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Functional genomics of plant chitinase-like genes Johnston, David Morris 2006

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FUNCTIONAL GENOMICS OF PLANT CHITINASE-LIKE GENES by DAVID MORRIS JOHNSTON  B.Sc. Honours, The University of Ottawa, 2001  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Botany)  THE UNIVERSITY OF BRITISH COLUMBIA September 2006 © David Morris Johnston, 2006  Abstract The Arabidopsis chitinase-like1 (Atctl1) mutant, pom1 is compromised in primary cell wall development, resulting in short roots when grown on high sucrose and shortened hypocotyls when grown in darkness. To better understand this phenotype and the evolution of AtCTL1 and its homologue, AtCTL2, we obtained a large number of CTL sequences and determined the phylogenetic relationships among them. Since microarray analysis had suggested a change in auxin response or homeostasis in pom1, I used the auxin reporter DR5::GUS in the pom1 background to assess changes in distribution. To assess whether the biochemical functions of AtCTL1 homologues in Arabidopsis and other plants are conserved, I transformed pom1 with AtCTL2 and CTLs from poplar (Populus trichocarpa x Populus deltoides clone H-11) and from Picea glauca (spruce) and assessed rescue of the pom1 phenotype. To further understand CTL expression and function, Arabidopsis and poplar CTL promoter::GUS fusions were also expressed in Arabidopsis, PopCTL1 overexpressed  in Arabidopsis, and CTL  expression down regulated in poplar by RNAi. Our results indicate that CTL genes represent an ancient family encoding proteins of conserved biochemical function. In dicots, represented by Arabidopsis and poplar) duplicated CTL genes are differentially expressed in conjunction with primary and secondary cell wall development, respectively. Mutation of these genes results in improperly formed primary walls in certain cell types in the case of AtCTL1, and an impairment in the differentiation of vascular bundles for AtCTL2. Overexpression of PopCTL1 in Arabidopsis seems to over stimulate the differentiation of vascular bundles, and our studies show that auxin distribution is altered in the Atctl1 mutant. Down regulation of PopCTL1 and PopCTL2 in poplar appears to phenocopy aspects of these mutations, resulting in secondary cell walls that appear to have less deposition of lignin and an accelerated production of secondary xylem respectively. While specific biochemical function(s) of CTL genes were not studied, potential functions are discussed.  ii  Table of Contents Abstract................................................................................ ii Table of Contents……………………..……………………....iii List of Tables ....................................................................... v List of Figures .................................................................... vi Acknowledgements………………………………………….viii Chapter 1 Introduction........................................................ 1 1.1 Plant Cell Expansion ................................................................................................................................ 1 1.2 Mechanisms and mutants in plant cell expansion..................................................................................... 2 1.3 Secondary Cell Wall Formation in xylem development........................................................................... 4 1.4 Mechanisms and mutants in xylem secondary cell wall formation .......................................................... 5 1.5 Chitinase like Gene involvement in development .................................................................................... 6 1.6 CTL expression in Arabidopsis and poplar .............................................................................................. 9 1.7 Evolutionary and Phylogenetic considerations in functional elucidation ............................................... 10 1.8 Predicting CTL function......................................................................................................................... 11 1.9 Thesis objectives and summary .............................................................................................................. 12  Chapter 2 Materials/Methods .......................................... 13 2.1 CTL Phylogenetics, homologue discovery, and protein modelling .......................................... 13 2.1.1 BLAST analysis and homologue discovery....................................................................... 13 2.1.2 Tree construction using Phyml and Treeview ................................................................... 13 2.1.3 Protein modelling and trafficking prediction ...................................................................... 14 2.2 General Nucleic Acid Methods................................................................................................. 14 2.2.1 Plasmid DNA preparation and DNA sequencing .............................................................. 14 2.1.2 DNA restriction, gel purification and ligation ..................................................................... 14 2.1.3 Genomic DNA and total RNA isolation.............................................................................. 15 2.2.4 Primer design and PCR based genotyping ....................................................................... 16 2.2.5 Reverse transcriptase PCR (RT PCR) and expression analysis ...................................... 18 2.3 Cloning procedures.................................................................................................................. 18 2.3.1 Host bacterial strains, plasmids, and plant materials........................................................ 18 2.3.2 Site directed mutagenesis, cloning, and sequencing of PCR products into the pDRIVE vector.......................................................................................................................................... 21 2.3.3 Construction of AtCTL1 promoter::AtCTL1 coding region fusion construct in pART27.... 23 2.3.4 Construction of AtCTL1 promoter::PopCTL1 coding region fusion construct in pART27. 23 2.3.5 Construction of PopCTL1 coding region overexpression construct in pART27................ 24 2.3.6 Construction of AtCTL1 coding region overexpression construct in pCAMBIA 1305.1 .... 24 2.3.7 Construction of AtCTL1 and PopCTL1 promoter::GUS fusions in pCAMBIA 1305.1 ....... 24 2.3.8 Construction of PopCTL1 RNAi constructs in pART27..................................................... 25 2.4 Arabidopsis germination, growth conditions, transformation, and selection............................ 25 2.4.1 Seed sterilization, germination, transplantation, and growth conditions ........................... 25 2.4.2 Arabidopsis floral dip genetic transformation .................................................................... 26 2.4.3 Arabidopsis transgenic seed selection.............................................................................. 26 2.4.4. Seedling growth assays ................................................................................................... 26 2.5 poplar growth conditions, transformation, and tissue culture .................................................. 27 iii  2.5.1. Agrobacterium mediated poplar transformation in tissue culture..................................... 27 2.5.2. poplar growing conditions................................................................................................. 29 2.6 Dissecting, light, UV fluorescence microscopy analysis.......................................................... 29 2.7 GUS expression analysis........................................................................................................ 30  Chapter 3: Bioinformatic analysis of CTLs..................... 31 3.1 Introduction .............................................................................................................................. 31 3.2 Results ..................................................................................................................................... 34 3.3 Discussion................................................................................................................................ 39  Chapter 4 Functional analysis of AtCTL1, AtCTL2, PopCTL1 and PopCTL2 .................................................... 42 4.1 Introduction .............................................................................................................................. 42 4.2 Results ..................................................................................................................................... 45 4.2.1 A knockout phenotype for AtCTL2 mutant Arabidopsis .................................................... 45 4.2.2 Auxin involvement in the pom1 phenotype as studied using the DR5::GUS reporter construct in Col-0 and pom1 ...................................................................................................... 47 4.2.3 Arabidopsis and poplar CTL promoter ::GUS reporter constructs in Col-0....................... 50 4.2.4 pom1 phenotype rescue using AtCTL1, AtCTL2, PopCTL1, and PopCTL2 transgenes.. 54 4.2.5 CTL overexpression in Arabidopsis ................................................................................. 56 4.2.6 RNAi of PopCTL1 and PopCTL2 in transgenic poplar...................................................... 59 4.2.7 Pith lignification in wild-type poplar ...................................................................................65 4.3 Discussion................................................................................................................................ 66  Chapter 5 Conclusions and Future Perspectives .......... 74 References......................................................................... 77 Appendix A ........................................................................ 87  iv  List of Tables Table 1: PCR protocols used in testing for transgenes in genomic DNA.......................................... 17 Table 2: Semiquantitative PCR parameters and primers used in gene expression analysis............ 18 Table 3: CTL promoters and coding regions, PCR conditions, and primers used for their amplification....................................................................................................................................... 22 Table 4: CTL proteins included in phylogenetic analysis and their Genbank ID/publication………..35  v  List of Figures Figure 1: Wild type and pom1 phenotypes of Arabidopsis thaliana (Columbia-0 ecotype)................. 8 Figure 2: mRNA expression profiles of Arabidopsis and poplar CTLs in floral stems and developing secondary xylem.. ........................................................................................................... 10 Figure 3: Phylogenetic alignment of all known Arabidopsis chitinases and chitinase-likes performed by M.-T. Hauser (unpublished).. ......................................................................................11 Figure 4: Plasmids used in all cloning and transformation proceedures........................................... 21 Figure 5: Co-expression analysis of AtCTL1 and AtCTL2.. .............................................................. 33 Figure 6: Phylogenetic analysis of plant chitinase like genes, based on alignment of a conserved 290 amino acid region...................................................................................................... 37 Figure 7: Primary amino acid structure alignment of Arabidopsis, poplar, and Picea glauca. .......... 38 Figure 8: Gene structure comparison of Arabidopsis and poplar CTLs.. .......................................... 39 Figure 9: Sections of Col-0, Atctl1, and Atctl2 root/hypocotyls and inflorescences taken at 1 cm from rosette level from 1.5 month old inflorescences........................................................................ 46 Figure 10: Handcut sections of Wt (A, B) and Atctl2 (C, D) inflorescences.. .................................... 47 Figure 11: GUS assay testing activity of the DR5::GUS construct in Col-0 by wounding................. 48 Figure 12: Comparison of DR5 driven GUS expression in 6 day old Col-0 or pom1 seedlings grown in light, in darkness, or in light with 4.5% sucrose.................................................................. 49 Figure 13: Comparison of handcut sections of floral stem and root/hypocotyl from DR5::GUS fusions in Col-0 or pom1 backgrounds.............................................................................................. 50 Figure 14: Comparison of AtCTL1, AtCTL2, PopCTL1, PopCTL2 promoter::GUS fusions in Col-0 at the 4 day old seedling stage grown in darkness, light or in light with 4.5% w/v sucrose. ... 51 Figure 15: Comparison of AtCTL1, AtCTL2, PopCTL1, PopCTL2 promoter::GUS fusion expression in cotyledons and wounded leaves.................................................................................53 Figure 16: Comparison of root/hypocotyl and basal inflorescence sections from AtCTL1, AtCTL2, PopCTL1, PopCTL2 promoter::GUS fusions in Col-0......................................................... 53 Figure 17: Phenotype rescue assays using 4.5% sucrose or dark on transgenic pom1 plants for four days. ........................................................................................................................... 54 Figure 18: Measurements of transgenic pom1 phenotype rescue. ................................................... 56 Figure 19: Expression levels of 35S::PopCTL1 in Col-0 and basal inflorescence sections.............. 58 Figure 20: Hand sections of Col-0 and 35S::PopCTL1 from three different lines. ............................ 58 Figure 21: CTL expression in wild type and RNAi lines of poplar. .................................................... 60  vi  Figure 22: Greenhouse grown poplar heights and branch number................................................... 62 Figure 23: Handcut sections of Wt, PopCTL1 RNAi, and PopCTL2 RNAi downregulated poplar.... 63 Figure 24: Longitudinal sections of Wt poplar and PopCTL2 RNAi plant 3J1................................... 64 Figure 25: Sections of Wt poplar highlighting pith tissue development............................................. 65  vii  Acknowledgements I would like to thank a number of people without whom this study would not have been possible: My parents, brothers, and extended family who sometimes don’t appreciate plant genetics as much as I, but who’ve supported the work I’ve done with love, phone calls, visits, council and muffins/cookies/beers throughout these busy years. Dr. John Arnason and Dr. Douglas Johnson at the University of Ottawa for encouraging my interest and enthusiasm for botanical research. Eileen Hu, John Shin, and Huang-Ju Chen for great help in sectioning, microscopy and genotyping of Arabidopsis and poplar. My labmates for invaluable support and friendship in all aspects of wet lab work and interpretation, with special intellectual mention of Dr. Bahram Soltani for helping to identify alterations in vascular bundles in transgenic plants, Dr. Lee Johnson who helped get me started in construct production, June Kim who conspired with me to create leadership, and Clarice Souza for interpretation of phylogenetic results. Dr. Craig Berezowsky who gave me TA mentorship and comraderie as I relearned the ropes of molecular genetics while teaching others. The numerous friends who let me couch surf during this MSc process, especially Ed McCormick, David Llewelan and Cori Johnston. My committee members, Dr. Lacey Samuels, Dr. Shawn Mansfield and their labs for their intellectual input and use of their facilities for microscopy and poplar transformation and tissue culture respectively. Dr. Carl Douglas for his role in initiating this project and providing critical analysis of my ideas and interpretations; helping me keep my head in the books and my feet on the ground. Most importantly, I would like to thank my grand colleague, Dr. Bjoern Hamberger who helped design, direct, and implement very important parts of this project. Moreover, his mentorship of this young geneticist/physiologist resulted in my ability to “catch fish” in molecular biology instead of being a brainless zombie. ;-)  viii  Chapter 1 Introduction 1.1 Plant Cell Expansion Plants grow through the organized processes of cell division, expansion, elongation, and differentiation. The radial pattern of tissues found in plant organs is first laid out during plant embryogenesis. Primary plant growth in seedlings then begins with cell division at apical meristems at both the root tip and the shoot tip. After cell division, new cells receive developmental information through biochemical gradients such as those provided by auxin and cytokinins, as well as cell-to-cell signalling. Plant cells use the turgor pressure they accumulate from absorption of water into their large vacuoles as the driving force behind cell expansion (Cosgrove 2005). Elongation is a specialized form of cell expansion, where plant cells undergo massive anti-isodiametric expansion to reach lengths many times greater than they are wide. The filamentous green algae Nitella for example, has internode cells that elongate to a length of 6 cm (average Arabidopsis cells are 20 - 100 um long), while remaining 0.1 cm wide! This regulation of this type of expansion is believed to be achieved by the properties of primary cell walls, which constrain and direct the nature of cell expansion, allowing for highly specific shapes and dimensions. Subsequent to elongation, cells undergo maturation where specialized programs of differentiation gives them different features and functions ranging from secretion of biochemicals to structural support through strengthened cell wall or nutrient storage in specialized plastids. A similar developmental progression takes place during secondary plant growth where cell division activity of the vascular and cork cambia creates new populations of cells that undergo expansion, elongation, and maturation, allowing the plant to extend radially after the basic plant form is produced by primary growth.  1  1.2 Mechanisms and mutants in plant cell expansion To regulate the development of diverse shapes and sizes, plants use different mechanisms to control cell expansion and elongation. The main way that plants control and direct this turgor-driven cell expansion is through the constraining potential of the primary cell wall. This wall is composed mostly of aligned cellulosic microfibrils and cross-linked xyloglucans amid of a matrix of homogalacturonan and rhamnogalacturonan pectins that help determine physiological properties of the wall (Yong et al. 2005). The primary wall can be remodelled to allow the cell further expansion and differentiation until a secondary cell wall and lignin are added in some specialized cells. Interference with the production of primary cell walls results in varying severities of cell expansion mutant phenotypes that loose some aspect of cell expansion control (Dolan and Davies 2004; Lloyd and Chan 2004) Synthesis of cellulose, which is a polymer of 1,4-glucan chains, is accomplished by cellulose synthases, which are composed of subunits termed CESA proteins, organized into plasma membrane localized complexes. CESA1, CESA3, and CESA6, are believed to encode protein subunits of the cellulose synthase complex involved in primary wall deposition (Gardiner et al. 2003). Mutants of these genes are rsw1 (Arioli et al. 1998), ixr1 (Scheible et al. 2001), and prc1 (Fagard et al. 2000; Schindelman et al. 2001), all of which show reduced cellulose synthesis in primary cell walls, and perturbations of cell expansion in a number of different tissues. Other mutants showing a link between cellulose synthesis and regulation of cell expansion, include cobra, a mutant in a GPI anchored protein affecting the orientation of cell expansion (Schindelman et al. 2001),  kobito1 which has  disorganized deposition of microfibrils (Pagant et al. 2002), knopf with a mutated alpha-glucosidase (Gillmor et al. 2002), the pom1 mutant for a chitinase-like gene (Pagant et al. 2002; Zhong et al. 2002), and botero1 which is involved in cytoskeletal organization mediating cellulose microfibril alignment (Bichet et al. 2001).  