IS------T AT4G38390 330 ------PEEIGLML-RGLGFGKEVHLYVASGEVYGGEDT-----LA------P AT5G01100 375 ------PREAAVFL-KAMGFPSTTNIYIVAGKIYGQ-NS-----MT------A AT5G15740 317 ------PEETALTL-TALGIDRNVQIYIAAGEIYGGQRR-----MK------A AT5G35570 414 ------PRETSLLL-RALEFPSSSRIYLVAGEAYGN-GS-----MD------P AT5G50420 416 PSCFYPI-PQAAECIA-RIVERSNGAVIYLSTDAAESETSL-----LQSLVVVDGKI-VP AT5G63390 372 ------VYEIARLL-KALGAPSNASIYIAGGEPFGGSRA-----LE------P AT5G64600 323 ------PKEVGIFL-KGLGYSQSTVIYIAAGEIYGGDDR-----LS------E AT5G65470 337 ------PEEMGLLL-AAFGFDNNTRLYLASHKVYGGEAR-----IS------T POFUT1_C 279 ------QQILEQIV-EKVGSIGAKSVFVASDKDHMID-----E------POFUT2_H 296 ------EGAVRKIR-SLMKTHRLDKVFVATDAVR---KE-----YE------E

III AT1G04910 362 -----GSAVDY----MVCL-LSDIFMPTYD------GPSNFANNLLGHRLYY--G-FR-T AT1G11990 471 -----LAALDF----IACA-AADAFAMTDS------GS-QLSSLVSGYRIYYGGG-KL-P AT1G14020 367 -----MAALDF----MVSV-ASNTFIPTYD------G--NMAKVVEGHRRYL--G-YK-K AT1G14970 435 -----MAAIDY----TVCL-HSEVFVTTQG------G--NFPHFLMGHRRYLFGG-HS-K AT1G17270 494 -----YAMLDK----TICA-MSSVFIGASG------S--TFTEDILRLRKDW--G-TS-S AT1G20550 384 -----MAALDF----LVCD-ESDVFVTNNN------G--NMARILAGRRRYF--G-HK-P AT1G22460 435 -----MAALDY----IVSI-ESDVFIPSYS------G--NMARAVEGHRRFL--G-HR-K AT1G29200 488 -----LAALDF----IACI-ASDVFAMTDS------GS-QLSSLVSGFRNYYGNG-QA-P AT1G35510 441 -----LAALDY----TVCL-HSEVFVSTQG------G--NFPHFLIGHRRYLYKG-HA-E AT1G38065 322 -----MAALDY----TVSL-LSEVFVTTQG------G--NFPHFLMGHRRFLFGG-HA-K AT1G38131 429 -----MAALDY----TVSL-LSEVFVTTQG------G--NFPHFLMGHRRFLFGG-HA-K AT1G51630 342 -----ENVIDF----YISS-RSDVFVPAIP------G-LFYANTVGKRIAL--G-KP-Q AT1G52630 343 -----KAALDY----HVSI-NSDAYVATYF------G--NMDKIVAAMRTYK--Q-MH-N AT1G53770 531 -----EAMLDK----TICA-LSSVFIGASG------S--TFTEDILRLRKDW--G-TA-S AT1G62330 525 -----LAVLDF----IACA-ASDAFAMTDS------GS-QLSSLVSGYRIYYGAG-KM-P AT1G76270 381 -----MAALDF----LVCD-ESDVFVTNNN------G--NMAKILAGRRRYL--G-HK-P AT2G01480 442 -----MAAIDY----TVCL-HSEVFVTTQG------G--NFPHFLMGHRRYMFGG-HS-K AT2G03280 389 -----MAALDF----IVSV-ASNTFIPTYY------G--NMAKVVEGHRRYL--G-FK-K AT2G37980 444 -----LAALDY----IVAL-ESDVFVYTYD------G--NMAKAVQGHRRFE--G-FK-K AT2G44500 446 -----MAAIDY----IVCE-KSDVFIPSHG------G-NMGHALQGQRAYA--G-HK-K AT3G02250 381 -----MAALDY----LISL-ESDIFVPTYY------G--NMAKVVEGHRRFL--G-FK-K AT3G03810 454 AHRLLWDALDF----AVSV-EADVFFPGFNNDGSGWP--DFSSLVMGQRLYE--RPSS-R AT3G05320 341 -----QSAIDY----EVCL-RADVFVGNSF------S--TFSSLIVLERTQK--A-R--- AT3G07900 459 -----MAAIDY----IVCK-ESDVFMASHG------G-NMGHAIQGHRAYE--G-HK-K AT3G21190 341 -----ENVIDF----YISS-RSDVFVPAIS------G-LFYANTVGKRIAL--G-KP-Q AT3G26370 445 -----LAALDF----LVCL-KSDVFVMTHG------G--NFAKLIIGARRYM--G-HRQK AT3G30300 458 AHKLLWEAIDY----VVSV-EADVFISGFDRDGKGHP--SFASLVMGHRLYQ--SASA-K AT3G54100 448 -----LAALDY----IVAL-ESDVFVYTYD------G--NMAKAVQGHRKFE--G-FR-K AT4G16650 392 -----LAAIDY----IVSD-ESDVFITNNN------G--NMAKILAGRRRYM--G-HK-R AT4G17430 455 -----QLYIEE----AVCSCASLGFVGTPG------S--TIADSVEMMRKYN--A-CS-S AT4G24530 409 -----MAAVDY----YVSM-KSDIFISASP------G--NMHNALQAHRAYL--N--L-K AT4G38390 394 -----MAALDF----IVCD-KSDAFVTNNN------G--NMARILAGRRRYL--G-HK-V AT5G01100 438 -----LAALDY----NLAL-ESDIFAYTYD------G--NMAKAVQGHRRFE--G-FR-K AT5G15740 381 -----MAALDY----LVAL-ESDIFVPTND------G--NMARVVEGHRRFL--G-FK-K AT5G35570 477 -----LAGLDY----IVAL-QSEVFLYTYD------G--NMAKAVQGHRRFE--D-FK-K AT5G50420 496 -----DAMLDK----TICA-MSSVFIGASG------S--TFTEDILRLRKDW--G-TS-S AT5G63390 436 -----LAAIDY----IVSL-SSDVFIPSHG------G-NMAKAMQGNRAYV--G-HR-K AT5G64600 387 -----TAALDY----IISV-ESDVFVPSHS------G--NMARAVEGHRRFL--G-HR-R AT5G65470 401 -----LAALDY----YVSM-HSDIFISASP------G--NMHNALVGHRTFE--N--L-K POFUT1_C 324 -----QEPDDMYTSLAIMG-RADLFVGNCV------S--TFSHIVKRERDHA--G-Q--- POFUT2_H 353 -----VAIIDQ----WICA-HARFFIGTSV------S-TFSFRIHEEREIL--G-LD-P

AT1G04910 362 -----GSAVDY----MVCL-LSDIFMPTYD------GPSNFANNLLGHRLYY--G-FR-T AT1G11990 471 -----LAALDF----IACA-AADAFAMTDS------GS-QLSSLVSGYRIYYGGG-KL-P AT1G14020 367 -----MAALDF----MVSV-ASNTFIPTYD------G--NMAKVVEGHRRYL--G-YK-K AT1G14970 435 -----MAAIDY----TVCL-HSEVFVTTQG------G--NFPHFLMGHRRYLFGG-HS-K AT1G17270 494 -----YAMLDK----TICA-MSSVFIGASG------S--TFTEDILRLRKDW--G-TS-S AT1G20550 384 -----MAALDF----LVCD-ESDVFVTNNN------G--NMARILAGRRRYF--G-HK-P AT1G22460 435 -----MAALDY----IVSI-ESDVFIPSYS------G--NMARAVEGHRRFL--G-HR-K AT1G29200 488 -----LAALDF----IACI-ASDVFAMTDS------GS-QLSSLVSGFRNYYGNG-QA-P AT1G35510 441 -----LAALDY----TVCL-HSEVFVSTQG------G--NFPHFLIGHRRYLYKG-HA-E AT1G38065 322 -----MAALDY----TVSL-LSEVFVTTQG------G--NFPHFLMGHRRFLFGG-HA-K AT1G38131 429 -----MAALDY----TVSL-LSEVFVTTQG------G--NFPHFLMGHRRFLFGG-HA-K AT1G51630 342 -----ENVIDF----YISS-RSDVFVPAIP------G-LFYANTVGKRIAL--G-KP-Q AT1G52630 343 -----KAALDY----HVSI-NSDAYVATYF------G--NMDKIVAAMRTYK--Q-MH-N AT1G53770 531 -----EAMLDK----TICA-LSSVFIGASG------S--TFTEDILRLRKDW--G-TA-S AT1G62330 525 -----LAVLDF----IACA-ASDAFAMTDS------GS-QLSSLVSGYRIYYGAG-KM-P AT1G76270 381 -----MAALDF----LVCD-ESDVFVTNNN------G--NMAKILAGRRRYL--G-HK-P AT2G01480 442 -----MAAIDY----TVCL-HSEVFVTTQG------G--NFPHFLMGHRRYMFGG-HS-K AT2G03280 389 -----MAALDF----IVSV-ASNTFIPTYY------G--NMAKVVEGHRRYL--G-FK-K AT2G37980 444 -----LAALDY----IVAL-ESDVFVYTYD------G--NMAKAVQGHRRFE--G-FK-K AT2G44500 446 -----MAAIDY----IVCE-KSDVFIPSHG------G-NMGHALQGQRAYA--G-HK-K AT3G02250 381 -----MAALDY----LISL-ESDIFVPTYY------G--NMAKVVEGHRRFL--G-FK-K AT3G03810 454 AHRLLWDALDF----AVSV-EADVFFPGFNNDGSGWP--DFSSLVMGQRLYE--RPSS-R AT3G05320 341 -----QSAIDY----EVCL-RADVFVGNSF------S--TFSSLIVLERTQK--A-R--- AT3G07900 459 -----MAAIDY----IVCK-ESDVFMASHG------G-NMGHAIQGHRAYE--G-HK-K AT3G21190 341 -----ENVIDF----YISS-RSDVFVPAIS------G-LFYANTVGKRIAL--G-KP-Q AT3G26370 445 -----LAALDF----LVCL-KSDVFVMTHG------G--NFAKLIIGARRYM--G-HRQK AT3G30300 458 AHKLLWEAIDY----VVSV-EADVFISGFDRDGKGHP--SFASLVMGHRLYQ--SASA-K AT3G54100 448 -----LAALDY----IVAL-ESDVFVYTYD------G--NMAKAVQGHRKFE--G-FR-K AT4G16650 392 -----LAAIDY----IVSD-ESDVFITNNN------G--NMAKILAGRRRYM--G-HK-R AT4G17430 455 -----QLYIEE----AVCSCASLGFVGTPG------S--TIADSVEMMRKYN--A-CS-S AT4G24530 409 -----MAAVDY----YVSM-KSDIFISASP------G--NMHNALQAHRAYL--N--L-K AT4G38390 394 -----MAALDF----IVCD-KSDAFVTNNN------G--NMARILAGRRRYL--G-HK-V AT5G01100 438 -----LAALDY----NLAL-ESDIFAYTYD------G--NMAKAVQGHRRFE--G-FR-K AT5G15740 381 -----MAALDY----LVAL-ESDIFVPTND------G--NMARVVEGHRRFL--G-FK-K AT5G35570 477 -----LAGLDY----IVAL-QSEVFLYTYD------G--NMAKAVQGHRRFE--D-FK-K AT5G50420 496 -----DAMLDK----TICA-MSSVFIGASG------S--TFTEDILRLRKDW--G-TS-S AT5G63390 436 -----LAAIDY----IVSL-SSDVFIPSHG------G-NMAKAMQGNRAYV--G-HR-K AT5G64600 387 -----TAALDY----IISV-ESDVFVPSHS------G--NMARAVEGHRRFL--G-HR-R AT5G65470 401 -----LAALDY----YVSM-HSDIFISASP------G--NMHNALVGHRTFE--N--L-K POFUT1_C 324 -----QEPDDMYTSLAIMG-RADLFVGNCV------S--TFSHIVKRERDHA--G-Q--- POFUT2_H 353 -----VAIIDQ----WICA-HARFFIGTSV------S-TFSFRIHEEREIL--G-LD-P

Figure 2.3 Phylogenetic relationships of all the members of the GT-65R/O-FuT family in Arabidopsis based on their protein sequences. Clades/sub-groups that have conserved gene structures are shaded by light blue. Exons: blue box; introns: line. At2G44500 and BnAt2G(1) group is marked by the red asterisk.

2.3.3 Expression Profiles of the GT-65R/ O-FuT Gene Family and At2G44500

2.3.3.1 Embryogenesis Profile

Figure 2.4 shows the expression pattern of all O-FuT members in the context of embryogenesis and during vegetative and reproductive growth stages. The analyses also clustered the genes based on the similarity of their expression profiles. Gene members of this family are expressed throughout development and their expressions are tissue-specific. Most members of the

GT-65R/O-FuT family have higher than the baseline expression in the early stages of embryogenesis, when the shoot apical meristematic (SAM) region is first established (Figure 2.4

A). Notably, there were three gene members, At2G44500, At3G30300 and At2G03280 that showed a high level of expression in the SAM (circle). At2g44500 is highly expressed in the rib meristem (RM) (Figure 2.5 A). The RM is a subjacent region of the shoot apical meristem and is composed of small dense and rapidly dividing cells that give rise to the leaf-stem units during indeterminate growth of the plant (Ruonala, 2008). This is supported by the observation that

At2G44500 is also highly expressed in young leaves and stems, specifically in the 2nd internode region as development progresses from embryogenesis to the vegetative stage (Figure 2.5 C).

Interestingly, At2445500 is not highly expressed in reproductive tissues such as the ovaries, microspores and mature pollen grains (Figure 2.5 D)

(A) Meristems SAM RQC

At1g22460 At1g29200 At3g07900 Figure 2.4 Expression profiles of GT- At1g11990 65R/O-FuT genes during A. thaliana At4g38390 development (A) Shoot Apical Meristems At2g44500 (SAM) and Root Quiescent Centre (RQC) At3g05320 (B) embryogenesis (C) seedling to flowering At5g63390 stage. Expression levels were measured via At2g03280 microarray analysis and analyzed by the At1g53770 GeneChip Operating Software (GCOS) At1g35510 obtained from the Bio-Analytic Resource and At1g14020 the AtGenExpress databases. Level of gene At4g16650 expression: yellow to red = baseline to At5g64600 upregulation; yellow to blue = baseline to At1g14970 downregulation (-) of gene expression. The At1g20550 gene of interest, At2G44500 is in red. At3g03810 At1g76270 At1g04910 At3g21190 At5g15740 At5g50420

At3g26370

At3g30300

At4g17430

At4g24530 At1g52630 At1g62330 At3g02250 At5g35570 At1g38131 At2g01480 At3g54100 At2g37980 At5g65470

(B) Globular Heart Torpedo Torpedo Apical Basal Cotyl Root Apical Basal Cotyl Root At1g11990 At1g22460 At2g01480 At1g14970 At2g37980 At1g76270 At2g44500 At1g62330 At3g30300 At3g21190 At5g15740 At5g35570 At1g35510 At5g64600 At1g29200 At3g02250 At1g5260 At1g20550 At5g63390 At3g03810 At1g38131 At3g07900 At4g17430 At5g65470 At1g14020 At1g53770 At2g03280 At5g50420 At1g04910 At3g26370 At3g05320 At3g54100 At4g38390 At4g16650 At4g24530

(C)

Figure 2.3 Phylogenetic relationship among members of the Arabidopsis GT-65R/O-FuT family. At2G44500 and BnAt2G(1) group is marked with a red asterisk. Clades/sub-groups that have conserved gene structures are shaded by light blue. Exons: blue box; introns: line.

(A)

(B)

Figure 2.5 Gene expression profile of At2G44500 in (A) embryonic (B) post- embryonic (C) vegetative and (D) reproductive tissues. Y-axes indicate the fold change values for each specific tissue in the x-axes. Fold change of 1 = no change in expression compared to the expression of the housekeeping (control) genes: AtACT2, AtUBQ10 and AtCBP20. Error bars denote standard deviation.

(C)

(D)

2.3.4 In silico Co-Expression and Network Profile of At2G44500

In order to attain further information with regard to its putative function, a gene network analysis with At2G44500 as ‘bait’ was conducted. A number of genes with known or putative function in embryogenesis, cell wall biosynthesis and signal transduction such as the Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein (At1G17620) and Glycosyl

Hydrolase (At2G27500) were found to be co-expressed (single solid line) and physically interact (double solid line) with At2G44500 (Figure 2.7). Genes directly connected with

At2G44500 involved a late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein gene (At1G17620), a glycosyltransferase that is a member of the GT61 family (At2G41640) and a Glycoside Hydrolase (At2G27500). At2G44500 was also shown to physically interact with

RACK1A (Receptor for Activated C Kinase1) through a yeast split-ubiquitin system (Ullah,

2013). RACK1A is a multifunctional WD-40 type scaffold protein that is involved in regulating different signal transduction pathways in eukaryotic cells. Interestingly, RACK1A also physically interacts with a glycoprotein (At5G2430) and a bHLH transcription factor

(AT1G09250)- both genes were also co-expressed with the glycoside hydrolase, At2G27500, which was also co-expressed with At2G44500 (Figure 2.7, upper left).

Figure 2.7 At2G44500 gene network. Information was compiled from The ATTED- II (http://atted.jp) and GeneMANIA (http://genemania.org) databases which use large functional association data (RNA-seq, genetic and protein interactions, co- localization, protein domain similarity, etc). At2G44500 (center, gray) was used as 'bait' in order to identify co-regulated genes (single line) and physically interacting proteins (double lines) in Arabidopsis.

(B)

(A)

2.4 Discussion (A) The plant fucosyltransferase family is a large superfamily of glycosyltransferases that

are divided into 3 groups namely: GT-10 (α1, 3/4 FuTs); GT-37 (α1,2-FuTs) and the GT-65R

(B) (O-FuTs). GT-65R/O-FuT is the largest group from this family yet most of its members are still

largely uncharacterized in plants. Here, it was shown that members of this family contained

conserved key amino acids essential for O-FuT catalytic activity (Figure 2.2) and that members

of this family are differentially expressed throughout plant development. At2G44500, a putative

O-FuT, is shown to be highly expressed in the shoot apical meristem (SAM) and young

vegetative tissues.

2.4.1 Phylogenetic and Protein Sequence Analyses

The phylogenetic analysis shows that the GT-37 and GT-65R/O-FuT families are more

closely related to each other than the GT-10 family. Members of the GT-10 family add fucose to

N-linked glycoproteins whereas members of the GT-37 family catalyze fucose to cell wall-related

polysaccharides such as xyloglucan (XyGs) and arabinogalactan proteins (AGPs). This suggests

that members of the GT-65R/O-FuT family may also be targeting polysaccharides that are related

to the cell wall.

At2G44500 is a member of a superfamily of GDP-O-Fucosyltransferases (O-FuTs). GDP

O-FuT is a family of conserved proteins representing the enzyme responsible for adding O-fucose

to target molecules such as the Epidermal Growth Factor-like proteins and proteins with

Thrombospondin repeats in mammalian cells (Haltiwanger, 2001; Neumetzler, 2012; Wang

2013). The present study shows that the putative O-FuTs in Arabidopsis contain the conserved

peptide regions found in characterized O-FuT proteins (Figure 2.2), which have been shown to

play important roles in their catalytic activity (Oriol, 1999; Maritnez-Duncker, 2003). In

59 particular, in C.elegans POFUT1, two amino acid residues located in motif I, called the ‘HxR’ motif are conserved in At2G44500 and most of the members of the GT-65R/O-FuT family

(Figure 2.2, red asterisks) were shown to have functional significance in the enzyme's catalytic activity (Lira-Navarrete, 2011). Although the enzymatic activities of the protein encoded by

At2G44500 remains to be determined, the presence of the conserved peptide motifs are strongly indicative of that they are members of the plant GT-65R/O-FuT family.

Some members of this family have recently been functionally characterized (Table 1.2).

For example, the At5G01100 gene and the AtFRB1 (FRIABLE1) protein it encodes has a putative

O-FuT domain. The AtFRB1/At5G01100 gene was shown to play a role in cell adhesion and cell wall integrity (Neumetzler, 2012). Loss of function mutants of this gene (frb1) exhibited cell dissociation phenotypes, characterized by sloughing off cells from tissues so that they appeared to crumble or seemed "friable". The cotyledons and hypocotyls in these mutants appeared to be fused with each other. frb1 mutants also had reduced levels of galactose and arabinose in the

Golgi, which consequently led to the decreased levels of galactose in pectic cell wall fractions

(Neumetzler, 2012). These phenotypes led to the proposal that FRB1 plays a role in cell wall architecture by a mechanism that involves addition of sugar moieties, such as galactose and arabinose, to an unknown target.

The mannan synthesis related genes (AtMSR1 and AtMSR2) form another sub-group in the

O-FuT phylogenetic tree (Figure 2.3). The Golgi-localized proteins they encode were shown to have indirect effects on hemicellulose synthesis, particularly in the addition of the polysaccharide, mannan. AtMSR1 knockout (msr1) and AtMSR1/AtMSR2 double mutants

(msr1/msr2) showed 40% and 50% decreases, respectively in the mannose levels of extracted cell walls from stems (Wang, 2013). The reduction in mannose levels in msr1/msr2 were rescued by

60 the expression of AtMSR1 and AtMSR2 under the control of the 35S promoter. Interestingly, in spite of this observed reduction in mannose levels in the cell wall there were no reported morphological defects observed in the embryo and adult plants.

It is interesting to note that the fucose levels were not directly affected in all the cell wall extracts analyzed in the frb1, msr1 and msr1/msr2 mutants (Neumeltzer, 2012; Wang, 2013). In other eukaryotic model systems such as in C.elegans and Drosophila melanogaster, GDP-O-FuTs add fucose directly to serine or threonine residues in glycoproteins (Wang, 2001). This family in

Arabidopsis is large and diverse and it is likely that each member is glycosylating unknown target/acceptor molecules (protein, lipid or carbohydrate) involved in processes such as in cell wall polysaccharide biosynthesis.

Overall, this suggests that different O-FuTs establish interactions with various cellular membranes and their broad sub-cellular distribution highlights the requirement for fucosylation in each cellular compartment in particular developmental contexts (Neumeltzer, 2012; Wang, 2013;

Wang, 2001). One big question that needs to be addressed is that if these proteins do indeed transfer fucose. The primary limitation is that the targets of these putative GTs have not yet been determined. Future work will therefore require systematic and exhaustive testing of both potential targets and substrates for this novel family of FuTs.

2.4.2 Gene Expression Profiles

Members of the O-FuT gene family are differentially expressed in various tissue types and developmental stages. The developmental expression analysis of At2G44500 showed that it is highly expressed in embryonic and post- embryonic tissues, specifically in the rib meristem, root cortex, endodermis and the lateral root cap. It is also highly expressed in vegetative tissues, particularly in young leaves and the 2nd stem internodes (Figures 2.4 and 2.5). These

61 observations reflect the dynamic nature of the cell walls in these different tissue types and the role that fucosylation plays in them. At2G44500 was one of the genes upregulated in embryogenic microspore cultures in B.napus (Chan, 2006).

2.4.3 Gene Network Analysis

The gene network analysis of At2G44500 showed that it was co-expressed with genes that are involved in cell wall biogenesis, embryogenesis and stress response, including: a late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein gene (At1G17620), a glycosyltransferase gene that encodes a member of the GT61 family (At2G41640) and a glycoside hydrolase (At2G27500) gene, respectively. LEA proteins are highly expressed during abiotic stress and in seeds and their expression is controlled by ABA (Hincha, 2008).

Hydroxypoline-rich glycoproteins play various important roles in growth and development, such as: in normal embryogenesis (Hall, 2002); plant defence (Deepak, 2010); and the overall structural integrity of the plant cell wall (Xu, 2011). Gycosyltransferases that are members of the GT61 family were found to play key roles in the synthesis of arabinoxylan in grass species, such as in rice and wheat (Mitchell, 2011; Ronald, 2012). Glycoside hydrolases are a widespread group of enzymes that hydrolyze the glycosidic bond between two or more carbohydrates or between a carbohydrate and a non-carbohydrate moiety (Henrissat, 2000).

RACK1A was previously shown to physically interact with the At2G44500 protein

(Ullah, 2013). RACK1A is a multifunctional WD-40 type scaffold protein that is involved in regulating different signal transduction pathways in eukaryotic cells. In Arabidopsis, RACK1A and RACK1B are functionally redundant proteins that act as negative regulators of ABA and mediate abiotic stress responses (Guo, 2009). It is likely that in the context of microspore embryogenesis and/or stress responses RACK1A functions like an adaptor protein in various

62 intracellular compartments (such as in the Golgi) and is able to interact with various proteins such as O-FuTs, cell surface receptors, protein kinases, transcription factors, and transcription- associated proteins (Patterson, 2004; Liu, 2007; Parent, 2008; Ullah, 2013).

3 Chapter 3

Molecular and In Silico Characterization of At2G44500 and Its B.napus Homolog, BnAt2G(1)

Abstract

Microspore embryogenesis is a developmental phenomenon in which microspores divert from their gametophytic pathway towards embryonic development when cultured and subjected to an inductive treatment such as mild heat stress. In Brassica napus this results in the formation of haploid embryos that can be used to generate doubled-haploid plants after chromosome doubling.

A genome-wide search for candidate genes involved in microspore embryogenesis was previously undertaken in B.napus microspore cultures. This search identified a transcript homologous to the

Arabidopsis gene, AT2G44500, which was consistently upregulated in embryogenic cells compared to pollen-like or non-responsive cells. In this study, the B. napus homolog [called

BnAt2G(1)] was isolated and characterized. Quantitative real-time PCR showed that BnAt2G(1) is highly expressed in embryogenic microspore cultures and in vegetative tissues, but not in reproductive tissues. In silico topology prediction programs suggest that BnAt2G(1)/At2G44500 encode type II-membrane proteins that are located in the Golgi. The sub-cellular localization of a

GFP::BnAt2G(1) fusion protein was visualized in root cells and leaf protoplasts. The transcription control elements that were identified in the in silico promoter analysis of the

At2g44500/BnAt2G(1) genes suggest that they bind various putative transcription factors that are involved in hormone-regulated stress responses.

3.1 Introduction

Haploid microspores of Brassica napus can be induced to switch their gametophytic development into embryogenesis by a mild stress in tissue culture (Barany, 2010). Ultimately, this

64 process can be used to produce doubled haploid plants which are important tools in plant breeding and biotechnology. Although at present, not all crops of agronomic interest and market-value respond efficiently to microspore embryogenesis (ME) induction, the knowledge obtained from species that are responsive enough to be considered as model systems (canola/rapeseed, tobacco, wheat and barley) can help us understand the molecular mechanisms of this process which may help apply it to recalcitrant genotypes or species. The search for molecular and cellular markers during the early stages of ME constitutes an important goal in the identification of cells committed to the embryogenic pathway as opposed to those cells that are non-responsive to ME induction.

Most of the analyses focus on the factors that are involved in the transcriptional switch from microspore to embryo development (Boutilier et al. 2006; Joosen et al. 2007; Malik et al.

2007). For example, different screening techniques such as subtractive hybridization (Boutilier,

2002); and differential display PCR (Reynolds, 1992; Custers et al. 2001) have been used to identify putative genes involved in early microspore embryogenesis such as FUSCA3; LEAF

COTYLEDON1 (LEC1); LEC2; BABYBOOM (Boutilier, 2002); WUSCHEL-Related

Homeobox (WOX2 and WOX9) and ABA Insensitive (ABI3). Another effort in the search for early embryogenic genes led to a genome-wide analysis of candidate genes that are involved in the early stages of B.napus microspore embryogenesis (Chan, 2006). Since embryogenic cells can be separated from those committed to pollen formation in the B. napus microspore culture system by flow sorting (Pauls, 1998), a differential microarray experiment can then be used to characterize the relative expression levels of genes in these two types of cells. In this study, an

Arabidopsis cDNA array was used to compare the transcriptomes of embryogenic cells with pollen-like microspores to identify genes that were related to the early stages of microspore embryogenesis. The transcriptome profiles that were obtained from this study identified genes that

65 were (1) upregulated in microspore-derived pollen-like cells; (2) consistently upregulated in microspore-derived embryogenic cells; and (3) down-regulated in embryogenic versus pollen-like cells (Chan, 2006). Of special interest is the transcript with homology to the Arabidopsis gene

AT2G44500, which is highly expressed in embryogenic microspore culture cells.