2  Other less well-defined mechanisms in cell expansion control are believed to be involved not directly in cellulose production, but at the level of signal production or perception. In addition to cellulose synthase like genes (Csl) and cellulases, Arabidopsis thaliana cell expansion mutants have implicated genes playing roles in coordination and communication in cell expansion, such those encoding Nglycosylases, membrane bound receptors, extra cellular matrix enzymes, and regulators of cell wall sugar composition, gibberellic acid (GA), ethylene regulation, auxin and cytokinin regulation, abscisic acid (ABA) regulation, and brassinosteroid regulation (Dolan and Davies 2004). Of particular interest are carbohydrate active enzymes (and not themselves cellulose synthases) involved in regulation of cell expansion such as those characterized by korrigan, a mutant in a gene encoding an endo-1,4-D-glucanase (Nicol et al. 1998), rsw3 a mutant in a gene encoding an enzyme putatively processing N-glycans during ER quality control (Burn et al. 2002) and rdh1, a mutant in a gene encoding an arabinogalactan protein (Seifert et al. 2002). Expansins, a class of plant proteins that permit primary cell walls to expand during turgor-driven growth, are believed to disrupt non-covalent binding between cellulose and hemicellulose polysaccharides that constrain cell wall expansion, suggesting non catalytic interactions between wall components may also be important in development (Choi et al. 2003). Arabidopsis contains over 800 genes or 3.3% of all of its genes, that are annotated as glycosidase or glycosyltransferase-related; far more than any other non-plant organism studied thus far (Coutinho et al. 2003). That a large number of mutants in wall synthesis have been reported and that such a large fraction of the Arabidopsis genome is involved in wall production and remodelling, supports the idea that the plant cell wall is a complex and dynamic structure, that is involved in complex processes such as control of morphogenesis in addition to providing structural integrity.  3  1.3 Secondary Cell Wall Formation in xylem development Specialized plant cells such as xylem and fiber cells undergo an additional process of maturation after primary cell wall development called secondary wall formation, whereby additional cellulose layers and phenolic compounds are laid down inside the primary cell wall (Bourquin et al. 2002). There are different cell types within xylem tissue that carry out this program in different ways. Primary xylem is derived from procambium near the apex of the primary plant body and consists first of elongated protoxylem cells, which undergo secondary cell wall development and the formation of thick, lignified annular rings, followed by cell death. Metaxylem vessels are also dead at  maturity, differentiate after protoxylem, and are  characterized by scalariform, reticulate and pitted secondary walls impregnated with lignin (Fukuda 2004).  Secondary xylem in angiosperms such as poplar  consists of vessels similar to metaxylem as well as fiber and ray parenchyma cells, and is formed through the cell division and differentiation activity of a vascular cambium, which forms in stems at some distance below the apex at the onset of secondary growth (Israelsson et al. 2003). Formation of secondarily thickened cells in the secondary xylem cells progresses through four broad phases of development: 1) Cell division from cambial cells, with some cells retaining capacity for continued division, 2) Radial and longitudinal cell expansion,  3)  Secondary  cell  wall  formation  (cellulose  and  lignification  continues/commences, and finally 4) programmed cell death, protoplast lysis, and selective cell wall digestion (Hertzberg et al. 2001). Selective cell wall digestion taking place in xylem tracheids and vessel elements creates pitted walls and pipelike cells with perforated end walls for the movement of water and minerals throughout the plant body. These stages of xylem development are depicted schematically in Figure 2B, below. Secondarily thickened cell walls are stronger and more impermeable to water, allowing xylem cells to function as vascular tissues which also provide the strength for which woody plants are known.  4  1.4 Mechanisms and mutants in xylem secondary cell wall formation To form a secondary wall, a developing vessel or fiber cell must synthesize a large amount of additional cellulose. Since secondary cell walls are laid down inside the primary cell wall, perturbation of cellulose synthesis does not usually result in cell expansion phenotypes typical of defects in primary cell wall production. This is shown by analysis of the mutants irx1 (Taylor et al. 2000), irx3 (Taylor et al. 1999), and irx5 (Taylor et al. 2003) which are null mutants in cellulose synthase genes encoding the CESA subunits involved in secondary wall formation, CESA4, CESA7, and CESA8, respectively. Rather than having isodiametric growth or broken walls, these mutants all show severe reduction in cellulose content of secondary cell walls have xylem vessels which collapse under the negative pressure inside. All three cellulose synthase subunits have been shown to be required for the formation of a cellulose synthase complex crucial for secondary cell wall production, and have also been shown to co-localize with microtubules which presumably orient cellulose microfibril production (Gardiner et al. 2003). Microtubule mutants such as botero1 (Bichet et al. 2001) and mor1 (Sugimoto et al. 2003) likely show cell expansion phenotypes because of microtubule involvement in primary cell wall development, masking potential cell wall defects specific to secondary cell wall synthesis. There are however mutants such as fra3 (Zhong et al. 2004) where perturbations in cytoskeletal organization compromise secondary cell wall specific formation in fiber cells and do not have defects in primary cell wall expansion. In addition to more extensive deposition of cellulose, secondary cell wall growth involves deposition of other secondary wall specific polysaccharides, proteins, and the complex phenylpropanoid polymer, lignin. Mutants showing reductions in lignin production often have xylem with compromised structural integrity (Anterola and Lewis 2002). Downregulation of lignin production may sometimes result in elevated  5  cellulose production (Anterola and Lewis 2002; Li et al. 2003), and conversely the downregulation of cellulose production has been shown to stimulate increased lignification (Cano-Delgado et al. 2003). Lignin production takes place as part of secondary cell wall development, but also during stress and pathogen responses mediated by jasmonate and ethylene production resulting from interference with cellulose synthesis in primary cell walls (Ellis et al. 2002)  1.5 Chitinase like gene involvement in development Lipo-chitooligosaccharides (NOD factors) are chitin-like molecules that have long been known as growth promoting compounds used by nitrogen fixing bacteria such as Rhizobium meliloti in plant communication during the formation and maintainance of root nodules (Denarie et al. 1996). Purified NOD factors from R. meliloti and a wide range of taxonomically unrelated bacterial species can be applied to their host plant’s roots where they trigger root hair deformations and often full nodule growth that is not inducible by any known class of plant hormone or hormone inhibitor. That similar lipo-chitooligosaccharide signals from diverse bacterial species can induce changes of plant development suggests convergent evolution, and hints that plants may use endogenous chitin-like signals in the regulation of their growth. Proteoglycans such as xylogen, a glycosylated protein involved in xylem formation, may provide plants with a regulatory link between such oligosaccharide signaling, and spatial/temporal control of development (Motose et al. 2004). Strong support for this idea comes from studies of the temperature sensitive carrot mutant, ts11, which is arrested in cell expansion and embryo development at the globular stage when grown at 32°C, but grows normally when supplied with a bacterial NOD factor (De Jong et al. 1993). This same mutant was shown to be rescued by application of a glycosylated endochitinase enzyme secreted extracellularly in wild type carrot, suggesting the developmental function missing is the modification of an endogenous chitin-like molecule or proteoglycan to produce N-acetylglucosamine containing signals (De Jong et al. 1992; Kragh et al. 1996). 6  The importance of chitinase-like genes in development and cell expansion in the model plant Arabidopsis thaliana was first revealed by the discovery that mutations in a chitinase-like gene (AtCTL1) lead to cell expansion and other defects (Zhong et al., 2002), as first described for the pom1 mutant. Eleven different pom1 alleles of AtCTL1 were described based on cell expansion defects (Benfey et al. 1993; Hauser et al. 1995). Under normal growth conditions, pom1 plants are somewhat stunted in growth, however when grown in 4.5 % sucrose media, the seedlings show radial swelling of roots with greater numbers of root hairs (Figure 1), and etiolated hypocotyls grow to half the length of wild-type. These mutant plants also have isodiametric root epidermis and cortex cells, implying loss of cell expansion regulation (Figure 2). In the root, part of these observed differences might be accounted for by reduction of cell division, as the comparison of cell numbers in the plant’s isodiametric growth zone show 20 in wild-type vs. 7 in pom1 (Hauser et al., 1995). Another 12 alleles of pom1/Atctl1 have since been isolated in screens for phenotypes to unrelated to cell expansion, including pom1-22 called esr1 enhanced shoot regeneration in tissue culture (Cary et al. 2001) and elp1, ectopic deposition of lignin (Zhong et al. 2002). Characterization of the esr1 mutation showed plant cells to be hyper responsive to cytokinins, having elevated shoot regeneration in Arabidopsis tissue culture. Additionally Atctl1 mutants have malformed pith cell walls and a mosaic pattern of ectopic deposition of lignin throughout the pith and vascular tissues, totalling a 20% increase in total deposition of lignin (Zhong et al., 2002). This ectopic lignification correlates to a 50% increase in expression of genes encoding the lignin biosynthetic enzymes CCoAOMT, PAL, and CCR in the same tissues, in the absence of secondary cell wall thickening (Zhong et al. 2000). The POM1 gene (At1g05850) was cloned on the basis of the elp1 mutant phenotype and is predicted to encode a 321 amino acid protein with a predicted molecular mass of 35,556 Da. The gene was named AtCTL1, CHITINASE LIKE 1, because of the similarity of the predicted protein to chitinases (Zhong et al. 2002). A second AtCTL gene (At3g16920), AtCTL2,  7  annotated as having glycosyl transferase activity has been identified based on sequence similarity to ATCTL1, but until now has no described mutant phenotype (Hauser et al., unpublished). AtCTL2 has 66% amino acid identity to AtCTL1. Both AtCTL1 and AtCTL2 are predicted to be cell wall localized, based on N-terminal signal sequences (Zhong et al., 2002; M. Hauser, unpublished).  Figure 1: Wild type and pom1 phenotypes of Arabidopsis thaliana (Columbia-0 ecotype). Plants were grown in the dark or on 4.5% sucrose for four days. Hand sections were taken from the bottom of 20 cm tall floral stems and stained with toluidine blue. Micrographs of root sections from root hair growth zones of seedlings grown on 4.5% sucrose are adapted from (Schneider et al. 1997). st, stele; en, endodermis; co, cortex; ep, epidermis. Scale bars 100 µm.  8  1.6 CTL expression in Arabidopsis and poplar AtCTL gene expression patterns have been determined by microarray transcript profiling in Arabidopsis. Ehlting et al. (2005) profiled the expression of all Arabidopsis genes over the course of inflorescence stem development and interfascicular fiber differentiation. Data from this experiment on the expression of AtCTL1 and AtCTL2 are shown in Figure 2A. While AtCTL1 is not differentially regulated, expression of AtCTL2 is strongly upregulated, along with a large number of secondary cell wall biosynthetic genes (Ehlting et al., 2005), during stem development and fiber formation. The first microarray–based gene profiling experiment in poplar used a 2,995element EST array to profile to gene expression changes during secondary xylem differentiation (Hertzberg et al., 2001). Two ESTs encoding poplar chitinase-like proteins were present on the microarray (EST AI163580 and EST AI164688), and data from this study showed that one of chitinase-like mRNAs was upregulated during the late stages of xylem formation related to cell wall thickening and lignification (Figure 2B). Work in the Douglas lab (unpublished) used PCR based cloning to isolate the full length cDNA clones corresponding to the two poplar chitinase-like genes from P. trichocarpa x P. deltoides hybrid clone H11. The poplar PopCTL1 and PopCTL2 genes encode proteins with 66% to 68% amino acid identity to AtCTL1 and AtCTL2. Analysis of PopCTL1 sequence using the PSORT program has shown the presence of a signal secretion peptide at the N-terminus, suggesting it is cell wall localized.  9  Figure 2: A) mRNA expression profiles of AtCTL1 and AtCTL2 using 26K gene longmer arrays during primary inflorescence development. RNA was extracted from sections illustrated in the UV photos of cross-sections taken at 1, 3, 5, 7 cm from SAM. B) mRNA expression profiles of PopCTL1 and PopCTL2 across developing xylem tissue in poplar trees. Samples were taken at stages A to E as shown and normalized by comparison to undifferentiated cambium. Cell division takes place in A, expansion and elongation in A, B, and C, secondary cell wall deposition (s1, s2, and s3 layers) in D and E, and programmed cell death in E. Adapted from Hertzberg et al. (2001).  1.7 Evolutionary and Phylogenetic considerations in functional elucidation Phylogenetic reconstruction/comparison shows that AtCTL1 and AtCTL2 are members of a clade of chitinase-like genes distinct from a large number of other true chitinases in the Arabidopsis genome (M.T. Hauser, unpublished; Figure 3). Preliminary analysis of ESTs from the model non-vascular plant Physcomitrella patens, has shown it to also contain CTL sequences (B. Hamberger, unpublished) implying that plants possessed this family of genes for millions of years before evolution of vascular plants. It is also interesting to note that in all three plants with  10  fully sequenced genomes (Arabidopsis, poplar and rice), there are two CTL genes (B. Hamberger, unpublished), suggesting not only that two copies are sufficient for plant development, but that duplication is not tolerated. From a functional point of view, it is not clear why two CTL genes are apparently required in plants. While expression of PopCTL1 during xylem development suggests a role there, the original CTL function may be in primary growth based on apparent conservation of CTL genes in a non-vascular plant (Physcomitrella). Identification and phylogenetic and functional comparisons between expected CTL proteins in other plant clades, should allow further hypothesis to be made with regard to CTL function and evolution in land plants.  Figure 3: Phylogenetic alignment of all known Arabidopsis chitinases and chitinase-likes performed by M.-T. Hauser (unpublished). Branches are drawn to scale, with the scale bar showing 0.1 substitutions per site.  1.8 Predicting CTL function Since PopCTL1 is greatly upregulated in poplar secondary xylem, while PopCTL2 is not differentially expressed across this developmental gradient, it seems likely that PopCTL1 is the more important gene of the two in secondary wall formation or some other aspect of xylem development. Ehlting et al. (2005) and Figure 2 show that a similar CTL expression pattern exists in Arabidopsis thaliana with regards to AtCTL1 and AtCTL2 in floral stems. Consistent with this, other Arabidopsis 11  expression data mining studies (Brown 2005; Persson et al. 2005; Yokoyama and Nishitani 2006) also show that AtCTL1 expression is correlated with expression of genes encoding primary cell wall cellulose synthase subunits, while AtCTL2 expression is correlated with expression of genes encoding cellulose synthase subunits involved in secondary cell wall biosynthesis.  This work suggests the  hypothesis that CTL proteins in vascular plants have evolved two distinct functions, one associated with primary wall development, and one with secondary cell wall development, and that these two functions are conserved in different land plant lineages.  1.9 Thesis objectives and summary The objectives of this thesis were to obtain functional information for poplar and Arabidopsis CTL genes, to test the hypothesis that conserved duplicated CTL genes are present in diverse land plant lineages, and to test the hypothesis that proteins encoded by duplicated CTL genes have distinct functions in primary and secondary growth and/or primary and secondary wall formation. This thesis reports the results from bioinformatic analysis of CTL genes from a large number of plants having significant sequence information. This is followed by the comparative analysis of function between AtCTL1 and PopCTL2, and AtCTL2 and PopCTL1, as well as two CTL genes from Picea glauca. Information about PopCTL and AtCTL function and functional relationships between these CTL genes was obtained from Arabidopsis pom1 phenotypic rescue experiments, analysis of the phenotype of an AtCTL2 T-DNA insertion mutant, CTL overexpression, and analysis of CTL promoter::GUS expression in Arabidopsis for both Arabidopsis and poplar CTLs. To study PopCTL1 and PopCTL2 function in vivo, RNAi techniques were used to generate transgenic poplar lines with down regulated PopCTL1 and PopCTL2 expression, and the phenotypes of these lines investigated.  12  Chapter 2. Materials and Methods 2.1 CTL Phylogenetics, homologue discovery, and protein modelling 2.1.1 BLAST analysis and homologue discovery The coding sequences of the AtCTL1 and AtCTL2 genes were submitted to the nucleotide-nucleotide BLAST function in the Genome BC Forestry Genomics EST database at http://treenomix0.forestry.ubc.ca/ to search for poplar, Picea glauca, and Pinus taeda CTLs before public release. To ensure all possible homologues were found, a number of different 200 bp long sequences from conserved regions of AtCTL1 and AtCTL2 were also submitted as queries against the EST database hosted by the Treenomix group for the Forestry Genome BC lab at http://treenomix0.forestry.ubc.ca/blast/index.html. Subsequent partial EST regions were resubmitted, allowing a number of contiguous fragments to be found. The CAP function within the BioEdit program (Hall 1999) was used on these fragments to form full length contigs. Contig sequences were analyzed for coding regions and predicted proteins were then aligned to AtCTL1 and AtCTL2 sequences for comparison. This was process was repeated using Arabidopsis, poplar, Picea glauca and Physcomitrella patens CTLs  as queries of publicly available EST  databases at: http://www.ncbi.nlm.nih.gov/, http://www.tigr.org/tdb/tgi/plant.shtml http://www.kazusa.or.jp/en/plant/porphyra/EST/  2.1.2 Tree construction using Phyml and Treeview Translated CTL coding regions were aligned using the CLUSTALW Multiple alignment function in the BioEdit program (Hall 1999), then truncated to a strongly conserved region corresponding to amino acid 40 to amino acid 314 in AtCTL1. 32 putative CTLs were saved in FASTA format, along with CHI26, a family 19 chitinase from Hordeum vulgare (Song and Suh 1996). Phylogenetic predictions were generated by running these sequences through Phyml Version 2.4.4 (Guindon and Gascuel 2003) with 100 parametric bootstrap replicates, optimized  13  proportions of invariable sites, 4 categories of substitution rate, and optimized gamma distribution parameters; other parameters were left at their defaults. Finished, unrooted trees were then visualized using Treeview version 1.6.6 (Page 1996) available at http://taxonomy.zoology.gla.ac.uk/rod/rod.html  2.1.3 Protein modelling and trafficking prediction To predict conserved protein domains, Arabidopsis, poplar, and Picea glauca CTL proteins  were  submitted  to  the  NCBI  protein  domain  database  (http://www.ncbi.nlm.nih.gov/Structure/lexington/lexington.cgi?cmd=rps).  at  Protein  targeting prediction was accomplished by submission of Arabidopsis, poplar, and Picea  glauca  CTLs  to  the  WOLF  PSORT  program  at  the  website  http://wolfpsort.seq.cbrc.jp/ (Horton et al. 2006).  2.2 General Nucleic Acid Methods 2.2.1 Plasmid DNA preparation and DNA sequencing Plasmid DNA was prepared by alkaline lysis by use of Qiaprep spin Plasmid Miniprep kit or Midiprep kit (Qiagen), following the manufacturer’s protocol. All DNA sequencing was carried out using M13 forward and reverse primers on PCR products cloned directly into pDRIVE (Quiagen). Sequencing chemistry was Big Dye 3.1 and reads were determined by automated Prism Cycle Sequencing with at the University of British Columbia Nucleic Acid and Protein Service unit.  2.1.2 DNA restriction, gel purification and ligation Sequences to be digested were tested in silico using the Restriction Mapper software  available  at:  http://www.bioinformatics.vg/biolinks/bioinformatics/Restriction%2520Analysis.shtml.  All restriction reactions took place at 37 ˚C using Roche and New England Biolabs enzymes following manufacturer protocols for single, double, and triple digests. Complete or partial digests were run on 1% agarose gels and gel purified with Qiagen PCR purification kits or Minielute kits if under 10 kb in size, or Qiagen 14  QiaXII kits if larger than 10 kb. To concentrate the DNA, samples were vacuum dried for 5 min at room temperature and then assayed for quantity using a 1% agarose gel electrophoresis and EtBr staining and UV fluorescence imaging. Visual comparison of band intensity under UV allowed an estimate of molecular concentration. Vector and insert DNA was ligated in a ratio of 1 vector molecule to 3 insert molecules, using T4 ligase (Roche) and buffer following the manufacturer’s protocols. Ligations were left for 48 h at 4 ˚C before transformation of half the volume into chemically competent DH5α E. coli cells.  2.1.3 Genomic DNA and total RNA isolation Poplar and Arabidopsis tissue was disrupted by bead milling with 1 mm zirconiasilica beads and 0.09 g of fresh leaves in 2.0 mL Eppendorf tubes, then using a kit and following manufacturer’s instructions from step 4 (Nucleon Phytopure Plant DNA Extraction Kit, Amersham). DNA pellets were eluted in 30 µl of distilled, filtered water, and then further diluted 1:15 in distilled water for PCR. Poplar and Arabidopsis RNA was extracted by grinding 0.09 g of frozen leaf, whole seedling, or stem tissue, in liquid nitrogen then following a modified protocol of Qiagen’s Rneasy kit developed by O. Schevchenko and A. Bruner (unpublished). Briefly this involved mixing 0.01 g of soluble polyvinylpyrrolidone (PVP-40,Sigma) per mL of Qiagen RLT buffer including beta mercaptoethanol. 500 µl of this was added to 0.1 g of tissue ground in liquid nitrogen within a 1.5 mL eppendorf micro centrifuge tube. This mixture was shaken for 1 minute, then vortexed at high speed for 30 seconds. 200 µl of Rnase free KoAC (pH 6.5) was then added, mixed by inversion, and then incubated on ice for 15 min. After incubation, tubes were spun at 12,000 rpm for 15 min at 4 ˚C, and the supernatant transferred to a clean 1.5 mL tube. 350 µl of 100% ethanol was added and mixed by pipetting, then this mixture was applied to pink Qiagen Rneasy columns and the Rneasy kit protocol followed from step 6 onward.  15  DNA and RNA quality and concentration was checked by analysis of 260 and 280 nm absorbance in a spectrophotometer, as well as direct visualization using EtBr UV fluorescence on a 1.5% agarose gel.  2.2.4 Primer design and PCR based genotyping Primers were designed in the Bioedit program by visually selecting high G or C content sequences from desired regions and then testing them for primer dimers, optimal size, and annealing temperature in the virtual PCR program Amplifx, freely available at http://jullien.n.free.fr/article.php3?id_article=10. PCR was performed in a Thermocycler PCR machine using the following protocol in a 50 µl volume plus a 20 ul volume of mineral oil: Step 1. 94 ˚C for 2 minutes Step 2. 94 ˚C for 30 seconds Step 3. X ˚C for 30 seconds (annealing step that is reaction specific) Step 4. 72 ˚C for Y seconds (template extension step that is reaction specific) Step 5. Go to step two Z number of times (cycles number step that is reaction specific) Step 6. 94 ˚C for 30 seconds Step 7. X ˚C for 30 seconds (annealing step that is reaction specific) Step 8. 72 ˚C for 7 minutes Step 9. 12 ˚C for 1 hour Arabidopsis DNA was used in a PCR reaction amplifying a 940 base pair region of the Actin2 gene using primers: AtActin2F (CTGAGGCTGATGATATTCAACCAATCG) and, AtActin2R (GATCCTTCCTGATATCCACATCACAC) PCR was performed with 35 cycles at 55 ˚C annealing temperatures and 1 minute extension time. Plants were tested for transgenes using the information presented in Table 1.  16  Table 1: PCR protocols used in testing for transgenes in genomic DNA. Transgene construct Parameters Primers used AtCTL1 promoter with PopCTL1 coding region in pART27  58 ˚C, 1 min ext, 40 cycles  PopCTL1.20 GTGCTACAACAAGGAAATGAGTCCCA Han-R CAACGTGCACAACAGAATTGAA  CaMV 35S promoter with AtCTL1 coding region in pCAMBIA 1305.1  57 ˚C, 1 min 15 sec ext, 35 cycles  AtCTL1.3 Last Exon Fwd CACTTGCATTCCAAGCTGCAATCTG pCAMBIA1305.1 Nos Term Rev GATTAATCATCGCAAGACCGGCAACAGG  CaMV 35S promoter with AtCTL2 coding region in pCAMBIA 1305.1  57 ˚C, 1 min 15 sec ext, 35 cycles  AtCTL2.5 Last Exon Fwd CTGCAACAGCGGATTCGATAACGATGAG pCAMBIA1305.1 Nos Term Rev GATTAATCATCGCAAGACCGGCAACAGG  CaMV 35S promoter with PopCTL1 coding region in pART27  58 ˚C, 1 min ext, 40 cycles  PopCTL1.20 GTGCTACAACAAGGAAATGAGTCCCA Han-R CAACGTGCACAACAGAATTGAA  PopCTL2 promoter with PopCTL2 coding region in pCAMBIA 1305.1  57 ˚C, 1 min ext, 30 cycles  PopCTL2.3 Last Exon Fwd CTATGGGGATCAAGTTTGCGGCCAGG pCAMBIA1305.1 Nos Term Rev GATTAATCATCGCAAGACCGGCAACAGG  AtCTL1 promoter with GUS coding region in pCAMBIA 1305.1  52 ˚C, 1 min ext, 35 cycles  AtCTL1.11B GGGAGCTCACGCCATTGTTACTGTTGG GUS Up GGGTCCTAACCAAGAAAATGAAGGAG  PopCTL1 promoter with GUS coding region in pCAMBIA 1305.1  52 ˚C, 1 min ext, 35 cycles  PopCTL1.19D CCCATCTGAGCCTCAACGAAAa GUS Up GGGTCCTAACCAAGAAAATGAAGGAG  PopCTL1 150 base pair RNAi construct in pART27  54 ˚C, 45 sec ext, 35 cycles  CAMV 35S Forward CTGGCGAACAGTTCATACAGAGTC CAMV 35S Reverse GTGTTCTCTCCAAATGAAATGAACTTCC  PopCTL1 400 base pair RNAi construct in pART27  54 ˚C, 45 sec ext, 35 cycles  CAMV 35S Forward CTGGCGAACAGTTCATACAGAGTC CAMV 35S Reverse GTGTTCTCTCCAAATGAAATGAACTTCC  PopCTL2 150 base pair RNAi construct in pART27  54 ˚C, 45 sec ext, 35 cycles  CAMV 35S Forward CTGGCGAACAGTTCATACAGAGTC CAMV 35S Reverse GTGTTCTCTCCAAATGAAATGAACTTCC  PopCTL2 400 base pair RNAi construct in pART27  54 ˚C, 45 sec ext, 35 cycles  CAMV 35S Forward CTGGCGAACAGTTCATACAGAGTC CAMV 35S Reverse GTGTTCTCTCCAAATGAAATGAACTTCC  17  2.2.5 Reverse transcriptase PCR (RT PCR) and expression analysis 2 µg of total RNA was reverse transcribed in a 40 µl volume using Invitrogen Superscript II enzyme and following the manufacturer’s protocol for first and second strand synthesis (Invitrogen). Semi quantitative PCR was conducted on 2µl samples of cDNA in a 50 ul volume reaction (with an addition of 20 µl of mineral oil) to determine gene expression levels. As a control in Arabidopsis cDNA, Actin2 expression levels were assayed by using the primers AtActin2F RT (CTTCCGCTCTTTCTTTCCAAGCTC) and AtActin2R RT (CCATCACCAGAATCCAGCACAATACCG) with 53 ˚C annealing temperature, 1 min extension time, and 27 cycles. As a control in poplar cDNA, TRANSLATION INITIATION FACTOR 5A (TIF-5A) expression levels were assayed by using the primers PopTIF-5A F RT (CTCATCTCACAACTGTGATGTTCCC) and PopTIF-5A R RT (CCATTTTCAGTCAAAAGACTCACAAAAC) with 53 ˚C annealing temperature, 1 min extension time, and 27 cycles. For both poplar and Arabidopsis housekeeping genes, optimal PCR sub saturation levels were determined to occur at 27 cycles. Primers were designed to selectively amplify cDNA despite gDNA contamination, and this was confirmed by testing these primers against genomic DNA negative controls. Table 2 lists the genes and conditions used to assay CTL mRNA levels in Arabidopsis and poplar.  2.3 Cloning procedures 2.3.1 Host bacterial strains, plasmids, and plant materials All cloning was done in Escherichia coli strain DH5α which is a mutant for recombination and RNase enzymes, which was grown overnight in liquid LB broth at 37˚C and constant shaking at 200 rpm. Transformation of calcium chloride/rubidium  competent  cells  was  carried  out  as  described  in  (Sambrook et al. 1982).  18  Table 2: Semiquantitative PCR parameters and primers used in gene expression analysis. Gene expression SemicDNA specific primers tested quantitative PCR conditions AtCTL1  52 ˚C, 1 min ext, 27 cycles  AtCTL1.25RT GATCTGCATTGCGGCTTGTAG AtCTL1.26RT GTTGCAACTCCATAGCCACAGGAC  AtCTL2  52 ˚C, 2 min ext, 27 cycles  AtCTL2.11 RT Forward GGGAG4CAAAACCTCATGCGGGTACG AtCTL2.2 CCAGAGGTTATAAAACTTTCACTCAAG  PopCTL1  52 ˚C, 1 min ext, 27 cycles  PopCTL2  52 ˚C, 1 min ext, 27 cycles  PopCTL1.25RT GAAGCAAAACCTCATGTGGTTAT PopCTL1.26RT CAGTTTTGCCATAGTTGTAGTTCCA PopCTL2.25RT CGTTGGCTGCAAAACCTCCTGTGGTTA PopCTL2.26RT CAGCTCCATAGTTATAGTTCCAGAAGAT  Agrobacterium strains used for transformation were GV3101 for Arabidopsis floral dip and EHA-105 for poplar leaf disc infection. It was found that 12 h of shaking at 200 rpm at 29˚C was enough to generate an OD600 of 1.8 in 2 mL of LB broth. The GV3101 strain used was found to be spectinomycin and streptomycin resistant, forcing pART27 selection to be carried out using kanamycin at a concentration of 100 µg/mL instead. Transformation of chemically competent Agrobacterium was carried out as described in (An 1987). Arabidopsis plants were all ecotype Columbia-0. The AtCTL1 knock out allele used was pom1-9 an Atctl1 EMS alllele containing a premature stop codon at amino acid 11 (M.-T.Hauser, unpublished). The AtCTL2 knock out allele used was the T-DNA insertion line SAIL_319_A05 ordered from the Syngenta Arabidopsis Insertion Library  (SAIL)  collection  available  http://www.arabidopsis.org/servlets/TairObject?type=organization&id=211883.  at It  was confirmed to contain the T-DNA by BASTA selection and Thermal Asymmetric Interlaced-Polymerase Chain Reaction (TAIL-PCR) was used to amplify DNA fragments flanking the T-DNA left border from this transformed line (B. Hamberger, 19  person communication). RNA extraction and cDNA synthesis followed by semiquantitative PCR (as outlined in Table 2) were used to confirm down regulation of AtCTL2. Seeds for the DR5::GUS artificial auxin response promoter-GUS fusion in a Columbia-0 background were kindly provided by Dr. Thomas Berleth (University of Toronto, Toronto, ON). DR5::GUS in Col-0 was crossed with pom1 to produce two lines of homozygous DR5::GUS in pom1 mutant seeds (B. Hamberger, unpublished). Two lines of AtCTL2::GUS in Col-0, PopCTL2::GUS in Col-0, CaMV 35S:AtCTL2 in pom1, and PopCTL2:PopCTL2 in pom1 seeds were also received from B. Hamberger (unpublished results) and included in the study.  The poplar clone used for transformation and all subsequent experiment was in clone 717-1B4, a hybrid of Populus tremula and Populous alba kindly provided by Dr. Shawn Mansfield. Plasmids used in this study were Qiagen’s pDRIVE PCR cloning vector, CAMBIA’s pCAMBIA 1305.1 (www.CAMBIA.org) , and RNAi vectors pHANNIBAL (Wesley et al. 2001) and pART27 (Gleave 1992). 150 bp and 500 bp PopCTL2 RNAi constructs in pART27, as well as CaMV 35S:AtCTL2 and PopCTL2:PopCTL2  constructs in pCAMBIA 1305.1 were received from B.  Hamberger and were used for plant transformation. General plasmid maps are shown in Figure 4.  20  Figure 4: Plasmids used in all cloning and transformation procedures. Qiagen’s pDRIVE was used to clone PCR products, which were then cut and ligated into either pHANNIBAL or pCAMBIA 1305.1. The binary plasmids pART27 and pCAMBIA 1305.1 were the final destination for cloned fragments which were intended for Agrobacterium mediated plant transformation.  2.3.2 Site directed mutagenesis, cloning, and sequencing of PCR products into the pDRIVE vector. Site directed mutagenesis was employed to introduce appropriate restriction sites to CTL promoter and coding regions. The specific primers and conditions used are shown in Table 3. PCR products were run on a 1.5% agarose gel, along with a 1 kb DNA mass ladder (Invitrogen). No more than 10 seconds of UV light was used to visualize ethidium bromide stained nucleic acid, and appropriately sized bands were cut out and the DNA purified using either Qiagen PCR Purification Kits or Qiagen Minielute Purification kits. 4 ul of the resulting gel purified fragment was  21  Table 3: CTL promoters and coding regions, PCR conditions, and primers used for their amplification. Non-complimentary base pairs in primers are small letters, and restriction sites are underlined. Region amplified  PCR conditions  Primers used  AtCTL1 promoter (955 bp)  55 ˚C, 1 min extension, 30 cycles  AtCTL1.11B (with Sac I site) GGgAGCTcACGCCATTGTTACTGTTGG AtCTL1.12 (with Nar I site) CAACTATgGcgCCAATACCAGCTAC  AtCTL1 promoter (957 bp)  55 ˚C, 1 min extension, 30 cycles  AtCTL1.11B (with Sac I site) GGgAGCTcACGCCATTGTTACTGTTGG AtCTL1.12 (with Nco I site) GCTTACAACCATgGAACAAATACCAGCTAC  AtCTL1 coding region (1645 bp)  52 ˚C, 1 min 30 seconds extension, 30 cycles  AtCTL1.13B (with Nar I and Nco I site) GTTGggcGCcATGGTGACAATCAGGAG AtCTL1.14B (with Spe I site) GAATAGAAAcTaGTATCATTCGCAAACC  PopCTL1 promoter (1722 bp)  55 ˚C annealing, 1 min 30 seconds extension, 30 cycles  PopCTL1.11C (with Sac I site) CATATAGAGCTCCAAGGGTAGTTGGCG PopCTL1.12E (with Nco I site) CATTTGGCCTCCATccTGGCTATGCCTG  PopCTL1 coding region (1794 bp)  54 ˚C annealing, 1 min 30 seconds extension, 30 cycles  PopCTL1.13B (with Nar I site) ggcGccATAGCCAAGATGGAGGCCAAATG PopCTL1.14C (with Spe I site) CACActAGTGACCCAATATACACTCTCTACATAG  PopCTL1 coding region (1797 bp)  50 ˚C annealing, 1 min 30 seconds extension, 30 cycles  PopCTL1.15 (with Sac I site) CGTACAGGCcTcgagAAGATGGAGGC PopCTL1.14C (with Spe I site) CACActAGTGACCCAATATACACTCTCTACATAG  PopCTL1 fragment of exon 1 (146 bp)  55 ˚C annealing, 45 seconds extension, 30 cycles  Fwd54 (Xho I and Xba I sites) GGGcTcgagtctagaGTAGTGGTAAATGGAGATGAGTC Rev200 (Kpn I and Hind III sites) gggGgTacCaagcTtGTAGGTCTGGAAAAAATCAGAA  PopCTL1 fragment of exon 1 (415 bp)  55 ˚C annealing, 45 seconds extension, 30 cycles  PopCTL1.17 (Xho I and Xba I sites) CAGctcgAGtCtAGATGGAGGCCAAATGGTGTTC PopCTL1.18 (EcoR I and Hind III sites) TACgaAttcaAGcTTTTGCTTCCAACATGCCCAAGGAAAGC  22  ligated into the linearized pDRIVE vector following the protocol included in the Qiagen PCR Cloning kit (Qiagen). 300 ng of pDRIVE plus PCR product was then sequenced using M13 forward or M13 reverse primers to get pair end reads covering the whole fragment. Where fragments were larger than 1000 bp, a fragment specific primer was used to achieve full coverage in the middle of inserts (generally priming 600 bp into the fragment)  2.3.3 Construction of AtCTL1 promoter::AtCTL1 coding region fusion construct in pART27 A number of pDRIVE plasmids containing either AtCTL1 coding region in pDRIVE or AtCTL1 promoter were screened by digestion with NarI and BamHI for clones with the insert having the BamHI near to its 3’ end. Positive AtCTL1 coding region clones, had the excised insert gel purified and ligated to NarI/ BamHI pDRIVE molecules containing the promoter in the correct orientation. This promoter: coding region fusion was then cut out using BamHI and a partial NotI digest, and ligated to a completely BamHI and partially NotI digested pHANNIBAL vector backbone of around 3500 bp. This ligation added the OCS terminator to the 3‘ UTR of the AtCTL1 coding region. Finally, the whole cassette was removed by NotI digestion and ligated into NotI cut pART27.  2.3.4 Construction of AtCTL1 promoter::PopCTL1 coding region fusion construct in pART27 A pDRIVE plasmid containing the PopCTL1 coding region with the vector’s Sac I restriction site in the 5’ end was cut with NarI and SacI and ligated to NarI and SacI fragment of AtCTL1 promoter. This promoter::coding region fusion was then cut out of pDRIVE using NotI and BamHI, and ligated into the pHANNIBAL vector such that it replaced the CaMV 35S promoter and intron region bounded by these two enzymes. From pHANNIBAL, the cassette of AtCTL1 promoter, PopCTL1 coding region, and OCS terminator was then cut out by NotI digestion and ligated into NotI digested pART27.  23  2.3.5  Construction  of  PopCTL1  coding  region  overexpression  construct in pART27 pDRIVE plasmids with PopCTL1 coding regions containing SacI and SpeI sites, were screened by test digestion with BamHI and SpeI. Plasmids with the vector BamHI restriction site in the 3’ end of the insert were first sequenced, then digested with XhoI and BamHI, and the PopCTL1 fragment was ligated into XhoI and BamHI cut pHANNIBAL.  From pHANNIBAL, the cassette of CaMV 35S  promoter,  PopCTL1 coding region, and OCS terminator was then cut out by NotI and SpeI digestion and ligated into NotI and SpeI digested pART27.  2.3.6 Construction of AtCTL1 coding region overexpression construct in pCAMBIA 1305.1 The AtCTL1 coding region was cloned into the pDRIVE vector in an orientation with the vector’s NheI restriction site in its 3 prime end. This clone was digested to completion with NcoI and NheI and run on a 1% agarose gel to yield a 1680 bp band. pCAMBIA 1305.1 was also digested with NcoI to completion, but only partially with NheI, allowing isolation of vector fragments of 9820 bp in size and only missing the GUSPlus coding region. Both digested insert and vector were purified in 40 ul of distilled water using a Qiagen QiaXII kit (Qiagen) and ligated together over night as described in 2.1.2.  2.3.7 Construction of AtCTL1 and PopCTL1 promoter::GUS fusions in pCAMBIA 1305.1 pDRIVE plasmids with AtCTL1 coding regions containing sites were sequenced and digested with SacI and NcoI. The resulting fragment was digested with SacI and NcoI, gel extracted, and the resulting fragment was ligated into SacI and NcoI digested pCAMBIA 1305.1. pDRIVE plasmids with PopCTL1 coding regions containing SacI and NcoI sites, were sequenced and found to contain one of two allelic promoters both slightly divergent from the sequence reported in the published genome. One of these was 24  digested with SacI and NcoI, and the resulting fragment was ligated into SacI and NcoI digested pCAMBIA 1305.1.  