The present work describes cloning of the homolog of the Arabidopsis gene, At2G44500 from B. napus. The gene sequence and protein domains are characterized, the number of homologs in B.napus was determined by Southern blotting and the expression levels of the gene in embryogenic microspore cultures at different stages were determined by quantitative real-time

(qRT)- PCR. In silico methods were used to determine their gene regulatory elements, protein structures (as well as their functional domains) and to show that it encodes a conserved GDP-O-

Fucosyltransferase domain. An in silico analyses showed that it is upregulated in cells located in the rib meristem of the shoot apical meristem (Chapter 2). The current work also describes the syntenic relationships between chromosomal segments surrounding the gene in Arabidopsis and the Brassica members (B.rapa, B.oleracea and B.napus) of the triangle of U (U, 1935). Lastly, transgenic plants expressing GFP fused with the Brassica protein were generated to determine the sub-cellular localization of this novel plant O-Fucosyltransferase.

3.2 Materials and Methods

3.2.1 Plant Materials and Growth Conditions

Plants of Brassica napus cultivar ‘Topas’ (a highly ME responsive line) were grown in 15- cm pots in a growth cabinet (BioChamber) set to 20 oC/15 oC (day/night) under a 16-hour photoperiod regime with a light intensity of 160 µmol/m2sec and 70% relative humidity. The plants were irrigated with tap water and were fertilized with an all purpose 20-20-20 fertilizer

66 every two days. When plants began bolting, buds of approximately 2 to 3 mm in length and 70 to

85% petal to anther length ratio were selected for microspore culture.

3.2.2 Microspore culture

Culture media and other solutions needed for microspore washing and culture were chilled in 4°C. The entire microspore isolation procedure was usually accomplished within one hour while the solutions were still cool. Embryogenesis was induced in microspores isolated at the late-uninucleate to early binucleate stage. 70 to 80 buds were collected and were surface sterilized in 70% ethanol for 5 minutes and bleach for 20 minutes. The sterilized buds were rinsed three times with sterile double distilled water and were macerated in a blender with a B5 wash (Gamborg with 13% sucrose) (Phytotechnology, Cat# G398,) solution. The crude suspension was filtered consecutively through 64 µm then 40 µm Nitex nylon sieves (Tetko Co.,

Elmsford, NY). The filtrate was pelleted by centrifugation for 5 min at 200 x g. The supernatant was decanted and the microspore pellets were re-suspended in 13% sucrose wash solution. The centrifugation and washing steps were repeated, three times, and the final pellet was resuspended in 50 mL NLN-13 media (Nitsch and Nitsch, 1967) (Phytotechnology, Cat# N479).

For the heat shock treatment, the suspension culture was incubated in a 32 ⁰C water bath for 30 minutes while non-induced cultures were incubated in room temperature (25 ⁰C).

The isolated microspores were plated at a final density of 50,000 mL-1 in room temperature NLN-13 media. 10 mL of the microspore suspension was dispensed into Petri plates

(150 mm wide, 10 mm deep). The plates were incubated at 32 ⁰C (induced treatment) or 25 ⁰C

(non-induced treatment) in the dark for 14 - 17 days, when embryos in the globular and heart stages appeared in the induced but not in the non-induced cultures. Samples for RNA extraction

67 were taken from four time points: 0, 3, 5 and 7 day old microspore cultures for both induced

(heat-treated) and non-induced treatments.

3.2.3 Homologous Gene Isolation

3.2.3.1 DNA Extraction

Genomic DNA (gDNA) was isolated from young leaves using a modified CTAB method

(Doyle, 1989; Clarke, 2009; Springer, 2010). Briefly, 700 uL of the preheated CTAB extraction buffer (3% CTAB - cetyltrimethylammonium bromide; 1.4 M NaCl; 100 mM Tris-HCl, pH 8.0;

20 mM EDTA, pH 8.0; 1% PVP; 0.2% β-mercaptoethanol) were added to 0.8 g – 1 g of ground tissue and were incubated in 65ᴼC for 60 min. After incubation, 400 uL of 24:1 chloroform:isoamyl alcohol were added, mixed by vortexing and centrifuged at 10,000 x g for 5 minutes. The top aqueous phase was then transferred to fresh tubes. 0.7 volume of ice cold isopropanol was added, mixed by inversion and incubated in room temperature for 10 minutes.

The DNA was pelleted in a microcentrifuge at 10,000 x g for 10 min in room temperature. The resulting pellet was washed with 500 uL of 70% ethanol and centrifuged for 2 min at 10,000 x g.

The supernatant was carefully discarded and the pellet was air dried. The DNA was dissolved in

50 uL of sterile water. Alternatively, a DNeasy Plant Mini Kit (QIAGEN Inc., CA, USA) was used for routine DNA extraction, according to the manufacturer's instructions.

3.2.3.2 Southern Blot

Southern blot analysis was used to determine the copy number of the At2g44500 homologs in B. napus. DNA was extracted from leaves of 3 - 4 week old plants using the modified CTAB method mentioned above. The genomic DNA was quantified using a Nanodrop

ND1000 Spectrophotometer (Nanodrop Technologies) and a Qubit Fluorometer (Life

Technologies). Probes were prepared using the PCR DIG probe synthesis kit (Roche)

68 amplifying the template: pTopo2.1-BnAt2G_cDNA. Approximately 20 μg of genomic DNA was digested with restriction enzymes HindIII, SpeI and SwaI (New England Biolabs, Beverly,

MA). The digested DNA was separated by electrophoresis on a 1% agarose ethidium bromide gel at 90 V for 2.5 hours. The DNA was transferred onto a charged nylon membrane (Roche,

Cat# 11417240001 ) and was left overnight. Pre-hybridization and hybridization (with the synthesized probe) was performed using the DIG Easy Hyb Kit (Roche,Cat# 11603558001).

The hybridized probe was detected with an anti-digoxigenin-AP conjugate (Roche, Cat#

11093274910). The DNA blot (membrane) was covered with a CDP-star solution (Roche, Cat#

11685627001) and sealed and incubated with a Kodak X-OMAT film (Kodak Inc, Cat# F1274) in cassettes. The films were then exposed for 15 min to 1 hr. Subsequently, exposed films were developed in the dark room by alternately submerging them for 2 to 5 minutes in the following order: developer (Kodak HC-110), water and fixation (Kodak Kodafix- 200) solutions. The films were air dried for 30 minutes and bands were detected and recorded using the ChemiDoc

XRS (epi white illumination) and the Image Lab image acquisition and analysis software

(BioRad).

3.2.3.3 RNA Extraction and cDNA Synthesis

Total RNA samples from 0, 3, 5, and 7 day old microspore cultures were isolated using a modified glass bead disruption/RNeasy kit protocol. Briefly, the cells from induced (32 ⁰C, embryogenic) and non-induced (25 ⁰C, non-embyrogenic) cultures were harvested for each time point by low speed centrifugation. The cell pellets were lysed by resuspending them with RLT buffer (Qiagen) containing beta-mercaptoethanol (with a ratio of 10 uL of beta-mercaptoethanol per 1 mL of RLT buffer). The suspension was transferred to Lysing Matrix C tubes (MP

Biomedicals), which contain 1 mm silica beads, and the cells were mechanically disrupted with a

FastPrep homogenizer (Savant). The lysate was decanted onto a QIAshredder (Qiagen Inc.,

Mississauga) and centrifuged for 2 min at ~12,000 x g. A half volume of 96% ethanol was added and mixed by pipetting. The mixture was loaded onto to the RNeasy mini column and centrifuged at 8000 x g for 15 s. The column was washed with RW1 buffer (RNeasy Midi Kit, QIAGEN) and centrifuged at 8000 x g for 15 s. Another washing buffer (RPE) (RNeasy Midi Kit, QIAGEN) was added to the RNeasy column and it was centrifuged for 2 min at ≥ 8000 × g. The RNA was eluted with 30 µl or 50 µl of RNase-free water by centrifugation at 8000 x g for 2 min. RNA was also isolated from seedlings, young leaves, old leaves, stem and buds by using the RNeasy Mini kit

(Qiagen). To remove genomic DNA, either an on-column DNAse digestion (Qiagen) was performed or samples were treated with Turbo DNAse, according to the manufacturer’s specifications (Ambion, Austin, TX). 10X TURBO DNase [5 μL (0.1 volumes)] buffer and 1 μL of TURBO DNase were added to the 50 μL RNA. The solution was gently mixed, centrifuged and incubated at 37oC for 20–30 min. Re-suspended DNase inactivation reagent was added [5 μL (0.1 volumes)], and the mixtures was incubated for 2 min at room temp, with occasional agitation of the tubes by flicking. The samples were centrifuged at 10,000 x g for 1.5 min and the supernatants

(containing RNA) were transferred to new tubes and frozen at -80 ºC. RNA quality and quantity were measured with a Nanodrop. Approximately 2 ug of RNA was separated by electrophoresis at

100V using a 1% EtBr agarose gel to check the quality and integrity. cDNA was synthesized from

1 ug of RNA using the qScript cDNA synthesis kit (Quanta BioSciences), according to the manufacturer's instructions. After its completion, 1/5th of the 1st strand reaction was used for

PCR amplification, using gene-specific primers, to determine if the cDNA synthesis worked.

3.2.3.4 Rapid Amplification cDNA Ends (RACE) PCR

The SMARTer RACE cDNA Amplification Kit (Clontech, Palo Alto, CA) was used to amplify the 5' and 3' untranslated regions (UTR) of BnAt2G(1), according to the manufacturer’s instructions. Briefly, two cDNA populations namely, 5' and 3' RACE-ready cDNA, were synthesized from 1 ug of total RNA by the joint action of the SMARTer II A Oligonucleotide (5'-

AAGCAGTGGTATCAACGCAGAGTACXXXXX–3') and SMARTScribe Reverse

Transcriptase. The 5'-ends were amplified by RACE PCR using the 5'RACE-ready cDNA as a template, a gene-specific primer in the anti-sense direction (GSP1: 5'-

ACATCCTTCCCACCTCTACCCGTTCTCAAC-3') and a universal primer mix (UPM:5'-

CTAATACGACTCACTATAGGGC-3'). The 3'UTR was PCR-amplified using 3'-RACE-ready cDNA as the template, a gene-specific primer in the sense-direction (GSP2: 5'-

CCCTCCCTTTCTCACGTCACCCTTGCTC-3') and UPM. The resulting PCR products were gel purified (PureLink Gel Extraction Kit, Invitrogen) and were cloned into a TOPO-vector for the subsequent sequencing reactions.

3.2.3.5 Quantitative PCR (qPCR)

For qPCR reactions, the BnAt2G(1)- specific primers BnAt2G_fwd1 (5'-

ATCATCGCTAGGATCCTTGGTGCTT-3') and BnAt2G_rev1 (5'-

GGAGTCCTTTTCTCTTCCACAGGTC-3') were used. The Brassica napus Actin2 (Act2) gene

(primers BnActin_fwd 5'- TTCTCCACCGAAGAACTGCT-3' and BnActin_rev 5'-

TTCTCCACCGAAGAACTGCT-3') was used as an endogenous control in order to normalize relative amounts of total RNA. qPCR reaction mixtures consisted of 2.5 µl of cDNA, 12.5 µl of

Power SYBR Green PCR Master Mix (Applied Biosystems) and 5 µl of each primer in a total volume of 25 µl were added to 48-Well Optical Reaction Plates for amplification and

71 quantification in a StepOne Real-Time PCR System (Applied Biosystems). The experiment was repeated three times (biological replicates) and the delta-delta Ct method (Livak, 2001) was used in order to make a relative quantification comparing induced and non-induced transcript levels. Due to the nonparametric distribution of data, statistical analysis of delta-delta Ct values was performed using ANOVA and Bonferonni-Holm post-hoc analysis with significance set at p<0.05.

3.2.4 Gene Cloning

3.2.4.1 PCR Product Purification

PCR fragments of BnAt2G were purified using the PureLink Quick Gel Extraction Kit

(Invitrogen) or the PureLink PCR Purification Kit (Invitrogen), according to the manufacturer's instructions. For both methods, DNA was purified using a centrifuge. For the gel extractions, the correctly-sized fragments, corresponding to different lengths of the BnAt2G gene, were excised from 1% TBE agarose gels, stained with ethidium bromide (EtBr) and were transferred into 1.5 ml Eppendorf tubes. Gel Solubilization Buffer (1.2 mL) was added to one volume of the excised

DNA gel (approximately 400 mg in weight) and the mixture was incubated in a in 50 oC water bath for 15 min. For optimal DNA yields, 400 uL of isopropanol was added to the dissolved gel solution. The resulting solution was pipetted onto extraction columns and washed with buffer

W1. The DNA was eluted with 25 µl of pre-warmed (55-60oC) Molecular Biology Grade

(MBG) water. For direct PCR purification, 240 uL of the PureLink Binding Buffer +

Isopropanol was added to 60 uL of PCR product and were loaded onto spin columns. The subsequent wash steps were done and the DNA was eluted with 30 uL of pre-warmed MBG water. The concentrations of the eluted DNA were measured with the Nanodrop.

3.2.4.2 Cloning into TOPO® Easy Vector

Purified PCR products were cloned into the pCR®2.1-TOPO® vector with the TOPO

TA Cloning® Kit (Invitrogen), according to the manufacturer’s instructions. Briefly, 1 µl of salt solution (1.2 M NaCl and 0.06 M MgCl2), 1 µl of TOPO® vector (50 ng/µl) and 4 µl of DNA

(50-60 ng) were used to set up a ligation reaction of 6 µl. The reaction mixture was incubated for 15 min, at room temperature. Chemically competent E. coli (DH5α) cells were transformed with the plasmid DNA by heat shock in a 42 oC water bath for 90 s. Transformed cells were plated onto an LB selective medium containing 5 mg/mL yeast extract, 10 mg/ml bactotryptone,

10 mg/mL NaCl, 15 mg/mL bactoagar and 50 μg/mL kanamycin. The plates were then incubated at 37 oC for 24 hours. Positive clones were screened by PCR to test whether they contained full length inserts using the insert or gene-specific primers. PCR-verified positive colonies were then selected for overnight cultures in a 37 ºC shaker.

3.2.4.3 Cloning of GFP Constructs

The BnAT2G(1) cDNA was cloned into the pENTR™/TEV/D-TOPO vector

(Invitrogen) according to the manufacturer's instructions (Figure 3.1 A). Successful cloning of the cDNA into this vector generated entry (pENTRY) clones that could subsequently be inserted into pMDC43 (Curtis, 2003) which is a binary destination vector (pDEST) that expresses GFP

(pENTRY+pDEST = expression clone) (Figure 3.1 B). This was performed through an LR reaction in which an LR clonase was used to facilitate the recombination of attL substrate in pENTRY clones with the attR substrate in pMDC43 (pDEST) in order to create a clone that expresses GFP::BnAt2G. Positive pENTRY and expression clones were verified with colony

PCR and sequenced using primers specific to the insert and vectors- pENTR™/TEV/D-TOPO and pMDC43, respectively.

(A) (B)

Figure 3.1 Maps of the vectors used in creating GFP::BnAt2G(1) expression constructs. (A) The pENTR™/TEV/D-TOPO vector was used to generate entry clones containing the BnAt2G(1) cDNA. Positive clones are resistant to kanamycin. (B) The binary destination vector, pMDC43 contain the coding sequence of GFP (yellow highlight). Succesful insertion of the entry clone to pMDC43 results in a GFP::BnAt2G(1) expression construct whose positive clones are resistant to hygromycin.

3.2.4.4 Plasmid Extraction

Plasmids were extracted from 5 ml overnight cultures using PureLink Quick Plasmid

Mini-Prep Kits (Invitrogen), as prescribed by the manufacturer's instructions. Briefly, cells were harvested from overnight LB cultures via centrifugation. Cell pellets were then resuspended with 250 uL of the R3 (resuspension) Buffer+ RNAse A solution and were lysed by adding 250 uL of an alkalinic L7 (lysing) buffer. Plasmid DNA samples were precipitated using silica membrane columns and were eluted in pre-warmed (55-60oC) MBG water. DNA concentrations were measured by Nanodrop.

3.2.4.5 Plasmid DNA Sequencing

The purified plasmids were sequenced using the Applied Biosystems 3730 DNA analyzer (Applied Biosystems) using the BigDye Terminator (Applied Biosystems) label with approximately 30 ng/1 kb of DNA template; 1 µl (l0 µM) of sequencing primer; 2 ul of the 5x sequencing buffer in a total reaction volume of l2 µl. The clones were sequenced in both orientations using forward and reverse M13 primers provided by the TOPO TA and pENTR™/TEV/D-TOPO Cloning Kits.

3.2.5 Agrobacterium and Arabidopsis Transformation

3.2.5.1 Generation of Competent Agrobacterium

Generation of competent Agrobacterium EHA105 strain and its transformation were achieved by adapting the methods established by Hofgen and Willmitzer (1988) and Weigel and

Glazebrook (2002). Briefly, single colonies from plated-EHA105 (rifampicin plates) were inoculated into 50 mL of LB containing rifampicin (25 ug/mL). The cultures were then incubated in a shaker at 28 ºC for 48 hours. 2 mL of the 50 mL culture was then used to inoculate 50 mL of fresh LB media with rifampicin (25 ug/mL) and the culture was incubated in

28 ºC until the OD600 reached between 0.5-1.0. The culture was then chilled on ice for five minutes and was pelleted in a bench top centrifuge at 5000 rpm for 10 minutes in 4 ºC. The supernatant was discarded and the pelleted cells were resuspended with 1 mL of ice-cold CaCl2

(20 mM). 100-150 uL of the cell solution was aliquoted into chilled Eppendorf tubes. These tubes were then flash frozen in liquid nitrogen and were either used immediately for transformation or stored in -80 ºC for two months.

3.2.5.2 EHA105 Transformation and Preparation for Arabidopsis Transformation

One microgram of purified plasmid DNA was added to EHA105 competent cells. The cells were incubated for 5 min on ice and were subsequently frozen in liquid nitrogen for another

5 min. The cells were immediately thawed by incubating them in a 37 ºC water bath for 5 min.

After thawing, 2 mL of fresh LB was added and the culture was incubated at 28 ºC for 4 h with gentle shaking. The cells were pelleted by centrifuging the culture at high speed at room temperature for 2 min. The harvested cells were re-suspended in 100 uL LB medium and dispensed onto LB agar plates containing 50 ug/mL kanamycin and 25 ug/ml rifampicin (for pMDC and pFGC vectors) and were incubated at 28 ºC for 48 h. Positive transformants were verified by colony PCR and were grown in LB media containing appropriate antibiotics in 28 ºC for 48 h.

3.2.5.3 Arabidopsis Transformation

Arabidopsis plants were transformed by the "Floral Dip" method (Clough and Bent, 1998).

Briefly, the bacterial cell suspensions harbouring the vector of interest were poured into two liter disposable plastic autoclave bags containing 120 mL of 5% sucrose solution supplemented with

0.03% Silwet L-77. Inflorescences of healthy Arabidopsis plants were dipped into the bacterial solution for approximately 10 seconds with gentle agitation. Dipped plants were placed sideways under a plastic cover for 24 h to maintain high humidity in growth chambers and were grown normally until seed harvesting. Plants were grown in growth chambers (BioChambers) with a 16 h light and 8 h dark cycle (with a light intensity of 120 µmol/m2sec from white fluorescent tubes) with day and night temperatures of 24 °C and 20 °C, respectively and with a relative humidity of approximately 70%.

3.2.6 In silico analysis

3.2.6.1 Promoter Analysis

For the promoter analysis, sequences 1 kb upstream of the start codon of both the

Arabidopsis and Brassica genes were analyzed using three publicly available databases

(ATHENA: http://www.bioinformatics2.wsu.edu/cgi-bin/Athena/cgi/visualize_select.pl; PLACE: http://www.dna.affrc.go.jp/PLACE/ , AGris (http://arabidopsis.med.ohio-state.edu/AtcisDB/) and

PlantCARE: http://bioinformatics.psb.ugent.be/webtools/plantcare/html/).

3.2.6.2 Protein Structure and Topology Prediction

The secondary and tertiary protein structures for both At2G44500 and BnAt2G(1) were predicted using Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index) and RaptorX

(http://raptorx.uchicago.edu/). Protein Topology was determined using the programs listed on

Table 4.2.

3.2.7 Leaf Protoplast Isolation

Leaf protoplasts were isolated using a modified version of a novel and simple technique called "Tape- Arabidopsis Sandwich" (Wu et al, 2009). Briefly, the adaxial and abaxial surfaces of 10-15 leaves from 3-5 weeks old plants were affixed by a strip of time tape and 3M "Magic" tape, respectively. The lower epidermal layers (in the abaxial surface) were removed by carefully peeling away the Magic tape. The resulting upper epidermis layers from the adaxial surface

(which is still adhered to the time tape), were transferred to petri plates containing 10 mL of the enzyme solution [1% Cellulase 'Onozuka' R10 (Yakult, Japan); 0.25% macerozyme 'Onozuka'

R10 (Yakult, Japan); 0.4 M mannitol; 10 mM CaCl2; 20 mM KCl; 0.1% BSA (Bovine Serum

Albumin); and 20 mM MES (4-Morpholinoethanesulfonic acid hydrate), pH = 5.7].

The peeled leaves were gently shaken at the speed of 80 rpm in dark and in room temperature for 1-2 hours. The enzyme solution containing the released protoplasts were collected and centrifuged at 100 x g for 3 minutes. The supernatant was removed and the resulting pellets were resuspended in an ice-cold W5 solution (150 mM NaCl; 125 mM CaCl2; 5 mM KCl; 5 mM glucose and 2 mM MES, pH = 5.7). After performing the wash procedure twice, the isolated protoplasts were incubated on ice for 30 minutes. After incubation, the protoplasts were centrifuged and resuspended in 1 mL of MMg solution (0.4 mM mannitol; 15 mM MgCl2 and 4 mM MES, pH = 5.7).

3.2.8 BODIPY-TR and Concanavlin A (ConA) Staining

Four-day old roots of transgenic Arabidopsis expressing GFP::BnAt2G(1) were washed in HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer, pH = 7. They were then incubated in 5 µM BODIPY TR Ceramide complexed to BSA (Molecular Probes,

Life Technologies) for 30 minutes at 4 °C. Roots were washed three times with ice-cold HEPES and incubated in fresh medium at 37 °C for 30 minutes in the dark.

Protoplasts were washed in PBS and stained with 100 µg/mL of concanavalin A (conA) conjugated with Texas Red for 30 minutes at room temperature. ConA selectively binds to α- mannopyranosyl and α-glucopyranosyl residues that are commonly found in the ER.

3.2.9 Brefeldin A (BFA) Treatments

Control (wt) and GFP::BnAt2G(1) expressing roots were incubated for 2 hours with 100 ug/mL BFA (Sigma) or water as the control treatment.

3.2.10 Microscopy

GFP::BnAt2G(1) expressing roots were sliced with razor blades and mounted between a slide and cover slip in doubled distilled water. The slides were examined using a Leica DM 6000B

78 microscope connected to a Leica TCS SP5 system equipped with 8 visible laser excitations, a

Radius 405 nm laser and a Chameleon Ultra Infrared Laser. The absorption/emission filters used were 488/509 nm (488 nm argon/krypton laser) for GFP and 589/617 nm (633 nm - helium/neon laser) for BODIPY-TR and 595/615 for conA.

3.3 Results

3.3.1 Isolation of Brassica napus genes that are homologous to a putative Arabidopsis O-

fucosyltransferase, At2G44500

Two genes orthologous to At2G44500, namely BnAt2G(1) and BnAt2G(2), were amplified from B. napus cv Topas by PCR using primers designed from the conserved sequence regions (csr) between genomic regions of At2G44500 and a B. napus EST sequence (accession # ca12f) homologous to At2G44500 (Table 3.1). Two bands (1.25 kb and 1.15 kb) (Figure 3.2 A, lane 2 and 3) were amplified when the Bn-csr1f and At-csr1r primer pair was used. Similarly, when primers designed from the conserved regions between BnAt2G(1) and BnAt2G(2) were used (Bn-csr1f and Bn-csr2r), two bands (1.3 kb and 1.2 kb) were amplified when gDNA was used as template (Figure 3.2 A, lane 3). These bands were cloned and sequenced. Subsequently, gene specific primers were designed from the resulting sequences. Single bands (2 kb and 1.8 kb) were amplified when gene-specific primer pair sets (Bn-pp1f/Bn-pp1r and Bn-pp2f/ Bn-pp2r, respectively) were used in the PCR reactions with gDNA as a template (Figure 3.2 A, lanes 4 and

6). The first copy was annotated as BnAt2G(1) and was amplified using primer pairs, Bn-pp1f and

Bn-pp1r from leaf gDNA (Figure 3.2 A, lane 4). The BnAt2G(1) coding region (1.5 kb) was also amplified using Bn-pp1f and Bn-pp1r using leaf cDNA as a template (Figure 3.2 A, lane 5). The second genomic fragment amplified by Bn-pp2f/ Bn-pp2r was annotated as BnAt2G(2). Several attempts to amplify the coding region of this particular copy using gene specific primer pair sets

(such as Bn-pp2f/ Bn-pp2r) from leaf cDNA did not yield any results (Figure 3.2 A, lane 7). The genomic fragments, BnAt2G(1) and BnAt2G(2), were approximately 85% and 84% homologous to At2G44500, respectively.

(A)

(B)

Figure 3.2 Amplification of B.napus genes homologous to At2G44500. (A) PCR gel showing amplified bands using primers designed from conserved sequence regions (Bn-csr) and gene specific primer pairs (Bn-pp1f/Bn-pp1r; Bn-pp2f/Bnpp2r). (B) Overview of the multiple sequence alignment (MSA) of At2G44500 coding sequence (CDS) and genomic DNA sequence (gDNA) of its B. napus homologs. Arrow heads indicate the different primer positions within the gene sequences. Bar graph indicates the level of sequence conservation. Numbers on the left side indicate sequence lengths.

Table 3.1 - Primers used to amplify BnAt2G(1) and BnAt2g(2) Primer Name Sequence Tm (˚C) Bn-pp1f AACATGGCGTTACCCAAGAA 56 Bn-pp1r CACTCACTCTTTGAGACTTA 54 Bn-pp2f ATGCTAAAACTCGGACTCAA 55 Bn-pp2r TTATTGTTCTTGTTGTTGTTGC 54 Bn-cs1f CTCAAAAAACGACACGCA 54

At-cs1r CTTATCACGAGAGAAAACT 54

Bn-cs2r GAGCGGTGAGAGGACAAA 56

The following sequences were examined using multiple sequence alignment (Figure 3.3

A): At2G44500 coding sequence; isolated gDNA sequences of BnAt2G(1) and BnAt2g(2); and the gDNA sequences from the reference genomes, BnA05 (BnaA0504070D) and BnC04

(BnaC04g03810D). The alignments show that BnAt2G(2) and BnA05 do not contain an ATG

(Methionine) at the position that corresponds to the translation start site in BnAt2G(1) and BnC04, but codons for Asparagine (AAT) (Figure 3.3 A, grey highlight). This suggests that BnAt2G(2) may not be a functional gene.

Figure 3.3 B shows the alignment of cDNA sequences derived from the 5' and 3' RACE reactions with the isolated gDNA sequence of BnAt2g(1); the sequence from the reference genome, annotated as BnaC04 and the complete mRNA sequence assembled by G-Mo.R-Se

(Gene Modelling Using RNA-Seq - http://www.genoscope.cns.fr/externe/gmorse/). G-Mo.R-Se uses short RNA-seq reads to generate de novo gene models by building candidate exons from the positions of reads that were mapped on the reference genome. Thus, the resulting gene structure is based on the information from the RNA-seq database rather than a priori knowledge about the gene structure. Based on the alignment, the gene structure of BnAt2G(1) was found to be similar to At2G44500, which consists of three exons and two introns. However, attempts to amplify the 5' and 3' RACE cDNAs for BnAt2G(2), based on the primers designed from its putative start site

(Bn-pp2f and Bn-pp2r) failed to produce amplicons (data not shown). Also, based on the reference genome database, G-Mo.R-Se failed to assemble a complete mRNA sequence and gene model for BnAt2G(2) (not shown).