2.3.8 Construction of PopCTL1 RNAi constructs in pART27. The 146 bp PopCTL1 fragment was digested with XhoI and KpnI and ligated into pHANNIBAL that had been cut with the same enzymes. This ligation product was sub cloned, and digested with XbaI and HindIII to receive the similarly digested 146 bp PopCTL1 fragment. This pHANNIBAL now contained two copies of the insert but in opposite orientations and separated by an intron. A cassette composed of the CaMV 35S promoter, OCS terminator, and these two inserts separated by an intron, was cut out by digestion with NotI and inserted into NotI digested pART27. A nearly identical procedure was followed for 416 bp fragments, except EcoRI was used in place of KpnI.  2.4 Arabidopsis germination, growth conditions, transformation, and selection 2.4.1 Seed sterilization, germination, transplantation, and growth conditions Arabidopsis seeds were sterilized by a five-min incubation in 70% ethanol, followed by a 1 min spin at 13,000 rpm in an eppendorf micro centrifuge in discarded Qiagen Spin Miniprep columns. In the same column, seeds were then incubated in 100% ethanol for additional five min and again spun twice for one minute at 13,000 rpm to dryness. Seeds were then sprinkled on a sterile media of 2.35 g/L MurashiSkoog salts (Sigma), 6 g/L agar (Sigma), 0.1% w/v sucrose and pH 5.7. Seeds were then stratified by placement at 4 ˚C for 72 h. After stratification, seeds were then placed in a light chamber where they received 18 h light per day at a constant 25 ˚C. After about two weeks of growth, plantlets developing true leaves were transferred to small pots filled with Sunshine mix number 5 thoroughly watered with All Purpose Miracle Grow at the recommended concentration. These pots were 25  covered with a transparent plastic dome, then transferred to a larger phytochamber where they received 18 h of light at constant temperature of 21 ˚C. Plants typically flowered one month after stratification.  2.4.2 Arabidopsis floral dip genetic transformation Arabidopsis transformation was carried out using a modified floral dip protocol that allowed for up to five treatments per flowering plant and resulted in a high number of subsequent transformants (Martinez-Trujillo et al. 2004). After 12 h of darkness and humidity, dipped plants were transferred back to the phytochamber where they were allowed to further develop before another dip one week later. Siliques were harvested for seed as they opened while the plant continued to flower, and dry seeds were stored at room temperature in 1.5 mL eppendorf tubes.  2.4.3 Arabidopsis transgenic seed selection Depending on the binary vector used, transgenic Arabidopsis seed was selected for by either 50 µg/mL of hygromycin B (Sigma) or kanaymicin sulfate (Sigma) supplemented in media used for the seed sterilization and germination protocol described above in 2.4.1. Positive transformants were transferred to soil after two weeks on the media and grown under a dome using the same protocol described in 2.4.1.  2.4.4. Seedling growth assays To test for complementation of the pom1 mutant phenotype, or changes in seedling growth rate in darkness or light, Arabidopsis seed from the T2 generation were plated in thin lines on grided square petri dishes containing only 2.35 g/L MurashiSkoog salts (Sigma) and 6 g/L agar (Sigma), at pH 5.7 and stratified for 72 h at 4 ˚C. Seedlings tested for their sucrose enhanced phenotype were plated on similar media with 4.5% w/v sucrose added. Plates to undergo dark treatment were  26  carefully wrapped in tinfoil, and all plants were placed sideways in a growth chamber for four days under conditions of 18 h light per day at a constant 25 ˚C. Plants were imaged at the end of four days, using an Epson flatbed scanner.  2.5 Poplar growth conditions, transformation, and tissue culture 2.5.1. Agrobacterium mediated poplar transformation in tissue culture Preparation of woody plant media (WPM) was accomplished by following the protocol described by McCown and Lloyd (1980). The following protocol kindly provided by Dr. Shawn Mansfield was then followed, using agro bacterium EHA105 (Hood et al. 1993) and poplar clone 717-1B4. Day 1 1. Overnight cultures of Agrobacterium containing large and small PopCTL1 or PopCTL2 RNAi constructs in pART27 were grown in liquid LB medium containing 25 ug/mL of rifampicin, 25 ug/mL of gentomycin, and 50 ug/ul of Kanamycin. 2. Filter papers were wrapped in tin foil and autoclaved. Media was made for each of the next 9 steps, including: -  2 litres of liquid WPM (step 4 and 5) 25 petri dishes containing WPM .1/.1/.1 (0.1 μM of NAA, BA, and TDZ) agar (step 7)  -  25 growth jars with WPM .1/.1/.1carb500(ug/mL)cef250(ug/mL) (step 8)  -  25 growth jars with WPM .1/.1/.1carb500cef250kan25 (step 9)  Day 2 3. Agrobacterium was grown overnight in WPM with 100μM filtered acetosyringone in ethanol. 4. Leaf discs were cut from 5 week old wild type 717 plants in sterile water using a #4 cork borer and placed immediately into liquid WPM in petri plates (~25 per plate).  27  Day 3 5. Agrobacterium grown overnight was diluted with WPM to an Optical Density of 0.1 – 0.2 at 600 nm absorbance. 6. 10ml Agrobacterium solution was placed in a 50ml falcon tube and cocultivate with 25 discs for about ½ an hour with 75 to 100 rpm shaking at 28˚C. 7. Leaf discs were blotted dry with sterile filter paper and place abaxial side up (upside down) on WPM .1/.1/.1 in the dark. Day 6 8. Leaf discs were moved to WPM .1/.1/.1 for callus growth and carb500cef250 antibiotics to kill Agrobacterium and again placed in the dark along with appropriate controls. Day 9 9. Leaf discs were moved to WPM .1/.1/.1carb500cef250kan25 in subdued light Weekly 10. Culture jars were checked weekly for Agro or fungal contamination, and if found were transfered immediately to fresh WPM .1/.1/.1carb500cef250kan25 5 Weeks 11. Distinct shoots or clumps from different discs were transferred to WPM0/0.01/0carb500cef250kan25 to induce shoot formation. 9 Weeks 12. Elongated shoots were transferred to WPM0.01/0carb500cef250kan50 for rooting. 13 Weeks 13. Rooted and good looking non-rooted tips were transferred to WPM0.01/0/0carb500cef250kan75. 14. This was continued until a good number of shoots were rooting on both concentrations of kanamycin.  28  15. To clone lines and bulk up plant numbers, shoots were cut into sections with at least two nodes and put back into the rooting media.  2.5.2. Poplar growing conditions An initial test trial took healthy wild type and PopCTL2 400 bp RNAi in vitro grown poplar plants of at least 5 cm in height and with well developed root systems, and planted them in 2 liter pots filled with well watered but unfertilized Sunshine Mix 5 soil. These were then grown for three months in a phytochamber with high air flow where they received water every second day,18 hours of light at constant temperature of 21 ˚C. A more extensive trial transferred healthy in vitro grown poplar plants of at least 5 cm in height and with well developed root systems, and planted them in 1 liter pots filled with well watered Sunshine Mix 5 soil thoroughly soaked with All Purpose Miracle Grow at the recommended concentration. These plants were kept under transparent plastic domes at 18 h of light at constant temperature of 21 ˚C for one month as they became established. Healthy saplings were then transferred to a greenhouse in mid February where they received daily watering by flooding over a period of 3 to 10 months. After sectioning, poplars that were harvested for microscopy were transferred to a phytochamber with high air flow where they received water every second day,18 h of light at constant temperature of 21 ˚C . Only one branch was allowed to re-grow per tree and the rest were pruned with scissors. PopCTL2 RNAi photographs in Figure 23 come exclusively from these regrown trees.  2.6 Dissecting, light, UV fluorescence microscopy analysis Thin hand sections were made using double edged Wilkinson Sword razor blades across and in transverse of Arabidopsis floral stems and hypocotyl/roots, or poplar stems. For increased resolution, a Leica vibratome was also used where possible  29  to cut 40 and 100 um thick sections of these tissues. Both poplar and Arabidopsis sections were stained for 30 s with 1% toluidine blue in 1% sodium borate before addition of distilled water and immediate viewing. Lignified cell walls appeared turquoise coloured, while non-lignified walls stained different shades of purple. UV, bright and dark field illuminated cross and transverse sections were taken using a Zeiss Axioplan light microscope attached to a digital camera at the UBC Bioimaging facility and in the lab of Dr. Lacey Samuels. Phloroglucinol staining of sections of Arabidopsis and poplar were performed as follows: samples were placed in a 1% phloroglucinol-HCl (1:1 water: EtOH solvent) solution for 10 min, and mounted in a 1:1 mixture of glycerol:6 N HCl and observed immediately. Sections were analyzed with bright and dark field illumination using a Zeiss Axioplan microscope at the UBC Bioimaging facility. When observing large plant organs, GUS stained seedlings or thick poplar sections bright field microscopy using a Leica dissecting microscope and Spot32 camera and software were used in the UBC Bioimaging facility.  2.7 GUS expression analysis GUS reagent solution was prepared by mixing a water based solution with concentrations of: 100 mM NaPO4, pH 7.0 0.1% Triton X-100 0.5% X-GLUC (bromochloroindoyl-b-glucuronide) in dimethyl formamide 2 mM potassium ferricyanide (K3Fe(CN)6) 2 mM potassium ferrocyanide (K4Fe(CN)6) Whole seedlings, plant parts, or handcut floral stem or root sections were placed in the cold GUS solution, then put under full vacuum for 15 minutes as described in (Stomp 1992). Samples were incubated at 37 ˚C for 14 h or until a strong blue  30  colour developed. Large Arabidopsis organs were visualized using a Leica dissecting microscope and Spot32 camera and software, while thin GUS stained floral stem or root sections were visualized using a Zeiss Axioplan microscope and Q-Capture software at the UBC Bioimaging facility.  Chapter 3: Bioinformatic analysis of CTLs 3.1 Introduction Bioinformatic approaches can allow gene discovery and predictions about gene function using electronically available sequence, expression, structural data and various tools. These in silico analyses can thus guide “wet lab” based work. For example, as previously mentioned, AtCTL2 was discovered by sequence homology to AtCTL1 using BLAST analysis in Genebank. These genes were both shown to be distinct from true chitinases by phylogenetic analysis (Figure 3; M.T. Hauser, unpublished) Before I started my project, BLAST and MEGABLAST analysis against GenBank, using the coding regions of the AtCTL1 gene yielded a number of homologous CTL sequences  including  most  notably,  PintaCTL1  from  Pinus  taeda,  both  Physcomatrella patens homologues PhypaCTL1 and PhypaCTL2, five new dicot CTLs, and five new monocot CTLs. (B. Hamberger, unpublished). Blast analysis of poplar EST databases had shown the presence of two poplar CTL genes, and analysis of the Genome BC Treenomix EST collection confirmed the presence of a second chitinase-like in poplar PopCTL2 (B. Hamberger, unpublished). While the biochemical function of AtCTL1 or any CTL protein is not yet known, unpublished results on the recombinant protein expressed in E. coli indicates it does not have in vitro catalytic activity against a chitin substrate (C. Douglas, unpublished) This finding is supported by amino acid alignments amongst known chitinases and CTLs, suggesting the glutamate residues are important for hydrolytic activity in chitinases are not conserved in CTLs (Zhang et al. 2004). 31  As previously discussed, AtCTL1 is implicated in primary cell wall development based on the pom1 mutant phenotypes. A major function for AtCTL2 has not been revealed by phenotypes in available Atctl2 mutants (Brown 2005; Persson et al. 2005), although it has been predicted to be involved in secondary cell wall formation based on microarray analysis in developing Arabidopsis floral stems (Ehlting et al. 2005). Further, AtCTL expression data from the e-FP program from the University of Toronto (Vinegar et al. 2006) suggests wide expression of this gene. AtCTL1 seems to be expressed at moderate levels in all Arabidopsis tissues at all stages of development except mature pollen, senescing leaves or sepals. Although also expressed in most tissues, AtCTL2 is predicted to be most strongly expressed in inflorescences, roots, petioles, heart stage embryos, siliques and flowers, but has no expression in mature seeds, senescing leaves, meristems, or cotyledons. Primary cell walls are found in every cell of Arabidopsis so it makes sense to see near global expression of AtCTL1 in growing plants, but this predicted pattern of AtCTL2 expression does not lead to any clear predictions regarding function, aside from the high expression in inflorescence stems. To help clarify a relationship between expression and function, tissue and cell specific expression should be assayed, since in silico approaches only show organ specific data. It is likely that these genes are not expressed in all cell types within the organs described above and thus our picture of CTL expression is incomplete. The hypothesis that CTLs have distinct cell type-specific expression patterns is tested in Chapter 4 of this thesis. A different approach to elucidate gene function using gene expression uses genes sharing similar expression patterns with our genes of interest. Dr. Bjoern Hamberger in the Douglas lab carried out a co-expression analysis by submitting AtCTL1 and AtCTL2 as queries to the GenExpress program (Segal et al. 2005) and identified the top 10 genes with most similar expression patterns. Reiteration of this process, by the same analysis of the 10 genes generated a network of genes that share expression patterns with AtCTL1 and AtCTL2 (Figure 5).  32  According to this analysis, AtCTL1 is closely co-expressed with genes encoding KORRIGAN, COBRA, ACC oxidase and most interestingly, the primary cell wall cellulose synthases CesA3 and CesA5 (Cosgrove 2005), re-enforcing the idea that it is involved in primary cell wall development. AtCTL2 on the other hand is coexpressed with IRX1, IRX3, and IRX5,  genes encoding cellulose synthase  subunits that have all been previously shown to be involved in secondary cell wall development (Taylor et al. 2000). Based on this analysis, and supporting expression data, we make the prediction that AtCTL2 is a protein involved in secondary cell wall synthesis and possibly with cellulose production or modification in the layers of the secondary wall, while AtCTL1 is involved in primary cell wall development. Similar predictions have been made for both genes by other groups working independently (Brown 2005; Persson et al. 2005).  Figure 5: Co-expression analysis of AtCTL1 and AtCTL2. The analysis was performed by Dr. Hamberger using using GenExpress (Segal et al. 2005). CTL genes are highlighted by a pink square and the immediate cellulose synthase co-expression network by a black circle.  Additional CTL gene discovery and phylogenetic analyses should help establish relationships between Arabidopsis CTLs with predicted functions based on in silico  33  analyses, and CTL genes found within other plant lineages, including poplar. This would also help to reveal patterns of CTL evolution and divergence amongst all plants and possibly other organisms. Finally, amino acid alignment of selected chitinases and CTLs from these lineages could shed light on potential biochemical properties of CTLs in relation to known chitinases.  3.2 Results To increase the number of CTL sequences available for phylogenetic analysis, I screened public and private EST databases as listed in 2.1.1 using BLAST for possible CTL genes. Table 4 gives the name, species, publication source, and Genebank ID for each EST, as available. Two major phyla of land plants were discovered to contain CTLs; Bryophyta represented by Physcomitrella patens, and Spermatopsida represented by a number of gymnosperms and angiosperms. Of the other extant phyla of land plants, only Lycopsida represented by Selaginella moellendorffii has any appreciable amount of public genomic information, but only class I chitinases with greater similarity to the true Hordeum vulgare chitinase CHI26 than CTLs were found (data not shown). No CTL sequences were found in the single cell green algae Chlamydomonas reinhardtii, nor the multicellular red algae, Porphyra yezoensis, for which complete genome sequence information is available, suggesting that CTL genes encoding proteins with significant similarity to Arabidopsis CTLs, evolved after the evolution of the land plant habit. Within the Spermatopsida, most species were found to contain a maximum of two CTL genes. Gossypium hirsutum was the exception and was found to contain at least three CTLs, including GoshiCTL1 and GoshiCTL2 which have been previously reported to be associated with secondary cell wall development (Zhang et al. 2004). That only two CTLs were usually observed in any single species is an especially important observation in Arabidopsis, poplar, and rice since these plants have fully sequenced genomes. Identification of two Pinus taeda and two Picea glauca CTLs, in addition to a CTL in Cyclas rumphii and Ginko biloba, was significant, since no Gymnosperm CTLs had been reported to this date.  34  Table 4: CTL genes/ESTs included in phylogenetic analysis and their Genbank ID.  Name  Species  Genebank ID and publication  HorvuCHI26 PhypaCTL1 PhypaCTL2 PicglCTL1 PicglCTL2 PintaCTL1 PintaCTL2 GinbiCTL CycruCTL OrysaCTL1 OrysaCTL2 SacofCTL ZeamaCTL HorvuCTL TriaeCTL AtCTL1 AtCTL2 PopCTL1 PopCTL2 VinviCTL1 VinviCTL2 GoshiCTL1 GoshiCTL2 GoshiCTL3 SoltuCTL PissaCTL BranaCTL CitsiCTL1 CitsiCTL2 GlymaCTL PontrCTL MaldoCTL  Hordeum vulgare Physcomitrella patens Physcomitrella patens Picea glauca Picea glauca Pinus taeda Pinus taeda Ginko biloba Cyclas rumphii Oryza sativa Oryza sativa Saccharum officinarum Zea mays Hordeum vulgare Triticum aestivum Arabidopsis thaliana Arabidopsis thaliana Populus trichocarpa Populus trichocarpa Vitis vinifera Vitis vinifera Gossypium hirsutum Gossypium hirsutum Gossypium hirsutum Solanum tuberosum Pisum sativum Brassica napus Citrus sinensis Citrus sinensis Glycine max Ponc trifolia Malus domestica  576565 (Leah et al. 1991) 100362431 67723837 83392172, 83381286, 83390845, 70633925 49061832, 70658804, 83380632, 83377852 67048305, 67048374 48936546, 48936546, 49451360 67051174, 67050807, 27918838, 66989050 27915704, 27914102, 66986591, 66986572 32979588 51979696 35023090, 35941565, 35012997, 35116451 110530097, 76290204 16333065 70969400, 110427949 17226330 (Zhong et al. 2002) 42564264 3854865, 33452044, 33455768, 33455966,33456434 46839267, 73870349 110694702 33407264, 110364977 32401252 (Zhang et al. 2004) 34016875 (Zhang et al. 2004) 31072749 39820453, 39825041, 39831061, 39834279 37051095 56842897, 32501124 45449003, 46209309, 34519941, 34520272 21650175, 21650413, 21651525, 42477697 26048416, 26048212, 55286693, 55288819, 55293044, 55287218 71818780  MedtrCTL  Medicago trunculata  83667010, 27403940  Many CTLs in non-fully sequenced genomes were represented by multiple ESTs, allowing in silico sequence contigs to be generated that represent predicted full length or near full length CTL sequences to be deduced I aligned the predicted amino acid sequences using a conserved 290 amino acid region from these 32 CTL proteins, excluding a variable N-terminal region of 23 to 47 amino acids pertaining to AtCTL1 residues 1 to 48 (Figure 7). This alignment included the true Hordeum vulgare chitinase, CHI26. The alignments were used for phylogenetic  35  reconstructions, as described in 2.1.2. Predicted partial protein sequences used in this analysis are given in Appendix A. As shown in Figure 6, the reconstructed CTL phylogeny grouped the proteins into clades that correspond to known land plant lineages, with separate Bryophytes and Spermatopsida clades, gymnosperm and angiosperm clades, and monocot and dicot clades. Within the dicot clade, there was support for a further phylogenetic distinction between CTLs potentially involved in primary vs. secondary wall formation. AtCTL1 and AtCTL2 were found within distinct dicot clades with moderate bootstrap support (73). Both cotton CTLs, GhCTL1 and GhCTL2 which were previously characterized as being involved with secondary cell wall development (Zhang et al. 2004) grouped with AtCTL2, while a new cotton CTL identified from EST data, GhCTL3 was placed in the same clade as AtCTL1 further suggesting differences in amino acid sequence may be useful in distinguishing CTL function. Functional relationships of poplar CTLs to other CTLs were more difficult to predict, since there was no support for a close relationship of PopCTL2 with either AtCTL1 or AtCTL2. PopCTL1 grouped with AtCTL2, but was separated from PopCTL2. Lack of bootstrap support (38) for this topology makes the prediction of orthology to AtCTL2 difficult. Given that limited sequence information is available, gymnosperm CTLs were not as well represented in this analysis as angiosperm CTLs, but the two duplicated CTLs in Picea glauca and Pinus taeda, respectively were clearly in different clades. This was supported by a high bootstrap value of 95, suggesting independent evolution of two CTL genes within the gymnosperm lineage. Monocots also displayed this clear grouping of CTLs into distinct clades, with a very significant bootstrap value of 95 and rice representation in each indicating independent evolution after an initial duplication in monocotyledons. The topology of the reconstructed tree, with the monocot clade placed very close to the dicot clade containing AtCTL1 suggests a possible origin of the monocot CTLs from a single ancestral gene common to monocots and dicots.  