(A) At2G44500-CDS ------BnAt2G(1) ------TTTTTTTATTTTCTGCCTTCTTCATCG BnaC04 CGCCCACCTCTCACTTTTTCTCCCTCTCACACGTTTTTTTATTTTCTGCCTTCTTCATCG BnAt2G(2) ------ATAGGTTGATGTAATTAT BnA05 ------ATAGGTTGATGTAATTAT

At2G44500-CDS ------BnAt2G(1) CCGGAGACTTTTGACTATTCACTTTCTTTAAACCTCTCTTTCTCCATTACCCAA--ACCA BnaC04 CCGGAGACTTTTGACTATTCACTTTCTTTAAACCTCTCTTTCTCCATTACCCAA--ACCA BnAt2G(2) TCGGAATGGTTCCACTAGTCGAAGCGATGTAATCACTGTAGAATAACTGAATAAGTATGG BnA05 TCGGAATGGTTCCACTAGTCGAAGCGATGTAATCACTGTAGAATAACTGAATAAGTATGG

Bn-pp1f At2G44500-CDS ------ATGGCGTTATCGAAAAACAGTAA

BnAt2G(1) CAAGATCTCTCTCTCTCTCTCTCTTCTTTCTTCGAACATGGCGTTACCCAAGAACGGTGG BnaC04 CAAGATCTCTCTCTCTCTCTCTCTTCTTTCTTCGAACATGGCGTTACCCAAGAACGGTGG BnAt2G(2) TCGTGCAATCAAACCAATCGTTGTGATAGAATTGAGTAATGAAATGAAATTGTTAGTGTG BnA05 TCGTGCAATCAAACCAATCGTTGTGATAGAATTGAGTAATGAAATGAAATTGTTAGTGTG * * *

At2G44500-CDS CAGTAACAGCTTTAACAAGAAGAAAGTTTCGTATATCTCAGTCCCTTCTCAGATCATAAA BnAt2G(1) TAACAGCAGCAGCACCAAGAAGAAAGTTTCCTACATCTCAGTCCCTTCTCAGATCATAAA BnaC04 TAACAGCAGCAGCACCAAGAAGAAAGTTTCCTACATCTCAGTCCCTTCTCAGATCATAAA BnAt2G(2) TAACAGCAGCAGCACCAAGAAGAAAGTCTCCTACATCTCAGTCCCTTCTCAGATCATAAA BnA05 TAACAGCAGCAGCACCAAGAAGAAAGTCTCCTACATCTCAGTCCCTTCTCAGATCATAAA * * **** * ************ ** ** **************************

At2G44500-CDS CTCTTTATCATCTTCCTCATTACAATCTCTTCTTGTTTCTCCCAAGAAATCATCAAGAAG BnAt2G(1) CTCTCTATCTTCCTCCTCTCTACAATCCCTCCTCGTCTCCCCCAAGAAGTCATCGAGATG BnaC04 CTCTCTATCTTCCTCCTCTCTACAATCCCTCCTCGTCTCCCCCAAGAAGTCATCGAGATG BnAt2G(2) CTCTCTATCTTCCTCCTCCTTACAATCCCTCCTCGTCTCCCCCAAGAAGTCATCGAGATG BnA05 CTCTCTATCTTCCTCCTCCTTACAATCCCTCCTCGTCTCCCCCAAGAAGTCATCGAGATG **** **** ** ***** ******* ** ** ** ** ******** ***** *** *

At2G44500-CDS CACCAACAGATTCAGCTTCTCTTATAGGAACCCTAGAATCTGGTTCTTTACTCTCTTCCT BnAt2G(1) CACCAACAGGTTCAG------CTACCGAAACCCAAGAATCTGGTTCTTGACTCTCTTCCT BnaC04 CACCAACAGGTTCAG------CTACCGAAACCCAAGAATCTGGTTCTTGACTCTCTTCCT BnAt2G(2) CACCAACAGGTTCAC------CTACCGAAACCCAAGAATCTGGTTCTTGACTCTCTTCCT BnA05 CACCAACAGGTTCAC------CTACCGAAACCCAAGAATCTGGTTCTTGACTCTCTTCCT ********* **** ** * ***** ************** ***********

At2G44500-CDS CGTCTCACTCTTTGGAATGTTGAAACTCGGTTTCAACGTTGACCCAATCTCTCTCCCTTT BnAt2G(1) CGTCTCACTCTTCGGCATGCTGAAACTCGGACTCAACGTTGACCCGATCTCCCTCCCTTT BnaC04 CGTCTCACTCTTCGGCATGCTGAAACTCGGACTCAACGTTGACCCGATCTCCCTCCCTTT

Bn-pp2f BnAt2G(2) CGTCTCACTCTTCGGCATGCTAAAACTCGGACTCAACGTTGACCCGATCTCCCTCCCTTT BnA05 CGTCTCACTCTTCGGCATGCTAAAACTCGGACTCAACGTTGACCCGATCTCCCTCCCTTT ************ ** *** * ******** ************* ***** ********

At2G44500-CDS CTCACGTTACCCTTGTTCAACGACCCAACAACCACTAAGCTTCGACGGAGAACAAAACGC BnAt2G(1) CTCACGTTACCCTTGCTCCACGGGTAGCTTCGACGAACACCACGCCGTTTCCCATCTCGC BnaC04 CTCACGTTACCCTTGCTCCACGGGTAGCTTCGACGAACACCACGCCGTTTCCCATCTCGC BnAt2G(2) CTCACGTCACCCTTGCTCCACGGGTAGCTTCGACGAACACCACGCCGTTTCCCATCTCGC BnA05 CTCACGTCACCCTTGCTCCACGGGTAGCTTCGACGAACACCACGCCGTTTCCCATCTCGC ******* ******* ** *** * * ** ** ** ***

At2G44500-CDS TGCATCCCATCTTGGTTTGGCTCAAGAGCCGATTTTGTCTACTGGGTCATCGAATTCGAA

Bn-csf1 BnAt2G(1) TTTCGC------CTCAAAAAACGACACGCA BnaC04 TTTCGC------CTCAAAAAACGACACGCA BnAt2G(2) TTTCGC------TTCAGAAAACGACACGCA BnA05 TTTCGC------TTCAGAAAACGACACGCA * * * * ** *

At2G44500-CDS TGCGATTATACAATTAAACGGTGGAAAGAACGAGACTTTGTTAACGGAAGGTGATTTCTG BnAt2G(1) GTCCAGTTCGTCTTCTGAGCATCGAAAGAACGAGACTTTGCCCACGGAAGGCGACTTCTG BnaC04 GTCCAGTTCGTCTTCTGAGCATCGAAAGAACGAGACTTTGCCCACGGAAGGCGACTTCTG BnAt2G(2) GTCCAACTCGTCTTCTGTGTATCCAAAGAACGAGACTTTGCCCACGGAAGGTGACTTCTG BnA05 GTCCAACTCGTCTTCTGTGTATCCAAAGAACGAGACTTTGCCCACGGAAGGTGACTTCTG * * * * **************** ******** ** *****

At2G44500-CDS GAAGCAACCTGATGGGTTAGGGTTTAAGCCCTGTTTGGGATTTACAAGTCAATATAGAAA BnAt2G(1) GAAGCAACCTGATGGGCTAGGGTTTAAGCCGTGTCTTGGATTCAGCAGACAGTATAGAAA BnaC04 GAAGCAACCTGATGGGCTAGGGTTTAAGCCGTGTCTTGGATTCAGCAGACAGTATAGAAA BnAt2G(2) GAAGCAACCTGATGGGTTAGGGTTTAAGCCGTGTCTTGGGTTCAGCAGACAGTATCGAAA BnA05 GAAGCAACCTGATGGGTTAGGGTTTAAGCCGTGTCTTGGGTTCAGCAGACAGTATCGAAA **************** ************* *** * ** ** * ** ** *** ****

At2G44500-CDS AGATAGCAATTCGATTTTGAAGAACAGATGGAAGTATTTGCTTGTTGTTGTCTCTGGTGG BnAt2G(1) AGACAGCAACTCGATTCTCAAGAACAGGTGGAAGTATCTTCTCGTTGTTGTCGCCGGCGG BnaC04 AGACAGCAACTCGATTCTCAAGAACAGGTGGAAGTATCTTCTCGTTGTTGTCGCCGGTGG BnAt2G(2) AGACAGCAACTCGATTCTCAAGAACAGGTGGAAGTATCTTCTCGTTGTTGTCGCTGGTGG BnA05 AGACAGCAACTCGATTCTCAAGAACAGGTGGAAGTATCTTCTCGTTGTTGTCGCTGGTGG *** ***** ****** * ******** ********* * ** ********* * ** **

At2G44500-CDS GATGAATCAGCAAAGGAATCAGATTGTTGATGCTGTTGTGATCGCTAGGATCCTTGGAGC BnAt2G(1) GATGAATCAGCAGAGGAACCAGATCGTTGATGCAGTGATCATGGCTAGGATCCTTGGTGC BnaC04 GATGAATCAGCAGAGGAACCAGATCGTTGATGCAGTGATCATGGCTAGGATCCTTGGTGC BnAt2G(2) GCTGAATCAGCAGAGGAACCAGATCGTTGATGCAGTGATCATCGCTAGGATCCTTGGTGC BnA05 GCTGAATCAGCAGAGGAACCAGATCGTTGATGCAGTGATCATCGCTAGGATCCTTGGTGC * ********** ***** ***** ******** ** * ** ************** **

At2G44500-CDS TTCTTTAGTTGTTCCTGTTTTGCAGGTTAATGTCATTTGGGGAGACGAA------BnAt2G(1) GTCTCTCGTTGTCCCTGTTCTGCAAGTCAACGTCATCTGGGGAGACGAAaggtatata-- BnaC04 GTCTCTCGTTGTCCCTGTTCTGCAAGTCAACGTCATCTGGGGAGACGAAaggtatata-- BnAt2G(2) TTCTCTCGTTGTACCTGTTCTGCAAGTCAACGTCATCTGGGGAGACGAAaggtatatata BnA05 TTCTCTCGTTGTACCTGTTCTGCAAGTCAACGTCATCTGGGGAGACGAAaggtatatata *** * ***** ****** **** ** ** ***** ************

At2G44500-CDS ------BnAt2G(1) --acataagctatttaatattcatagttaatgttatgtgacatttgttgattataattgt BnaC04 --acataagctatttaatattcatagttaatgttatgtgacatttgttgattataattgt BnAt2G(2) acacataagctctttaatattcatatttaatgttatgtgacatttgctgattacaattgt BnA05 acacataagctctttaatattcatatttaatgttatgtgacatttgctgattacaattgt

At2G44500-CDS ------AGTGAATTTGCGGATATATTCGATTTGGAGCATTTCAAGGATGtuTTT BnAt2G(1) -GtgtttcaattatAGTGAGTTTGCGGATATATTCGATTTGGAGCATTTCAAGAACGTTT BnaC04 -gtgtttcaattatAGTGAGTTTGCGGATATATTCGATTTGGAGCATTTCAAGAACGTTT BnAt2G(2) aatgtttgaattatAGTGAGTTTGCGGATATATTCGATTTGGAGCATTTCAAGAACGTTT BnA05 aatgtttgaattatAGTGAGTTTGCGGATATATTCGATTTGGAGCATTTCAAGAACGTTT ***** ********************************* * ****

At2G44500-CDS TAGCAGATGATGTTCATATAGTTTCGTCTTTACCTTCTACACATGTTATGACGAGACCTG BnAt2G(1) TAGCCGATGATGTTCATATAGTTTCGTCTTTACCTTCAACACATGTAATGACGAGACCTG BnaC04 TAGCCGATGATGTTCATATAGTTTCGTCTTTACCTTCAACACATGTAATGACGAGACCTG BnAt2G(2) TAGCGGATGATGTTCATATAGTTTCGTCTTTACCTTCAACACATGTAATGACGAGACCTG BnA05 TAGCGGATGATGTTCATATAGTTTCGTCTTTACCTTCAACACATGTAATGACGAGACCTG **** ******************************** ******** *************

At2G44500-CDS TTGAAGAGAAGAGGACTCCACTTCATGCTTCTCCTCAATGGATTCGTGCTCATTATCTTA BnAt2G(1) CGGAAGAGAAAAGGACTCCACTTCACGCTTCTCCTCAATGGATTCGTGCTCATTACCTCA BnaC04 TGGAAGAGAAAAGGACTCCACTTCACGCTTCTCCTCAATGGATTCGTGCTCATTACCTCA BnAt2G(2) TGGAAGAGAAAAGGACTCCCCTTCACGCTTCTCCTCAATGGATTCGCGCTCATTACCTCA BnA05 TGGAAGAGAAAAGGACTCCCCTTCACGCTTCTCCTCAATGGATTCGCGCTCATTACCTCA ******** ******** ***** ******************** ******** ** *

At2G44500-CDS AG------BnAt2G(1) AGcgagtaagtactctgtttctttactctgttttattaagctaactctgtttcctctgtt BnaC04 AGcgagtaagtactctgtttctttactctgttttattaagctaactctgtttcctctgtt BnAt2G(2) AGcgagtaagtactctgtttctttgctctgttttattaagctaactctgtttcctctgtt BnA05 AGcgagtaagtactctgtttctttgctctgttttattaagctaactctgtttcctctgtt **

At2G44500-CDS ------BnAt2G(1) tcttcgccctgtttattaagctaactctgtttattaagctaactctgtttcctctgtt-- BnaC04 tcttcgccctgtttattaagctaactctgtttattaagctaactctgtttcctctgtt-- BnAt2G(2) tctttgctctgttttattaagctaactctgtttccattgtttcttcgctttgtttattaa BnA05 tctttgctctgttttattaagctaactctgtttccattgtttcttcgctttgtttattaa

At2G44500-CDS ------BnAt2G(1) ------tct------BnaC04 ------tct------BnAt2G(2) gctaactctgtttcttcgctctgtttattaagctaactctgtttcttcgctctgtttatt BnA05 gctaactctgtttcttcgctctgtttattaagctaactctgtttcttcgctctgtttatt

At2G44500-CDS ------BnAt2G(1) ------tcgctctgttttattaagccaactctgtttcctctgtttctttg BnaC04 ------tcgctctgttttattaagccaactctgtttcctctgtttctttg BnAt2G(2) aagctaactctgtttcttcactctgtttactaagctaactctgtttcctctgtttctgtg BnA05 aagctaactctgtttcttcactctgtttactaagctaactctgtttcctctgtttctgtg

At2G44500-CDS ------CGAATTAACAGAGAAAGAG BnAt2G(1) ctct--gttttgtcaagctaactctgtttcctctgtttctgCAGATTAACAGAGAAAGAG BnaC04 ctct--gttttgtcaagctaactctgtttcctctgtttctgCAGATTAACAGAGAAAGAG BnAt2G(2) ctctgtttttattaagctaactctgttttcctctgtttctgCAGATAAACAGAGAAAGAG BnA05 ctctgtttttattaagctaactctgttttcctctgtttctgCAGATAAACAGAGAAAGAG * ** *************

At2G44500-CDS TTTTACTTCTCCGTGGACTCGATTCAAGACTCTCCAAAGACCTCCCTTCAGATCTTCAGA BnAt2G(1) TGTTACTTCTCCGTGGCCTCGATTCAAGGCTCTCCAACGACCTTCCTTCTGATCTCCAGA BnaC04 TGTTACTTCTCCGTGGCCTCGATTCAAGGCTCTCCAACGACCTTCCTTCTGATCTCCAGA BnAt2G(2) TGTTACTTCTCCGTGGCCTCGATTCAAGGCTCTCCAACGATCTTCCTTCTGATCTCCAGA BnA05 TGTTACTTCTCCGTGGCCTCGATTCAAGGCTCTCCAACGATCTTCCTTCTGATCTCCAGA * ************** *********** ******** ** ** ***** ***** ****

At2G44500-CDS AACTCCGATGCAAGGTTGCTTTCCAAGCGCTTAGATTTTCTCCGAGGATTCTTGAACTCG BnAt2G(1) AACTCCGATGCAAGGTAGCTTCCCAAGCGCTGAGATTCTCACCGAGGATACTCGAACTCG BnaC04 AACTCCGATGCAAGGTAGCTTTCCAAGCGCTGAGATTCTCACCGAGGATACTCGAACTCG BnAt2G(2) AACTCCGATGCAAGGTAGCTTTCCAAGCTTTGAGATTCTCACCGAGAATCCTCGAACTCG BnA05 AACTCCGATGCAAGGTAGCTTTCCAAGCTTTGAGATTCTCACCGAGAATCCTCGAACTCG **************** **** ****** * ***** ** ***** ** ** *******

At2G44500-CDS GTAATAAACTAGCTTCAAGAATGCGTAACCAAGGACAGTATCTCTCACTTCATCTAAGAA BnAt2G(1) GCAACAAGCTAGCTTCGAGAATGCTTAGCGAAGGACAGTACCTCTCACTCCATCTACGTA BnaC04 GCAACAAGCTAGCTTCGAGAATGCTTAGCGAAGGACAGTACCTCTCACTCCATCTACGTA BnAt2G(2) GCAACAAGCTAGCTTCGAGAATGCTTAGCGAAGGACAGTACCTCTCACTCCATCTACGTA BnA05 GCAACAAGCTAGCTTCGAGAATGCTTAGCGAAGGACAGTACCTCTCACTCCATCTACGTA * ** ** ******** ******* ** * ********** ******** ****** * *

At2G44500-CDS TGGAGAAAGATGTTTGGGTTAGAACTGGTTGTCTCCCTGGTTTAACTCCCGAGTACGATG BnAt2G(1) TGGAGAAAGACGTTTGGGTCAGAACCGGTAGTCTCCCCGGTTTAACCCCCGAGTACGACG BnaC04 TGGAGAAAGACGTTTGGGTCAGAACCGGTTGTCTCCCCGGTTTAACCCCCGAGTACGACG BnAt2G(2) TGGAGAAAGACGTTTGGGTCAGAACCGGTTGTCTCCCCGGTTTAACCCCCGAGTACGACG BnA05 TGGAGAAAGACGTTTGGGTCAGAACCGGTTGTCTCCCCGGTTTAACCCCCGAGTACGACG ********** ******** ***** *** ******* ******** *********** *

At2G44500-CDS AAATAGTCAACAGCGAAAGAGAACGACACCCTGAGCTTCTTACCGGTAGATCAAACATGA BnAt2G(1) AGATAGTCAACAGCGAGAGACAACGCCACCCGGAGCTCCTCACGGGCCGTTCAAACATGA BnaC04 AGATAGTCAACAGCGAGAGACAACGCCACCCGGAGCTTCTCACGGGCCGTTCAAACATGA BnAt2G(2) AGATAGTCAACAGCGAGAGACAACGCCACCCGGAGCTTCTCACCGGCCGTTCAAACATGA BnA05 AGATAGTCAACAGCGAGAGACAACGCCACCCGGAGCTTCTCACCGGCCGTTCAAACATGA * ************** *** **** ***** ***** ** ** ** * **********

At-csr1 At2G44500-CDS CTTATCACGAGAGAAAACTTGCCGGTTTATGCCCTTTAACTGCTCTAGAAGTCACAAGGC BnAt2G(1) CTTACAACGAGAGAAAACTCGCTGGGCTTTGTCCTTTAACCGCTCTAGAAGTCACGAGGC BnaC04 CTTACAACGAGAGAAAACTCGCTGGGCTTTGTCCTTTAACCGCTCTAGAAGTCACGAGGC

Bn-csr2 BnAt2G(2) CTTACAACGAGAGAAAACTCGCTGGCCTTTGTCCTCTCACCGCTCTAGAAGTCACGAGGC BnA05 CTTACAACGAGAGAAAACTCGCTGGCCTTTGTCCTCTCACCGCTCTAGAAGTCACGAGGC **** ************* ** ** * ** *** * ** ************** ****

At2G44500-CDS TGCTTAAAGCATTAGAAGCTCCAAAGGATGCGAGAATCTATTGGGCAGGAGGAGAGCCTC BnAt2G(1) TGCTTAAGGCCTTAGAGGCACCAAAGGACGCGAGAATCTACTGGGCGGGAGGAGAGCCTC BnaC04 TGCTTAAGGCCTTAGAGGCACCAAAGGACGCGAGAATCTACTGGGCGGGAGGAGAGCCTC BnAt2G(2) TTCTCAAAGCCTTGGAAGCACCGAAGGACGCGAGAATCTACTGGGCGGGAGGAGAGCCTC BnA05 TTCTCAAAGCCTTGGAAGCACCGAAGGACGCGAGAATCTACTGGGCGGGAGGAGAGCCTC * ** ** ** ** ** ** ** ***** *********** ***** *************

At2G44500-CDS TAGGAGGAAAAGAGGTTTTGGAGCCACTAACTAAAGAATTCCCTCAGTTCTACAACAAGC BnAt2G(1) TAGGTGGGAAAGAGGCTTTGGAGCCGTTGACCAAAGAGTTCCCTCACCTCTACAACAAAC BnaC04 TAGGTGGGAAAGAGGCTTTGGAGCCGTTGACCAAAGAGTTCCCTCACCTCTACAACAAAC BnAt2G(2) TAGGTGGGAAAGAGGCTTTGGAGCCGTTGACCAAAGAGTTCCCTCACCTCTACAACAAAC BnA05 TAGGTGGGAAAGAGGCTTTGGAGCCGTTGACCAAAGAGTTCCCTCACCTCTACAACAAAC **** ** ******* ********* * ** ***** ******** ********** *

At2G44500-CDS ACGATCTTGCTTTACCTGGCGAGCTTGAACCATTTGCCAACAAAGCTTCGGTTATGGCTG BnAt2G(1) ACGATCTCGCGTTACCCGGCGAGCTCGAACCCTTCGCCAAGAAAGCCTCGGTCATGGCTG BnaC04 ACGATCTCGCGTTACCCGGCGAGCTCGAACCCTTCGCCAAGAAAGCCTCGGTCATGGCTG BnAt2G(2) ACGATCTCGCGTTACCCGGCGAGCTAGAACCCTTCGCCAAGAAAGCCTCGGTCATGGCTG BnA05 ACGATCTCGCGTTACCCGGCGAGCTAGAACCCTTCGCCAAGAAAGCCTCGGTCATGGCTG ******* ** ***** ******** ***** ** ***** ***** ***** *******

At2G44500-CDS CTATAGACTACATTGTCTGCGAAAAGAGCGATGTTTTCATACCGTCTCATGGAGGAAATA BnAt2G(1) CGATAGACTACATCGTCTGCGAGAAGAGCGATGTCTTCATACCGTCTCACGGCGGGAACA BnaC04 CGATAGACTACATCGTCTGCGAGAAGAGCGATGTCTTCATACCGTCTCACGGCGGGAACA BnAt2G(2) CGATAGACTACATCGTCTGCGAGAAGAGCGACGTCTTCATACCGTCGCACGGTGGGAACA BnA05 CGATAGACTACATCGTCTGCGAGAAGAGCGACGTCTTCATACCGTCGCACGGTGGGAACA * *********** ******** ******** ** *********** ** ** ** ** *

At2G44500-CDS TGGGACACGCGTTACAAGGGCAAAGAGCTTACGCTGGTCACAAGAAATACATCACTCCTA BnAt2G(1) TGGGACACGCGTTGCAAGGGCAGAGAGCTTACGCTGGTCACAAGAAGTACATCACTCCTA BnaC04 TGGGACACGCGTTGCAAGGGCAGAGAGCTTACGCTGGTCACAAGAAGTACATCACTCCTA BnAt2G(2) TGGGACACGCGTTGCAAGGGCAGAGAGCTTACGCTGGTCACAAGAAGTACATTACGCCTA BnA05 TGGGACACGCGTTGCAAGGGCAGAGAGCTTACGCTGGTCACAAGAAGTACATTACGCCTA ************* ******** *********************** ***** ** ****

At2G44500-CDS ATAAGAGACAGATGCTTCCTTATTTCATGAACTCGTCGCTTCCTGAATCGGATTTTAACA BnAt2G(1) ACAAGAGACATATGCTTCCTTACTTCATGAATGCCTCTCTGCCTGAATCGGAGTTTAATA BnaC04 ACAAGAGACATATGCTTCCTTACTTCATGAATGCCTCTCTGCCTGAATCGGAGTTTAATA BnAt2G(2) ACAAGAGACATATGCTTCTTTACTTCATGAATGCTTCGCTGCCTGAGTCGGAGTTTAACA BnA05 ACAAGAGACATATGCTTCTTTACTTCATGAATGCTTCGCTGCCTGAGTCGGAGTTTAACA * ******** ******* *** ******** * ** ** ***** ***** ***** *

At2G44500-CDS GAATTGTGAAAGATCTTCATCGAGAATCGCTAGGACAGCCGGAGTTGAGAATGAGCAAAG BnAt2G(1) GGATTGTGAAGGATTTTCATCGAGAGTCTTTGGGGCAGCCGGAGTTGAGAACGGGTAGAG BnaC04 GGATTGTGAAGGATTTTCATCGAGAGTCTTTGGGGCAGCCGGAGTTGAGAACGGGTAGAG BnAt2G(2) GGATTGTGAAGGATCTTCATCGAGAGTCTTTGGGACAACCGGTGTTGAGAACGGGTAGAG BnA05 GGATTGTGAAGGATCTTCATCGAGAGTCTTTGGGACAACCGGTGTTGAGAACGGGTAGAG * ******** *** ********** ** * ** ** **** ******** * * * **

At2G44500-CDS CTGGTAAAGATGTTACAAAACATCCTGTTCCAGAGTGTATGTGCTCGGATAGACAACAAC BnAt2G(1) GTGGGAAGGATGTTACTAAGCATCCTGTTTCAGAGTGTATGTGTTCAGATAGACGACAAC BnaC04 GTGGTAAGGATGTTACTAAGCATCCTGTTTCAGAGTGTATGTGTTCAGATAGACGACAAC BnAt2G(2) GTGGGAAGGATGTTACTAAGCATCCTGTTTCAGAGTGTATGTGTTCAGATAGACAGCAAC BnA05 GTGGGAAGGATGTTACTAAGCATCCTGTTTCAGAGTGTATGTGTTCAGATAGACAGCAAC *** ** ******** ** ********* ************* ** ******* ****

At2G44500-CDS AAGAACAACAGTCCGATGCTTAA------

Bn-pp1r BnAt2G(1) AACAACAATAAGTCTCAAAGAGTGAGTGGTTCGTGCAGAGTATTCTTTTAGTTCTTTTTT BnaC04 AACAACAATAAGTCTCAAAGAGTGAGTGGTTCGTGCAGAGTATTCTTTTAGTTCTTTTTT

Bn-pp2r BnAt2G(2) AACAACAAGAACAATAAGTCTTGAAGAGTGGTTGGTTCGTGCAGAAAGGGC------BnA05 AACAACAAGAACAATAAGTCTTGAAGAGTGGTTCGTAGTATTCTTTTAGTTCTTTTTTTT ** ***** *

At2G44500-CDS ------BnAt2G(1) TTTGGCCTTGATTTGGAGTATATATTATGCGTTTGTATTCTTTCAAGTGAGGTTATTTAC BnaC04 TTTGGCCTTGATTTGGAGTATATATTATGCGTTTGTATTCTTTCAAGTGAGGTTATTTAC BnAt2G(2) ------BnA05 T------

At2G44500-CDS ------BnAt2G(1) GTGGGGGAAGTAGAATAGGGATGAGATTATGATATGATTCTTTAATTTGATTTATTTGAA BnaC04 GTGGGGGAAGTAGAATAGGGATGAGATTATGATATGATTCTTTAATTTGATTTATTTGAA BnAt2G(2) ------BnA05 ------

At2G44500-CDS ------BnAt2G(1) ACTGTAATTATATATTACAGTGTTTGTATACGGTTATACTTGTACCATACATAAATAAAA BnaC04 ACTGTAATTATATATTACAGTGTTTGTATACGGTTATACTTGTACCATACATAAATAAAA BnAt2G(2) ------BnA05 ------

At2G44500-CDS ------BnAt2G(1) AGCA------BnaC04 AGCAACATTTG BnAt2G(2) ------BnA05 ------

5'+3'RACE-cDNA ------(B) BnAt2G(1)_gDNA ------TTTTTTTATTTTCTGCCTTCTTCATCG mRNA_gMORSE_943.4223.1 CGCCCACCTCTCACTTTTTCTCCCTCTCACACGTTTTTTTATTTTCTGCCTTCTTCATCG BnaC04-ref CGCCCACCTCTCACTTTTTCTCCCTCTCACACGTTTTTTTATTTTCTGCCTTCTTCATCG

5'+3'RACE-cDNA ------ACTTTCTTTAAACCTCTCTTTCTCCATTACCCAAACCACA BnAt2G(1)_gDNA CCGGAGACTTTTGACTATTCACTTTCTTTAAACCTCTCTTTCTCCATTACCCAAACCACA mRNA_gMORSE_943.4223.1 CCGGAGACTTTTGACTATTCACTTTCTTTAAACCTCTCTTTCTCCATTACCCAAACCACA BnaC04-ref CCGGAGACTTTTGACTATTCACTTTCTTTAAACCTCTCTTTCTCCATTACCCAAACCACA **************************************** 5'+3'RACE-cDNA AGATCTCTCTCTCTCTCTCTCTTCTTTCTTCGAACATGGCGTTACCCAAGAACGGTGGTA BnAt2G(1)_gDNA AGATCTCTCTCTCTCTCTCTCTTCTTTCTTCGAACATGGCGTTACCCAAGAACGGTGGTA mRNA_gMORSE_943.4223.1 AGATCTCTCTCTCTCTCTCTCTTCTTTCTTCGAACATGGCGTTACCCAAGAACGGTGGTA BnaC04-ref AGATCTCTCTCTCTCTCTCTCTTCTTTCTTCGAACATGGCGTTACCCAAGAACGGTGGTA ************************************************************ 5'+3'RACE-cDNA ACAGCAGCAGCACCAAGAAGAAAGTTTCCTACATCTCAGTCCCTTCTCAGATCATAAACT BnAt2G(1)_gDNA ACAGCAGCAGCACCAAGAAGAAAGTTTCCTACATCTCAGTCCCTTCTCAGATCATAAACT mRNA_gMORSE_943.4223.1 ACAGCAGCAGCACCAAGAAGAAAGTTTCCTACATCTCAGTCCCTTCTCAGATCATAAACT BnaC04-ref ACAGCAGCAGCACCAAGAAGAAAGTTTCCTACATCTCAGTCCCTTCTCAGATCATAAACT ************************************************************ 5'+3'RACE-cDNA CTCTATCTTCCTCCTCTCTACAATCCCTCCTCGTCTCCCCCAAGAAGTCATCGAGATGCA BnAt2G(1)_gDNA CTCTATCTTCCTCCTCTCTACAATCCCTCCTCGTCTCCCCCAAGAAGTCATCGAGATGCA mRNA_gMORSE_943.4223.1 CTCTATCTTCCTCCTCTCTACAATCCCTCCTCGTCTCCCCCAAGAAGTCATCGAGATGCA BnaC04-ref CTCTATCTTCCTCCTCTCTACAATCCCTCCTCGTCTCCCCCAAGAAGTCATCGAGATGCA ************************************************************ 5'+3'RACE-cDNA CCAACAGGTTCAGCTACCGAAACCCAAGAATCTGGTTCTTGACTCTCTTCCTCGTCTCAC BnAt2G(1)_gDNA CCAACAGGTTCAGCTACCGAAACCCAAGAATCTGGTTCTTGACTCTCTTCCTCGTCTCAC mRNA_gMORSE_943.4223.1 CCAACAGGTTCAGCTACCGAAACCCAAGAATCTGGTTCTTGACTCTCTTCCTCGTCTCAC BnaC04-ref CCAACAGGTTCAGCTACCGAAACCCAAGAATCTGGTTCTTGACTCTCTTCCTCGTCTCAC ************************************************************ 5'+3'RACE-cDNA TCTTCGGCATGCTGAAACTCGGACTCAACGTTGACCCGATCTCCCTCCCTTTCTCACGTC BnAt2G(1)_gDNA TCTTCGGCATGCTGAAACTCGGACTCAACGTTGACCCGATCTCCCTCCCTTTCTCACGTT mRNA_gMORSE_943.4223.1 TCTTCGGCATGCTGAAACTCGGACTCAACGTTGACCCGATCTCCCTCCCTTTCTCACGTT BnaC04-ref TCTTCGGCATGCTGAAACTCGGACTCAACGTTGACCCGATCTCCCTCCCTTTCTCACGTT *********************************************************** 5'+3'RACE-cDNA ACCCTTGCTCCACGGGTAGCTTCGACGAACACCACGCCGTTTCCCATCTCGCTTTCGCCT BnAt2G(1)_gDNA ACCCTTGCTCCACGGGTAGCTTCGACGAACACCACGCCGTTTCCCATCTCGCTTTCGCCT mRNA_gMORSE_943.4223.1 ACCCTTGCTCCACGGGTAGCTTCGACGAACACCACGCCGTTTCCCATCTCGCTTTCGCCT BnaC04-ref ACCCTTGCTCCACGGGTAGCTTCGACGAACACCACGCCGTTTCCCATCTCGCTTTCGCCT ************************************************************ 5'+3'RACE-cDNA CAAAAAACGACACGCAGTCCAGTTCGTCTTCTGAGCATCGAAAGAACGAGACTTTGCCCA BnAt2G(1)_gDNA CAAAAAACGACACGCAGTCCAGTTCGTCTTCTGAGCATCGAAAGAACGAGACTTTGCCCA mRNA_gMORSE_943.4223.1 CAAAAAACGACACGCAGTCCAGTTCGTCTTCTGAGCATCGAAAGAACGAGACTTTGCCCA BnaC04-ref CAAAAAACGACACGCAGTCCAGTTCGTCTTCTGAGCATCGAAAGAACGAGACTTTGCCCA ************************************************************ 5'+3'RACE-cDNA CGGAAGGCGACTTCTGGAAGCAACCTGATGGGCTAGGGTTTAAGCCGTGTCTTGGATTCA BnAt2G(1)_gDNA CGGAAGGCGACTTCTGGAAGCAACCTGATGGGCTAGGGTTTAAGCCGTGTCTTGGATTCA mRNA_gMORSE_943.4223.1 CGGAAGGCGACTTCTGGAAGCAACCTGATGGGCTAGGGTTTAAGCCGTGTCTTGGATTCA BnaC04-ref CGGAAGGCGACTTCTGGAAGCAACCTGATGGGCTAGGGTTTAAGCCGTGTCTTGGATTCA ************************************************************ 5'+3'RACE-cDNA GCAGACAGTATAGAAAAGACAGCAACTCGATTCTCAAGAACAGGTGGAAGTATCTTCTCG BnAt2G(1)_gDNA GCAGACAGTATAGAAAAGACAGCAACTCGATTCTCAAGAACAGGTGGAAGTATCTTCTCG mRNA_gMORSE_943.4223.1 GCAGACAGTATAGAAAAGACAGCAACTCGATTCTCAAGAACAGGTGGAAGTATCTTCTCG BnaC04-ref GCAGACAGTATAGAAAAGACAGCAACTCGATTCTCAAGAACAGGTGGAAGTATCTTCTCG ************************************************************ 5'+3'RACE-cDNA TTGTTGTCGCCGGCGGGATGAATCAGCAGAGGAACCAGATCGTTGATGCAGTGATCATGG BnAt2G(1)_gDNA TTGTTGTCGCCGGCGGGATGAATCAGCAGAGGAACCAGATCGTTGATGCAGTGATCATGG mRNA_gMORSE_943.4223.1 TTGTTGTCGCCGGTGGGATGAATCAGCAGAGGAACCAGATCGTTGATGCAGTGATCATGG BnaC04-ref TTGTTGTCGCCGGTGGGATGAATCAGCAGAGGAACCAGATCGTTGATGCAGTGATCATGG ************* ********************************************** 5'+3'RACE-cDNA CTAGGATCCTTGGTGCGTCTCTCGTTGTCCCTGTTCTGCAAGTCAACGTCATCTGGGGAG BnAt2G(1)_gDNA CTAGGATCCTTGGTGCGTCTCTCGTTGTCCCTGTTCTGCAAGTCAACGTCATCTGGGGAG mRNA_gMORSE_943.4223.1 CTAGGATCCTTGGTGCGTCTCTCGTTGTCCCTGTTCTGCAAGTCAACGTCATCTGGGGAG BnaC04-ref CTAGGATCCTTGGTGCGTCTCTCGTTGTCCCTGTTCTGCAAGTCAACGTCATCTGGGGAG ************************************************************ 5'+3'RACE-cDNA ACGAAA------BnAt2G(1)_gDNA ACGAAAGGTATATAACATAAGCTATTTAATATTCATAGTTAATGTTATGTGACATTTGTT mRNA_gMORSE_943.4223.1 ACGAAAGGTATATAACATAAGCTATTTAATATTCATAGTTAATGTTATGTGACATTTGTT BnaC04-ref ACGAAAGGTATATAACATAAGCTATTTAATATTCATAGTTAATGTTATGTGACATTTGTT ****** 5'+3'RACE-cDNA ------GTGAGTTTGCGGATATATTCGATTTGGAGCATTT BnAt2G(1)_gDNA GATTATAATTGTGTGTTTCAATTATAGTGAGTTTGCGGATATATTCGATTTGGAGCATTT mRNA_gMORSE_943.4223.1 GATTATAATTGTGTGTTTCAATTATAGTGAGTTTGCGGATATATTCGATTTGGAGCATTT BnaC04-ref GATTATAATTGTGTGTTTCAATTATAGTGAGTTTGCGGATATATTCGATTTGGAGCATTT **********************************

5'+3'RACE-cDNA CAAGAACGTTTTAGCCGATGATGTTCATATAGTTTCGTCTTTACCTTCAACACATGTAAT BnAt2G(1)_gDNA CAAGAACGTTTTAGCCGATGATGTTCATATAGTTTCGTCTTTACCTTCAACACATGTAAT mRNA_gMORSE_943.4223.1 CAAGAACGTTTTAGCCGATGATGTTCATATAGTTTCGTCTTTACCTTCAACACATGTAAT BnaC04-ref CAAGAACGTTTTAGCCGATGATGTTCATATAGTTTCGTCTTTACCTTCAACACATGTAAT ************************************************************ 5'+3'RACE-cDNA GACGAGACCTGCGGAAGAGAAAAGGACTCCACTTCACGCTTCTCCTCAATGGATTCGTGC BnAt2G(1)_gDNA GACGAGACCTGCGGAAGAGAAAAGGACTCCACTTCACGCTTCTCCTCAATGGATTCGTGC mRNA_gMORSE_943.4223.1 GACGAGACCTGTGGAAGAGAAAAGGACTCCACTTCACGCTTCTCCTCAATGGATTCGTGC BnaC04-ref GACGAGACCTGTGGAAGAGAAAAGGACTCCACTTCACGCTTCTCCTCAATGGATTCGTGC *********** ************************************************ 5'+3'RACE-cDNA TCATTACCTCAAGCG------BnAt2G(1)_gDNA TCATTACCTCAAGCGAGTAAGTACTCTGTTTCTTTACTCTGTTTTATTAAGCTAACTCTG mRNA_gMORSE_943.4223.1 TCATTACCTCAAGCGAGTAAGTACTCTGTTTCTTTACTCTGTTTTATTAAGCTAACTCTG BnaC04-ref TCATTACCTCAAGCGAGTAAGTACTCTGTTTCTTTACTCTGTTTTATTAAGCTAACTCTG *************** 5'+3'RACE-cDNA ------BnAt2G(1)_gDNA TTTCCTCTGTTTCTTCGCCCTGTTTATTAAGCTAACTCTGTTTATTAAGCTAACTCTGTT mRNA_gMORSE_943.4223.1 TTTCCTCTGTTTCTTCGCCCTGTTTATTAAGCTAACTCTGTTTATTAAGCTAACTCTGTT BnaC04-ref TTTCCTCTGTTTCTTCGCCCTGTTTATTAAGCTAACTCTGTTTATTAAGCTAACTCTGTT

5'+3'RACE-cDNA ------BnAt2G(1)_gDNA TCCTCTGTTTCTTCGCTCTGTTTTATTAAGCCAACTCTGTTTCCTCTGTTTCTTTGCTCT mRNA_gMORSE_943.4223.1 TCCTCTGTTTCTTCGCTCTGTTTTATTAAGCCAACTCTGTTTCCTCTGTTTCTTTGCTCT BnaC04-ref TCCTCTGTTTCTTCGCTCTGTTTTATTAAGCCAACTCTGTTTCCTCTGTTTCTTTGCTCT

5'+3'RACE-cDNA ------AATTAACAGAGAAAGAGTGTTAC BnAt2G(1)_gDNA GTTTTGTCAAGCTAACTCTGTTTCCTCTGTTTCTGCAGATTAACAGAGAAAGAGTGTTAC mRNA_gMORSE_943.4223.1 GTTTTGTCAAGCTAACTCTGTTTCCTCTGTTTCTGCAGATTAACAGAGAAAGAGTGTTAC BnaC04-ref GTTTTGTCAAGCTAACTCTGTTTCCTCTGTTTCTGCAGATTAACAGAGAAAGAGTGTTAC ********************** 5'+3'RACE-cDNA TTCTCCGTGGCCTCGATTCAAGGCTCTCCAACGACCTTCCTTCTGATCTCCAGAAACTCC BnAt2G(1)_gDNA TTCTCCGTGGCCTCGATTCAAGGCTCTCCAACGACCTTCCTTCTGATCTCCAGAAACTCC mRNA_gMORSE_943.4223.1 TTCTCCGTGGCCTCGATTCAAGGCTCTCCAACGACCTTCCTTCTGATCTCCAGAAACTCC BnaC04-ref TTCTCCGTGGCCTCGATTCAAGGCTCTCCAACGACCTTCCTTCTGATCTCCAGAAACTCC ************************************************************ 5'+3'RACE-cDNA GATGCAAGGTAGCTTCCCAAGCGCTGAGATTCTCACCGAGGATACTCGAACTCGGCAACA BnAt2G(1)_gDNA GATGCAAGGTAGCTTCCCAAGCGCTGAGATTCTCACCGAGGATACTCGAACTCGGCAACA mRNA_gMORSE_943.4223.1 GATGCAAGGTAGCTTTCCAAGCGCTGAGATTCTCACCGAGGATACTCGAACTCGGCAACA BnaC04-ref GATGCAAGGTAGCTTTCCAAGCGCTGAGATTCTCACCGAGGATACTCGAACTCGGCAACA *************** ******************************************** 5'+3'RACE-cDNA AGCTAGCTTCGAGAATGCTTAGCGAAGGACAGTACCTCTCACTCCATCTACGTATGGAGA BnAt2G(1)_gDNA AGCTAGCTTCGAGAATGCTTAGCGAAGGACAGTACCTCTCACTCCATCTACGTATGGAGA mRNA_gMORSE_943.4223.1 AGCTAGCTTCGAGAATGCTTAGCGAAGGACAGTACCTCTCACTCCATCTACGTATGGAGA BnaC04-ref AGCTAGCTTCGAGAATGCTTAGCGAAGGACAGTACCTCTCACTCCATCTACGTATGGAGA ************************************************************ 5'+3'RACE-cDNA AAGACGTTTGGGTCAGAACCGGTAGTCTCCCCGGTTTAACCCCCGAGTACGACGAGATAG BnAt2G(1)_gDNA AAGACGTTTGGGTCAGAACCGGTAGTCTCCCCGGTTTAACCCCCGAGTACGACGAGATAG mRNA_gMORSE_943.4223.1 AAGACGTTTGGGTCAGAACCGGTTGTCTCCCCGGTTTAACCCCCGAGTACGACGAGATAG BnaC04-ref AAGACGTTTGGGTCAGAACCGGTTGTCTCCCCGGTTTAACCCCCGAGTACGACGAGATAG *********************** ************************************ 5'+3'RACE-cDNA TCAACAGCGAGAGACAACGCCACCCGGAGCTCCTCACGGGCCGTTCAAACATGACTTACA BnAt2G(1)_gDNA TCAACAGCGAGAGACAACGCCACCCGGAGCTCCTCACGGGCCGTTCAAACATGACTTACA mRNA_gMORSE_943.4223.1 TCAACAGCGAGAGACAACGCCACCCGGAGCTTCTCACGGGCCGTTCAAACATGACTTACA BnaC04-ref TCAACAGCGAGAGACAACGCCACCCGGAGCTTCTCACGGGCCGTTCAAACATGACTTACA ******************************* **************************** 5'+3'RACE-cDNA ACGAGAGAAAACTCGCTGGGCTTTGTCCTTTAACCGCTCTAGAAGTCACGAGGCTGCTTA BnAt2G(1)_gDNA ACGAGAGAAAACTCGCTGGGCTTTGTCCTTTAACCGCTCTAGAAGTCACGAGGCTGCTTA mRNA_gMORSE_943.4223.1 ACGAGAGAAAACTCGCTGGGCTTTGTCCTTTAACCGCTCTAGAAGTCACGAGGCTGCTTA BnaC04-ref ACGAGAGAAAACTCGCTGGGCTTTGTCCTTTAACCGCTCTAGAAGTCACGAGGCTGCTTA ************************************************************ 5'+3'RACE-cDNA AGGCCTTAGAGGCACCAAAGGACGCGAGAATCTACTGGGCGGGAGGAGAGCCTCTAGGTG BnAt2G(1)_gDNA AGGCCTTAGAGGCACCAAAGGACGCGAGAATCTACTGGGCGGGAGGAGAGCCTCTAGGTG mRNA_gMORSE_943.4223.1 AGGCCTTAGAGGCACCAAAGGACGCGAGAATCTACTGGGCGGGAGGAGAGCCTCTAGGTG BnaC04-ref AGGCCTTAGAGGCACCAAAGGACGCGAGAATCTACTGGGCGGGAGGAGAGCCTCTAGGTG ************************************************************ 5'+3'RACE-cDNA GGAAAGAGGCTTTGGAGCCGTTGACCAAAGAGTTCCCTCACCTCTACAACAAACACGATC BnAt2G(1)_gDNA GGAAAGAGGCTTTGGAGCCGTTGACCAAAGAGTTCCCTCACCTCTACAACAAACACGATC mRNA_gMORSE_943.4223.1 GGAAAGAGGCTTTGGAGCCGTTGACCAAAGAGTTCCCTCACCTCTACAACAAACACGATC BnaC04-ref GGAAAGAGGCTTTGGAGCCGTTGACCAAAGAGTTCCCTCACCTCTACAACAAACACGATC ************************************************************ 5'+3'RACE-cDNA TCGCGTTACCCGGCGAGCTCGAACCCTTCGCCAAGAAAGCCTCGGTCATGGCTGCGATAG BnAt2G(1)_gDNA TCGCGTTACCCGGCGAGCTCGAACCCTTCGCCAAGAAAGCCTCGGTCATGGCTGCGATAG mRNA_gMORSE_943.4223.1 TCGCGTTACCCGGCGAGCTCGAACCCTTCGCCAAGAAAGCCTCGGTCATGGCTGCGATAG BnaC04-ref TCGCGTTACCCGGCGAGCTCGAACCCTTCGCCAAGAAAGCCTCGGTCATGGCTGCGATAG ************************************************************

5'+3'RACE-cDNA ACTACATCGTCTGCGAGAAGAGCGATGTCTTCATACCGTCTCACGGCGGGAACATGGGAC BnAt2G(1)_gDNA ACTACATCGTCTGCGAGAAGAGCGATGTCTTCATACCGTCTCACGGCGGGAACATGGGAC mRNA_gMORSE_943.4223.1 ACTACATCGTCTGCGAGAAGAGCGATGTCTTCATACCGTCTCACGGCGGGAACATGGGAC BnaC04-ref ACTACATCGTCTGCGAGAAGAGCGATGTCTTCATACCGTCTCACGGCGGGAACATGGGAC ************************************************************

5'+3'RACE-cDNA ACGCGTTGCAAGGGCAGAGAGCTTACGCTGGTCACAAGAAGTACATCACTCCTAACAAGA BnAt2G(1)_gDNA ACGCGTTGCAAGGGCAGAGAGCTTACGCTGGTCACAAGAAGTACATCACTCCTAACAAGA mRNA_gMORSE_943.4223.1 ACGCGTTGCAAGGGCAGAGAGCTTACGCTGGTCACAAGAAGTACATCACTCCTAACAAGA BnaC04-ref ACGCGTTGCAAGGGCAGAGAGCTTACGCTGGTCACAAGAAGTACATCACTCCTAACAAGA ************************************************************

5'+3'RACE-cDNA GACATATGCTTCCTTACTTCATGAATGCCTCTCTGCCTGAATCGGAGTTTAATAGGATTG BnAt2G(1)_gDNA GACATATGCTTCCTTACTTCATGAATGCCTCTCTGCCTGAATCGGAGTTTAATAGGATTG mRNA_gMORSE_943.4223.1 GACATATGCTTCCTTACTTCATGAATGCCTCTCTGCCTGAATCGGAGTTTAATAGGATTG BnaC04-ref GACATATGCTTCCTTACTTCATGAATGCCTCTCTGCCTGAATCGGAGTTTAATAGGATTG ************************************************************

5'+3'RACE-cDNA TGAAGGATTTTCATCGAGAGTCTTTGGGGCAGCCGGAGTTGAGAACGGGTAGAGGTGGGA BnAt2G(1)_gDNA TGAAGGATTTTCATCGAGAGTCTTTGGGGCAGCCGGAGTTGAGAACGGGTAGAGGTGGGA mRNA_gMORSE_943.4223.1 TGAAGGATTTTCATCGAGAGTCTTTGGGGCAGCCGGAGTTGAGAACGGGTAGAGGTGGTA BnaC04-ref TGAAGGATTTTCATCGAGAGTCTTTGGGGCAGCCGGAGTTGAGAACGGGTAGAGGTGGTA ********************************************************** *

5'+3'RACE-cDNA AGGATGTTACTAAGCATCCTGTTTCAGAGTGTATGTGTTCAGATAGACGACAACAACAAC BnAt2G(1)_gDNA AGGATGTTACTAAGCATCCTGTTTCAGAGTGTATGTGTTCAGATAGACGACAACAACAAC mRNA_gMORSE_943.4223.1 AGGATGTTACTAAGCATCCTGTTTCAGAGTGTATGTGTTCAGATAGACGACAACAACAAC BnaC04-ref AGGATGTTACTAAGCATCCTGTTTCAGAGTGTATGTGTTCAGATAGACGACAACAACAAC ************************************************************

5'+3'RACE-cDNA AATAAGTCTCAAAGAGTGAGTGGTTCGTGCAGAGTATTCTTTTAGTTCTTTTTTTTTGGC BnAt2G(1)_gDNA AATAAGTCTCAAAGAGTGAGTGGTTCGTGCAGAGTATTCTTTTAGTTCTTTTTTTTTGGC mRNA_gMORSE_943.4223.1 AATAAGTCTCAAAGAGTGAGTGGTTCGTGCAGAGTATTCTTTTAGTTCTTTTTTTTTGGC BnaC04-ref AATAAGTCTCAAAGAGTGAGTGGTTCGTGCAGAGTATTCTTTTAGTTCTTTTTTTTTGGC ************************************************************

5'+3'RACE-cDNA CTTGATTTGGAGTATATATTATGCGTTTGTATTCTTTCAAGTGAGGTTATTTACGTGGGG BnAt2G(1)_gDNA CTTGATTTGGAGTATATATTATGCGTTTGTATTCTTTCAAGTGAGGTTATTTACGTGGGG mRNA_gMORSE_943.4223.1 CTTGATTTGGAGTATATATTATGCGTTTGTATTCTTTCAAGTGAGGTTATTTACGTGGGG BnaC04-ref CTTGATTTGGAGTATATATTATGCGTTTGTATTCTTTCAAGTGAGGTTATTTACGTGGGG ************************************************************

5'+3'RACE-cDNA GAAGTAGAATAGGGATGAGATTATGATATGATTCTTTAATTTGATTTATTTGAAACTGTA BnAt2G(1)_gDNA GAAGTAGAATAGGGATGAGATTATGATATGATTCTTTAATTTGATTTATTTGAAACTGTA mRNA_gMORSE_943.4223.1 GAAGTAGAATAGGGATGAGATTATGATATGATTCTTTAATTTGATTTATTTGAAACTGTA BnaC04-ref GAAGTAGAATAGGGATGAGATTATGATATGATTCTTTAATTTGATTTATTTGAAACTGTA ************************************************************

5'+3'RACE-cDNA ATTATATATTACAGTGTTTGTATACGGTTATACTTGTACCATACATAAATAAAAAGCAAC BnAt2G(1)_gDNA ATTATATATTACAGTGTTTGTATACGGTTATACTTGTACCATACATAAATAAAAAGCA-- mRNA_gMORSE_943.4223.1 ATTATATATTACAGTGTTTGTATACGGTTATACTTGTACCATACATAAATAAAAAGCAAC BnaC04-ref ATTATATATTACAGTGTTTGTATACGGTTATACTTGTACCATACATAAATAAAAAGCAAC **********************************************************

5'+3'RACE-cDNA ATTTGGC BnAt2G(1)_gDNA ------mRNA_gMORSE_943.4223.1 ATTTG-- BnaC04-ref ATTTG--

Figure 3.3 DNA sequence alignments of BnAt2G(1) and BnAt2G(2). (A) Multiple sequence alignment (MSA) of the gDNA sequences of At2G44500, BnAt2G(1), BnAt2G(2) and homologous sequences from the reference genome (BnaC04g03810D [BnaC04] and BnaA0504070D [BnA05]). (B) MSA of the isolated BnAt2G(1) cDNA with the mRNA sequence from the reference genome.

The region enclosed by brackets is where the Southern Blot probe hybridized. Transcription start and stop sites are indicated by green and red lines, respectively.

Templates used for PCR: gDNA = genomic DNA; cDNA = complementary DNA from B.napus leaf tissue. Yellow highlight = exons; light blue = introns. Primer location, orientation and names (in Table 1.1) are indicated by arrows.

3.3.2 Gene Copy Number

A Southern blot analysis gave restriction patterns that are consistent wih the DNA sequences in and around the two homologs of At2G44500 in the B. napus genome (Figure 3.4).

When Topas gDNA was double digested with SpeI and SwaI [enzymes that cut at restriction sites that border BnAt2G(1) and BnAt2G(2) ] a probe that binds to an internal conserved region the in genes (Figure 3.3 C) hybridized to a 4 kb band (Figure 3.4 A, lane 3), which is consistent with the predicted fragments of 4 kb and 4.2 kb from the BnAt2G(1) and BnAt2G(2) genomic sequences, respectively (Figure 3.4 B). It is likely that the fragment sizes are too similar for the electrophoresis to resolve into two bands. To confirm the presence of two copies of At2G44500 homologs in B. napus, the genomic DNA was cut with SpeI and an enzyme (HindIII) for which

BnAt2G(2) has a unique internal restriction site (Figure 3.4 B). When gDNA was double digested with SpeI and HindIII, the probe hybridized to three DNA fragments that were 1.4kb, 7kb and

10kb in size (Figure 3.4 A, lane 2). The 1.4 kb and 7 kb bands correspond to fragments predicted by the maps produced by the RestrictionMapper database (http://www.restrictionmapper.org/) after HindIII + SpeI digestions, of the BnAt2G(2) copy (Figure 3.4 B). Whereas, the 10kb band corresponds to a predicted fragment resulting from HindIII digestion of BnAt2G(1) (Figure 3.4

B).

(A)

(B)

Figure 3.4 Southern Blot of BnAt2G gene copies from cv. Topas leaf genomic DNA (gDNA). A) DNA fragments resulting from double digestion of SpeI, HindIII and SpeI, SwaI. B) Restriction map of the Brassica homologs which indicates the position of the restriction sites used in the analysis and shows the origins of the bands observed from the respective restriction digest reactions. Probe hybridization location is shown in the gray box.

3.3.3 At2G44500 locus is syntenic to regions located in B.napus chromosomes C04 and A05

Comparisons of the B. napus and A. thaliana genomes show highly conserved syntenic blocks that contain more than ten gene pairs (Chalhoub, 2014). To determine if the genomic region that contains At2G44500 is syntenic to particular chromosomal regions of the B. napus genome, the full length genomic DNA sequence of the gene was used to BLAT (BLAST-like

Alignment Tool) search the whole B. napus genome. The resulting hits that had significant BLAT-

92 scores were located in chromosome C04 and A05 and show 87% and 86% similarity to the reference genome, respectively. The BnAt2G(1) genomic sequence that was isolated and characterized in the current work is homologous to a B. napus gene that is located on chromosome

C04, which is annotated as BnaC04g03810D, while the BnAt2G(2) sequence is similar to a gene located in chromosome A05, which is annotated as BnaA0504070D. Multiple sequence alignments between the isolated B. napus homologs from cv. Topas and the reference genome cv.

‘Darmor-bzh’ (Chalhoub, 2014) show that the coding regions are conserved and the differences are seen in the intronic regions (Figure 3.3 A). Interestingly, the mutation in the putative translation start site observed in BnAt2G(2) is also seen in its homolog, BnA0504070D on chromosome A05 (Figure 3.3 A, grey highlight).

Figure 3.5 shows the syntenic relationships between a portion of the A. thaliana chromosome 2 containing At2G44500 and the B. napus chromosome C04 and A05 regions containing the homologous genes BnAt2G(1) [BnaC04g03810D] and BnAt2G(2)

[BnaA0504070D]. Overall, most of the Arabidopsis genes were colinearly located and show sequence homology with genes in both chromosomes C04 and A05, with some evidence of gene rearrangements and duplications in B. napus (of homologs of At2G44460,

At2G44470,At2G44540, At2G44550, At2G44570).

Figure 3.5 Syntenic genes located on the partial segments of the A.thaliana and B. napus chromosomes. Syntenic relationship between the A. thaliana and B. napus using regions surrounding the A. thaliana gene, At2G44500. Genes located in the A. thaliana chromosome 2 (red) and its B. napus homologs, BnAt2G(1) and BnAt2G(2) located in chromosomes C04 (blue) and A05 (green), respectively, show position co-linearity and homology (as indicated by vertical lines). Gene orientation is indicated by arrow heads.