36  Figure 6: Phylogenetic analysis of plant chitinase like genes, based on alignment of conserved 290 amino acid region. A UPGMA tree was reconstructed from aligned protein sequences that were mined from NCBI and Genome BC EST libraries. Branches are drawn to scale, with the scale bar showing 0.1 substitutions per site. Major plant taxonomic divisions are indicated by coloured balloons and coloured bars highlight the position of poplar, rice, spruce and Arabidopsis CTL pairs. Bootstrap values are shown for major divisions. The outgroup is a barley (Hordeum vulgare) family 19 chitinase called CHI26/1cnsA. For this analysis, AtCTL and PopCTL were changed to ArathCTL and PoptrCTL respectively.  The obvious phylogenetic differences between gymnosperm and angiosperm CTLs are apparent in the amino acid alignment depicted in Figure 7. To qualitatively guide the alignment, data on amino acid conservation in chitinase evolution were included (Bishop et al. 2000). Focusing on the most highly conserved region between position 70 and 327 in CHI26, one can see that some amino acids important for chitinase function such as the catalytic glutamic acid at position 169, the tyrosine at position 200, and the asparagine at 201, are shared between the representative chitinase and chitinase likes.  37  Barley CHI26 AtCTL1 AtCTL2 PopCTL1 PopCTL2 PicglCTL1 PicglCTL2  10 20 30 40 50 60 70 80 90 100 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| MRSLA-------VVVAVVATVAMAIG------------TARG-------------------------SVSSIVSRAQFDRMLLHRNDGACQAKGFYTYDA MVTIRS-GSIVILVLLAVSFLALVANGEDK-------TIKVKKVRGNKV-CTQGWECSWWSKYCCNQTISDYFQVYQFEQLFSKRNTPIAHAVGFWDYQS MVSKPLFSLLLLTVALVVFQTGTLVNAEDSEPSSSTRKPLVKIVKGKKL-CDKGWECKGWSEYCCNHTISDFFETYQFENLFSKRNSPVAHAVGFWDYRS MEAK---WCSLVTMALLVMLV-VVVNGDES----SQVKTVVKIVKGKKV-CDKGWECKGLSAYCCNQTISDFFQTYQFENLFSKRNTPVAHASGFWDYHS MMKIS----IWIILALALVNLALVVNGDDS------EKTIVKTVKGKKL-CKRGWECLDWSEYCCNETISDIFQPYQFENLFSNRNSPVAHAVGFWDYRS METKQIIG--SVNRMLVVVGICLVISSWLCCIEPAAAADEDQTTKSKKISCIKGAECKN-------KTISELFTVDQFESLFSHRNAPMAHAQGFWDYHS MAPGR---------VFLVVLLALAVSFNVS-------------LAKTKI-CDKGWECKG--VYCCNQTISQIFTVDNFEELFSKRNSPVAHAVGFWDYYS  110  Barley CHI26 AtCTL1 AtCTL2 PopCTL1 PopCTL2 PicglCTL1 PicglCTL2  130  220  230  150  160  170  180  190  200  * *T H * 240  *W  250  260  270  280  *Q *S *Y  290  300  ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| NYGPAGRAIGVDLLANPDLVATDATVGFKTAIWFWMTAQPPK-PSSHAVIAGQWSPSGADRAAGRVPGFGVITNIINGGIECGHG-QDSRVADRIGFYKR NYGAAGEALKADLLNHPEYIEQNATLAFQAAIWRWMTPIKRAQPSAHDIFVGNWKPTKNDTLSKRGPTFGSTMNVLYGEYTCGQG-SIDPMNNIISHYLY NYGQTGEALKVDLLSHPEYLENNATLAFQAAIWRWMTPPKKHLPSAHDVFVGKWKPTKNDTAAKRTPGFGATINVLYGDQICNSGFDNDEMNNIVSHYLY NYGKTGEALKTDLLNHPEYLENNATLAFQAAIWKWMTPEKKHLPSAHDVFVGKWKPTKNDTLAKRVPGFGTTMNVLYGDQVCGKG-DDESMNNIVSHYLY NYGAAGEALKEDLLSHPEYIEQNATLAFKAAMWRWYTPIKKSQPSAHEAFLGKWKPTKNDTLAKRVPGFGTTMNVLYGDQVCGQG-DIDAMNNFISHYLY NYGQLGQALKVDLLHHAEDLSQNATLAFQAAMWRWMNPIKVKQPSAHQVMVGKWVPTKNDTNSLRHPGFGMTINILKGEAECGAGSDDKQMNKRIAHYLY NYGQIGEALKLDLLNYPDLLSNNATIGFQTAIWRWMNPIKPKQPSAHDVLVGNWKPTKNDTASFRFPGFGMTINVLNGGLECGQG-DIEAMSNRVSHYLH  **N I  *Q *K  *N  310  Barley CHI26 AtCTL1 AtCTL2 PopCTL1 PopCTL2 PicglCTL1 PicglCTL2  140  ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| FVAAAAAFP--GFGTTGSADAQKREVAAFLAQTSHETTGG------WATAPDGAFAWGYCFKQERGASSDYCTPS--AQWPCAPGKRYYGRGPIQLSHNY FITAAALFEPLGFGTTGGKLMGQKEMAAFLGHVASKTSCG------YGVATGGPLAWGLCYNREMSPMQSYCDESWKFKYPCSPGAEYYGRGALPIYWNF FITAAAEYQPLGFGTAGEKLQGMKEVAAFLGHVGSKTSCG------YGVATGGPLAWGLCYNKEMSPDQLYCDDYYKLTYPCTPGVSYHGRGALPVYWNY FITAAAEYQPHGFGTSGGKLTGQKEVAAFLGHVGSKTSCG------YGVATGGPLAWGLCYNKEMSPSKTYCDDYYKYTYPCTPGVSYHGRGALPLYWNY FILASTSFQHLGFCTTGGKATQMKELAAFLAHVGCKTSCG------YGVATGGPLAWGLCYNKEMSPSQTYCDDFYKYTYPCTPGAEYYGRGAIPIFWNY FITAAAHFEPKGFGATGGDLVQKKELAAFFAHVATETSCESLMAQSSTATTDSPTKWGLCYKEELSPDSTYCESS--LVYPCAPGVSYHGRGALPVYWNY FINAAAQFEGIGFGTTGGQLMQQKELAAFFGHVAAETSCG------YSVAVGGPYAWGLCYKEEMCPDQLYCDQN--YLFPCSPGASYHGRGALPLYWNY  210  Barley CHI26 AtCTL1 AtCTL2 PopCTL1 PopCTL2 PicglCTL1 PicglCTL2  120  320  330  340  350  360  370  *R  380  390  400  ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| YCDILGVGYG-----NNLDCYSQRPFA------------------------------------------------------------------------FLDLMGIGREDAGPNDELSCAEQKPFNPSTVPSSSSS--------------------------------------------------------------YLDLIGVGREEAGPHEKLSCADQEPFSSSSSAPPSSGSSS-----------------------------------------------------------YLDLMGVGREEAGSHDVLSCAEQLPFNQASASASSS---------------------------------------------------------------YLDLLGLNREDAGPHEYLTCAEQVAFNPSTSSPSASS--------------------------------------------------------------FLDQLDVGRDNAG--DNLDCSDQKVLNPSSA--------------------------------------------------------------------FLDLMGVGRELAG--DHLDCGEQVALNPVSN---------------------------------------------------------------------  Figure 7: Primary structure alignment of Arabidopsis, poplar, and Picea glauca CTLs. Putative substrate binding residues (Bishop et al. 2000) are indicated with an asterix and the expected amino acid below it. The position of the two catalytic glutamic acids at residues 136 and 164 are indicated with arrows.  As previously noted (Passarinho and de Vries 2002; Zhang et al. 2004) substitution of a second conserved (acidic) catalytic glutamic acid residue at position 136 with a (basic) lysine residue likely abolishes chitinase activity. It is very interesting to note, that this substitution is seen in all Arabidopsis, poplar, and other angiosperm CTLs but not in Picea glauca, Pinus taeda, Ginko biloba, Cyclas rumphii or Physcomitrella patens CTLs. This suggests the latter species may retain CTLs with catalytic potential while angiosperm CTLs have lost that biochemical function. This idea is further supported by a similar pattern of amino acid residue retention in 38  predicted substrate binding sites shared between Picea glauca CTLs and the true chitinase CHI26 at positions 242, 276, and 294; positions with amino acid substitution in Arabidopsis and poplar CTLs. This finding suggests evolutionary relatedness of Physcomitrella CTLs and gymnosperm CTLs that that is not obvious in Figure 6. To gain further possible insights into functional similarity between duplicated CTL genes, the gene structures of Arabidopsis and poplar CTLs were compared (Figure 8). Both sets of Arabidopsis and poplar CTLs have similar intron-exon patterns, with introns of similar sizes and locations in AtCTL1 and AtCTL2, further suggesting evolution from a common ancestral gene. It was interesting to observe that AtCTL1 has an intron of about 250 bp in its 5’ UTR. Since none of the other genes shared this feature, this suggests recent acquisition of an intron in the Arabidopsis lineage.  Figure 8: Gene structure comparison of genomic DNA representing Arabidopsis and poplar CTL genes. Models are drawn to scale in terms of nucleic acid base pairs.  3.3 Discussion While chitinases were found in the single celled alga Chlamydomonas reinhardtii, and multicellular red alga, Porphyra yezoensis (data not shown) no homologous CTLs were observed. My analysis thus shows that CTL genes are likely to be a land plant specific family of genes. Furthermore, the presence of two CTL genes (three in cotton) appears to be a trait common to all land plant lineages examined. This suggests that two copies were found in early land plants that gave rise to  39  gymosperms and bryophytes. It further suggests that both modern angiosperm CTLs are derived from a single CTL after the divergence from gymnosperms. Evolution generally favors functional diversification of gene families following gene or genome duplication and this is supported in Arabidopsis by the observation that gene families tend to be about twice as big as in Physcomitrella patens (Rensing et al. 2002), and further by the observed retention of duplicated genes in poplar following a whole genome duplication event 60 million years ago (Tuskan et al. 2006). This trend is presumably more pronounced in plants like poplar which have undergone additional whole genome duplication events (Tuskan et al. 2006). However, the fully sequenced genome of poplar, like Arabidopsis and Physcomytrella, still only has only two CTLs.  The nearly universal trait of a two-member CTL gene family in land plants suggests that there emerged two CTL subfunctions as early as 400 million years ago, and that the loss of one copy has been generally selected against over time. Conversely, it shows that there has been no selection for gain of an extra gene product from a third or fourth copy of the gene. The only observed exception was the presence of two AtCTL2-like CTLs in cotton. Since this plant devotes a large amount of resources to secondary wall formation during cotton fiber differentiation over a short time, this could be an exceptional case in which there has been selection for a second AtCTL2-like gene.  The roles played by the duplicated CTL genes are hinted at by co-expression analysis done by the Douglas lab and two others independently (Brown 2005; Persson et al. 2005). This data strongly suggests that AtCTL1 is involved in primary cell wall development, and that AtCTL2 has a function in the formation of secondary cell walls. Expression patterns in developing secondary xylem in poplar seem to suggest the same division of primary and secondary cell wall development for PopCTL2 and PopCTL1 respectively (Figure 2). However, since PopCTL1 did not clearly group with Arabidopsis CTLs in the phylogenetic reconstruction (Figure 40  6), and there were no unique commonalities in amino acid alignment (Figure 7) or gene structure comparison (Figure 8) between these genes, it is not possible on the basis of this data to make additional inferences about orthology. It is also difficult to speculate on evolutionary or functional relationships between characterized Angiosperm CTLs and Physcomitrella CTLs, but it is interesting to ask what the functions of two CTLs may be in a species which does not develop a specialized vascular system requiring secondary cell walls (Schaefer and Zryd 2001). Physcomitrella does share commonalities in hormone signaling (Decker et al. 2006) and primary cell wall development (Schipper et al. 2002; Lee et al. 2005), but without more knowledge of CTL function or expression in Physcomitrella, it is impossible to speculate whether the two PhypaCTLs serve the same function(s) as those in higher plants.  CTL genes are observed to exist in gymnosperms as well, a lineage that is estimated to have diverged from angiosperms about 300 million years ago (Kenrick and Crane 1997). Not surprisingly, gymnosperm CTLs appear quite different in amino acid sequence to Angiosperm CTLs, with PicglCTL1 having 43% amino acid identity to AtCTL1, relative to 58% for PopCTL2 to AtCTL1. A significant part of this difference arises from stronger homology to true chitinases and specifically, what appears to be an intact chitinase catalytic site, and a number of conserved residues in the putative substrate binding sites. While this does not mean that the gymnosperm CTLs do indeed retain chitinase activity, it does suggest that mechanisms in cell wall formation have functionally diverged in angiosperms relative to gymnosperms, such that distinct CTL proteins, are used. Interpretation is further complicated by the observation that of a number of genes with similarity to chitinases exist in diverse plant and animal species that lack an intact chitinase catalytic site, but still have a developmental function. One example is the NOD factor upregulated chitinase homologue Srchi24. While this gene is involved in plant nodule formation in Sesbania rostrata, was shown to lack hydrolytic activity on the NOD factor isolated from the bacterial symbiont due to the change in a catalytic glutamate into a lysine (Goormachtig et al. 2001). In a contrasting 41  experiment, mutation of either catalytic glutamate in a tobacco class 1 chitinase was shown to transform it into a chitin binding lectin-like molecule completely devoid of chitinase activity (Iseli-Gamboni et al. 1998) which strongly suggests a CTL without both glutamates is catalytically inactive against chitin, as we suggest. It will be interesting to test whether gymnosperm and bryophyte CTLs have chitinase activity, and to test whether they play the same role in wall development that CTLs seem to play in Arabidopsis.  Chapter 4 Functional analysis of AtCTL1, AtCTL2, PopCTL1 and PopCTL2 4.1 Introduction As discussed in Chapters 1 and 3, Arabidopsis and poplar CTLs have similar developmental expression patterns, and thus potentially similar developmental functions. AtCTL2 and PopCTL1 have similar expression patterns which associate with secondary wall formation and are thus putatively orthologous. Amino acid sequence alignments and phylogenetic reconstructions, showing conservation of two CTL genes in diverse taxa are also consistent with the prediction of subspecialized CTL function. In this chapter, I present experiments to test these hypotheses and help establish CTL function in primary and secondary wall development. It has been previously suggested that mutants for AtCTL2 have no observable cell wall phenotype although there is evidenced for alterations in non-cellulosic components of the wall (Brown 2005; Persson et al. 2005). This might be explained if secondary cell wall development is only partially compromised in these mutants. Presumably, only severely weakened secondary cell walls, or cells under heightened stress need the additional structural support provided by a normally thickened secondary wall before collapsing. However, even normally thickened vascular cell secondary walls may collapse under heightened stress as evidenced 42  by a study of tracheid structure under drought stress in pine needles (Cochard et al. 2004). It is also possible that partial functional redundancy exists between AtCTL1 and AtCTL2, making an AtCTL2 mutant phenotype difficult to observe. A knockout phenotype for a poplar gene orthologous to AtCTL2 might show much more obvious phenotypes, since poplar makes a much larger commitment to secondary wall formation during secondary xylem formation. In this chapter, a poplar AtCTL2 orthologue (e.g., PopCTL1) was downregulated to test for novel phenotypes not present or obvious in Arabidopsis, which does not rely intensively on vascular cambium activity to complete its life cycle. In an effort to better characterize the AtCTL1 phenotype, microarray experiments performed in our lab were performed comparing global gene expression profiles in wild-type vs. the pom1 mutant in 4-day old etiolated seedlings. Genes differentially expressed (down regulated) in pom1 included a significant number of genes related to auxin responses (B. Hamberger and C. Douglas, unpublished). This finding led to the hypothesis that auxin homeostasis, synthesis, transport or early steps in the signal transduction cascade may be affected by the mutation. Since auxin has been shown to affect patterning in root and inflorescences in Arabidopsis (Hobbie et al. 2000; Hamann et al. 2002) changes in auxin transport or function in the pom1 mutant might help explain aspects of its phenotype. Auxin is also very important in secondary growth as a regulator of cambial activity and xylem formation (Uggla et al. 1996). As a way to further investigate this observation by indirectly assessing auxin distribution, a DR5 synthetic auxin-responsive promoter::GUS fusion was crossed into the pom1 background by B. Hamberger (unpublished), and in this chapter I analyzed GUS expression in these plants in parallel to DR5::GUS expression in wild-type plants. Although AtCTL1 and AtCTL2, as well as PopCTL1 and PopCTL2 have different expression patterns based on microarray expression profiling, little is known about the cell and tissue specific expression patterns of these genes. Also, since phylogenetic analysis did not generate a clear prediction of orthology between  43  CTLs in Arabidopsis and poplar, a comparison of these gene expression patterns in Arabidopsis using promoter::GUS fusions for promoters from AtCTL1, AtCTL2, PopCTL1, and PopCTL2 might help to support putative orthology (e.g. by revealing common expression patterns). Another approach to establishing whether proteins have common biochemical functions is phenotype complementation. To test conservation of CTL function, I transformed the pom1 mutant with Arabidopsis and poplar CTL transgenes and tested for phenotype rescue. Since all Arabidopsis and poplar CTLs are predicted to have extracellular secretion peptide signals they should all be secreted to the cell wall where they could potentially replace the AtCTL1 (POM1) function. This could be further extended to the two Picea glauca CTLs included in this study, however PicglCTL1 was not predicted by WOLF PSORT to have an extracellular targeting signal, while PicglCTL2 was (data not shown). In addition to mutant analysis to help determine gene function, overexpression can yield important clues about gene function. Overexpression of the Arabidopsis gene IFL1/AVB1 results in an incredible change in vascular bundle formation and distribution in floral stems (Zhong and Ye 2004), while its null mutant results in reduced auxin transport down the inflorescence and is accompanied by reduced xylem and interfascicular fiber formation (Zhong and Ye 2001). Because they may have distinct developmental functions, deregulation of AtCTL1 and AtCTL2 expression through CaMV 35S promoter overexpression could result in contrasting phenotypes, which are different from loss of function (mutant) phenotypes.. In order to test these predictions, I carried out a series of experiments on Arabidopsis and poplar CTL genes in both plants, in collaboration and in parallel with my colleague Dr. Bjoern Hamberger. Dr. Hamberger generated the following materials and data used in my experiments; ƒ  A homozygous AtCTL2 T-DNA insertion line, in which the location of the insertion was verified.  44  ƒ  Construction of 35S::AtCTL2 constructs and generation of transformants in the pom1 mutant background  ƒ  Construction of AtCTL2::GUS fusions and generation of transformants in wild-type, and initial GUS expression analysis  ƒ  DR5::GUS lines in the pom1 background and initial GUS expression analysis  ƒ  Generation of PopCTL2 RNAi plasmids of two different insert sizes  All other materials and experiments in this chapter were generated and carried out by me, and the results shown are from my own experiments, in some cases using materials generated by Dr. Hamberger (as above).  