3.3.4 BnAt2G(1) is overexpressed in embryogenic microspore cultures

To determine the tissue specificity of BnAt2G(1) expression in B. napus, reverse transcriptase PCR (rt-PCR) was performed using gene specific primers, Bn-pp1f and Bn-pp1r, and cDNA prepared from leaf, stem, seedlings, buds and microspore RNA (Figure 3.6 A). BnAt2G(1) was expressed in B. napus leaves, stems and seedlings but not in mature seeds, buds and non- induced microspores. Quantitative real time PCR (qRT-PCR) was performed to measure gene expression levels of BnAt2G(1) in noninduced and induced B. napus microspore cultures (MCs), using the same gene specific primers as above and cDNA from 0 d, 3 d, 5 d and 7 d non-induced cultures and from cell cultures 3 d, 5 d and 7 d after mild heat induction. Three, five and seven day old embryogenic (induced) cells showed 2.5-, 1.75- and 3-fold increases, respectively, in

BnAt2G(1) expression compared to 0 day old and non-induced microspore cultures (Figure 3.6

B).

(A)

(E)

(B)

(F)

Figure 3.6 BnAt2G(1) expression in vegetative tissues and microspore cultures. (A) Reverse transcriptase (RT) - PCR using BnAt2G(1) specific primers and cDNA synthesized from leaf, stem, bud, seedling, seed and pollen tissues. (B) Quantitative real- time PCR (qRT- PCR) of induced and non-induced microspore cultures show that there is a 2, 1.75 and 3 fold increase in its expression in 3, 5 and 7 day old induced/embryogenic microspore cultures, respectively. Mean relative expression was determined with calculations according to the Comparative CT method (Livak and Schmittgen, 2001) based on comparisons to transcript levels of 0d microspore cultures cv. Topas with BnActin as the internal control for normalization. Error bar represents standard deviation.

3.3.5 Functional POFUT motifs are conserved in At2G44500 paralogs and in its B. napus

homologs

At2G44500 and its ortholog in Arabidopsis, At3g07900 were predicted to contain domains annotated as “GDP-fucose protein O-fucosyltransferase (O-FuT) ”

(InterPro:IPR019378), which was formerly known as an auxin-like membrane protein of unknown function (DUF246; PF10250; InterPro:IPR004348). The protein sequences that belong to this group are classified based on the conservation of amino residues that play a role in GDP-

Fucose binding as well as the 'H-X-R' motif which was shown to play an important role in the catalytic (O-FuT) activity in C. elegans (Lira-Navarrete, 2011). The conserved peptide regions of the O-FuT superfamily have been previously described (Oriol, 1999; Martinez-Duncker,

2003) and these conserved regions motifs were reported to be located in the C-terminal region and contribute to the binding of the nucleotide donor, based on the protein crystal structures of

C. elegans POFUT1 (Lira-Navarrete, 2011) and H. sapien POFUT2 (Chen, 2012; Voxeur,

2012). This O-FuT domain is also present on the C-terminal region of BnAt2G(1) (Figure 3.7).

The phylogenetic tree of all putative O-FuTs in Arabidopsis (see chapter 2) shows that there is a high level of diversity at the protein level within the same species.

3.3.6 Protein Structure Prediction and Topology

Both the Phyre and RaptorX analyses predicted that the protein sequence of both

At2G44500 and BnAt2G(1) are highly similar to the crystallized form of POFUT1 - PDB annotation: 3ZY2 (Lira-Navarette, 2011). The bar graph represents the exact distribution of the secondary structure classes for each amino acid residue in the polypeptide sequence (Figure 3.7, blue = alpha helix; yellow = beta strands; red = coiled structures). Based on this analysis, the secondary structure of At2G44500 is composed of: 36% alpha- helix, 8%; beta sheet; and 55%

97 loop (Figure 3.7 A). For BnAt2G(1), its secondary structure is composed of: 38% alpha- helices;

7% beta sheets and 53% loops (Figure 3.7 B). The transmembrane (TM) domains of both

At2G44500 and BnAt2G(1) were found to have alpha-helix structures and the predicted amino acid residues responsible for the O-FuT catalytic activity are found in the beta-sheets (Figures 3.7

A and 3.7 B). The 3D structures of both proteins are shown in Figure 3.7 C. Table 3.2 summarizes the predicted protein topologies of At2G44500 and BnAt2G(1) from various TM prediction databases. The consensus predictions for both homologs are that their respective protein structures contain a TM domain with the cytosolic N-terminal and a C-terminal facing the lumen (Figure 3.7 D).

(A) At2G44500 MALSKNSNSNSFNKKKVSYISVPSQIINSLSSSSLQSLLVSPKKSSRSTNRFSFSYRNPR BnAt2G(1) MALPKNGGNSSSTKKKVSYISVPSQIINSLSSSSLQSLLVSPKKSSRCTNRF--SYRNPR *** **. ..* .**********************************.**** ******

At2G44500 IWFFTLFLVSLFGMLKLGFNVDPISLPFSRYPCSTTQQPLSFDGEQNAASHLGLAQEPIL BnAt2G(1) IWFLTLFLVSLFGMLKLGLNVDPISLPFSRYPCSTGSFD-----EHHAVSHLAFASKNDT ***:**************:**************** . *:.*.***.:*.:

At2G44500 STGSSNSNAIIQLNGGKNETLLTEGDFWKQPDGLGFKPCLGFTSQYRKDSNSILKNRWKY BnAt2G(1) ------QSSSSSEHRKNETLPTEGDFWKQPDGLGFKPCLGFSRQYRKDSNSILKNRWKY :: . : ***** ********************: ****************

At2G44500 LLVVVSGGMNQQRNQIVDAVVIARILGASLVVPVLQVNVIWGDESEFADIFDLEHFKDVL BnAt2G(1) LLVVVAGGMNQQRNQIVDAVIMARILGASLVVPVLQVNVIWGDESEFADIFDLEHFKNVL *****:**************::***********************************:**

At2G44500 ADDVHIVSSLPSTHVMTRPVEEKRTPLHASPQWIRAHYLKRINRERVLLLRGLDSRLSKD BnAt2G(1) ADDVHIVSSLPSTHVMTRPAEEKRTPLHASPQWIRAHYLKRINRERVLLLRGLDSRLSND *******************.**************************************:*

At2G44500 LPSDLQKLRCKVAFQALRFSPRILELGNKLASRMRNQGQYLSLHLRMEKDVWVRTGCLPG BnAt2G(1) LPSDLQKLRCKVASQALRFSPRILELGNKLASRMLSEGQYLSLHLRMEKDVWVRTGSLPG ************* ******************** .:*******************.***

At2G44500 LTPEYDEIVNSERERHPELLTGRSNMTYHERKLAGLCPLTALEVTRLLKALEAPKDARIY BnAt2G(1) LTPEYDEIVNSERQRHPELLTGRSNMTYNERKLAGLCPLTALEVTRLLKALEAPKDARIY *************:**************.*******************************

At2G44500 WAGGEPLGGKEVLEPLTKEFPQFYNKHDLALPGELEPFANKASVMAAIDYIVCEKSDVFI BnAt2G(1) WAGGEPLGGKEALEPLTKEFPHLYNKHDLALPGELEPFAKKASVMAAIDYIVCEKSDVFI ***********.*********::****************:********************

At2G44500 PSHGGNMGHALQGQRAYAGHKKYITPNKRQMLPYFMNSSLPESDFNRIVKDLHRESLGQP BnAt2G(1) PSHGGNMGHALQGQRAYAGHKKYITPNKRHMLPYFMNASLPESEFNRIVKDFHRESLGQP *****************************:*******:*****:*******:********

At2G44500 ELRMSKAGKDVTKHPVPECMCSDRQQQEQQSDA BnAt2G(1) ELRTGRGGKDVTKHPVSECMCSDRRQQQQ---- *** .:.********* *******:**:*

Figure 3.7 Protein structure and characteristics of Arabidopsis and B. napus O - Fucosyltransferases (POFUTs). (A) Multiple protein sequence alignment of At2G44500 and BnAt2G(1). The peptide motifs conserved in POFUTs are indicated inside the box. The amino acid residues, HLR (red) were shown to have catalytic significance in C. elegans and H. sapiens. The transmembrane (TM) domain is highlighted in gray. Red triangles represent the conserved GDP-Fucose binding sites in POFUTs. Protein secondary structure prediction of (B) At2g44500 and (C) BnAt2G(1). The bar graphs represent the exact distribution of the secondary structure classes for each of the amino acid residue in the polypeptide sequence (blue = alpha helix; yellow = beta strands; red = coiled structures). The blue box indicates the TM and the red box indicates conserved catalytic residues in POFUTs. Predicted 3D structures for (D) At2g44500 and (E) BnAt2G(1). The predicted protein topology for both proteins is indicated in (F).

(B)

100

(C)

101

(D) (E)

(F)

(D) (E)

(F)

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Table 3.2 At2G44500 and BnAt2G(1) Protein Topology Predictions. TM:Transmembrane Domain

>At2G44500 Program TM Domains N- C- URL Name terminus Terminus HMMMTO 62-79 OUT IN http://www.enzim.hu/hmmtop/i P ndex.php SOSUI 61-83 OUT IN http://harrier.nagahama-i- bio.ac.jp/sosui/ TMPred 61-81 OUT IN http://embnet.vital- it.ch/software/TMPRED_form. html TOPPRED 59-79 OUT IN http://mobyle.pasteur.fr/cgi- bin/portal.py?#forms::toppred Phobius 61-79 OUT IN http://phobius.sbc.su.se/

>BnAt2G(1) Program TM Domains N- C- URL Name terminus Terminus HMMMTO 59-77 OUT IN http://www.enzim.hu/hmmtop/i P ndex.php SOSUI 1-58 OUT http://harrier.nagahama-i- bio.ac.jp/sosui/ TMPred 59-79 OUT IN http://embnet.vital- it.ch/software/TMPRED_form. html TOPPRED 57-77 OUT IN http://mobyle.pasteur.fr/cgi- bin/portal.py?#forms::toppred Phobius 59-77 OUT IN http://phobius.sbc.su.se/

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3.3.7 Subcellular Localization of BnAt2G(1)

Fucosyltransferases are type-II transmembrane proteins with a large C-terminal globular catalytic domain facing the luminal side (Nilsson et al., 1993, 1996). To validate the protein topology prediction data generated above, stably transformed Arabidopsis plants expressing

BnAt2G(1) tagged with GFP were generated. GFP that was N-terminally fused with BnAt2G(1) appeared as small punctates surrounding the nucleus (Figure 3.8 D and 3.9 A). Although some of the green punctates colocalized with a Golgi marker, BODIPY-TR, some did not (Figure 3.8

F and 3.9 C). The fluorescent structures were investigated further by treatment with Brefeldin A

(BFA), which is known to block vesicular transport in the secretory pathway and induces vesiculation and disassembly of the Golgi apparatus. The fluorescence pattern of BFA-treated roots changed from well-defined vesicles (Figures 3.9 A to 3.9 C) to a pattern of diffused and disassembled structures (Figure 3.9 D).

GFP::BnAt2G(1) expressing protoplasts were also treated with BFA and showed a similar pattern of punctate disassembly (Figure 3.9 F and 3.9 G) compared to the non-treated cells (Figure 3.9 E). Concanavlin A conjugated to AlexaFluor 594 (conA) was used to mark the endoplasmic reticulum (ER). GFP::BnAt2G(1) structures and conA did not show any co- localization in BFA treated cells (Figure 3.9 H).

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Figure 3.8 Subcellular localization of GFP::BnAt2G(1). (A-C) Control (wild type) roots show minimal green fluorescence. (B) Control roots stained with

BODIPY-TR (red). (C) Bright field merged image of panels A and B (control). (D)

Root cells that were stably expressing GFP::BnAt2G(1). (E) Cells that were stained with BODIPY-TR (red). (F) Merged image of panels D and E. Scale bars: A to C =

20 microns; D to F = 5 microns.

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(A) (B) (C) (D)

(E) (F) (G) (H)

Figure 3.9 Brefeldin A (BFA) treatment of GFP::BnAt2G(1) expressing root cells and leaf protoplasts. (A) BnAt2G(1) associated with GFP fluorescence appear as small punctates. (B) BODIPY-TR stain of GFP::BnAt2G(1) expressing root cells. (C) Merged image of panels A and B. (D) GFP::BnAt2g(1) expressing cell that was treated with BFA. (E) Leaf protplasts with no BFA treatment. (F-H). BFA- treated protoplasts expressing (E) GFP::BnAt2G(1) and stained with the ER marker, Concanavlin A (ConA). (G) (H) Merged image of BFA treated protoplast expressing GFP::BnAt2G(1) and stained with ConA. Scale bars = 5 microns.

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3.3.8 In silico Promoter Analysis of At2G44500 and BnAt2g(1)

To identify the elements that control the transcription of At2G44500 and BnAt2G(1), an in silico promoter analysis was conducted. Three publicly available databases (ATHENA,

PLACE and PlantCARE) were used to analyze the sequences, approximately 1 kb upstream from the start codon. Table 3.4 and Figure 3.10 show the regulatory elements and motifs that are present in both Arabidopsis and Brassica homologs on their respective promoter regions. The highlighted rows signify that the particular motif was conserved in both genes. In general, the shared motifs contain stress-responsive elements (MYB1AT, W-Box, GCC-Box, CGTCA- motif) as well as motifs that are related to plant growth and development such as ARF (Auxin

Response Factor), ARE (Auxin Responsive Element) and Skn1 motifs. There are three W-Box motifs present in the B.napus homolog compared to one found in the Arabidopsis gene.

Interestingly, an equal number (two) of auxin response and jasmonic acid Response elements were found in both genes.

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(A)

(B)

Figure 3.10 Regulatory elements found in the promoter regions of (A) BnAt2G(1) and (B) At2G44500. Each element was colour coded to see the similarities in the upstream sequences between the homologs.

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Table 3.3 Putative cis acting elements identified from the At2g44500 and BnAt2G(1) promoter regions.

Cis Element Sequence Function Skn1- motif GTCAT Cis-element required for endosperm expression GARE TAACAA Giberellic Acid Response Element ERE Ethylene Response Element W-BOX TTGACC/T WRKY Binding Site Motif MYB MYB recognition site found in the promoters of the dehydration-responsive gene rd22 A/TAACCA - Involved in regulation of genes that are responsive to water stress in Arabidopsis MBS MYB Binding Site CTAACCA - Plays a role in drought- and abscisic acid- regulated gene expression MYC2 BS Binding site for MYC2 BHLH TF - this TF cross talks with light, ABA and jasmonic acid signaling pathways. -plays a role in drought- ABA-regulated gene CACATG expression. ARF TGTCTC Auxin Response Factor AUX-RR CORE GGTCCAT Involved in auxin response due to stress. HSE AGAANNTTCT Heat Shock Element

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3.4 Discussion

The present study identified two homologs of At2G44500 in the B. napus genome, named

BnAt2G(1) and BnAt2G(2), respectively. These Brassica napus homologs of At2G44500 were

significantly upregulated in embryogenic microspore cultures in B. napus, thus confirming the

observation by Chan and Pauls (2006) that the expression of this gene is developmentally

regulated and occurs during embryogenesis in this model system.

3.4.1 Homologous Gene Isolation

Although it was possible to isolate the genomic sequences of the two orthologous genes in

B. napus, only BnAT2G(1) was expressed in leaf, stem, seedlings and embryogenic cells from

microspore cultures (Figure 3.2 A and Figure 3.6). There was no amplification observed when

BnAt2G(2)-specific primers were used in RT –PCR reactions using leaf cDNA (Figure 3.2 A) and

cDNA from other tissues such as leaf, stem and seedlings (data not shown). A closer look at the

genomic sequence of BnAt2G(2) showed that it contains the codon "AAT", which encodes for

asparagine instead of "ATG" (methionine), in the position that corresponds to the translation start

site in BnAt2G(1) (Figure 3.3 A). This sequence difference was also observed in the sequence for

BnAt2G(2) in the reference gDNA sequence, BnA0504070D (Figure 3.3 A). These results suggest

that BnAt2G(2) is a non-functional gene or may encode a shorter protein. If BnAt2G(2) was a

shorter protein, in silico translation from a downstream start site should result in a protein with a

complete O-FuT motif. However, when this was done, the resulting amino acid sequence was

shorter (238 AA) than the protein sequence translated from the full coding sequence isolated from

BnAt2G(1) (550 AA) and it was missing the transmembrane domain and also had a truncated O-

FuT motif.

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B. napus is an allopolyploid in which its genome originated from independent hybridization events between its ancestor - the diploid B. rapa (A genome donor) and B. oleracea

(C genome donor) (U, 1935; Palmer et al, 1983; Parkin et al, 1995). The synteny analyses shows that BnAt2G(1) (or BnaC04g03810D) is found in a the C04 chromosome contributed from B. oleracea (Chalhoub et al, 2014) and that BnAt2G(2) (or BnaA0504070D) is found in the A05 chromosome contributed by B. rapa (Chalhoub et al, 2014). In an allopolyploid genome, natural selection and stochastic processes govern the changes in gene paralogs. Genetic modifications such as redundancy, subfunctionalization, neofunctionalization, gene deletion and gene silencing

(Ohno 1970; Hughes and Hughes 1993; Force et al. 1999; Lynch and Force 2000; Lynch et al.

2001; Gu 2003) can occur within generations after allopolyploidization and can be biased by ancestry (Volkov et al. 1999; Ozkan et al. 2001; Shaked et al. 2001; Adams et al. 2003, 2004;

Wang et al. 2004; Adams and Wendel 2005). The results of the current indicate that the

BnAt2G(1) (or BnaC04g03810D) paralog has been functionally conserved, whereas the other paralog [BnAt2G(2) or BnaA0504070D] has been silenced by mutations that included the mutation of the start codon.

3.4.2 Predicted Protein Topology and Subcellular Localization

All of these genes appear to be members of a large family of O-Fucosyltransferases (O-

FuT) that have been described in Arabidopsis. The predicted protein topologies of At2G44500 and BnAt2G(1) resemble other known fucosyltransferases - which are a type II-membrane protein with a C-terminal catalytic domain facing the luminal side (Nilsson et al., 1993, 1996).

Generally, these enzymes are localized in the Golgi.

The predicted location for the BnAt2G(1) protein in the endomembrane system was supported by the microscopic observations made with the transgenic Arabidopsis plants stably

111 expressing GFP::BnAt2G(1). The green fluorescence was visualized as punctates, which are believed to be vesicles in the secretory pathway (Figure 3.9 A and C). The location of the fluorescent proteins in the transgenic plants was further investigated by examining is location relative to BODIPY-TR dye, which is believed to be a marker for Golgi (Pagano, 1991; Ralston,

1993). This dye consists of a sphingolipid analog, called ceramide, conjugated with BODIPY and accumulates in the Golgi. The current results indicate that some of the GFP punctates co-localized with the Golgi marker, BODIPY-TR (Figure 3.8 and 3.9 C ). This finding is consistent with numerous studies that indicate that the Golgi is where most protein post-translational fucosylations occur in plants.

The Golgi also plays a fundamental role in generating new cell walls in higher plants.

During cytokinesis, a new cell wall is formed and starts to assemble in Golgi-derived secretory vesicles to the centre of the dividing cell. Fusion of these vesicles gives rise to the cell plate, which in turn undergoes an elaborate process of maturation leading to a fully functional cell wall

(Hepler, 1996; Cutler, 2002; Segui-Simarro, 2004).

Although some of the GFP::BnAt2G(1) punctates colocalized with the Golgi marker, some did not. The relationship between the BnAt2G(1) protein and the secretory pathway was further explored by treating the transgenic tissue with Brefeldin A. Brefeldin A (BFA) is a fungal metabolite that blocks vesicular transport in the cell's secretory pathway, causes the formation of endosomal aggregates and movement of Golgi proteins to the ER (Contento,

2012). The BFA-treated GFP::BnAt2G(1) roots in the current study had patterns of diffused and disassembled structures (Figure 3.9 D) as opposed to non-treated roots which showed distinct punctate structures (Figure 3.9 A). This observation is consistent with other plant systems

(Ritzethaler, 2002). For example, in tobacco BY-2 suspension cells, the first effect of BFA is the

112 loss of COPI coats from the Golgi. This is followed by a loss of the oldest, trans-Golgi network cisterna and the simultaneous maturation of the younger cisternae, consistent with the cisternal progression/maturation model of intra-Golgi trafficking (Pfeffer, 2010). Shortly after, the ER begins to fuse to individual cisternae in the stack which eventually results in a region of stacked

ER that contains patches of Golgi proteins. Some stacks that do not fuse with the ER form the so-called BFA compartments. It is possible that the structures seen in the BFA-treated cells in the current study are the ER-Golgi hybrid structures that resulted from the BFA-treatments.

However, it is possible that BnAt2G(1) is being transported to or is localized in other compartments that are part of the secretory pathway. In particular, because ceramide is a lipid analog, there is a possibility that it might be bound by lipid bodies in the cell and not be a specific marker for Golgi. Lipid bodies are unique spherical organelles which bud off from the

ER and are surrounded by a single phospholipid layer (van der Schoot, 2011). To address this possibility, the stained BFA-treated GFP::BnAt2G(1) protoplasts were stained with the ER marker, Concanavlin A (conA). However, the GFP::BnAt2G(1) punctates did not colocalize with conA (Figure 3.9 H), which suggests that BnAt2G(1) is neither localized in the ER nor in the ER-derived lipid bodies and that it maybe trafficked to a different compartment in the cell's secretory pathway through Golgi-derived vesicles.

GDP-fucose is the donor substrate for all known fucosyltransferases (Becker, 2003). It is synthesized in the cytoplasm and is shown to be transported into the Golgi lumen by a GDP- fucose transporter (Hirschberg, 1998; Wuff, 2000). The current observations about the co- localization of GFP::BnAt2G(1) and BODIPY-TR suggest that this novel plant O-FuT is localized in the Golgi and may be sorted to another compartment in the cell in Golgi-derived vesicles.

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3.4.3 In silico Promoter Analysis

The transcription control elements that were identified in the in silico promoter analysis of the At2g44500 and BnAt2G(1) genes indicate that they may be responsive to stress effectors such as MYBs, MYCs and heat shock and may be regulated by hormones such as giberellic acid (GA), abscisic acid (ABA), jasmonic acid (JA), ethylene and auxin. In addition, the W-boxes they contain indicate that the genes involved in signal transduction pathways in abiotic stress reponse.

The MYB binding sites in the At2G44500 and BnAt2G(1) promoters suggest that they are regulated by this large family of transcription factors in plants that are key factors regulating networks controlling: development, metabolism and responses to biotic and abiotic stresses (Abe,

2003). For example, MYB2 controls the ABA induction of salt and dehydration responsive genes in A. thaliana (Abe, 2003), consistent with the observation that At2G44500 expression is increased in A. thaliana plants that have undergone salt and drought stress, particularly in the roots as well as in ABA-treated seedlings, 3 hours after hormone induction even though

At2G44500 and BnAt2G(1) do not have the ABA response element in their promoter sequences. .

The W-Box promoter sequence found in At2G44500 and BnAt2G(1), to which WRKY transcription factors (TFs) bind, suggest that they are responsive to one of the largest families of transcriptional regulators in plants, that form integral parts of signalling webs, modulating many plant processes including: the innate immune system of the plant, elicitor-, microbe-, pathogen - associated molecular pattern triggered immunity (ETI, MTI and PTI, respectively) and systemic acquired resistance (SAR) (Eulgem, 2007). Furthermore, WRKY TFs regulate NPR1 gene expression (Yu, 2001) which encodes the receptor for the hormone, salicylic acid (SA), which is essential for plant immunity (Wu, 2012).

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Although At2G44500 and BnAt2G(1) contain the cis acting elements for response factors for various hormones, including auxin, GA and ethylene, their functions remain unclear because auxin, GA and ethylene treatments of seedlings for various lengths of time did not show significant increases in At2G44500 expression. This suggests that there might be cross-talk interactions with these different hormone networks.

3.4.4 Conclusions

At2G44500 is a member of the plant O-Fucosyltransferases (O-FuTs) family (Chapter 2), which is a type of glycosyltransferase that adds fucose to their target molecules. In the current work, its homologous genes in B. napus were cloned and sequenced. Protein sequence analyses of both the Arabidopsis and B. napus homologs showed that the O-FuT domain and amino acid residues essential for O-FuT activity are conserved. The protein structure and topology predictions showed that they are type II membrane proteins. The subcellular localization study suggested that

BnAt2G(1) is localized in the Golgi and is most likely trafficked through the cell's secretory pathway via Golgi-derived vesicles. The in silico promoter analyses of these homologs suggest that their expression may be controlled by different hormones as responses to biotic and abiotic stresses.

Taken together, At2G44500 and BnAt2G(1) are plant O-FuTs that play roles in microspore embryogenesis as well as in other contexts, such as in stress responses. It would be of great interest to identify the target substrates of these O-FuTs and elucidate their function in the context of cell growth, development and stress response.

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4 Chapter 4

Functional Characterization of At2G44500 and BnAt2G(1)

Abstract

Cell walls are essential components that provide structural integrity and physical support to plants.

They are also vital for proper growth and development and play important roles in plant defences and responses to environmental stresses. Their structures are highly complex and largely composed of polysaccharides (such as cellulose, hemicelluloses and pectins), modifying enzymes and structural proteins. The biosynthesis of these polysaccharides are catalyzed by various glycosyltransferases (GTs). O-fucosyltransferases (O-FuTs) are a type of GT’s that add fucose to their target substrates. Here, a member of the Arabidopsis GT-65R/O-FuT gene family and its B. napus homolog were functionally characterized. Knockdown mutants of At2G44500/BnAt2G(1) showed phenotypes that are indicative of cell wall loosening and affected lateral root and stem development. Overexpression of At2G44500/BnAt2G(1) resulted in a smaller stem diameter and decreased number of lateral roots compared to wild type and knockdown lines. The FT-IR analysis suggests that the phenotypes observed in the mutants are caused by changes in cell wall components particularly, pectin. The results show the potential role of these putative O-FuTs in cell adhesion and control of cell wall biomechanics.

4.1 Introduction

The cell wall has a polysaccharide-rich extracellular matrix composed of complex interlinked networks of glycoproteins, cellulose, non-cellulosic and pectic polysaccharides. They play important roles in providing structural stability and flexibility in the plant and critically influence growth and development. It is a dynamic and constantly changing structure that reacts to intrinsic and extrinsic cues and its supramolecular structure is constantly rearranged by the

116 integration of new cell wall materials and modification of glycodisic linkages. Interfering with either the biosynthesis or post- translational modification of cell wall components can cause changes in the cell wall structure/organization and can thereby influence cell growth processes.

The addition of sugar moieties to cell wall carbohydrates and glycoproteins are mainly catalyzed by glycosyltransferases (GTs). In plants, a large number of distinct GTs are required because of the diversity in each of the glycosidic linkage types that connect all monosaccharides within these polymers. Until recently, GTs were thought to only have a limited influence on the basic physiology of plants. However, the identiﬁcation of large numbers of GT genes and their functional characterization has changed that view.

The addition of the sugar fucose (Fuc) is catalyzed by the fucosyltransferase (FuT) family of GTs. In Arabidopsis thaliana, fucosylation of N-linked glycans is done by α-1,3/4 FuTs while

Fuc is added to xyloglucan and arabiogalacatan proteins (AGPs) by members of the α-1,6 FuT family. Another family, belonging to the FuT family, is the largely uncharacterized O- fucosyltransferase (O-FuTs) group. The current study characterizes a putative Arabidopsis O-FuT,

At2g44500 and its Brassica homolog, BnAt2G(1) using molecular, genetic and biochemical approaches. Knockdown and over expression of this gene and its B. napus homolog in

Arabidopsis affected lateral root and stem development. Mutants were found to have altered cell wall compositions as revealed by FT-IR analysis. Changes in the expression of these proteins not only altered cell wall composition but also strongly affected plant growth, providing evidence that these novel O-FuTs play important roles in cell wall development.

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4.2 Materials and Methods:

4.2.1 Plant Material

A. thaliana lines (Col-0 background) with T-DNA insertions in the promoter

(SALK_74450) and coding regions (SALK_20490) of At2G44500 were obtained from the Salk

Institute (www. http://signal.salk.edu). A population of T1 plants segregating for the T-DNA insertion were self-pollinated for four generations to obtain stable insertion lines.

4.2.2 Genotyping of T-DNA Insertion Mutants

T4 seeds for SALK_74450 and SALK_20490 were grown and the progeny were screened for homozygosity by PCR using primers specific to the At2g44500 (RP and LP) and to the TDNA left border sequence (LBb1.3). Leaf tissues from randomly selected individuals were used for genotyping (A-J for SALK_704450; E-L for SALK_020490 and wt for wild type).

4.2.3 Generation of RNAi Construct

The pFGC RNAi vector was used to generate an RNAi silencing construct containing a

450bp fragment from the BnAt2G(1) coding region that has 90% homology to At2g44500. The silencing mechanism caused by the expression of this vector is due to the production of double stranded RNA (dsRNA) driven by a CaMV 35S promoter. The dsRNA results from the sequence complementation of the insert (which is specific to the target gene). The hairpin loop is due to the petunia chalcone synthase (chsA) intron flanking the target gene fragment. The target gene region was amplified from B. napus cDNA using the following primers: RNAi:BnAt2G-Fwd (5' –

TTAATTAAGGCGCGCCCTCAAGAACA GGTGGAAGTA – 3') containing the restriction sites

AscI and PacI and RNAi:BnAt2G-Rev (5' –

CCTAGGATTTAAATGGAGAAGTAACACTCTTTCT – 3') containing the restriction sites

AvrII and SwaI. These same sites flank the ChsA intron in pFGC5941 and thus allow for the

118 directional cloning of the PCR fragment containing the same compatible ends. In order to clone the PCR generated fragment in the sense orientation, first, the pFGC vector and the fragment were digested with AscI and SwaI and the fragments were ligated. The second step was to sub-clone the

PCR fragment into the anti-sense orientation. This was done by digesting the pFGC vector and the

BnAt2G(1) gene fragment with AvrII and PacI and ligating them together.

The plasmid constructs for each step were used to transform E. coli and kanamycin resistant colonies were selected. PCR positive clones were cultured in LB overnight at 37° C and were followed by plasmid extraction and purification. The plasmids were sequenced to confirm the ligation success and insert orientation using primers specific to the BnAt2G(1) insert and the chsA intron.

4.2.4 Generation of 35S:: BnAt2G(1) Overexpression Construct

The BnAT2G(1) cDNA was cloned into the pENTR™/TEV/D-TOPO vector (Invitrogen) according to the manufacturer's instructions. Successful cloning of the cDNA into this vector generated entry (pENTRY) clones that could subsequently be inserted into pMDC32 (Curtis,

2003) which is a binary destination vector (pDEST) driven by the CaMV 35S promoter. This was performed through a LR reaction in which an LR clonase was used to facilitate the recombination of attL substrate in pENTRY clones with the attR substrate in pMDC32 (pDEST) in order to create a clone that overexpresses BnAt2G(1). Positive pENTRY and expression clones were sequenced and verified by colony PCR using primers specific to the insert and vectors- pENTR™/TEV/D-TOPO and pMDC32, respectively

4.2.5 Generation of Competent Agrobacterium

Competent Agrobacterium strain EHA105 cells were produced and transformed with the gene fragments by adapting the methods established by Hofgen and Willmitzer (1988) and

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Weigel and Glazebrooke (2006). Briefly, single colonies from plated-EHA105 (Rifampicin plates) were inoculated into 50 mL of LB containing Rifampicin (25 ug/mL). The cultures were incubated in a shaker at 28 ºC for 48 hours. 2 mL of the 50 mL culture was used to inoculate 50 mL of fresh LB media with rifampicin (25 ug/mL) and the culture was incubated at 28ºC until the OD600 reached between 0.5-1.0. The culture was chilled on ice for 5 minutes and the cells were pelleted in a bench top centrifuge at 5000 rpm for 10 minutes in 4 ºC. The supernatant was discarded and the pelleted cells were resuspended with 1mL of ice-cold CaCl2 (20mM). 100-150 uL of the cell solution was aliquoted into chilled Eppindorf tubes. These tubes were flash frozen in liquid Nitrogen and were either used immediately for transformation or stored at -80 ºC for two months.

4.2.6 EHA105 Transformation and Preparation for Arabidopsis Transformation

One microgram of purified plasmid DNA was added to EHA105 competent cells. The cells were incubated for five minutes on ice and were subsequently frozen in liquid nitrogen for another 5 minutes. The cells were immediately thawed by incubating them in a 37 ºC water bath for 5 minutes. After thawing, 2mL of fresh LB was added and the culture was incubated at 28 ºC for 4 hours with gentle shaking. The cells were pelleted by centrifuging the culture at high speed

(3,000 x g) at room temperature for 10 minutes. The harvested cells were subsequently resuspended in 100 uL LB medium and dispensed onto LB agar plates containing 50 ug/ml kanamycin and 25 ug/mL rifampicin (for pMDC and pFGC vectors) and were incubated at 28 ºC for 48 hours. Positive transformants were verified by colony PCR and were subsequently grown in

LB media containing appropriate antibiotics in 28 ºC for 48 hours.

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4.2.7 Arabidopsis Transformation

Arabidopsis plants were transformed by the "Floral Dip" method (Clough and Bent, 1998).

Briefly, the bacterial cell suspensions harbouring the vector of interest were poured into two liter disposable plastic autoclave bags containing 120 mL of 5% sucrose solution supplemented with

0.03% Silwet L-77. Inflorescences of healthy Arabidopsis plants were dipped into the bacterial solution for approximately ten seconds with gentle agitation. Dipped plants were then placed sideways under a plastic cover for 24 hours to maintain high humidity in growth chambers and were grown normally until seed harvesting. Plants were grown in growth chambers

(BioChambers) with a 16 h light and 8 h dark cycle (with a light intensity of 120 µmol/m2sec from white fluorescent tubes) with day and night temperatures of 24 °C and 20 °C, respectively. The relative humidity was maintained at approximately 70%.

4.2.8 RNAExtraction and qRT-PCR

RNA isolation and cDNA synthesis were performed (as in section 3.2.3.3) from leaves of wild type (wt), g-20490, g-74450, RNAi:BnAt2G(1) and Oex plants. For qPCR reactions, the

At2G44500 specific primers At44500-fwd1 (5’ ATGACGAGACCTGTTGAAGAGAA-3' and

At44500-rev1 (5’-GAGGTCTTTGGAGAGTCTTGAAT-3’) were used. The A. thaliana genes

Actin2 (Act2) and EF1-alpha genes (AtACTIN2-fwd 5’-TTACCCGATGGGCAAGTC-3’

AtACTIN2-rev 5’-GCTCATACGGTCAGCGATAC-3’) and (AtEF1-alpha fwd 5'-

TGAGCACGCTCTTCTTGCTT-3' and AtEF1-alpha rev 5'-GGTGGTGGCATCCATCTTGT-

3') were used as housekeeping genes. qPCR reaction mixtures consisted of 2.5 µl of cDNA, 12.5

µl of Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) and 5 µl of each primer in a total volume of 25 µl were added to 48-Well Optical Reaction Plates for amplification and quantification in a StepOne Real-Time PCR System (Applied Biosystems).

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The experiment was repeated three times (biological replicates) and the delta-delta Ct method

(Livak, 2001) was used in order to make a relative quantification comparing induced and non- induced transcript levels. Due to the nonparametric distribution of data, statistical analysis of delta-delta Ct values was performed using ANOVA and Bonferonni-Holm post-hoc analysis with significance set at P<0.05.

4.2.9 Phenotyping

Seeds were surface sterilized with 70% ethanol for 2 minutes followed by 50% bleach for

5 minutes and rinsed three times with sterile water. They were stratified for 3 days in 4 °C and were either imbibed in 0.5 MS agar plates (for early development analysis) or directly sown to soil. The plates were subsequently placed in the tissue culture room under 16h/8h 24 °C light/20

°C dark cycle with a light intensity of 120 µmol/m2sec from cool white fluorescent tubes for 15 days. The seedlings were transferred to 24 cell trays with LA4 Sunshine Mix soil and with a slow release granular fertilizer applied to each cell of the tray. Soil based phenotypic analyses were conducted in growth chambers

(BioChambers) with a 16 h light and 8 h dark cycle (with a light intensity of 120 µmol/m2sec from white fluorescent tubes) with day and night temperatures of 24 °C and 20 °C, respectively and with a relative humidity of approximately 70%. All experiments were set up with a

Randomized Complete Block Design (RCBD). For soil-based phenotyping, measurements were made every 2 days after seedling transplant and were continued throughout the life of the plant.

Table 4.2 shows the traits that were measured in different developmental stages as established by

Boyes et al (2001).

To determine if the mutant and transformed lines have any phenotypic defects, a growth- stage-based phenotypic analysis method was adopted from Boyes et al (2001). The plant traits that

122 were measured were: germination rate (n=8), rosette diameter (n=34), plant height (n=35), branch numbers (BRAN; n=35), silique number (SILN; n=35), seed number per silique (SN; n=54), root length (n=15), number of lateral roots (n=15), stem diameter (n=15) and above ground biomass

(n=6). Figure 4.1 summarizes the phenoptyping methodology conducted. Seed number per silique was determined from 24 separate inflorescences per plant and from 8 plants in total.

4.2.10 Stem Stains

Stems from five wt and five mutant lines were hand sectioned (total of 20 sections per line) and stained with 0.02% Toluidine Blue and 3% Phloroglucinol-HCl (Wiesner Staining).

Stained stems were then visualized under a light microscope.

Figure 4.1 Phenotyping methodology

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4.2.11 Fourier Transformed Infrared Spectroscopy

For the FT-IR analysis, three whole plants from each line were dried in 80 °C for 3 days.

Dried tissues were ground to fine powder. Spectra were collected with the use of Thermo

Scientific Nicolet™ iS™10 FT-IR Spectrometer and the OMNIC Spectra software.

4.2.12 Image Analysis

All image analyses were done using ImageJ (http://imagej.nih.gov/ij/).

4.2.13 Statistical Analysis

Anova and the Bonferroni-Holm Post Hoc analyses were performed using a Microsoft

Excel Statistical add-in called, XL tool box. Principal Component Analysis (PCA) was carried out using Minitab 17 and SAS.

4.3 Results

4.3.1 Confirmation and Genotyping of T-DNA Insertion in SALK Lines

To ensure that the SALK_74450 and SALK_20490 insertion lines that were used for the phenotypic analyses were homozygous at the At2G44500 locus and were disrupted by the T-

DNA insertion, T4 plants were genotyped using two sets of primer pairs. The first pair contained gene-specific sequences (GSPPs) that hybridized to regions flanking the known insertion site for each line (Figure 4.2). The large size of the insertion prevented PCR amplification and the absence of PCR fragments in the analyses performed with the gDNA of individual plants from

SALK_74450 and SALK_20490 genetic backgrounds, as seen in Figure 4.2 A (lanes 3 to 7) and

4.2 C (lanes 3 to 9) and its presence in the wild type (wt) controls (where there is no T-DNA disruption in the genome; Figure 4.2 A, lane 2; Figure 4.2 C, lane 2) confirm that both copies of

At2G44500 are interrupted in the SALK lines. To confirm the presence of the T-DNA a second primer pair consisting of one T-DNA specific primer (LBb1.3) and one gene specific

124 primer (20490_RP for SALK_20490 or 74450_RP for SALK_74450) was used. Individual plants that contain the insertion were expected to amplify a single band when this particular primer pair is used with gDNA as a template. As seen in figure 4.2 A individual plants from SALK_74450

(lanes 9-13) and SALK_20490 in Figure 4.2 C (lanes 11 to 17) amplified single PCR products with sizes 0.75 and 0.95 kb, respectively. The wild type controls did not show any PCR amplification when this primer pair is used because it did not contain the T-DNA insertion (Figure

4.2 A, lane 8; Figure 4.2 C, lane 10). Taken together, the At2G44500 gene was disrupted by T-

DNA in the mutants and that the individual plants from the T4 generation were also homozygous for the insertion. These lines were used for the subsequent gene expression and phenotyping analyses to further characterize the gene function of At2G44500.

Name Sequence 744550_RP GAACGCGAAGTCTGATCAAAG 744550_LP GGGTATATTCGAGGAGCCAAAG 20490_RP AAACCCTAACCCATCAGGTTG 20490_LP GCCAGTCTTTGGCTATAAGCC LBb1.3 ATTTTGCCGATTTCGGAAC

RNAi:BnAt2G(1)_fwd TTAATTAAGGCGCGCCAACCCCCG CHSa_rev1 TCCTCCCCTCTTTCTACCTTCC

CHSa_fwd AGCGAGCTAGCAAAATTCCA rev-antisense TCCCGGGTCTTAATTAACTCTCTAG pMDC-NOSf GCGGGACTCTAATCATAAAAACCC pMDC-35Sr AAGTTCATTTCATTTGGAGAGGACC At2G-Iso2r TTATTGTTGTTGTTGTCGTCT

Table 4.1 Primers used to identify and genotype T-DNA insertion lines, generate and validate RNAi::BnAt2G(1) and 35S::BnAt2G(1) plants

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Figure 4.2 Identification and validation of At2G44500 TDNA insertion mutants. (a) Genoyping of wild-type (wt) and T-DNA insertion mutant line 74450 by PCR using genomic DNA (gDNA) as template. The expected size amplified using gene-specific primer pair (GSPP) (lanes E-L) was 1kb and 0.75kb for the T-DNA insertion. (b) Schematic diagram of T-DNA insertion locations for each mutant line. Primer locations are indicated by arrows. RP, right primer; LP, left primer; LBb1.3, TDNA insertion primer. (c) The expected product size for line 20490 using its GSPPs was 1.2kb (lanes A-J) while the T-DNA insertion was 0.95kb. Trackit DNA ladder (Invitrogen) was used as a molecular weight marker.

20490 LP+RP LBb1.3+ 20490_RP (C) (GSPP) (TDNA insertion-specific) wt A B C D H I J wt A B C D H I J 1.3 kb 0.9 kb 0.95 kb 0.6 kb LBb1.3

(B) 20490 LP 20490 RP

74450 LP 74450 RP At2G44500

LBb1.3

(A) 74450 LP+RP LBb1.3+ 74450_RP GSPP (TDNA insertion-specific) wt E F G K L wt E F G K L 1.5kb 1 kb 0.75 kb

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4.3.2 Generation of RNAi Lines

The RNAi silencing construct contained a 350 bp fragment specific to the BnAt2G(1) coding sequence. This region also had a 90% level of homology with At2G44500. To check if the insert was ligated in the proper orientation for each step of the subcloning process, PCR was performed using vector - specific (chsA_rev1) and insert-specific (RNAi-BnAt2G(1)_fwd) primers (Figure 4.3 B). PCRs with five out of thirteen clones resulted in the amplification of a band of the expected size (0.55kb) indicating the BnAt2G(1) fragment was ligated in the correct orientation (Figure 4.3 A, lanes 2,3,5,9 and 11). The resulting plasmid was then digested and used to ligate in the second BnAt2G(1) insert in the anti-sense orientation. The resulting clones were verified for proper orientation using primers chsA-fwd (vector-specific) and rev-antisense (gene insert-specific). Three out of twelve clones were positive for the insertion in the anti-sense orientation with the expected band size of 1.15kb (Figure 4.3 C, lanes 8,12 and 13).

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  ×  × × ×  ×  × × ×

× × × × × ×  × × ×  

Figure 4.3 Colony PCR of E.coli clones harbouring the plasmid pFGC5941+RNAi insert in the (A) sense and (C) anti-sense orientations. (B) Partial vector map indicates the location of primers used for orientation verification. Checkmarks indicate the clones that were positive for the insertions.

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The plasmid from kanamycin resistant clones were digested with AscI and PacI to check if the BnAt2G(1) inserts ligated in the correct orientation (Figure 4.4 A). Double digested positive clones are expected to yield bands with sizes of approximately 2.15 kb and a 10 kb. Samples used for this analysis were: control plasmid (ctrl) which does not contain the complete RNAi construct and plasmid extracted from clones G, K, L which harbor the complete RNAi construct. Lanes 1-4 show the gel of uncut plasmids to test the efficiency of the double digestion. Lanes 6 – 9 show

AscI+PacI digested control and plasmids from clones G,K,L. The control plasmid yielded a smaller band (~1.8 kb) as it only contains the BnAt2g(1)-sense insert. Plasmid double digested from clones G, K, and L yielded the expected band sizes if the inserts in the RNAi construct were ligated in the proper orientation (lanes 7-9).

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Figure 4.4 BnAt2G(1) insert orientation verification. (A) Vector map of RNAi::BnAt2G(1) construct indicating its main features such as the spacer sequence, chsA intron (red); basta resistance gene (light blue); kanamycin resistance (gray) and the left and right borders (LB and RB). (B) AscI and PacI digestion of plasmids (lanes 6-9) from control (ctrl) and kanamycin- resistant clones G, K, L. Lanes 1-4 show uncut plasmids to test the efficacy of the double digestion.

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The completed constructs were used to transform the A. tumefaciens strain, EHA105.

Colony PCR of EHA105 clones show that one out of six clones was a positive transformant as it yielded the expected 2 kb amplicon when RNAi construct- specific primer pairs (RNAi-

BnAt2G(1)_fwd and rev-antisense) were used (Figure 4.5 A, lane 2). The positive clone was cultured and used for Arabidopsis transformation using the floral dip method (Clough and Bent,

1998). Five independent primary transformants (T0) were selected based on Basta resistance.

Seeds were collected from individual T0 plants and were again subjected to kanamycin selection.

The resulting T1 plants that survived were grown and self-pollinated to generate T2 seeds.

Segregation analysis for a single locus insertion was done in T2 plants using Basta selection and

PCR with primers specific to the RNAi::BnAt2G(1) construct (chsA-fwd and rev-antisense).

Single insertion mutants segregated in Basta plates in a 3:1 ratio (resistant:sensitive) and were positive for the construct-specific primers (Figure 4.5 C, lanes 5,8 and 9). The plants with a single locus insertion were grown and self-pollinated until the T4 generation. T4 plants retained their

Basta resistance (Figure 4.5 D) and were used for the subsequent phenotyping analyses.

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×  × × × ×

Figure 4.5 Generation of RNAi::BnAt2G(1) in A. thaliana.

(A) Colony PCR of transformed A. tumefaciens EHA105. Checkmark indicates the clone that was positive for the RNAi construct

(lane 2, 2kb). (B) Partial vector map of the RNAi::BnAt2G(1) construct, indicating primer positions. Double arrows indicate the region amplified by the primers and the expected band size. (C)

PCR screening for transgenic plants expressing the

RNAi::BnAt2G(1) construct. Asterisks indicate positive RNAi transgenics which (D) show resistance to 50ug/mL Basta (upper half) as opposed to the wild type control (lower half) of the plate.

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Because the gene specific insert of the RNAi construct was designed from the BnAt2G(1) cDNA sequence, it was possible that it may have silenced other members of the O-FuT family that had high sequence similarity to BnAt2G(1) and/or At2G44500. To confirm that the gene silencing of the RNAi::BnAt2g(1) was specific to the endogenous At2G44500 gene, a multiple sequence alignment of all the gene members of the A. thaliana O-FuT family and the BnAt2G(1) gene insert sequence of the RNAi::BnAt2G(1) construct was performed (Figure 4.6). The red triangles indicate the locations of the gene specific primers used to design the RNAi construct and the bar graph represents the overall sequence conservation from the alignment. As seen in the graph, the average conservation is approximately 70 - 75%. When a phylogenetic tree was generated based from this alignment, At2G44500 and the RNAi sequence clustered in the same group (Figure 4.6 J), indicating that it is likely that the RNAi construct silenced At2G44500.

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(A)

(B)

Figure 4.6 Comparison of the BnAt2G(1) RNAi gene insert sequence of the RNAi::BnAt2G(1) construct to the the A. thaliana O-FuT family. (A) Multiple sequence alignment of the A. thaliana O-FuT family and the BnAt2G(1) gene insert sequence of the RNAi::BnAt2G(1) construct. (B) Phylogenetic tree generated from the alignment. At2G44500 and the RNAi sequences are clustered in the same group as indicated by the red box. Red arrowheads indicate the location of the primers used to amplify the BnAt2G(1) insert used in generation the RNAi::BnAt2G(1) construct.

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4.3.3 Generation of 35S::BnAt2G(1) Lines

To determine if clones harboured the complete overexpression 35S::BnAt2G(1) construct, PCR was done using primers specific to its Nos-terminator (pMDC-NOSf) and the

35S promoter (pMDC-35Sr). Positive clones showed the expected PCR amplicon with a size of approximately 1.5 kb (Figure 4.7 B). Additionally, when purified plasmids were double digested with EcorI and HindIII, they yielded 2.3 and 8.9 kb bands (Figure 4.7 E) which indicated that the cDNA was inserted in the proper orientation, based on the restriction map in figure 4.7 D.

The resulting constructs were subsequently used to transform EHA105 cells.

Transgenic lines overexpressing BnAt2G(1) were generated by dipping Arabidopsis flowers into cultures of transformed EHA105 cells. Independent transgenic lines were selected for hygromycin resistance (50 ug/mL). Genomic DNA (gDNA) was extracted from leaves of hygromycin-resistant T2 plants. The integration of the 35S::BnAt2G(1) construct was tested by

PCR, using primers specific to the Nos terminator (pMDC-NOSf) and BnAt2G(1) (At2G-Iso2r).

As seen in Figure 4.7 E, the expected 1.4 kb band was amplified from genomic DNA isolated from the hygromycin resistant plants. These T2 plants were selfed to produce T3 seeds. The T3 plants were then subjected to hygromycin selection in which they conferred 100% hygromycin resistance.

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Figure 4.7 Generation of 35S::BnAt2G(1) overexpression Lines. (A) Partial and complete (D) vector maps of 35S::BnAt2G(1), indicating PCR primer (red triangles) and restriction site locations. Double arrows indicate the regions amplified by the primer pairs and the expected band sizes. (B) Colony PCR of E. coli clones harbouring the expression vector, 35S::BnAt2G resulting from the LR reactions with the pDEST vector, pMDC32. Positive clones amplified an expected band of 1.5 kb using primers, NOS-f and pMDC-35Sr. (D) PCR screen of 35S::BnAt2G(1) expressing plants using gDNA from T3 plants with primers pMDC-NOSf and BnAt2G-Iso2r. (D) Restriction digestion of purified expression vectors to verify correct plasmid insertion and orientation. (E) 35S::BnAt2G(1) is expected to yield 8.9 kb and 2.3 kb bands (red arrows) when double digested with EcoRI and HindIII.

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4.3.4 Gene Expression Level of Mutants

ANOVA and post-hoc statistical analyses of the delta Ct values for the expression of

At2g44500/BnAt2G(1) (measured by quantitative real-time PCR) in the control plants, the mutant lines and the transgenic lines used in this study [wild type control, g74450, g20490,

RNAi::BnAt2G(1), 35S::BnAt2G(1)] showed that the gene was expressed at statistically different levels in mutant and transgenic lines compared to the wild type control (Figure 4.8, Table 4.1). In particular, the mutant lines that have T-DNA insertions in the promoter region (g-74450; light green bars) and in the first exon (g-20490; red bars) had expression levels reduced to approximately 30% and 50% of wild type levels, respectively (Figure 4.8). The expression of

At2g44500/ BnAt2G(1) in the RNAi-silenced lines (purple bars) was also similarly decreased, to

40% of the control levels. This level was not significantly different than the level of expression observed for the T-DNA knock down lines, based on the post-hoc statistical analyses (Table 4.1).

The largest effect was observed in the 35S::BnAt2G(1) overexpression lines (blue bars), which had an average 6-fold increase in expression compared to wild type (Figure 4.8, Table 4.1).

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(A) avg = 6.39

avg = 0.329 avg = 0.507 avg = 0.398

(B)

Figure 4.8 At2G44500 gene expression levels in mutant lines. (A) Average fold change is indicated for three biological replicates from each mutant or transgenic line: g74450, g20490, RNAi::BnAt2G(1) and 35S::BnAt2G(1). (B) RNA quality as assessed from ribosomal RNA banding patterns for samples from individuals used in the qPCR analysis.

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Table 4.2 ANOVA and Bonferroni-Holm analysis of Ct values of mutant lines compared to the wild type (wt) control.

ANOVA SS DF Between 82.43191 4 Within 0.42365 10

F 486.4392 P 2.09E-11 ****

Post-Hoc: Bonferroni-Holm Group 1 Group 2 Critical P Significant? g20490 wt 0.005 3.75E-06 Yes RNAi::BnAt2G(1) 35S::BnAt2G(1) 0.005556 2.07E-05 Yes g74450 35S::BnAt2G(1) 0.00625 2.13E-05 Yes g20490 35S::BnAt2G(1) 0.007143 2.18E-05 Yes RNAi::BnAt2G(1) wt 0.008333 2.82E-05 Yes 35S::BnAt2G(1) wt 0.01 3.07E-05 Yes g74450 wt 0.0125 0.000325 Yes g20490 RNAi::BnAt2G(1) 0.016667 0.025078 No g74450 g20490 0.025 0.040977 No g74450 RNAi::BnAt2G(1) 0.05 0.34892 No

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4.3.5 Phenotyping

Significant differences were found in the number of branches (BRAN), seed number per silique (SN), root length, number of lateral roots (n), and diameter of the stem second internode between the mutant lines, transgenic lines and the wild type ( Figure 4.10). However, there were no statistical differences observed in the germination rates, rosette diameters, plant heights, total seed weights (TSW), silique numbers (SILN) and above ground biomass in the knockdown mutants and transgenic, compared to the wt for all time points taken (Figure 4.9).

(A) (B)

(C)

Figure 4. 9 Phenotypic traits measured for wt, At2G44500 mutant and transgenic lines. (A) Germination rate (B) stem length (C) rosette diameter (D) biomass and (E) yield related traits such as the number of branches in the main inflorescence (BRAN), total number of siliques (SILN) and seed number per silique (SILN) (continued)

140

(D) (E)

(F)

(D)

(G)

(D)

Figure 4. 9 Phenotypic traits measured for wt, At2G44500 mutant and transgenic lines (continued) Root growth related traits such as (F) the number of lateral roots from 8 to 15 days after germination. (G) and root length. Asterisks identify significantly different values for traits in the mutants and transgenic lines in comparison with the wild type control. Each bar colour represents the plant genotype as seen in the legend. The error bars denote the standard deviation.

141

(A )

(D ) (B)

(D )

(C )

(D )

* (D

) *

(D )

(E)

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Figure 4.10 Stem traits of wt, At2G44500 mutant and transgenic Arabidopsis plants. Representative pictures of the (A) whole stem (B) 5x (C) 10x magnification of the cross sections (D) diameter measurement from each genotype (n = 15). Each bar colour represents the plant genotype as seen in the legend. Asterisks identify significantly different values in the mutants and transgenic lines in comparison with the wild type control.The error bars denote the standard deviation.

4.3.6 FTIR Results

Baseline-corrected and area-normalized FT-IR spectra of wild type (wt), g-20490, g-

74450, RNAi::BnAt2G(1) and 35S::BnAt2G(1) are shown in Figure 4.11. The wave number region ranging from 900 – 1800 has been identified as the "fingerprint region" for cell wall components such as cellulose, hemicelluloses and pectins (Alonso-Simon, 2004; Mouille, 2003;

Kacurakova, 2002) (Figure 4.11, green rectangle). This region is characterized by strong absorption bands at specific wave numbers, which can be assigned to pectic (1745 cm-1, 1620 cm-1, 1397 cm-1 and 1250 cm-1), hemicellulosic (1375 cm-1) and cellulosic (1201 cm-1) polysaccharides (Table 4.3).

Because it is difficult to interpret the spectra based on visual observation alone, a principal component analysis (PCA) was conducted to compare the cell wall compositions of the mutant and wild type plants. When the whole spectrum for the fingerprint region (wave numbers 900 to 1800 cm-1) was used in the analysis, the first and second principle components

(PC1 and PC2) accounted for 56.4% and 23.5% of the variance, respectively (Figure 4.12 A).

Altogether, PC1 and PC2 accounted for 80% of the observed variance between the lines.

The PC scores plot shows a clear separation of the mutant lines from the wt line and variation among the mutant lines (Figure 4.12 A). The T-DNA insertion lines g-20490 and g-

74450 were primarily separated from the wild type by PC2, whereas, RNAi::BnAt2G(1) has

143 negative values for both PC1 and PC2. The 35S::BnAt2G(1) overexpression line (Oex) deviated from the wt primarily due to the negative value of the PC1 component.

The PC1 loadings plot had peaks corresponding to highly methylated pectin (>80% degree of methylation), namely at 1600, 1410-1430,1230 and 1073 cm-1 (Figure 4.11 B) while

PC2 loadings plot showed peaks for pectin with low levels of methylation (<30% degree of methylation) at 1745, 1600, 1410, 1430 and 1014 cm-1 (Figure 4.12 C). The reference spectra from purified pectin were adapted from Zdunek et al. (2012) (Figure 4.12 D and E).

Interestingly, the PC2 loading plot (Figure 4.12 C) also shows a strong negative peak at wave number 1745, which correspond to esterified pectins (Table I). The negative PC scores of

RNAi::BnAt2G(2), relative to wt (Figure 4.12 A), suggests a decrease in both forms of pectin in the knockdown mutants. The 35S::BnAt2G(1) overexpression line (Oex) had a negative PC1 value compared to wt, which suggests that the Oex lines have a decreased level of highly methylated pectin in the cell wall. G-74450 had a negative PC2 value, which suggests a decrease in low methylated pectin levels.

ANOVA and Bonferronni-Holm post hoc analyses showed that the absorption values of wave numbers corresponding to specific cell wall components are statistically different from the wild type (Table 4.6). Interestingly, a comparison of the absorption values of the knockdown, g-

74450 and RNAi::BnAt2G(1) and the over expression (Oex) lines showed that their respective cell wall compositions are also statistically different (Table 4.7). In particular, the absorption values in wave numbers 1550 cm-1, 1650 cm-1 (for Amide/Proteins) and 1600 to 1630 cm-1 (for non-esterified uronic acid side chain of pectin) were significantly higher in the knockdown mutants compared to the Oex lines but are not different from the wild type.