4.2 Results 4.2.1 A knockout phenotype for AtCTL2 mutant Arabidopsis Plants from a homozygous line with a T-DNA insertion in AtCTL2 (Atctl2-1) were grown on MS media in darkness and in light supplemented with 4.5% sucrose in an assay similar to that performed for the original assay used to identify the pom1 mutation. No difference was observed between Col-0 and Atctl2 root development on high sucrose or hypocotyls growth in the dark (data not shown). To confirm a null allele for AtCTL2 in this line, semiquantitative RT-PCR was performed on RNA from this line, as described in Table 2. This confirmed that AtCTL2 expression was completely eliminated from these plants (data not shown). Atctl2 plants that were grown on soil had no obvious phenotypic difference from Col-0, except for a possibly thinner stems (data not shown). However, sectioning of the bases of these inflorescence stems and staining with phloroglucinol indicate that there was a reduction in number of vascular bundles (Figure 9). The mean number of bundles in Atctl2 (n= 13) was 6.0 and mean stem diameter was 0.75 mm. Wild type plants (n= 14) had an average of 7.6 bundles and diameter of 1.01 mm. Statistical confidence was shown to be greater than 95% for both averages, using both a parametric (two tailed student t-test) and non-parametric Kriskal-Wallis tests (Figure 9). Three of the 13 plants displayed ectopic lignification in pith cells in 45  interfascicular regions, and increased formation of phloem fibers in a pattern similar to that seen in pom1, but there was no observed associated change in cell expansion. These cells displayed thickened secondary cells walls in cross section and appeared to occur in proximity to underdeveloped vascular bundles with interfascicular fiber development over the region between the protoxylem and the pith (Figure 10).  Figure 9: Sections of Col-0, Atctl2, and Atctl1 root/hypocotyls and inflorescences taken at 1 cm from rosette level from 1.5 month old inflorescences. Sections were stained with phloroglucinol. Ph, phloem; X, xylem, P, pith; IF, interfascicular fibers, PX, primary xylem; SX, secondary xylem; C, cambium; Co, cortex. Scale bars: 100 um.  46  Figure 10: Handcut sections of Wt (A, B) and Atclt2 (C, D) inflorescences. Sections were taken at 1 cm above rosette level from 1.5 month old plants. A and B are Col-0, C and D are Atctl2 sections from different individuals. Sections were stained with phloroglucinol or toluidine blue. Ph, phloem; X, xylem, P, pith; IF, interfascicular fibers, Ph F, phloem fiber; VB, vascular bundle; Scale bars: 100 um.  4.2.2 Auxin involvement in the pom1 phenotype as studied using the DR5::GUS reporter construct in Col-0 and pom1 In order to test the auxin responsiveness of DR5::GUS in the wild-type background, a proof of concept experiment was performed on 15 cm tall inflorescences by cutting halfway through the stem 1 cm above the level of rosette, followed by staining for GUS activity one week later as described in 2.7. Having severed four or more vascular bundles, GUS staining was observed above the wound site, but not below, confirming that auxin flow had been altered by the cut and that the transgene was working well (Figure 11). As a side note, it was interesting to observe that auxin accumulation correlated with ectopic deposition of lignin in the pith, cell expansion and division in the cortex, and what appeared to be greatly increased formation of xylem around the outside of the normal stele.  47  Figure 11: GUS assay testing activity of the DR5::GUS construct in Col-0. 25cm tall floral stems were cut with a razor blade, and sectioned above and below the site of wounding one week later. Photos were taken using both dark field and UV autofluoresence. Scale bars: 100 µm.  A seedling based assay was then carried out using the DR5::GUS reporter gene in both Col-0 and pom1 backgrounds. Plants were grown on plain MS media, MS media supplemented with 4.5% sucrose, and in complete darkness for 4 days before staining for GUS activity. Results are shown in Figure 12. DR5::GUS expression in Col-0 was observed throughout the cotyledons and roots, with local maxima at the root tip, SAM, and root/hypocotyl transition zone. pom1 seedlings had a dramatically lowered DR5::GUS expression in roots, where only the above maxima and patchy GUS expression in cotyledons were visible. Under normal conditions, pom1 plants appear to be reduced in either auxin production, translocation, or sensing since DR5 is a synthetic promoter element sensitive to changes in all three (Ulmasov et al. 1997). High sucrose was observed to reduce GUS expression in cotyledons of both Col-0 and pom1. Sucrose also altered root 48  DR5::GUS expression in pom1, but not in Col-0. In sucrose-treated pom1 roots, patchy DR5::GUS activity observed in small local maxima within the swollen roots, which suggests a defect in auxin distribution. The pom1 etiolated hypocotyls were observed to be characteristically short as expected, and, in contrast to Col-0, DR5::GUS expression was observed thoughout the hypocotyls. However, this since the pom1 hypocotyl is much shorter, this could be interpreted as compression of the normal developmental gradient observed in Col-0. To assess whether disruption of auxin distribution might be responsible for the pom1 phenotypes later in development, I sectioned one month old plants at the base of the inflorescences and at the root/hypocotyl junction. Results are shown in Figure 13.  Figure 12: Comparison of DR5 driven GUS expression in 6 day old Col-0 or pom1 seedlings grown in light, in darkness, or in light with 4.5% sucrose. All seedlings were stained for 14 hours at 37°C. Scale bars: 1 mm.  49  As predicted, GUS staining was observed in vascular bundles of Col-0, but was localized to the primary xylem and phloem at the base of the inflorescence and at the root/hypocotyls junction. In the pom1 background, no GUS expression was detected in inflorescences or at the root/hypocotyl junction. This correlates with the reduced secondary xylem production seen at the root/hypocotyls junction and might partly explain the phenotype.  Figure 13: Comparison of handcut sections of floral stem and root/hypocotyl from DR5::GUS fusions in Col-0 or pom1 backgrounds. One month old floral stems were sectioned at 1 cm from the base. Hypocotyls were sectioned from the region above the soil and below the rosette. Scale bars: 100 µm.  4.2.3 Arabidopsis and poplar CTL promoter ::GUS reporter constructs in Col-0 To compare CTL gene expression using an in vivo system, promoter::GUS constructs were prepared for AtCTL1 and PopCTL1 promoter regions as described in 2.3.7 and transformed into Col-0 Arabidopsis plants via floral dip. Selection and screening produced two lines for AtCTL1::GUS which displayed strong GUS expression, while multiple PopCTL1::GUS lines did not show any significant developmental expression in Arabidopsis. AtCTL2 and PopCTL2, promoter::GUS lines were produced by Bjoern Hamberger, and two strongly expressing lines for each construct were included in this study. 50  Figure 14 shows the results of an initial assay for GUS expression using all four constructs. Grown for four days in the dark or in the light, AtCTL1::GUS plants displayed diffuse dark blue staining at the growing ends of the seedling, namely cotyledons and top and bottom of the hypocotyl and the entire root. Growth of these plants on a high sucrose media resulted in a reduction of cotyledon expression and did not affect root expression. Leaves showed a wound inducible response as shown in Figure 15. This pattern is strikingly similar to that of that directed by the PopCTL2::GUS construct, which showed much stronger expression and a slightly greater range of expression levels. A majority of PopCTL2::GUS seedlings accumulated a strong blue colour at local maxima part way up along the hypocotyl and this did not change with the addition of sucrose to the media.  Figure 14: Comparison of AtCTL1, AtCTL2, PopCTL1, PopCTL2 promoter::GUS fusions in Col-0 at the 4 day old seedling stage grown in darkness, light or in light with 4.5% w/v sucrose. Scale bars: 1 mm.  51  In contrast to AtCTL1::GUS and PopCTL2::GUS, AtCTL2::GUS lines showed expression that co-localized with plant vasculature, including veins in the cotyledons, hypocotyls and root systems (Figure 14, 15). An interesting pattern observed in these plants was the discontinuous expression of GUS reporter along hypocotyl vasculature of etiolated seedlings, and similar expression along root vasculature of light grown seedlings. Little or no PopCTL1::GUS expression was observed in multiple lines, although PCR based genotyping confirmed 6 independent lines assayed did indeed contain the construct (data not shown), and occasional GUS expression in trichomes and epidermal cells was observed in mature stems (see below). To assay CTL::GUS expression patterns at the cellular level in mature plant tissues and in response to wounding, CTL::GUS plants were grown in soil and 15-cm tall inflorescences were harvested, sectioned and stained for GUS activity. Results are shown in Figure 16. Again, AtCTL1 seems to have a similar pattern of expression to that of PopCTL2, with GUS expression in cortex, phloem, vascular bundle cambium, xylem and some pith cells near to vascular bundles. At the root/hypocotyls junction, both GUS constructs showed expression in the region  Figure 15: DR5::GUS expression in cotyledons and wounded leaves. Cotyledons from 6 day old CTL::GUS plants and leaves from 3 week old plants, one hour after wounding with forceps were stained with X-GLUC for 14 hours at 37°C. Scale bars: 1 mm.  52  corresponding to the vascular cambium, cortex, and some expression in xylem tissues, presumably xylem parenchyma cells. In contrast, AtCTL2::GUS expression was confined to cells in which secondary cell walls develop. Inflorescences were observed to accumulate staining in xylem side of vascular bundles and in developing interfascicular fibers. Figure 16 shows that AtCTL1 and AtCTL2 seem to have overlapping expression only in the regions of vascular bundle cambium and developing xylem in inflorescences.  In  root/hypocotyls, the pattern of AtCTL2 expression was very much more clearly localized to the secondary xylem at the root/hypocotyls junction, which may be another region of overlapping expression between AtCTL1 and AtCTL2. No reproducible PopCTL1::GUS expression was observed and occasional expression was only seen sometimes in trichomes and epidermal cells.  Figure 16: Comparison of root/hypocotyl and basal inflorescence sections from AtCTL1, AtCTL2, PopCTL1, PopCTL2 promoter::GUS fusions in Col-0 15 cm tall floral stems were sectioned at 1 cm from the bottom and both 1 5 cm from the shoot apical meristem. Hypocotyls were sectioned from the region above the soil and below the rosette. Scale bars: 100 µm. .  53  4.2.4 pom1 phenotype rescue using AtCTL1, AtCTL2, PopCTL1, and PopCTL2 transgenes Phenotype rescue assays were performed to test the ability of different CTLs under the control of different promoters to rescue the pom1 phenotype when grown in darkness or on sucrose. Constructs were made as described in section 2.3.2 to 2.3.6 and transformed into the pom1 mutant by floral dip. Two lines each of 35S::AtCTL2 and PopCTL2::PopCTL2 plants were obtained from Bjoern Hamberger and included in this study. Seed selection on either kanamycin or hygromycin plates produced 14 putative lines for AtCTL1::AtCTL1, of which genotyping showed 2 to be false positives (data  Figure 17: Phenotype rescue assays using 4.5% sucrose or dark on transgenic pom1 plants for four days. Promoter::coding region fusions tested were from left to right, AtCTL1 promoter::AtCTL1, CaMV35S::AtCTL2, CaMV35S::PopCTL1, PopCTL2::PopCTL2. Statistics were done on 10 seedlings per line. For segregating populations in transgenic lines, only long rescued roots or hypocotyls were included in this analysis. Scale bars: 1 mm.  54  not shown). All four AtCTL1::PopCTL1 lines were genotyped by PCR as containing the rescue construct, and 9 of 10 lines proved to contain the 35S::PopCTL1 construct. Seeds from two lines per construct were sterilized and plated on MS media, stratified at 4 degrees, and then allowed to grow vertically in a phytochamber for a period of four days. Plates were scanned on a bench-top scanner and measurements done in Adobe Photoshop Version 7.0. The results are displayed qualitatively in Figure 17 and quantitatively in Figure 18. After four days of growth in the light on sucrose media, 30 Col-0 plants were found to have a mean root length of 0.77 cm. This was greater than the mean root length on sucrose for pom1 (0.26 mm; n=29). pom1 plants which contained the AtCTL1::AtCTL1 construct were found to very closely match the mean root lengths of Col-0, which confirmed that this method of transgenic phenotype rescue works (Figures 17 and 18). Measurement of root lengths of pom1 plants containing constructs,  35S::PopCTL1  35S::AtCTL2  constructs,  PopCTL2::PopCTL2  constructs, likewise showed mean root lengths that were all well with one standard deviation of wild-type and at least 0.5 cm longer than pom1 roots, showing that they are sufficient to substitute for the function of AtCTL1. I subsequently generated  a  spruce  CTL  complementation  construct  (35S::PicglCTL1).  Interestingly, when introduced in to the pom1 mutant background, this spruce CTL also was able to efficiently rescue the pom1 phenotype (Figures 17 and 18). All lines were also grown in darkness, and the phenotypes of etiolated seedlings observed (Figures 17 and 18). Col-0 plants had a mean hypocotyl length of 0.89 cm compared to 0.30 cm for pom1 seedlings. Again, pom1 plants which contained the AtCTL1::AtCTL1 construct were found to very closely match the mean hypocotyl lengths of Col-0 with mean values of 0.88 cm for two independent lines. Measurement  of  etiolated  hypocotyl  lengths  of  pom1  plants  containing  35S::PopCTL1 constructs, 35S::AtCTL2 constructs, and PopCTL2::PopCTL2  55  constructs likewise showed means that were within 0.1 cm of Col-0 hypocotyl lengths, and at least 0.6 cm taller than pom1 seedlings. Both tissue specific (AtCTL1 and PopCTL2 promoters) and constitutive, (CaMV 35S) regulatory regions were used in rescue the pom1 mutation, and no obvious difference in the effectiveness of one or the other was observed.  Figure 18: Measurements of transgenic pom1 phenotype rescue. Plants were measured under conditions of 4.5% sucrose or darkness. Two lines with 10 plants each were measured; plants are labeled by their genotype or transgene in the pom1 background.  4.2.5 CTL overexpression in Arabidopsis In section 4.2.1 it was shown that elimination of AtCTL2 function reduces the plant’s ability to form vascular bundles and can create an ectopic lignification phenotype somewhat similar to that observed in pom1. An ideal way to confirm this phenomenon would be to test for increased numbers of vascular bundles by overexpression of the same gene. Such plants have been produced but not yet analyzed. Instead, overexpression and phenotype analysis of the putatively orthologous gene PopCTL1 was accomplished by placing PopCTL1 under control of a 35S promoter region in Col-0 plants. Selection on kanamycin produced 10 lines which were screened by PCR for the presence of the transgene, and 8 of which were (Figure 19A). To check three of the more promising lines for transgene expression, semiquantitative RT-PCR was performed first for the Actin2 gene as a loading control, and then for the PopCTL1 gene using specifications in Table 2.  56  Col-0, pom1, and all three transgenic lines showed similar levels of Actin2 expression, but only lines 5 and 14 showed detectable levels of PopCTL1 expression. These two lines were then grown alongside Col-0 and pom1 on MS plates in the light, with added sucrose, or in the dark, but there was no discernable difference between these PopCTL1 expressing lines and Col-0 (data not shown). When grown on soil to maturity, these plants again showed no visible difference to Col-0, but sectioning of the floral stem when inflorescences were 25 cm tall and 1.5 months old showed several plants with an increased number of vascular bundles, from normal 8 in the wild type to as many as thirteen in overexpressing plants (Figure 19 and 20). The mean number of bundles in the 14 plants analyzed was 9.7, and it was found to be statistically significant by both parametric and nonparametric (Kruskal-Wallis) from the mean number of 7.6 in wild-type, with a p value of 0.01. Bundles appeared varied in sizes and shapes, and seemed to be in various stages of development in the pith. Closer inspection showed that some large bundles were bifurcated into sub-bundles (Figure 19), a process rarely observed in wild-type plants of this age. This process might be analogous to the bundle bifurcation observed in transition from root stele to eustele in Arabidopsis seedlings (Busse and Evert 1999). Impaired bundle development in PopCTL1 overexpressing plants seems to correlate with the bulbous, non-circular shape of the stems in cross section, suggesting deregulation of vascular bundle pattern can disrupt the normal shape of the inflorescence stem as it accommodates the extra tissues. This seems to be the case in clavata mutants, which have an enlarged apical meristem, produce many more vascular bundles than normal, and have flattened, axially distorted stems (Turner and Sieburth 2002). It is also possible that this distortion of stem shape is related to the unusual, parenchyma filled spaces observed between interfascicular fibers and cortex cells (Figure 19, 20). Root development was not extensively analyzed in these plants.  57  Figure 19: Expression of 35S::PopCTL1 in Col-0. A) PCR based genotyping of transgenic lines B) Semi-quantitative RT-PCR based expression profiling of selected lines C) Phenotype of a representative PopCTL1 expressing line under UV autofluoresence. Scale bars: 100 µm.  Figure 20: Hand sections of Col-0 and 35S::PopCTL1 plants. Sections were cut at 1 cm from soil and stained with either phloroglucinol or toluidine blue. Plants were 1.5 months old at the time of sectioning, but Lines 1 and 14 were sectioned earlier at three weeks of age. The hole in the pith of the 35S::PopCTL1 section is an artifact from sample preparation. Scale bars: 100 µm.  58  4.2.6 RNAi of PopCTL1 and PopCTL2 in transgenic poplar A very important aspect of this thesis was to analyze CTL function in a system that undergoes dramatic secondary growth and might have forms of cell expansion and cell wall development that Arabidopsis does not normally have. To knock down CTL expression in Populus alba X P. tremula clone 717, I made RNAi constructs of two different sizes, homologous to the first exon in the coding regions of PopCTL1 (see 2.3.8). A similar procedure was followed by Dr. Bjoern Hamberger for RNAi targeting the first exon of PopCTL2. For transformation of these constructs into poplar, leaf discs were cut from 6 week old leaves and the protocol listed in 2.5.1 was followed. Over the course of one year in the tissue culture facility in Dr. Shawn Mansfield’s lab at UBC, a large number of independent transformation events on separate discs were observed and cloned by tissue culture, of which 7 PopCTL2 RNAi showed an enhanced organ regeneration phenotype (shown in Figure 23B) and similar to that reported in (Cary et al. 2001). Leaf discs giving this phenotype gave rise to 400 bp PopCTL2 RNAi lines #A, E, F, I, J, and P as well as 150 bp RNAi line #H. No phenotypic differences from Wt were evident for PopCTL1 RNAi shoots or roots in tissue culture. When most lines had three individual plants which rooted successfully under kanamycin selection, these were transferred to soil in 1 liter pots, covered with plastic domes, and misted daily to reduce water stress. In total, five wild type survived soil transfer, in addition to plants representing 11 independent lines of PopCTL1 400 bp RNAi, 3 independent lines of PopCTL1 150 bp RNAi, 10 independent lines of PopCTL2 400 bp RNAi, and 3 independent lines of PopCTL2 150 bp RNAi.  59  Figure 21: CTL expression in wild type and RNAi lines. A) Wild-type PopCTL1 and PopCTL2 RNAi 5 month old, greenhouse grown poplar trees. RNA was extracted from the 6th leaf from the first 0.5 cm long internode. A genomic DNA control was included to show primer specificity for cDNA. RNA from the housekeeping TIF5A gene was used as a control for normalizing sample amounts. PopCTL1 RNAi 1A1, 1B1, and 1D4 contain 400 bp constructs, while 2G and 2M contain 150 bp constructs. PopCTL2 RNAi 3A2, 3F2, 3J1, and 3P1 contain 400 bp constructs, while 4C3,4H3, 4L1 contain 150 bp constructs. B) Results of additional screening for PopCTL1 RNAi knockdowns, from a second set of rooted transgenics. Smaller sized bands in PopCTL1 reactions are primer dimers. Conditions and primers used are described in table 2.  