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The knockdown line, g-74450 and the Oex mutants showed the most prominent stem phenotypes (Figure 4.10). Interestingly, these lines had higher levels of esterified pectin compared to wild type (Table 4.6, yellow box). Also, Oex lines had significantly lower levels of de-esterified pectin compared to wt (Table 4.6, yellow box) and knock down lines (Table 4.7, blue box).

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1021 1021

1620

wt 1620

1375 1375

1397 1397

1250

1745 1745

g-20490 g-20490

g-74450 g-74450

RNAi::BnAt2G(1)

35S::BnAt2G(1)

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Figure 4.11 FT-IR spectra of wild type (wt), mutant lines and transgenic lines: g-20490, g- 74450, RNAi::BnAt2G(1) and 35S::BnAt2G(1). Light green rectangle represents the polysaccharide fingerprint region used to identify plant cell wall components. Peaks that identified were: 1745, 1620, 1397, 1375, 1250 and 1021 cm-1 and are present in all the lines (n=3).

(A)

(B) (C)

(D) (E)

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Figure 4.12 Principal Component Analysis (PCA) of FT-IR spectra in the cell wall polysaccharide fingerprint region. (A) PCA scores plot of wt (grey triangle) and mutant lines: g-20490 (blue circle); g-74450 (red square); RNAi::BnAt2G(1) (purple triangle); and 35S::BnAt2G(1) (green diamond). (B) PC1 and (C) PC2 loadings plot. (D) Reference spectrum of purified forms of highly methylated and (E) low methylated pectin. Reference spectrum was from Zdunek et al, 2012.

4.4 Discussion

At2G44500 is a member of the large GT-65R/O-fucosyltransferase (O-FuT) family in

Arabidopsis of which only a few members have recently been characterized (Neumetzler, 2012;

Wang, 2013). The current results with At2G44500 mutants, summarized in Table 4.5, suggest that the O-Fut At2G44500 encodes plant cell wall components, particularly pectin, leading to defects in root and stem development.

Pectins are a highly heterogeneous group of polymers that contain as many as 14 different monosaccharides and have a high galacturonic acid content (Figure 4.13). Pectic polysaccharides can be classified as homogalacturonans (HGs), apiogalacturonans (AGs), xylogalacturonans (XGAs) and rhamnogalacturonans I and II (RGI and RGII). The typical distribution among the categories in the cell are: HG = 65%; RGI = 20-35%; XGA and RGII =

<10% (Mohnen, 2008). Together with hemicelluloses, pectins constitute the cell wall matrix in which cellulose microfibrils are embedded. These polymers are present in differing ratios and their interactions contribute to the strong yet dynamic and flexible properties of the cell wall.

In particular RGII is known to have glycosidic bonds with fucose (2-O-methyl fucose), but the glycosyltransferase(s) (GTs) responsible for its fucosylation are still unknown. While members of the GT-37 family are believed to catalyze the fucosylation of xyloglucan and arabinogalactan proteins (AGPs) and members of the GT-10 family were shown to fucosylate

N-linked glycoproteins (Chapter 2), the GT-65R family may serve that function, of which

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At2G44500 is a member. In fact, bioinformatic and structural modelling studies have suggested that members of this GT-65R/O-FuT family are putative RG-II related glycosyltransferases

(Hansen, 2012; Voxeur, 2012).

The phenotypic effects that were observed from the current work with the At2G44500 knock down and over expression mutants, on lateral root formation and changes in stem diameter, might be explained through their alteration of pectin properties in the cell walls of the stems and roots during development.

4.4.1 Lateral Roots and Stem Phenotypes

Lateral root formation represents an important element of the adaptability of the root system to its environment as it determines the final root architecture and the plants' ability to take up nutrients and water. Lateral roots (LR) are formed post-embryonically and LR primordia are initiated and protrude out of the primary root through the endodermal, cortical and epidermal layers (Atkinson, 2014). LR formation is mainly modulated by cell divisions in the pericycle regulated by the hormone auxin, but also requires modifications of overlying rigid cell walls of the epidermis through which the protruding LR must penetrate (Van Norman, 2013; Atkinson,

2014; Vilches-Barro, 2015). In Arabidopsis and other plant species, there are several mutants that have been reported to show defects in LR formation. Mutants such as: the auxin- overproducing mutant, superroot1/Atsur1 (Boerjan, 1995; Seo, 1998); a single amino acid mutation in the conserved domain of the transcription factor, Auxin Response Factor 2

AXR2/IAA7 (axr2-1) (Nagpal, 2000); elongated hypocotyl5/Athy5 (Sibout, 2006); cytokinin oxidase mutants (Dello, 2007); never ripe (nr) in tomatoes (Negi, 2010) - all showed an increased numbers of LRs as a result of insensitivity to ethylene (for nr mutants) and/or auxin overproduction. Some mutants have also been reported to produce fewer LRs, such as the

149 epinastic (epi) mutant in tomatoes, due to elevated ethylene levels (Negi, 2010) and a loss of function mutation in a jasmonate receptor in Arabidopsis called coronatine insensitive1 (COI1)

(Thatcher, 2012). Both At2G44500 and BnAt2G(1) contain transcription control elements that play roles in response to jasmonic acid (JA), ethylene and auxin (Chapter 3). The mechanism for these three pathways to interact with each other and control LR formation is still unclear and needs to be elucidated.

In the present study, the increases in lateral root formation in out of the knockdown mutants (g-74450) of the putative O-FuT, At2G44500, and the decrease in lateral roots in the

35S::BnAt2G(1) overexpression lines, compared to wild type controls, suggests that the former has weaker walls which allowed emerging LR primordia to precociously emerge from the primary root. Conversely, overexpression of the O-Fut [BnAt2G(1)] led to cell wall stiffening which resulted in fewer LRs protruding out of the main root.

Other studies have linked altered cell walls with increased LR formation. For example, knockdown mutants of an auxin importer, LAX3, show reduced LR emergence that was correlated with decreased expression of cell wall remodelling enzymes, such as: pectate lyases, xyloglucan xyloglucosyl transferases, glycosyl hydrolases and expansins (Swarup, 2008).

Previously characterized mutants that have been shown to have altered cell wall monosaccharide components also show defects in LR formation. For example, mutations in the Arabidopsis

GDP-D-mannose-4,6-dehydrogenase gene (GMD1), a key component in the synthesis of GDP- fucose (MUR1) (Bonin, 1997) and Cellulose Synthase 6 and 8 (CESA6/8) (Cutler, 1997), that rendered these proteins non-functional, showed an increase in LR formation (Roycewicz, 2014).

The glycosyltransferase family 77 (GT-77) is a family of putative arabinosyltransferase that have been implicated in the arabinosylation of cell wall components. Knockdown mutants of

150 members of GT-77 such as the Lateral Root Development 5 (LRD5)/Xyloglucotransferase 113

(XEG113) and Reduced Arabinose Yariv1 (RAY1) also showed increases in total lateral root length and contained reduced arabinose levels of extensins and arabinogalactan proteins (AGPs) in Arabidopsis (Roycewicz, 2014; Gille, 2013; Egelund, 2007).

The stem phenotypes observed in the overexpression and knock down lines suggest that the mutations may have affected vascular tisue development. It was shown that At2G44500 was highly expressed in the rib meristem (figure 2.5 A). The rib meristem is a region of the shoot apical meristem and is composed of rapidly dividing cells that give rise to vascular tissues during indeterminate growth of the plant (Ruonala, 2008). It is also interesting to note that the number of vascular bundles were different in the knockdown compared to the overexpression lines. In Arabidopsis, the de novo lateral root initiation requires tightly coordinated asymmetric division of pericycle cells located at the xylem pole (Beeckman, 2014; Van Norman, 2013).

Perhaps the decrease in the number of the xylem poles in the vascular bundles led to a change in nutrient transport capacity and affected lateral root initiation.

Other cell wall-related mutations that have led to changes in stem diameter, include the knockdown mutants of the leucine-rich extensin glycoproteins (Draeger, 2015) which have smaller stem diameters due to smaller cell sizes in their epidermal cell walls. Cell sizes and numbers in the mutants and transgenics were not examined in the current study but should be examined in future work.

4.4.2 FT-IR Results

Mutants and trangenics with altered At2G44500/BnAt2G expression were analyzed by

FT-IR. The results showed that the levels of methyl-esterified pectin in the cell walls may provide some insight into the connection between fucosylation and cell wall structure and

151 function. Most pectin forms (with the exception of RG-I) are made up of a poly-galacturonic acid (GalA) backbone (Figure 4.9). Homogalacturnonans (HG's) are the most abundant class of pectin (Wolf, 2009; Atmodjo, 2011; Braybrook, 2012; Peaucelle, 2011; Willats, 2001) and they are synthesized by various GTs, in the Golgi, and delivered by secretory vesicles to the cell wall

(Brandizzi, 2014) in highly methyl-esterified forms (Zhang, 1992; Sterling, 2001). They are subsequently modified by cell-wall modifying proteins, such as pectin methyl-esterases (PMEs), which are enzymes that catalyze the methyl de-esterification of HG's (Levesque-Tremblay,

2015; Lionetti, 2012; Micheli, 2001). De-esterified pectins form stiffer gels in vitro and are associated with cell wall stiffening in vivo (Zhao, 2008). The stiffening arises from cooperative calcium binding of contiguous carboxyl groups on two adjacent pectin chains (Braybrook, 2015;

Senechal, 2014; Micheli, 2001). The levels of esterified and de-esterified pectins can, therefore, be indirect indicators of PME activity and how they regulate cell wall rheology and biomechanics (Derbyshire, 2007; Peaucelle, 2008; Lionetti, 2012; Muller, 2013). Highly de- esterified pectin levels indicate increased PME activity and result in a favourable cross-linking with Ca2+ ions which leads to a more rigid/stiffer pectic gel network (Jolie, 2010).

Alternatively, low de-esterified pectin levels indicate decreased PME activity and promote the action of pectinases, such as polygalacturonases (PGs), which contribute to cell wall loosening.

Thus, one would expect that a cell wall containing a matrix that has high levels of de-esterified pectin would result to a stiffer, not softer cell wall. However, there are several observations that have been reported (including the phenotypes in this study) that go against this model. Table 4.8 summarizes these studies that suggest the control mechanisms for cell wall elasticity and growth are not very straightforward.

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4.4.3 Role of Fucosylation in Pectin Biosynthesis and Cell Adhesion

The precise molecular mechanisms of how At2G44500/BnAt2G(1) and other members of the O-FuT family affect cell wall biomechanics still remain unclear. However, our observations from the mutant analyses give a clearer picture on how O-FuTs and/or fucosylation could play an important role in cell wall adhesion, particularly in pectin synthesis.

The knowledge about fucose synthesis, metabolism and its distribution in the cell are continually evolving. However, there is evidence that defects in fucose synthesis could strongly affect plant growth and development. Arabidopsis mur1 mutants are defective in their de novo synthesis of fucose, and thus are completely lacking fucose in the cell wall (Reiter, 1993). mur1 plants exhibit a dwarfed growth (Reiter, 1993) and although the cell wall thickness in stems was not altered, the force required to break the primary walls upon longitudinal stretching was less than half compared to wild-type plants, indicating that mur1 has a defect in the intrinsic mechanical properties of the walls (Reiter, 1993). Loss of function mutants of a putative O-FuT,

FRIABLE1 (frb1), exhibited cell and organ dissociations that are indicative of defects in cell adhesion (Neumetzler, 2012). frb1 mutants also had a high degree of de-esterified pectins in their cell walls due to a 45–70% increase in PME activity in the mutants (Neumetzler, 2012). In contrast to the cell dissociation phenotypes observed in frb1 mutants, overexpression (OEx) of a pectin methylesterase inhibitor (PMEI5) displayed organ fusion phenotypes especially in stems

(Muller, 2013). PMEI5 overexpression led to aberrant growth of stems which have lateral shoots that failed to separate properly from the main stem. Interestingly, significantly higher amounts of fucose (and other neutral sugars such as rhamnose and arabinose) were found in the cell walls of the OEx plants compared to the wild type plants (Muller, 2013). This suggests that alterations

153 of methylesterified pectin levels (caused by PMEI overexpression) could result in changes in sugar distribution in the cell wall and ultimately lead to plant anatomical changes.

AtMSR1 and AtMSR2 were shown to be involved in mannan synthesis (Wang, 2013).

Although there were no obvious phenotypes found in msr1, msr2 single T-DNA insertion mutants and msr1 msr2 double mutants, the level of mannosyl residues in stem glucomannans decreased by approximately 40% for msr1 and by more than 50% for msr1 msr2 double mutants

(Wang, 2013). It is interesting to note that FRIABLE1, AtMSR1 and AtMSR2 belong to the same GT-65R/O-FuT family as At2G44500 (chapter 2).

4.4.4 Proposed Role of At2G44500/BnAt2G(1) in Pectin Synthesis and Cell Adhesion

In this study, knockdown expression of At2G44500/BnAt2G(1) led to increased lateral root formation, wider stem diameter and an increased level of de-esterified pectin in the cell wall compared to the overexpression mutants. The visible phenotypes observed in the knockdown mutants suggest that the inhibition of fucosylation of cell wall polysaccharides could affect cell wall components particularly, RG-II, which contains fucose residues on two of its side chains (Figure 4.13). In contrast, the overexpression of At2G44500/BnAt2G(1) showed a decreased number of lateral roots, smaller stem diameter and significantly lower levels of de- esterified pectins compared to the wild type and the knockdown mutants. These phenotypes are indicative of a compact and rigid cell wall perhaps due to a stronger adhesion between components in the polysaccharide matrix.

RG-II is a highly complex pectic polysaccharide composed of a polygalacturonic acid backbone and four distinct side chains (A–D; Figure 4.14). Boron (B) forms ester bonds with the two apiosyl residues of side chain A of two RG-II monomers to generate boron dimerized

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RG-II (Camacho-Cristobal, 2008; O'Neill, 2004). This dimerization results in the formation of the pectic polysaccharide network (O’Neill, 2004). In plant cell walls, more than 90% of RG-II are dimerized by boron, and without proper RG-II-interaction, the cell walls swell and increase in thickness (Fleischer, 1999). Increasing evidence supports that RG-II and its boron crosslinking are critical for plant growth and development by supporting cell adhesion and mechanical strength (Funakawa, 2015).

Based on the observations from this thesis, it is postulated that At2G44500/BnAt2G(1) may be involved in the synthesis of RG-II by catalyzing the fucose transfer in one of its side chains, particularly side chain A (Figure 4.14). Knocking down the expression of this fucosyltransferase led to a change in the RG-II structure and resulted in a limited or decreased boron-mediated RG-II dimerization (figure 4.13, left side). This result could potentially explain why phenotypes that are indicative of cell wall loosening, such as increased number of lateral roots and wider stem diameter were observed in the knockdown lines. Conversely, it is possible that the overexpression of BnAt2G(1)/At2g44500 may have caused ectopic fucosylation of RG-II and HG pectic polysaccharides and enhanced boron-mediated RG-II cross-linking (figure 4.14, right side). Consequently, this enhanced cross-linking of the pectic network may have caused the cells to strongly adhere to each another and lead to cell wall stiffening.

4.4.5 Study Limitations

The functional analyses of the different At2G44500/BnAt2G(1) mutants gave a clearer understanding of the putative function of this novel plant O-FuT. However, there were some methodological limitations in this study. The first limitation was related to the heterologous silencing of BnAt2g(1) in Arabidopsis through RNAi. Although the small interfering RNA

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(siRNA) resulting from the RNAi::BnAt2G(1) construct was approximately 90% similar to the endogenous Arabidopsis gene, At2G44500 expression was not completely knocked out. Instead there was a 60% reduction in expression (figure 4.8). The other T-DNA knockdown lines, g-

20490 and g-74450 showed a 50% and 70% At2G44500 expression reduction, respectively

(figure 4.8). The seeds from single insertion RNAi mutants that segregated in Basta plates in a

3:1 ratio (resistant: sensitive) that were also positive for the RNAi::BnAt2G(1) construct- specific primers in the T2 generation were pooled. This did not allow for the analysis of at least three independent RNAi lines. Nonetheless, the phenotypes observed from this RNAi knockdown population were generally consistent with the T-DNA knockdown lines particularly in their cell wall compositions as measured by FT-IR and analyzed with PCA (figure 4.12).

There was also a possibility that the siRNA may have silenced several genes belonging to the AtOFuT family that were paralogous to At2G44500. However, the alignment of the siRNA sequence with all the coding sequence of all the members of the AtOFuT family and the phylogenetic tree that was generated from this analysis showed that At2G44500 and the siRNA sequence clustered in the same group as At2G44500 (figure 4.6). This indicated that the siRNA which was designed from the B.napus sequence, silenced its Arabidopsis homolog, At2G44500.

A common feature of all T-DNA collections is the presence of multiple insertion events occurring in a single plant (O'Malley, 2010). Such events can occur at distinct genomic locations or occur as tandem events at a single site of integration. One important implication of a second, unlinked insertion is the possibility that it may be responsible for the observed phenotypes. Because of these additional potential genomic alterations, backcrossing of each T-

DNA insertion line to a wild type plant is recommended (Krysan, 1996; Alonso, 2003).

Alternatively, a second independent T-DNA insertion mutant of the same gene can also be used

156 to confirm that a phenotype was caused by the specific gene disruption if the second mutant recapitulates the phenotypes that were observed in the first one . This is because an independently derived T-DNA insertion line is very unlikely to share the same off-site genomic defects and/or secondary T-DNA inserts (O'Malley, 2010).

The two T-DNA lines that had insertion sites in the promoter (g-74450) and the first exon (g-20490) were homozygous for the insertion and showed a 70% and 50% reduction in

At2G44500 expression, respectively. Both independent T-DNA insertion lines had similar and consistent trends in their phenotypes such as in: germination, plant height, rosette diameter, number of branches, seed number per silique, total seed weights, silique numbers and in their cell wall compositions (figures 4.9 and 4.12). The differences seen in root length and lateral root emergence from these two lines (figure 4.9F and 4.9G) may be attributed to the location of the insertion sites within the gene and the different alleles may be functioning in a tissue-specific manner.

Taking into account all the data and information presented in this thesis, the methodological limitations listed above do not diminish the overall observation that At2G44500 could play a role towards cell wall matrix polysaccharide synthesis and manipulating the expression of this plant O-FuT affects various aspects of plant development.

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Figure 4.13 Schematic diagram of pectin polysaccharide structure. HG: Homogalacturonan; XGA: Xylogalacturonan; RG-I/II: Rhamnogalacturonan I/II

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Table 4.3 Phenotypic traits that were measured in mutant and wild type analyses.

Growth traits Reproductive traits (Yield- related traits)

Germination (%) Seed number per silique (SN)

Root length (mm) Number of siliques (SILN),

Number of lateral Number of branches on roots the main inflorescence (BRAN) Rosette diameter (mm) Stem length (mm)

Stem Diameter (um)

Biomass (mg)

Table 4.4 FT-IR absorption of cell walls and their assignment to main cell wall components.

Assigned Cell Wall Frequency Range found in literature References Component Cellulose 1000, 1033, 1040,1059, 1060, 1162, Diaz, 2012; Brown, 2005; 1115, 1313, 1317 Carpita, 2001; Wilson, 2001; Kacuráková, 2000 Xyloglucan 1019, 1041, 1075, 1078, 1120, 1130, Kacuráková, 2000; Coimbra, 1317, 1371, 1416 1999 Pectin 1014, 1019, 1097, 1100, 1104, 1143- Kacuráková, 2000; Coimbra, 1146, 1243, 1320, 1400 1999 Amide I (Protein) 1550, 1650 Wang, 2009; Sene, 1994 Phenolic Esters 1720 Carpita, 2001; Sene, 1994 Esterified Pectin 1740-1745 Mouille, 2003 De-esterified Pectin 1427,1420,1414,1600-1630 Mouille, 2003

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Table 4.5 ANOVA and Bonferroni-Holm analysis of the 2nd stem internodes of mutant lines compared to the wild type control and to each other. Stem - 2nd Internode

Name Count Avg. SD

wt 15 1165.75 75.69675 pMDC32 14 1223.967 46.80721 RNAi 15 1099.796 53.69225

g-20490 15 1375.248 51.21957 g-74450 15 1221.938 44.40829

ANOVA

SS DF Between 622130.1 4 Within 213399.4 69

F 50.28947 P 9.46E-20 ****

Post Hoc: Bonferonni-Holm

Group 1 Group 2 Critical P Significant? pMDC32 g-74450 0.005 1.88E-14 Yes wt g-74450 0.005556 1.24E-09 Yes

g-20490 0.00625 1.65E-09 g-74450 Yes RNAi g-74450 0.007143 6.83E-09 Yes pMDC32 g-20490 0.008333 2.25E-07 Yes RNAi pMDC32 0.01 4.23E-07 Yes

wt pMDC32 0.0125 0.010262 Yes wt g-20490 0.016667 0.019439 No wt RNAi 0.025 0.020167 No RNAi g-20490 0.05 0.905538 No

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Mutant Germination Rosette Plant Root # LR BRAN SILN Biomass Stem FT-IR Line Rate Diameter Height Length Diameter g-20490 nd nd nd longer - 8,9,10 More- on all less nd nd nd Higher days after time points level of de- germination esterified pectin g-74450 nd nd nd nd nd less nd nd larger Higher level of de- esterified pectin RNAi:: nd - nd longer - 14 and Less - 15 - - nd nd Higher BnAt2G(1) 15 days after days after level of de- germination germination esterified pectin 35S:: nd - nd longer - 14 and Less - on - - nd smaller Lower level BnAt2G(1) 15 days after all time of de- germination points esterified pectin Table 4.6 Summary of Results – nd: no statistical difference found compared to wild type.

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Table 4.7 ANOVA and post-hoc analyses of individual wave numbers with designated CW component for wt and mutant lines. Red letters indicate significance compared to the wild type (wt) control.

Significant compared to wildtype? (p<0.05) g- g- RNAi Oex WT 20490 74450 Wave Cell Wall AVG AVG AVG AVG AVG number Designation Abs Abs Abs Abs Abs 1039 Cellulose 0.97 no 0.95 yes 0.95 yes 0.95 yes 0.97 1059 Cellulose 0.90 no 0.90 no 0.89 no 0.88 yes 0.92 1061 Cellulose 0.89 no 0.89 no 0.88 no 0.87 yes 0.91 1161 Cellulose 0.46 no 0.49 no 0.48 no 0.45 no 0.47 1367 Cellulose 0.54 no 0.50 no 0.46 no 0.42 no 0.51 1041 Xyloglucan 0.96 no 0.95 no 0.94 yes 0.94 yes 0.96 1078 Xyloglucan 0.81 no 0.80 no 0.80 no 0.77 no 0.81 1120 Xyloglucan 0.57 no 0.58 no 0.58 no 0.55 no 0.59 1317 Xyloglucan 0.49 no 0.44 no 0.41 no 0.37 yes 0.47 1371 Xyloglucan 0.55 no 0.50 no 0.46 no 0.43 no 0.51 1005 Pectin 0.96 no 0.96 no 0.96 no 0.97 yes 0.95 1014 Pectin 0.99 no 0.99 no 0.99 no 1.00 yes 0.99 1400 De-esterified 0.56 no 0.49 no 0.47 no 0.41 yes 0.52 Pectin 1745 Esterified Pectin 0.15 no 0.24 yes 0.20 no 0.21 yes 0.13 1450 Amide/Proteins 0.44 no 0.38 no 0.39 no 0.34 no 0.39 1650 Amide/Proteins 0.42 no 0.44 no 0.43 no 0.38 no 0.46

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Table 4.8 Absorption values and the corresponding cell wall components in the knockdown, g-74450, RNAi::BnAt2G(1) compared with the over expression (Oex) lines. Red letters indicate significance.

Significant compared to Oex? (p<0.05) g- RNAi Oex 74450 Wave Cell Wall AVG AVG AVG number Designation Abs Abs Abs 1059 Cellulose 0.90 yes 0.89 no 0.88 1061 Cellulose 0.89 yes 0.88 no 0.87 1161 Cellulose 0.49 yes 0.48 no 0.45 1367 Cellulose 0.50 yes 0.45 no 0.42 1078 Xyloglucan 0.80 yes 0.80 no 0.77 1120 Xyloglucan 0.58 yes 0.58 no 0.55 1317 Xyloglucan 0.44 yes 0.41 no 0.37 1371 Xyloglucan 0.50 yes 0.46 no 0.43 1400 De-esterified 0.49 yes 0.47 yes 0.41 Pectin 1600 De-esterified 0.50 yes 0.51 yes 0.45 Pectin 1603 De-esterified 0.49 yes 0.51 yes 0.45 Pectin 1605 De-esterified 0.50 yes 0.51 yes 0.46 Pectin 1606 De-esterified 0.50 yes 0.51 yes 0.46 Pectin 1608 De-esterified 0.51 yes 0.51 yes 0.46 Pectin 1612 De-esterified 0.52 yes 0.52 yes 0.46

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Pectin 1614 De-esterified 0.52 yes 0.51 yes 0.46 Pectin 1616 De-esterified 0.53 yes 0.51 yes 0.46 Pectin 1618 De-esterified 0.54 yes 0.52 yes 0.47 Pectin 1620 De-esterified 0.54 yes 0.52 yes 0.47 Pectin 1622 De-esterified 0.54 yes 0.51 yes 0.47 Pectin 1624 De-esterified 0.54 yes 0.52 yes 0.47 Pectin 1626 De-esterified 0.54 yes 0.52 yes 0.46 Pectin 1628 De-esterified 0.53 yes 0.51 yes 0.46 Pectin 1630 De-esterified 0.52 yes 0.50 yes 0.45 Pectin 1414 De-esterified 0.48 yes 0.46 yes 0.41 Pectin 1419 De-esterified 0.47 yes 0.45 yes 0.40 Pectin 1427 De-esterified 0.44 yes 0.43 yes 0.38 Pectin 1550 Amide/Proteins 0.38 yes 0.39 yes 0.34 1650 Amide/Proteins 0.44 yes 0.43 yes 0.38

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Table 4.9 Summary of studies reporting the role pectin methylesterases (PMEs) in cell wall biomechanics.

Current Model:

PME Level of Pectin Cell wall phenotype Activity De-esterification High high Stiffening – restricts growth Low low Loosening – promotes growth

Reported phenotypes: Mutation and Level of Pectin Cell wall phenotypes Reference Description De-esterification pme3-1 low Loosening; increased Guenin et - knockdown mutant of hypocotyl length; al, 2011 PME3 increased number of - low PME acvtivity adventitious roots; decreased rosette diameter; decreased stem length pme35 low Loosening; decrease in Hongo, - knockdown mutant of mechanical stem 2012 PME35 strength - low PME acvtivity blr1 high reduced cell Peaucelle, - loss of function expansion and defects 2011a mutation of the in internode elongation transcription factor, BELLRINGER which resulted in the upregulation of PME5 35S::PMEI4 low Stiffening; delayed Pelletier, - overexpression of PME hypocotyl growth 2010 inhibitor (PMEI4) - low PME acvtivity 35S::PMEI3 low Stiffening; decreased Peaucelle, - overexpression of PME cell wall elasticity in 2008, inhibitor (PMEI3) apical meristems; lateral 2011a - low PME acvtivity organ formation suppression 35S::PME5 high Loosening; increased Peaucelle, - overexpression of PME5 cell wall elasticity in 2008, 2011 - high PME acvtivity apical meristems pin1 + exogenous IAA NR Loosening; increased Braybrook, - auxin transporter mutant cell wall elasticity 2013

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rescued by exogenous application of auxin, IAA pme48 low Stiffening; decreased Leorux, -knockdown of PME 48 germination, increased 2015 - low PME acvtivity pollen diameter 35S::BnAt2G(1) low Stiffening; smaller stem -overexpression of B. diameter; lower number napus homolog of of lateral roots At2G44500 g-20490 high Loosening; increased - knockdown mutant of number of lateral roots At2G44500 g-74450 high Loosening; wider stem - knockdown mutant of diameter At2G44500

166

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Figure 4.14 Proposed mechanism5 that explains how BnAt2G(1)/At2G44500 could affect cell wall extensibility. Pectin molecules can covalently bond with each other to form a pectic gel network. The wildtype (WT) structure of covalently bonded rhamnogalacturonan-II (RG-II) and homogalacturonan6 (HG) is shown in the middle. RG-II dimerization is mediated by Boron. The four distinct side chains of RG-II are indicated and labelled from A-D. (left) In BnAt2G(1)/At2G44500 knockdown lines, fucose linkages are non-existent (x), particularly in RG-II, thereby changing7 the overall structure and binding capabilities of the pectic network. The absence of fucose in side chain A of the RG-II structure can limit the Boron-mediated RG-II dimerization. This can then lead to a decrease in cell adhesion and result in cell wall loosening phenotypes such as an8 increased number of lateral roots and wider stem diameter. Lodging was also observed in RNAi::BnAt2G(1) lines. Pectin methylesterases (PMEs) can still act on the methyl-esters of HGs. (right) Overexpression of BnAt2G(1)/At2g445009 may have caused ectopic fucosylation of pectin as fucosyl linkages can be attached to methyl, rhamnose and galactose side chains. This can enhance and promote strong cross-linking or adhesion of the pectic network and result in stiffer/more rigid cell walls. 10

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14 Chapter 5

General Summary and Future Directions

At2G4450 and its B. napus homolog, BnAt2g(1), have been identified as plant O- fucosyltransferases (O-FuTs) that may play a role in the the biosynthesis of matrix polysaccharides and contribute to the biomechanics and development of the plant cell wall. In the present studies, the following features concerning At2G44500/BnAt2G(1) were shown:

First, they contain conserved key amino acids essential for O-FuT catalytic activity. Second, they have a protein structure and topology reminiscent of a type II membrane protein, which consists of a short cytoplasmic C-terminal domain, one transmembrane (TM) domain and a lumenal N-terminal domain. In general, most non-processive glycosyltransferases (which are enzymes that add monosaccharides to specific positions of polysaccharide chains or glycoproteins) fall under this type II membrane protein category. Third,

At2G44500/BnAt2G(1) proteins are localized in the secretory pathway (Golgi or Golgi-derived vesicles) where post-translational modifications and synthesis of matrx polysaccharides occur.

In silico analysis of At2G44500/BnAt2G(1) promoter sequences identified putative transcription control elements that are involved in hormone-regulated stress responses. The analyses of knockdown and overexpression mutants reveal that changing the expression of

At2G44500/BnAt2G(1) could lead to developmental phenotypes that could be related to the alteration of the cell wall polysaccharide matrix. It is suggested that the mutations affected the synthesis of the pectic polysaccharide, Rhamnogalacturonan-II (RG-II), and its ability to dimerize with other RG-II through boron.

The fucosyltransferases (FuTs) that are responsible for catalyzing the addition of fucose side chains to RG-II are still yet to be identified. At2G44500/BnAt2G(1) is a good candidate as

169 one of the FuTs that is involved in RG-II synthesis in the Golgi. The following future experiments are suggested to further understand the role of this FuT in RG-II synthesis and its contribution to cell wall development and biomechanics. First, it would be important to quantify the fucose levels in the cell walls of the knockdown and overexpression lines in order to verify this FuT's ability to catalyze fucose transfer. This can be done by extracting alcohol-insoluble residue (AIR) from the stems or roots of wildtype and mutant lines followed by analysis of the subsequent preparations by gas chromatography- mass spectometry (GC-MS) in order to obtain their non-cellulosic neutral monosaccharide profiles. Second, one could express the At2G44500 protein and perform a fucosyltransferase assay. One could also extract the At2G44500 protein from the knockdown and overexpression lines and measure the enzymatic activity in vitro.

At2G44500 was initially discovered to be upregulated in microspore cultures that were subjected to a mild heat stress. Based on these, it might be interesting to see how

At2G44500/BnAt2G(1) knockdown and overexpression lines respond to various abiotic and biotic stresses.

It is also interesting to note that the RNAi::BnAt2G(1) lines exhibited a lodging phenotype when the plants reached full maturity (Figure 4.14). It was also suggested here that an increase in boron-mediated RG-II dimerization can lead to stiffer cell walls. Plants with stiffer/more rigid cell walls are less prone to lodging (Hirano, 2014; Reiter, 1998). Establishing a conection between the role of O-FuTs in this important agronomic trait needs to be further explored. The contradictory reports of PME action on cell wall properties present a puzzle that still needs to be further studied. It is also important to elucidate the functional significance of the extensive interactions between cell wall matrix polysaccharides and pectin to understand their possible significance for cell wall mechanics and growth.

170

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Appendix 1- ANOVA of the fold change values of g74450 Oex 0.00625 2.13E-05 Yes knockdown and overexpression mutants based on comparisons g20490 Oex 0.007143 2.18E-05 Yes of transcript levels to wt with AtAct2 and AtEF1-alpha as the g20490 RNAi 0.016667 0.025078 No internal controls g74450 g20490 0.025 0.040977 No g74450 RNAi 0.05 0.34892 No Name Count Avg. SD g74450 3 0.32962 0.100872 g20490 3 0.507667 0.024007 RNAi 3 0.398173 0.048716 Oex 3 6.39 0.445758 wt 3 1 0

Test for equal variance (Levene's Test)

F DFn DFd P

2.857421773 4 10 0.081135

PASS - equal variance may be assumed (p > 0.05).

ANOVA SS DF Between 82.43191425 4 Within 0.423649629 10

F 486.4391973 P 2.08796E-11 ****

Post-hoc: Bonferronni-Holm Group 1 Group 2 Critical P Significant? g20490 wt 0.005 3.75E-06 Yes

RNAi wt 0.008333 2.82E-05 Yes

Oex wt 0.01 3.07E-05 Yes g74450 wt 0.0125 0.000325 Yes

RNAi Oex 0.005556 2.07E-05 Yes

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Appendix 2 – Statistical analysis for traits measured in A.thaliana wild type and mutants lines in different time points.

Germination Germination 5d after imbibtion 2d after imbibtion Name Count Avg. SD Name Count Avg. SD wt 10 0.93374 0.040761 wt 10 0.689686 0.071418 g-20490 8 0.886463 0.215925 g-20490 8 0.617213 0.177642 g-74450 8 0.905938 0.219121 g-74450 8 0.657438 0.225542

Test for equal variance (Levene's Test) Test for equal variance (Levene's Test) F DFn DFd P F DFn DFd P 1.484578 2 23 0.247517 3.053676 2 23 0.066656

PASS - equal variance may be assumed (p > 0.05). PASS - equal variance may be assumed (p > 0.05).

ANOVA ANOVA SS DF SS DF Between 0.010189 2 Between 0.023344 2 Within 0.677419 23 Within 0.622885 23

F 0.172978 F 0.430989 P 0.84224 P 0.655007

Post-hoc: Bonferronni-Holm Post-hoc: Bonferronni-Holm Group 1 Group 2 Critical P Significant? Group 1 Group 2 Critical P Significant? wt g-20490 0.016667 0.504743 No wt g-20490 0.016667 0.254017 No wt g-74450 0.025 0.697552 No wt g-74450 0.025 0.673704 No g-20490 g-74450 0.05 0.860459 No g-20490 g-74450 0.05 0.697867 No

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Germination Germination 6d after imbibtion 7d after imbibtion

Name Count Avg. SD Name Count Avg. SD wt 9 0.954762 0.049667 wt 9 0.956984 0.045469 g-20490 7 0.955129 0.067114 g-20490 7 0.956543 0.063605 g-74450 7 0.9832 0.044449 g-74450 7 0.9846 0.040745

Test for equal variance (Levene's Test) Test for equal variance (Levene's Test) F DFn DFd P F DFn DFd P 0.889745 2 20 0.426406 0.89883 2 20 0.422864

PASS - equal variance may be assumed (p > 0.05). PASS - equal variance may be assumed (p > 0.05).

ANOVA ANOVA SS DF SS DF Between 0.003894 2 Between 0.003767 2 Within 0.058614 20 Within 0.050774 20

F 0.664397 F 0.741822 P 0.525578 P 0.4889

Post-hoc: Bonferronni-Holm Post-hoc: Bonferronni-Holm Group 1 Group 2 Critical P Significant? Group 1 Group 2 Critical P Significant? wt g-74450 0.016667 0.254611 No wt g-74450 0.016667 0.22844 No g-20490 g-74450 0.025 0.374378 No g-20490 g-74450 0.025 0.345136 No wt g-20490 0.05 0.990142 No wt g-20490 0.05 0.987281 No

200

Rosette Diameter Rosette Diameter 25d 28d Name Count Avg. SD Name Count Avg. SD wt 33 46.95455 4.586604 wt 34 55.47059 4.157762 g-20490 32 46.95313 5.126401 g-20490 36 51.34722 5.588022 g-74450 35 45.65714 6.940685 g-74450 38 51.86842 7.70546

Test for equal variance (Levene's Test) Test for equal variance (Levene's Test)

F DFn DFd P F DFn DFd P 0.17504 2 97 0.839688 0.184173 2 105 0.83206

PASS - equal variance may be assumed (p > 0.05). PASS - equal variance may be assumed (p > 0.05).

ANOVA ANOVA SS DF SS DF Between 38.25278 2 Between 351.3586 2 Within 3125.747 97 Within 3860.222 105

F 0.593541 F 4.778566 P 0.554361 P 0.010322 *

Post-hoc: Bonferronni-Holm Post-hoc: Bonferronni-Holm Group 1 Group 2 Critical P Significant? Group 1 Group 2 Critical P Significant? wt g-74450 0.016667 0.369493 No wt g-20490 0.016667 0.000863 Yes g-20490 g-74450 0.025 0.391521 No wt g-74450 0.025 0.017807 Yes wt g-20490 0.05 0.999064 No g-20490 g-74450 0.05 0.741212 No

201

Rosette Diameter 30d Rosette Diameter Name Count Avg. SD 32d wt 33 59.01515 5.008563 Name Count Avg. SD g-20490 33 54.04545 5.844821 wt 31 60.33871 4.968882 g-74450 34 54.45588 6.690527 g-20490 31 54.53226 6.686972 g-74450 31 56.72581 6.616694 Test for equal variance (Levene's Test) Test for equal variance (Levene's Test) F DFn DFd P 0.14917 2 97 0.86162 F DFn DFd P 2.460866 2 90 0.091087 PASS - equal variance may be assumed (p > 0.05). PASS - equal variance may be assumed (p > 0.05).

ANOVA SS DF ANOVA Between 504.0794 2 SS DF Within 3373.108 97 Between 532.9892 2 Within 3395.581 90 F 7.247871 P 0.001165 ** F 7.063451 P 0.001414 ** Post-hoc: Bonferronni-Holm Group 1 Group 2 Critical P Significant? Post-hoc: Bonferronni-Holm wt g-20490 0.016667 0.000437 Yes Group 1 Group 2 Critical P Significant? wt g-74450 0.025 0.002466 Yes wt g-20490 0.016667 0.000262 Yes g-20490 g-74450 0.05 0.790247 No wt g-74450 0.025 0.018062 Yes g-20490 g-74450 0.05 0.199162 No

202

Rosette Diameter

35d Name Count Avg. SD wt 34 60.60294 5.100177 g-20490 34 55.86765 6.174617 g-74450 34 57.22059 6.424611

Test for equal variance (Levene's Test)

F DFn DFd P 0.878137 2 99 0.418768

PASS - equal variance may be assumed (p > 0.05).

ANOVA

SS DF

Between 404.5294 2 Within 3478.64 99

F 5.756332 P 0.004316 **

Post-hoc: Bonferronni-Holm Group 1 Group 2 Critical P Significant? wt g-20490 0.016667 0.000989 Yes wt g-74450 0.025 0.019017 Yes g-20490 g-74450 0.05 0.379194 No

203

Plant Height Plant Height 25d 28d Name Count Avg. SD Name Count Avg. SD wt 34 209.5588 36.70289 wt 34 209.5588 36.70289 g-20490 37 220 43.50862 g-20490 37 220 43.50862 g-74450 38 204.5 51.0351 g-74450 38 204.5 51.0351

Test for equal variance (Levene's Test) Test for equal variance (Levene's Test)

F DFn DFd P F DFn DFd P 0.319347 2 106 0.72732 0.319347 2 106 0.72732

PASS - equal variance may be assumed (p > 0.05). PASS - equal variance may be assumed (p > 0.05).

ANOVA ANOVA SS DF SS DF Between 4660.558 2 Between 4660.558 2 Within 208971.9 106 Within 208971.9 106

F 1.182023 F 1.182023 P 0.310667 P 0.310667

Post-hoc: Bonferronni-Holm Post-hoc: Bonferronni-Holm Group 1 Group 2 Critical P Significant? Group 1 Group 2 Critical P Significant? g-20490 g-74450 0.016667 0.161708 No g-20490 g-74450 0.016667 0.161708 No wt g-20490 0.025 0.2804 No wt g-20490 0.025 0.2804 No wt g-74450 0.05 0.634297 No wt g-74450 0.05 0.634297 No

204

Plant Height Plant Height 30d 32d Name Count Avg. SD Name Count Avg. SD wt 33 279.0455 29.93492 wt 33 329.3939 30.04886 g-20490 35 281.2571 38.04668 g-20490 37 322.027 39.88281 g-74450 38 265.1053 49.79419 g-74450 38 312.4079 51.76139

Test for equal variance (Levene's Test) Test for equal variance (Levene's Test)

F DFn DFd P F DFn DFd P 0.315976 2 103 0.729781 1.249967 2 105 0.290742

PASS - equal variance may be assumed (p > 0.05). PASS - equal variance may be assumed (p > 0.05).

ANOVA ANOVA SS DF SS DF Between 5625.594 2 Between 5168.246 2 Within 169631.9 103 Within 185288.8 105

F 1.707922 F 1.464379 P 0.186334 P 0.235905

Post-hoc: Bonferronni-Holm Post-hoc: Bonferronni-Holm Group 1 Group 2 Critical P Significant? Group 1 Group 2 Critical P Significant? g-20490 g-74450 0.016667 0.126238 No wt g-74450 0.016667 0.10201 No wt g-74450 0.025 0.165279 No g-20490 g-74450 0.025 0.371183 No wt g-20490 0.05 0.791582 No wt g-20490 0.05 0.390419 No

205

Plant Height BRAN – Number of Branches 35d Name Count Avg. SD Name Count Avg. SD wt 34 26.05882 4.880022 wt 36 388.6389 41.94156 g-20490 33 22 5.111262 g-20490 37 390.5946 40.30678 g-74450 38 21.28947 4.590872 g-74450 38 387.6842 57.24065 Test for equal variance (Levene's Test) Test for equal variance (Levene's Test) F DFn DFd P F DFn DFd P 2.263158 2 102 0.109221 0.3016 2 108 0.740255 PASS - equal variance may be assumed (p > 0.05). PASS - equal variance may be assumed (p > 0.05).

ANOVA ANOVA SS DF SS DF Between 461.959 2 Between 164.4209 2 Within 2401.698 102 Within 241285.4 108 F 9.809688 F 0.036798 P 0.000127 *** P 0.963883 Post-hoc: Bonferronni-Holm Post-hoc: Bonferronni-Holm Group 1 Group 2 Critical P Significant? Group 1 Group 2 Critical P Significant? wt g-74450 0.016667 6E-05 Yes g-20490 g-74450 0.016667 0.800234 No wt g-20490 0.025 0.001456 Yes wt g-20490 0.025 0.839593 No g-20490 g-74450 0.05 0.539225 No wt g-74450 0.05 0.935299 No

206

SILN- Silique Number/plant TSN – Total Seed Number/silique Name Count Avg. SD Name Count Avg. SD wt 34 207.1765 65.05917 wt 54 52.85185 7.400682 g-20490 33 181.9394 65.27726 g-20490 54 47.51852 7.914021 g-74450 38 165.9211 84.70884 g-74450 54 48.61111 7.810048

Test for equal variance (Levene's Test) Test for equal variance (Levene's Test)

F DFn DFd P F DFn DFd P 0.155301 2 102 0.85636 0.059865 2 159 0.941913

PASS - equal variance may be assumed (p > 0.05). PASS - equal variance may be assumed (p > 0.05).

ANOVA ANOVA SS DF SS DF Between 30813.05 2 Between 857.1975 2 Within 541531.6 102 Within 9455.13 159

F 2.90189 F 7.207432 P 0.059467 P 0.001009 **

Post-hoc: Bonferronni-Holm Post-hoc: Bonferronni-Holm Group 1 Group 2 Critical P Significant? Group 1 Group 2 Critical P Significant? wt g-74450 0.016667 0.02461 No wt g-20490 0.016667 0.000458 Yes wt g-20490 0.025 0.117867 No wt g-74450 0.025 0.004587 Yes g-20490 g-74450 0.05 0.380774 No g-20490 g-74450 0.05 0.471825 No

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Biomass g-74450 g-20490 0.008333 0.153498 No Name Count Avg. SD g-74450 wt 0.01 0.2661 No Oex 6 1.1233 0.180676 g-20490 wt 0.0125 0.322332 No RNAi 6 1.103167 0.115736 Oex RNAi 0.016667 0.822847 No g-74450 5 1.51754 0.255649 RNAi g-20490 0.025 0.896711 No g-20490 5 1.13028 0.486053 Oex g-20490 0.05 0.974511 No wt 6 1.355917 0.197302

Test for equal variance (Levene's Test)

F DFn DFd P 1.540995 4 23 0.223615

PASS - equal variance may be assumed (p > 0.05).

ANOVA SS DF Between 0.720002 4 Within 1.631247 23

F 2.537942 P 0.067504

Post-hoc: Bonferronni-Holm

Group 1 Group 2 Critical P Significant?

RNAi g-74450 0.005 0.005908 No Oex g-74450 0.005556 0.015021 No RNAi wt 0.00625 0.022063 No Oex wt 0.007143 0.059038 No

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Root Length Oex g-20490 0.007143 0.005156 Yes 8d Oex g-74450 0.008333 0.055109 No Name Count Avg. SD RNAi g-74450 0.01 0.069277 No wt 21 12.13686 1.985855 wt Oex 0.0125 0.256154 No Oex 21 12.96895 2.64808 wt g-74450 0.016667 0.263336 No RNAi 21 12.77662 2.292911 wt RNAi 0.025 0.339594 No g-20490 20 15.7882 3.412006 Oex RNAi 0.05 0.802623 No g-74450 20 11.26735 2.861996

Test for equal variance (Levene's Test)

F DFn DFd P 2.406091 4 98 0.054625

PASS - equal variance may be assumed (p > 0.05).

ANOVA SS DF Between 232.1822 4 Within 701.0912 98

F 8.113728 P 1.08E-05 ****

Post-hoc: Bonferronni-Holm

Group 1 Group 2 Critical P Significant? g-20490 g-74450 0.005 5.52E-05 Yes wt g-20490 0.005556 0.000144 Yes RNAi g-20490 0.00625 0.001895 Yes

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Root Length RNAi g-20490 0.007143 0.000135 Yes 9d wt g-74450 0.008333 0.275267 No Name Count Avg. SD RNAi g-74450 0.01 0.315309 No wt 21 17.02276 2.977348 wt Oex 0.0125 0.515565 No Oex 16 16.37181 2.998307 Oex RNAi 0.016667 0.583217 No RNAi 21 16.93929 3.153119 Oex g-74450 0.025 0.616026 No g-20490 20 21.395 3.578357 wt RNAi 0.05 0.93015 No g-74450 20 15.7056 4.523452

Test for equal variance (Levene's Test)

F DFn DFd P 1.980894 4 93 0.10383

PASS - equal variance may be assumed (p > 0.05).

ANOVA SS DF Between 399.5203 4 Within 1143.042 93

F 8.126429 P 1.15E-05 ****

Post-hoc: Bonferronni-Holm

Group 1 Group 2 Critical P Significant?

Oex g-20490 0.005 7.78E-05 Yes g-20490 g-74450 0.005556 8.18E-05 Yes wt g-20490 0.00625 0.000124 Yes

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Root Length wt g-20490 0.007143 0.000749 Yes 10d wt g-74450 0.008333 0.002256 Yes Name Count Avg. SD RNAi g-20490 0.01 0.032923 No wt 18 22.12 2.619358 wt Oex 0.0125 0.058043 No Oex 18 24.24294 3.771269 Oex g-20490 0.016667 0.067078 No RNAi 13 23.72631 2.465093 wt RNAi 0.025 0.094958 No g-20490 15 27.1 4.877792 Oex RNAi 0.05 0.669738 No g-74450 15 17.7 4.857983

Test for equal variance (Levene's Test)

F DFn DFd P 2.214524 4 74 0.075557

PASS - equal variance may be assumed (p > 0.05).

ANOVA SS DF Between 722.0535 4 Within 1094.84 74

F 12.20086 P 1.14E-07 ****

Post-hoc: Bonferronni-Holm

Group 1 Group 2 Critical P Significant? g-20490 g-74450 0.005 1.26E-05 Yes Oex g-74450 0.005556 0.000135 Yes RNAi g-74450 0.00625 0.000424 Yes

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Root Length wt g-74450 0.007143 0.012369 No 11d wt Oex 0.008333 0.014168 No Name Count Avg. SD wt g-20490 0.01 0.028491 No wt 16 28.66106 3.051655 Oex RNAi 0.0125 0.121937 No Oex 16 32.83006 5.628127 RNAi g-20490 0.016667 0.17796 No RNAi 13 30.07708 2.893101 wt RNAi 0.025 0.214351 No g-20490 15 32.7 6.264412 Oex g-20490 0.05 0.951868 No g-74450 15 22.83333 8.158402

Test for equal variance (Levene's Test)

F DFn DFd P 1.485876 4 70 0.215792

PASS - equal variance may be assumed (p > 0.05).

ANOVA SS DF Between 1012.999 4 Within 2196.5 70

F 8.070787 P 2.07E-05 ****

Post-hoc: Bonferronni-Holm

Group 1 Group 2 Critical P Significant?

Oex g-74450 0.005 0.000407 Yes g-20490 g-74450 0.005556 0.000897 Yes RNAi g-74450 0.00625 0.005421 Yes

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Root Length RNAi g-74450 0.007143 0.003 Yes 14d RNAi g-20490 0.008333 0.004965 Yes Name Count Avg. SD wt RNAi 0.01 0.007958 Yes wt 13 50.65869 5.309557 Oex RNAi 0.0125 0.01283 No Oex 13 64.22185 7.073539 wt g-74450 0.016667 0.107671 No RNAi 10 57.0482 5.002699 wt g-20490 0.025 0.21864 No g-20490 13 46.95538 9.139746 g-20490 g-74450 0.05 0.683247 No g-74450 13 45.40154 10.0225

Test for equal variance (Levene's Test)

F DFn DFd P 2.299476 4 57 0.069799

PASS - equal variance may be assumed (p > 0.05).

ANOVA SS DF Between 3090.383 4

Within 3371.786 57

F 13.06072 P 1.3E-07 ****

Post-hoc: Bonferronni-Holm

Group 1 Group 2 Critical P Significant?

Oex g-74450 0.005 1.09E-05 Yes wt Oex 0.005556 1.1E-05 Yes Oex g-20490 0.00625 1.57E-05 Yes

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Root Length Oex g-20490 0.007143 0.005773 Yes 15d RNAi g-74450 0.008333 0.010969 No Name Count Avg. SD RNAi g-20490 0.01 0.038288 No wt 13 58.82154 3.49537 wt g-74450 0.0125 0.086199 No Oex 13 65.623 4.56518 Oex RNAi 0.016667 0.233018 No RNAi 10 63.5086 3.362859 wt g-20490 0.025 0.259369 No g-20490 13 54.80715 12.03173 g-20490 g-74450 0.05 0.656503 No g-74450 13 52.69954 11.83081

Test for equal variance (Levene's Test)

F DFn DFd P 2.367739 4 57 0.063334

PASS - equal variance may be assumed (p > 0.05).

ANOVA SS DF Between 1517.589 4

Within 3915.249 57

F 5.523438 P 0.00079 ***

Post-hoc: Bonferronni-Holm

Group 1 Group 2 Critical P Significant? wt Oex 0.005 0.000269 Yes Oex g-74450 0.005556 0.001194 Yes wt RNAi 0.00625 0.003921 Yes

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# Lateral Roots g20490 RNAi 0.007143 0.000762 Yes 9d wt g74450 0.008333 0.00768 Yes Name Count Avg. SD g74450 RNAi 0.01 0.009122 Yes wt 12 1.666667 0.778499 Oex RNAi 0.0125 0.014848 No g-20490 12 4.583333 1.443376 wt Oex 0.016667 0.017832 No g-74450 12 0.666667 0.887625 wt RNAi 0.025 0.337553 No Oex 12 0.75 0.965307 g74450 Oex 0.05 0.827798 No RNAi 12 2.166667 1.585923

Test for equal variance (Levene's Test)

F DFn DFd P 1.48617 4 55 0.218963

PASS - equal variance may be assumed (p > 0.05).

ANOVA SS DF Between 121.7667 4

Within 76.16667 55

F 21.98195 P 7.04E-11 ****

Post-hoc: Bonferronni-Holm

Group 1 Group 2 Critical P Significant? g20490 g74450 0.005 5.82E-08 Yes g20490 Oex 0.005556 1.24E-07 Yes wt g20490 0.00625 3.35E-06 Yes

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# Lateral Roots Oex RNAi 0.007143 8.21E-05 Yes 10d wt Oex 0.008333 0.00021 Yes Name Count Avg. SD g74450 RNAi 0.01 0.000451 Yes wt 12 3.166667 0.834847 wt g74450 0.0125 0.003781 Yes g-20490 12 5.916667 1.505042 g20490 RNAi 0.016667 0.027758 No g-74450 12 1.666667 1.370689 wt RNAi 0.025 0.051409 No Oex 12 1.5 1 g74450 Oex 0.05 0.736874 No RNAi 12 4.333333 1.775251

Test for equal variance (Levene's Test)

F DFn DFd P 0.99018 4 55 0.420664

PASS - equal variance may be assumed (p > 0.05).

ANOVA SS DF Between 166.0667 4

Within 98.91667 55

F 23.08425 P 3.11E-11 ****

Post-hoc: Bonferronni-Holm

Group 1 Group 2 Critical P Significant? g20490 Oex 0.005 2.27E-08 Yes g20490 g74450 0.005556 3.02E-07 Yes wt g20490 0.00625 1.46E-05 Yes

216

# Lateral Roots wt g20490 0.007143 0.002144 Yes 13d g20490 RNAi 0.008333 0.003983 Yes Name Count Avg. SD Oex RNAi 0.01 0.006397 Yes wt 14 8.071429 2.730576 wt g74450 0.0125 0.018258 No g-20490 14 13.07143 4.763068 g74450 RNAi 0.016667 0.060891 No g-74450 14 5.857143 1.83375 g74450 Oex 0.025 0.111094 No Oex 14 4.5 2.47293 wt RNAi 0.05 0.953763 No RNAi 14 8 3.658499

Test for equal variance (Levene's Test)

F DFn DFd P 1.917641 4 65 0.118027

PASS - equal variance may be assumed (p > 0.05).

ANOVA SS DF Between 595.2286 4

Within 689.0714 65

F 14.03695 P 2.62E-08 ****

Post-hoc: Bonferronni-Holm

Group 1 Group 2 Critical P Significant? g20490 Oex 0.005 2.62E-06 Yes g20490 g74450 0.005556 1.57E-05 Yes wt Oex 0.00625 0.001226 Yes

217

# Lateral Roots wt g20490 0.007143 1.62E-06 Yes 15d wt RNAi 0.008333 6.08E-06 Yes Name Count Avg. SD wt Oex 0.01 0.000486 Yes wt 15 16.73333 2.711527 g74450 RNAi 0.0125 0.020998 No g-20490 15 24.73333 4.350151 wt g74450 0.016667 0.050865 No g-74450 15 14 4.423961 g74450 Oex 0.025 0.148289 No Oex 15 10.25 4.169047 Oex RNAi 0.05 0.341393 No RNAi 12 11.66667 3.194455

Test for equal variance (Levene's Test)

F DFn DFd P 1.400158 4 67 0.243448

PASS - equal variance may be assumed (p > 0.05).

ANOVA SS DF Between 1883.828 4

Within 997.45 67

F 31.63478 P 8.45E-15 ****

Post-hoc: Bonferronni-Holm

Group 1 Group 2 Critical P Significant? g20490 RNAi 0.005 6.86E-10 Yes g20490 Oex 0.005556 3.9E-09 Yes g20490 g74450 0.00625 2.84E-07 Yes

218

Stem Diameter wt Oex 0.0125 0.010262 Yes Name Count Avg. SD wt g-20490 0.016667 0.019439 No wt 15 1165.75 75.69675 wt RNAi 0.025 0.020167 Yes RNAi 14 1223.967 46.80721 RNAi g-20490 0.05 0.905538 No Oex 15 1099.796 53.69225 g-74450 15 1375.248 51.21957 g-20490 15 1221.938 44.40829

Test for equal variance (Levene's Test) F DFn DFd P 0.868895 4 69 0.487142

PASS - equal variance may be assumed (p > 0.05).

ANOVA SS DF Between 622130.1 4 Within 213399.4 69

F 50.28947 P 9.46E-20 ****

Group 1 Group 2 Critical P Significant? Oex g-74450 0.005 1.88E-14 Yes wt g-74450 0.005556 1.24E-09 Yes g-74450 g-20490 0.00625 1.65E-09 Yes RNAi g-74450 0.007143 6.83E-09 Yes Oex g-20490 0.008333 2.25E-07 Yes RNAi Oex 0.01 4.23E-07 Yes

219