After 5 months of growth in a greenhouse, RNAi mediated downregulation of CTL expression was monitored in individual trees by sampling the 6th leaf below the first 0.5 cm long internode, and isolating RNA for semi-quantitative RT-PCR analysis using the program in Table 2. Results are shown in Figure 21. Of the lines tested and shown in Figure 21A, 1B1 had slight knockdown of PopCTL1, while 1D4, 2G, and 2M had much more dramatic and specific reduction in PopCTL1 gene expression. PopCTL2 appeared effectively downregulated in 3A2, 3J1, 3P1, 4H3, and 4L1, and the downregulation was specific to PopCTL2. To find additional lines with PopCTL1 downregulation, RNA extraction was performed again on additional plants with results shown in Figure 21B. Lines 1H1, 1H3, 1X, and 2O were also significantly and specifically downregulated in PopCTL1 expression. Wt plants did not show any appreciable fluctuation in CTL expression based on this semi-quantitative RT-PCR assay. These CTL downregulated trees were measured for height and number of branches as shown in Figure 22. No clear trend was evident among trees based on height, but it is striking that about half of the RNAi lines with reduced CTL expression showed reduced apical dominance as evidenced by early branching 60  phenotypes. A number of PopCTL1 RNAi trees broke at the base of the stem early in development and had to be staked for further upright growth, but this phenotype was not further investigated.  To assay possible anatomical phenotypes, 2 Wt plant and 4 PopCTL1 downregulated RNAi lines were sectioned. In parallel, 2 Wt trees and 4 PopCTL2 downregulated plants were cut to the base, transferred to a phytochamber and allowed to regrow one main shoot. The trees were hand sectioned in the middle of each internode, from internodes 1 through 25, whereas after internode 25 stems became very thick and section quality dropped off sharply. Hand sections were stained with toluidine blue (TEB) or phloroglucinol and viewed immediately in a dissecting microscope in the UBC Bioimaging facility or in the laboratory of Lacey Samuels. Figure 23 shows the results of this analysis. Both TEB and phloroglucinol stains showed xylem, phloem fibers, and lignified pith cells very clearly as blue or red colours respectively, but TEB was also informative for its ability to stain pectin purple, while phloroglucinol was desirable for its ability to effectively diffuse through the thick woody sections. At very early stages of development (internode 2), it was possible to see major vascular bundles of protoxylem arranged in the vertices of a five point star eustele (Figure 23A). There was some variation observed in this pattern in PopCTL1 downregulated line 1H1 and 2O, which seemed to have 6 and 4 vertices at internode two. Looking at Wt, it is possible to see there was some variation in rates of xylem development in Wt as well. Despite this, PopCTL1 lines did all share what appears to be a reduction in red phloroglucinol staining of the xylem, evidenced especially well in internode 25 sections of 1D4 and 2O relative to Wt. This reduction in staining suggests a reduction in lignin content within these walls, and may be the cause for observed increased in breaking of PopCTL1 downregulated xylem during sectioning.  61  Figure 22: Greenhouse grown poplar tree heights and branch number. Trees were all grown for 5 months in soil within a greenhouse at the time of measurement. 2M died shortly after this data was recorded.  62  Figure 23: Handcut sections of wild type, A) PopCTL1 RNAi and B) PopCTL2 RNAi poplar. PopCTL1 RNAi trees were grown in greenhouse and were 5 (Wt2,1D4) or 8 (Wt3,1H1,2O,1x) months old, while PopCTL2 RNAi sections were derived from trees that were pruned at five months and regrown for two months in a phytochamber. SIM, shoot induction medium. Scale bars: 500 µm.  63  Figure 24: Longitudinal sections of Wt poplar and PopCTL2 RNAi plant 3J1. Sections were stained with phloroglucinol The IN6 section from 3J1 was cut at a thickness of 40 micrometers using a vibratome. The ectopic pith lignification phenotype of pom1 in an Arabidopsis inflorescence stem is shown for comparison. IN, internode. Scale bars: 1 mm.  Downregulation of PopCTL2 seemed to be correlated with an accelerated program of secondary xylem formation, and staining for lignin in the pith, progressing to complete staining as seen in the extreme example at internode 10 of plant 3J1 (Figure 23B). Visible secondary growth began much earlier in this individual (as early as internode 2) than in wild type, beginning as a complete blue staining vascular cylinder in internode 2, followed by waves of lignification emanating from the secondary xylem and progressing to complete staining of the pith (shown in longitudinal section in Figure 24) which might be a poplar variation on ectopic deposition of lignin in the pith seen in pom1 mutants. Other PopCTL2 RNAi plants 3P1, 3A2, and 4L1 were less dramatically affected by the downregulation but did show this trend, suggesting a larger analysis with more individuals be done to increase our confidence in correlating phenotype to genotype.  64  4.2.7 Pith lignification in wild-type poplar  Figure 25: Sections of poplar Wt magnified 50 times showing pith details from different stages of growth. Sections were hand cut and stained with toluidine blue. PF, phloem fibers; C, cambium; X, xylem; P, pith. Scale bars: 500 µm.  While examining sections from multiple wild-type plants, a very exciting and unexpected discovery was the observation of an island of lignified pith cells in the center of the poplar stems. These cells also appeared to contain a significant amount of air after internode 20, as evidenced by a large number of bubbles after both types of stain and in this tissue only. Lignification, starch accumulation, cell death, and accumulation of air in pith cells have all been previously observed in different species of tree (Metcalfe 1979). In Wt2 (Figure 25) this was observed to begin as a few slightly expanded pith cells staining either blue in TEB or red in phloroglucinol at internode 17 and by internode 25 it was a strongly blue staining tissue containing highly reflective cell walls. Viewing these internodes in transverse sections with phloroglucinol stain (Figure 24), it is possible to see that this lignified pith island is a continuous tissue distributed vertically throughout the pith. Whether the cell wall of these cells are actually lignified and what the function of this phenomena may be, remains to be determined. In some individuals, this “lignification” of the pith was observed to accompany much greater changes in shape of pith cells, suggesting cell expansion and lignification both play a role in normal pith development of this tree species.  65  4.3 Discussion A powerful test for characterizing the function of genes discovered by in silico and expression profiling approaches in poplar and other plants is complementation of mutations in potentially orthologous Arabidopsis genes. Rather than help determine functional orthology between poplar and Arabidopsis CTL genes however, the phenotype rescue experiments I performed showed that duplicated CTL genes in each species have apparently conserved biochemical functions despite significant differences in expression patterns, since all were able to complement the pom1 (Atctl1) mutant phenotype. Conversely, assay of tissue specific expression patterns specified by CTL promoter::GUS fusions did reveal strikingly similar patterns of AtCTL1 and PopCTL2 promoter driven expression in tissues developing primary walls, as predicted from expression profiling experiments (Hertzberg et al. 2001; Ehlting et al. 2005), suggesting possible similar roles in cell wall formation. It was not possible to draw the parallel conclusions with respect to AtCTL2 and PopCTL1, since for unknown reasons the PopCTL1 promoter region did not specify detectable developmental expression in Arabidopsis.  However,  the  AtCTL1  promoter::GUS  results  provided  an  independent confirmation that AtCTL1 is expressed in a pattern consistent with primary wall formation or modification, while AtCTL2 is expressed in cells undergoing secondary cell wall development, consistent with co-expression analyses carried out by Dr. Hamberger in the Douglas lab and others (Brown et al., 2005; Persson et al., 2005). One prediction based on expression patterns was that Arabidopsis Atctl2 mutant plants would show alterations in secondary wall formation. Instead, I observed a reduction in vascular bundle number in floral stems, and occasionally, ectopic lignification of the pith and enhanced phloem fiber formation, but no apparent defect in secondary wall formation, as observed by others (Persson et al. 2005). This might be interpreted as evidence that AtCTL2 activity is required for provascular cell type differentiation rather than directly in secondary wall formation. However, an alternative explanation for the Atctl2 phenotype, in which no apparent 66  secondary cell wall defect was observed, could be partially overlapping expression between AtCTL1 and AtCTL2, with residual AtCTL1 function in cells ungoing secondary wall development. Because the mode of action of AtCTL1 that underlies the Atctl1/pom1 loss of function phenotype is still not understood, the Douglas lab used global gene expression profiling to compare gene expression in pom1 mutant relative to wild type etiolated seedlings. This experiment showed that several auxin inducible genes are downregulated in pom1 (B. Hambereger, unpublished), suggesting a change in auxin levels, distribution, or response. The DR5::GUS reporter gene construct provides a powerful way to indirectly assay IAA levels in plants, since the DR5 promoter is highly responsive to auxin concentration (Ulmasov et al. 1997). Analysis of DR5::GUS expression patterns in wild-type and pom1 seedlings showed that levels of auxin may be reduced in pom1 mutants, and that the sucrose induced pom1 phenotype appears to disrupt auxin flow in the root (Figure 11). Sectioning of roots at the hypocotyl junction in fully grown pom1 plants revealed that DR5::GUS expression was low or absent in roots at later stages of plant development at the root-hypocotyl junction, correlating strongly with reduced secondary xylem formation at this location. These results are consistent with the microarray data showing alterations in auxin regulated gene expression in pom1, and suggest that some ctl mutant phenotypes could be due to alterations in auxin homeostasis or transport. Auxin has been shown to affect vascular patterning in root and inflorescence stems in Arabidopsis (Hobbie et al. 2000; Hamann et al. 2002) and to stimulate the formation of xylem fibers and vessels in both Arabidopsis and poplar. Auxin is also a well known stimulant of cell elongation in stems, while it inhibits cell elongation in roots (Taiz and Zeiger 1998). Its presence in a tissue has been shown to antagonize endogenous cytokinin induced cell division, and thus promote apical dominance in plants. One major way that auxin is believed to regulate cell development is through the regulation of auxin inducible genes, achieved by  67  stimulation of ubiquitination and degradation of negative regulators of Auxin Response Factor (ARF) transcription factors (Leyser 2001).The apparent changes in auxin levels and distribution based on DR5::GUS expression in pom1 may help explain the reduced number of vascular bundles formed in Atctl2 plants, and might be explained if the CTL gene product is normally involved in binding or modifying a chitin-like molecule involved directly or indirectly in regulating auxin response or distribution. Studies done in a symbiotic root nodule producing species of clover show that the application of symbiotic Rhizobium leguminosarum, lipo-chitin oligosaccharides (NOD factors), O acetylated chitin fragments, and flavonoid aglycones all produce a transient decrease in root auxin transport (Mathesius et al. 1998). If CTLs are involved in mediating a chemically similar signal molecule, absence of CTL activity may allow signal over accumulation and a resulting constitutive reduction in auxin transport. Why nodule formation would require reduced auxin transport is not clear, but it was shown that high levels are also needed for induction of cell division at the site of nodulation. Whether auxin production or distribution is altered in Atctl2 or in PopCTL1 overexpressing plants, or whether it directly plays a part in the phenotypes seen remains to be shown by DR5:GUS activity or direct assays on auxin levels in the mutant. Poplar plants with down regulated CTL expression were generated in order to test CTL function during primary and secondary growth in poplar, but the results remained somewhat inconclusive about this point. Down regulation of PopCTL2 seemed to phenocopy the ectopic deposition of lignin in the pith seen in pom1, and abnormal cell expansion may have been observed in some of these same cells, suggesting a link to primary cell wall development in poplar. Likewise what might be perturbation of secondary wall formation observed as putatively reduced lignification in secondary xylem of PopCTL1 down regulated trees, may be evidence of a function in secondary wall formation, as predicted by the PopCTL1 expression pattern which is associated with secondary wall formation during xylem development (Hertzberg et al. 2001). More detailed biochemical analysis of lignin and cellulose content and structure in the secondary cell walls in PopCTL1 down  68  regulated lines is necessary to further characterize this potential phenotype. The increased branching in both PopCTL1 and PopCTL2 downregulated poplar lines is consistent with the effect of the pom1 mutation on auxin levels or distribution. This phenotype suggests reduced apical dominance, which has long been understood as a characteristic effect of reduced basipetal auxin flow (Taiz and Zeiger 1998), consistent with the apparent affect of the pom1 mutation on auxin distribution. In trees, auxin is believed to be transported mostly through the vascular cambium where it promotes maintenance of cambial initials, radial expansion of cambial cells (in contrast to its effect in root), and reaction wood responses (Little and Savidge 1987). PopCTL2 downregulation appeared to accelerate secondary xylem formation and lignification or phenolic compound accumulation in the pith, which could be the result of a build up of auxin due to reduced translocation similar to that seen in auxin transport inhibitor treated poplar (Junghans et al. 2004) and Arabidopsis (Mattsson et al. 1999). As pom1 plants were reported to overproduce ethylene and be hyper-sensitive to cytokinins (Cary et al. 2001; Zhong et al. 2002), it is possible that the observed xylem phenotype in PopCTL2 RNAi poplars is connected to similar changes in hormone response in these plants. Reduced shoot auxin production in a tissue culture environment could account for the increased organ regeneration observed in PopCTL2 RNAi tissue culture, and that reported for Arabidopsis (Cary et al. 2001). Sucrose could play a role in the enhanced xylem formation observed in PopCTL2 downregulated poplars. The pom1 phenotype was originally described on the basis of a conditional root expansion phenotype when plants were grown  on high  sucrose (Benfey et al. 1993; Hauser et al. 1995). Cells in Arabidopsis roots and the base of the inflorescence are normally exposed to very low levels of glucose and sucrose (Deuschle et al. 2006). This is interesting to consider when growing a plant on high sucrose since it is known that plants use sugars as carbon sources in metabolism, and are very sensitive to their presence, with evidence suggesting roles as developmental signaling molecules (Moore et al. 2003; Yang et al. 2004;  69  Avonce et al. 2005; Rolland et al. 2006). Glucose for example has crosstalk with auxin, cytokinin, ethylene, and ABA hormone signaling pathways through the activity of the glucose hexokinase receptor, HXK1 (Xiao et al. 2000; Moore et al. 2003; Rolland et al. 2006). Pectins are also known to be important in developmental regulation (Ridley et al. 2001) and their production might be altered along with the apparent reduction in cellulose content of pom1 cell walls (Mouille et al. 2003). Sucrose is the major transport form of carbon in plants which flows through the phloem from sources of production to sinks such as root tissues where it is hydrolyzed into glucose and fructose (Taiz, 1998). Zebra-like (Malcolm Campbell, unpublished results) stripped patterns of cell expansion and lignification of the pith in pom1 inflorescences as seen in Fig 24 might be linked to altered source-sink relationships or changes in sensitivity/response to sugars whose production and transport are regulated by light and the circadian clock (Rogers et al. 2005a). pom1 mutants were shown to contain lower levels of cellulose in their cell walls, which would also suggest the possibility of channeling of excess carbon into other metabolites such as pectins. Whether auxin levels or transport are directly or indirectly reduced by the pom1 mutation, auxin plays a role in specifying source-sink relationships, helping to prevent sucrose efflux causing cell specific buildup in target cells (Maitra and Sen 1987). If auxin transport is compromised in PopCTL2 downregulated poplars , it is possible that in the primary cambial region below the sugar producing shoot apical meristem (SAM) in poplar, altered levels of auxin either directly, or through altered sensitivity to sucrose, lead to an accelerated growth of vascular tissue as seen in experiments with chemical transport inhibitors (Mattsson et al. 1999), and in auxin transport mutants such as pin1 and acl5 (Clay and Nelson 2005). Our GUS experiments showed expression of AtCTL1 preferentially in parenchyma cells of the stem, suggesting even at advanced stages of development, primary cell wall development or modification might be taking place within the pith and cortex of the plant. Exactly why these cells specifically loose control over primary cell wall growth remains to be shown, but ectopic lignification might be explained as a  70  response triggered by reduced cellulose in the primary cell wall of these abnormally expanded pith cells, similar to that observed in response to wounding and locally increased levels of auxin as shown in Figure 11 and reported elsewhere (Cano-Delgado et al. 2003). pom1 mutants have been previously shown not to be significantly altered in transcription factors regulating lignin metabolism (Rogers et al. 2005b) but rather are known to have lower levels of cellulose in their cell walls (Mouille et al. 2003), are upregulated in genes responsive to oxidative stress, and are downregulated in a number of primary cell wall and auxin inducible genes (Rogers et al. 2005b). Auxin is produced in young tissues such as the SAM and then transported basipetaly mostly through phloem and cambial parenchyma (Berleth et al. 2000). Based on our AtCTL1::GUS and DR5::GUS experiments, AtCTL1 and DR5 activity are similar, with GUS expression for both reporter constructs increasing near the SAM and the RAM. If AtCTL1 plays an important role in regulating primary wall development, and thus controlling cell expansion, improper expansion of pom1 mutant cells near the SAM or the RAM where auxin levels appear highest, may lead to altered auxin transport in these tissues. Improperly formed cells might mislocalize the auxin efflux transporter, PIN1 for example (Galweiler et al. 1998) resulting in non-polar, blocked flow of auxin. If these cells accumulate higher than normal levels of auxin, this may too feed into expansion phenotypes, as increased levels of auxin or heightened auxin response tends to increase cell size (Jones et al. 1998). Although nothing is known of auxin relations in Atctl2 mutants, AtCTL2::GUS expression shows a pattern consistent with a role in secondary wall formation in primary and secondary xylem formation and interfascicular fiber formation. Phenotype rescue does however suggest that the AtCTL2 protein is functionally equivalent to AtCTL1 (i.e., 35S::AtCTL2 expression can rescue the pom1 phenotype, Figure 17 and 18), suggesting that the functional diversification of CTLs in Arabidopsis is based only on differential regulation of the genes. This  71  suggests that, AtCTL2 is mediating the same process as AtCTL1, but doing so in cells undergoing secondary wall development. Auxin is very important in this process (Uggla et al. 1996), and is involved in vascular patterning, cell expansion and elongation, and xylem differentiation in the differentiation zone below the SAM (Turner  and  Sieburth  2002).  Auxin  transported  through  the  developing  inflorescence stem from young parts is likely involved in continued xylem and interfascicular fiber development as the stem matures. Based on the Atctl2 mutant phenotype (reduced numbers of vascular bundles), AtCTL2 may play a role in mediating the process of vascular bundle formation and development, perhaps in response to auxin transported via the vascular system. Alternatively, misexpression of these CTL genes could indirectly affect auxin regulated processes by altering the shape or function of cells involved in auxin transport, thus altering the auxin distribution during inflorescence stem development and leading to developmental anomalies. One way to further test to role of AtCTL2 in vascular patterning is test the effect of AtCTL2 overexpression. Such plants have been produced, but have not been analyzed at the time of writing. Instead, PopCTL1 overexpressing Col-0 plants were produced and studied. The phenotype of these plants (Figures 19 and 20), production of extra vascular bundles and a mis-shapen stem, supports the idea that CTLs can play a role, direct or indirect, in vascular development, provascular cell fate and cambial cell proliferation. It will be interesting to assay for auxin distribution in AtCTL2 and PopCTL1 overexpression lines relative to wild type plants using the DR5::GUS reporter system to determine if alterations in auxin distribution coincide with the observed developmental anomalies. Four other vascular patterning mutants avb1 (Zhong and Ye 2004), hca (Pineau et al. 2005), pin1 (Galweiler et al. 1998) and clavata1 (Brand et al. 2000) show changes in the formation and distribution of vascular bundles. IFL1/AVB1 is a very interesting transcription factor, since its overexpression completely transforms Arabidopsis vascular bundles from a collateral into an amphivasal arrangement,  72  while its null mutant has reduced basipetal auxin flow, reduced formation of interfascicular and xylem fibers in vascular bundles (Zhong and Ye 1999; Zhong and Ye 2001). hca (enhanced cambial activity), apparently like pom1, shows reduced levels of auxin, and increased sensitivity to cytokinins. Phenotypically, it also shows the reduced secondary xylem formation phenotype present in pom1 at the root/hypocotyls junction, and has premature and enhanced cambial cell activity leading to an altered pattern of vascular tissues. pin1 mutants also have reduced auxin transport, and while there is no change in vascular patterning, there is an increase in the size of vascular bundles (Okada et al. 1991). clavata 1 mutants have enlarged apical meristems that produce an increased number of provascular initials in the apical meristem, resulting in both many more vascular bundles and a very distorted stem axial shape (Brand et al. 2000; Turner and Sieburth 2002). Overexpression of the transcription factor ATHB-8 also increases cambial cell proliferation in Arabidopsis, resulting in increased and accelerated primary xylem formation (Baima et al. 2001). Reduced auxin in the acl5 mutant results in increased recruitment of cambial cells into xylem and phloem of vascular bundles (Clay and Nelson 2005). These alterations in vascular patterning show some commonalities with those observed in PopCTL1 overexpressing plants, but further analysis will have to be done to elucidate the precise mechanism behind the increase in bundle numbers. The biochemical function(s) of CTL proteins is still a mystery, although the fact that all poplar, Arabidopsis, and one spruce CTL gene are able to rescue the pom1 mutation suggests that despite their somewhat divergent amino acid sequences, these genes share a common biochemical function somehow tied to cell wall development and auxin, sucrose, ethylene, and cytokinin signaling (Hauser et al. 1995; Cary et al. 2001; Zhong et al. 2002). Previously, it has been shown that mutation of a chitin hydrolyzing catalytic site can change a chitinase into a lectinlike protein (Iseli-Gamboni et al. 1998) and that there are non CTL examples of such modified chitinases which nonetheless have developmental activity within plants (Goormachtig et al. 2001). It seems very likely that wall localized CTLs play  73  a direct role in developmental signaling by reversibly binding or modifying a chitinlike oligosaccharide ligand and releasing or degrading an effector molecule in response to other developmental signals. CTL activity could also involve physical interactions with the cellulose synthase complex and newly formed cellulose polymers, making most pom1 phenotypes secondary pleiotropic effects resulting from inappropriate cell wall development. I have shown that genes encoding biochemically similar CTL proteins persist as differentially expressed units in at least two different plant species (Arabidopsis and poplar). This suggests that two copies exist in angiosperms for the purpose of separating similar processes during development by allowing differential expression to evolve in a duplicated gene (subfunctionalization). Whatever the biochemical function of CTLs is, the presence of CTL genes in all land plants sampled, including Physcomitrella, and the ability of a spruce CTL gene to complement the pom1 phenotype shows that CTL proteins have maintained conserved biochemical function(s) since at least the divergence of gymnosperms and angiosperms, and possibly since the evolution of land plants.  Chapter 5 Conclusions and Future Perspectives In this thesis I showed that CTLs are a small family of genes that appear to have evolved specifically in land plants. As far as this study was able to ascertain, there appear to have been two copies present in a common ancestor of the bryophytes and Spermatopsida, but that there was only one ancestral angiosperm CTL which duplicated again into two CTL copies in most plants. While the two CTLs in Arabidopsis and poplar have different patterns of expression based on GUS analysis, and PicglCTL1 from Picea glauca has important differences in amino acid sequence, they all share a biochemical function which allows them to complement the pom1 mutation. In vivo, this suggests that CTLs are able might be able to complement each other’s function in tissues where there  74  is overlapping expression, which appears to be vascular bundle cambium in the inflorescence and secondary xylem or xylem parenchyma in the root/hypocotyl. This idea helps explain why Atctl1 and Atctl2 phenotypes also show similar ectopic lignification of the pith and enhanced phloem fiber development, indicating that reduced total CTL expression in vascular bundles might be more important than either individual AtCTL1 or AtCTL2 expression. Production of Atctl1/Atctl2 double mutants should allow better observation of what a plant looks like without any possible functional complementation. My finds suggest that the observed patterns of CTL expression, rather than biochemical function dictate their biological roles. Consistent with the idea that regulation is important in CTL function, constitutive overexpression of PopCTL1 resulted in altered vascular bundle formation and axial inflorescence shape. Transgenic plants for AtCTL1, AtCTL2, PopCTL2, PicglCTL1, and PicglCTL2 overexpression have been made and await both expression analysis and phenotyping to independently confirm the enhanced bundle formation phenotype. While our Atctl1 microarray and DR5::GUS studies show that auxin production, transport, or response is reduced in pom1, studies of auxin levels in Atctl2 mutants, CTL overexpressing plants and CTL downregulated poplar trees need to be pursued, and an explanation for altered auxin activity needs to be found. While alterations in auxin homeostasis as well as alterations in cell wall development, could help explain RNAi induced phenotypes in poplar. Further study into poplar CTL function will have to be done to be better able to make comparisons between species. Likewise, while PicgluCTL1 was able to rescue the pom1 sucrose induced phenotype, determining its function in that species is important to better understand evolution o fCTL biological function in land plants. Finally, the precise biochemical activity of CTL proteins needs to be discovered. As CTLs were originally annotated to be involved in carbohydrate modification, and there are a number of oligosaccharide molecules implicated in cell expansion and  75  differentiation, it still seems likely that CTLs are part of glycoprotein complex regulating cell wall development. Investigation into hormonal regulation of CTL expression and protein function, as well as discovery of its predicted carbohydrate ligand and its mode of action are all work that remains to be done to fully understand this developing developmental story.  76  References An, G. (1987). "Binary Ti vectors for plant transformation and promoter analysis." Methods Enzymol. 153: 292-305. Anterola, A. M. and N. G. Lewis (2002). "Trends in lignin modification: a comprehensive analysis of the effects of genetic manipulations/mutations on lignification and vascular integrity." Phytochemistry 61(3): 221-94. Arioli, T., L. Peng, et al. (1998). "Molecular analysis of cellulose biosynthesis in Arabidopsis." 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Plant Cell 11(11): 2139-52. Zhong, R. and Z. H. Ye (2001). "Alteration of auxin polar transport in the Arabidopsis ifl1 mutants." Plant Physiol 126(2): 549-63. Zhong, R. and Z. H. Ye (2004). "Amphivasal vascular bundle 1, a gain-of-function mutation of the IFL1/REV gene, is associated with alterations in the polarity of leaves, stems and carpels." Plant Cell Physiol 45(4): 369-85.  86  Appendix A Conserved portion of CTL protein sequences used to generate Figure 6.  HorvuCHI26 PhypaCTL1 PhypaCTL2 CycruCTL GinBiCTL PintaCTL1 PintaCTL2 PicglCTL1 PicglCTL2 ArathCTL2 ArathCTL1 PoptrCTL1 PissaCTL PoptrCTL2 MedtrCTL GlymaCTL GoshiCTL1 GoshiCTL2 GoshiCTL3 OrisaCTL1 OrisaCTL2 TriaeCTL VinviCTL1 VinviCTL2 CitsiCTL1 CitsiCTL2 SoltuCTL ZeamaCTL MaldoCTL BranaCTL HorvuCTL PontrCTL SacofCTL  ....|....| 5 ---------KGCNK----DGC------KICDRGWECKICDRGWECK --------CA KVCDKGWEC--------CI KICDKGWECKLCDKGWECK KVCTQGWECS KVCDKGWECK KYCTQGWECN KLCKRGWECL KLCDKGWECK KVCDKGWECK KLCDKGWECK KLCDKGWECK KQCIQGWECS KICDKGWECS KACDKGWECS TACDKGWECS KVCDKGWECK KVCDKGWECK KLCDKGWECK KVCIKGWECP KMCVKDWECN KICNKGWECS KLCDKGWECK KVCTQGWECV TACDKGWECS KVCIKGWECP KICNKGWECS  ....|....| 15 ---------S ---------T --------NT K-GIYCCNKT G--EYCCNKT K-ATDCKNKT K-GTYCCNQT K-GAECKNKT K-GVYCCNQT GWSEYCCNHT WWSKYCCNQT GLSAYCCNQT NWSIYCCNLT DWSEYCCNET GWSVYCCNET GWSAYCCNET GWSQFCCNQT GWSKYCCNHT YWSKYCCNKT G-SKYCCNDT G-SRFCCNDT G-SRFCCNET GWSEFCCNLT GWSKYCCNQT GWSEYCCNQT TWSKFCCNET KLSKFCCNLT G-SKYCCNDT GWSKYCCNLT WWSEYCCNQT G-SRFCCNET TWSKFCCNET G-SKYCCNDT  ....|....| 25 VSSIVSRAQF -SELFN-EMF ISDLFTVQTF ISEIFTVDQF ISQIFTVDQF ISELFTVDQF ISEIFTVDNF ISELFTVDQF ISQIFTVDNF ISDFFETYQF ISDYFQVYQF ISDFFQTYQF ISDYFQTYHF ISDIFQPYQF ISDYFQTYQF ISDYFQTYQF ISDYFRTYQF ISDYFQTYQF VSDVFQVYQF ITDFFKVYQF ITDYFKAYQF ITDYFKAYQF ISDYFQTYQF VSDFFQTYQF ISDYFQTYQF ISDYFQVYQF ITDYLDTDQF ITDFFKVYQF ISDYFQTYQF ISDYFQVYQF ITDYFKAYQF ISDYFQVYQF ITDFFKVYQF  ....|....| 35 DRMLLHRNDEAMFKHRNDK EDMFKHRNDR ESLFSHRNAESLFSKRNSESLFSHRNAEELFSKRNTESLFSHRNAEELFSKRNSENLFSKRNSEQLFSKRNTENLFSKRNTENLFSKRNTENLFSNRNSENLFSKRNDENLFAKRNSENLFAKRNTEDLFAKRNTEDLFAKRNSENLFSKRNSEELFAHRNDR EELFAKRNNENLFSKRNSENLFAKRNTENLFAKRNTENFFSKRNTELLFTKRNSENLFAKRNTENLFSKRNTEQLFAKRNTEELFAQRNNENFFSKRNTENLFAKRNT-  ....|....| 45 GACQAKGFYT -AAHAQGFWS -AAHAAGFWT PVAHAVGYWD PVAHAVGFWD PLAHAQGFWD PVAHAVGFWD PMAHAQGFWD PVAHAVGFWD PVAHAVGFWD PIAHAVGFWD PVAHASGFWD PVAHAVGFWD PVAHAVGFWD PTAHASGFWD PVAHAVGFWD PVAHAVGFWD PVAHAVGFWD PVAHAVGFWD PVAHAVGFWD SLAHAAGFWD SLAHAAGFWD PVAHAVGFWD PVAHAVGFWD PVAHAVGFWD PVAHAVGFWD PVAHAVGFWD PVAHAVGFWD PVAHAVGFWD PIAHAVGFWD SLAHAAGFWN PVAHAVGFWD PVAHAVGFWD  ....|....| 55 YDAFVAAAAA YDGFIAAAKM YDGFMAAAQM YHSFITAAAQ YHSFITAAAQ YHSFITAAAH YYSFINAAAQ YHSFITAAAH YYSFINAAAQ YRSFITAAAE YQSFITAAAL YHSFITAAAE YHSFINAAAL YRSFILASTS YRSFITAAAL YRSFITAAAL YHSFITAAAQ YHSFITAAAQ YHSFILAASI YQSFITAAAL YHAFITAAAL YKAFITAAAL YRSFILASAV YRSFITAAAV YHSFITAAAL YQSFITATVK YGSFIRATAL YQAFITAAAL YQAFITAAAL YQSFITAAAL YQAFITAASL YQSFITATVK YQAFITAAAL  87  HorvuCHI26 PhypaCTL1 PhypaCTL2 CycruCTL GinBiCTL PintaCTL1 PintaCTL2 PicglCTL1 PicglCTL2 ArathCTL2 ArathCTL1 PoptrCTL1 PissaCTL PoptrCTL2 MedtrCTL GlymaCTL GoshiCTL1 GoshiCTL2 GoshiCTL3 OrisaCTL1 OrisaCTL2 TriaeCTL VinviCTL1 VinviCTL2 CitsiCTL1 CitsiCTL2 SoltuCTL ZeamaCTL MaldoCTL BranaCTL HorvuCTL PontrCTL SacofCTL  ....|....| 65 FS--GFGTTG FEKDGFGMVG FEKDGFASVG YEGVGFGTTG YEGLGFGTTG YEPKGFGTTG FEGIGFGTTG FEPKGFGATG FEGIGFGTTG YQPLGFGTAG FEPLGFGTTG YQPHGFGTTG FEPQGFGTTG FQHLGFCTTG YQPLGFGTSG YQPHGFGTTG YQPHGFGTTG YQPHGFGTTG YEPLGFGTTG FEPLGFCTTG FEPRGFGTTG YEPRGFGTTG YQPLGFGTTG YQPHGFGTAG YQPHGFGTSA YQPLGFGTTG YQPLGFGTTG FEPQGFCTTG FEPLGFGTTG FEPLGFGTTG FEPRGFGTTG YQPLGFGTTG FEPQGFCTTG  ....|....| 75 SADVQKREVA GEDVQKRELS GDDMQKRELA GQLMQQRELA GDKMQQKEIA GDIVQKRELA GQVMQQKELA GDLVQKKELA GQLMQQKELA EKLQGMKEVA GKLMGQKEMA GKLTGQKEVA NKTMQMMEIA GKATQMKELA GKHGGQKEVA GKTSGQKELA GKLQSMKEVA EKLQNMKEVA GKRMQMKEVA GKQMQMMELC GKEVGMKEVA GREMSMKEVA GKVMQMKELA GKLMQMKEVA GKLMGQKEVA TKLDKMKEIC GKKMQMKEIA GKQMQMMELC GKLMQMKEIA GKLMGQKEMA GREMSMKEVA TKLDKMKEIC GKQMQMMELC  ....|....| 85 AFLAQTSHET AFFAHVAHET AFFAHVAHET AFLGHVASET AFLGHVASET AFFAHIATET AFLGNVAAET AFFAHVATET AFFGHVAAET AFLGHVGSKT AFLGHVASKT AFLGHVGSKT AFLGHVGSKT AFLAHVGCKT AFLGHVGSKT AFFGHVGSKT AFLGHVGSKT AFLGHVGSKT AFLAHVGAKT AFLGHVGSKT AFLGHVGAKT AFLGHVSAKT AFLGHVGCKT AFLGHVGSKT AFLGHVGSKT AFLAHVGCKT AFLGHVGSKT AFLGHVGAKT AFLGHVGSKT AFLGHVASKT AFLGHVGAKT AFLAHVGCKT AFLGHVGAKT  ....|....| 95 TGGWATA--SCGWSGA--SCGWSMA--SCGYSVA--SCGYSVA--SCESLMAQAA SCGYNVA--SCESLMAQSSCGYSVA--SCGYGVA--SCGYGVA--SCGYGVA--SCGYGVA--SCGYGVA--SCGYGVA--SCGYGVA--SCGYGVA--SCGYGVA--SCGDGVI--SCGFGVA--SCGYSVA--SCGYSLA--SCGYGVA--TCGYGVA--SCGYGVA--SCGYGVA--SCGYGVA--SCGYGVA--SCGYGVA--SCGYGVA--SCGYSLA--SCGYGVA--SCGYGVA---  ....|....| 105 ----PDGAFA ----KDGPTA ----KDGPTA ----VGGPLA ----VGGPLA STAPSDSPTK ----TGGPTA STATTDSPTK ----VGGPYA ----TGGPLA ----TGGPLA ----TGGPLA ----TGGPTA ----TGGPLA ----TGGPFA ----TGGPLA ----TGGPLA ----TGGPLA ----DGGPLA ----TGGPTA ----TGGPLA ----DGGSLA ----TGGPLS ----TGGPLA ----TGGPLA ----TGGPLA ----TGGPLA ----TGGPTA ----TGGPYA ----TGGPLA ----TGGSLA ----TGGPLA ----TGGPTA  ....|....| 115 WGYCFKQERG WGLCYNQELA WGLCYNQELA WGLCYREEMS WGLCYKEEMS WGLCYKEELS WGLCYKEEMS WGLCYKEELS WGLCYKEEMS WGLCYNKEMS WGLCYNREMS WGLCYNKEMS WGLCYNHEMS WGLCYNKEMS WGLCYNKELS WGLCYSKELS WGLCYNKEMS WGLCYNKEMS WGLCFKREMS WGLCYNHEMS WGLCYNHELS WGLCYNHEMS WGLCYNKEMS WGLCYNKEMS WGLCYNKEMS WGLCYNHEMS YGLCYNKEMS WGLCYNHEMS WGLCYNREMS WGLCYNREMS WGLCYNHEMS WGLCYNHEMS WGLCYNHEMS  88  HorvuCHI26 PhypaCTL1 PhypaCTL2 CycruCTL GinBiCTL PintaCTL1 PintaCTL2 PicglCTL1 PicglCTL2 ArathCTL2 ArathCTL1 PoptrCTL1 PissaCTL PoptrCTL2 MedtrCTL GlymaCTL GoshiCTL1 GoshiCTL2 GoshiCTL3 OrisaCTL1 OrisaCTL2 TriaeCTL VinviCTL1 VinviCTL2 CitsiCTL1 CitsiCTL2 SoltuCTL ZeamaCTL MaldoCTL BranaCTL HorvuCTL PontrCTL SacofCTL  ....|....| 125 ASSDYCTPSPEKDYC-KTG PMKDYC-KTG PDQLYCAQNY PDQLYCDPNY PDSTYCESSPDQLYCDQNPDSTYCESSPDQLYCDQNY PDQLYCDDYY PMQSYCDESPSKTYCDDYY PSQTYCDDYY PSQTYCDDFY PDKFYCDDYY PDKFYCDDYY PSKLYCDDYY PSKIYCDDYY PSQDYCDDYPKEDYCDKTN PSQSYCDNSN PSQSYCDDSN PSKSYCDDFPSKSYCDDDPNQIYCDDDPSQSYCDDSPSQDYCDDYPDQTYCDKTY PMQSYCDDYPMQSYCDETPSQSYCDDSN PSQSYCDDSPDQTYCDKTY  ....|....| 135 ---AQWPCAP D--LMYPCAP D--LLYPCAP ----LYPCAP ----LYPCSP ---LVYPCAP ---LLYPCAP ---LVYPCAP ----LFPCSP --KLTYPCTP -WKFKYPCSP --KYTYPCTP --KLTYPCTP --KYTYPCTP --KLTYPCSP --KLTYPCTP --KYTYPCTP --KYTYPCTP -YKYMYPCAP ---LQYPCVE E---LYPCVE E---LYRCAE -YKYTYPCTP -YKYTYPCTP -FKYTYPCTP -YKYTYPCTP -FKLTYPCTP ---TQYPCVE -YKYIYPCSP -WKYKYPCSP E---LYRCAE -YKYTYPCTP ---TQWPCVE  ....|....| 145 GKRYYGRGPI GAGYYGRGAF GAGYYGRGAF GASYHGRGAL GASYHGRGAL GVSYHGRGAL GASYHGRGAL GVSYHGRGAL GASYHGRGAL GVSYHGRGAL GAEYYGRGAL GVSYHGRGAL GAEYYGRGAI GAEYYGRGAI GAAYYGRGAI GAAYYGRGAI GVSYHGRGAL GVSYHGRGAL GAQYYGRGAL GAEYYGRGAI GVEYYGRGAL GVEYYGRGAL GADYYGRGAL GVEYFGRGAL GVSYHGRGAL GAEYYGRGAI GARYYGRGAL GAEYYGRGAI GAEYYGRGAL GAEYYGRGAL GVGYYGRGAL GAEYYGRGAI GAEYYGRGAI  ....|....| 155 QLSHNYNYGP PLYWNYNYGP PLYWNYNYGP PVYWNYNYGQ PVYWNYNYGQ PVYWNYNYGQ PIYWNFNYGP PVYWNYNYGQ PLYWNYNYGQ PVYWNYNYGQ PIYWNFNYGA PLYWNYNYGK PIYWNYNYGA PIFWNYNYGA PIYWNYNYGK PLYWNYNYGK PIYWNYNYGE PIYWNYNYGE PIYWNYNYGA PVFWNYNYGA PVYWNYNYGI PVYWNYNYGI PIFWNYNYGA PIYWNYNYGE PLYWNYNYGE PIYWNYNYGA PIYWNYNYGA PVYWNYNYGA PIYWNYNYGA PIYWNFNYGA PVYWNYNYGI PIYWNYNYGA PVYWNYNYGA  ....|....| 165 AGRAIGVDLL TGVALKQDLL TGKALKQDLL IGEALKVDLL IGEALKVDLL LGQALKVDLL IGEALKLDLL LGQALKVDLL IGEALKLDLL TGEALKVDLL AGEALKADLL TGEALKTDLL AGEALKVNLL AGEALKEDLL IGEALKVDLL AGEALKVDLL TGDALKVDLL TGEALKVDLL AGDGIKVDLL AGDGIHEDLL IGQGIKQDLL VGKGIKQDLL TGEALKVNLL AGEALKVDLL TGEALKVDIL TGEALKADLL IGDALKLNLL AGDGIKADLL AGDALKVDLL AGEALKADLL VGKGIKQDLL TGEALKADLL AGDGIKVDLL  ....|....| 175 ANPDLVATDA HHPEILSQNE HHPEILAQNE NHPEYLADNA THPEYLADNA HHAEYLSENA TSPDMVSNNA HHAEDLSQNA NYPDLLSNNA SHPEYLENNA NHPEYIEQNA NHPEYLENNA DHPEYIEQNA SHPEYIEQNA NHPEYIEQNA NHPEYIEQNA NHPEYIENNA NHPEYLEDNA HHPEYLEQNA HHPEYLEQNA NHPELLEQNA NHPELLEQNA DHPEYIEQNA NHPEYIEQNA NHPEYIENNA SHPEYIEQNA DHPEYIEQNA HHPEYLEQNA NHPEYIEQNA NHPEYIEQNA NHPELLEQNA SHPEYIEQNA HHPEYLEQNA  89  HorvuCHI26 PhypaCTL1 PhypaCTL2 CycruCTL GinBiCTL PintaCTL1 PintaCTL2 PicglCTL1 PicglCTL2 ArathCTL2 ArathCTL1 PoptrCTL1 PissaCTL PoptrCTL2 MedtrCTL GlymaCTL GoshiCTL1 GoshiCTL2 GoshiCTL3 OrisaCTL1 OrisaCTL2 TriaeCTL VinviCTL1 VinviCTL2 CitsiCTL1 CitsiCTL2 SoltuCTL ZeamaCTL MaldoCTL BranaCTL HorvuCTL PontrCTL SacofCTL  ....|....| 185 TVSFKTAMWF TIAWQAAVWY TIAWQAAIWY TLAFQAAIWR TLAFSAAIWR TLAFAAAIWR TIGFLTAMWR TLAFQAAMWR TIGFQTAIWR TLAFQAAIWR TLAFQAAIWR TLAFQAAIWK TLAFQAAIWK TLAFKAAMWR TLAFQAALWK TLAFQAALWQ TLAFQAALWR TLAFQTAMWR TIAFQAAIWR TMAFMAAMWR TLAFEAAIWR TLAFEAAIWR TLAFQAAIWR TLAFQAAIWR TLAFQAAMWR TLAFEAAIWK TMAFQAAIWR TLAFMAAMWR TLAFQAAVWR TLAFQAAIWR TLAFEAAIWR TLAFEAAIWK TLAFMAAMWR  ....|....| 195 WMT-AQPPKP WMTPAKTR-P WMTPAKTR-P WMNPIKPKQP WMTPIKRKQP WMTPMKVKQP WMNPIKPKQP WMNPIKVKQP WMNPIKPKQP WMTPPKKHLP WMTPIKRAQP WMTPEKKHLP WMTPVKKAQP WYTPIKKSQP WMTPPEKHIP WMTPPEKHLP WMTPVKKHQP WMTPMKKHQP WMTPIKKNQP WMTPMKKKQP WMTPMKRKQP WMTPMKRKQP WMTPVKKSQP WMTPVKKQQP WMTPVKKHQP WMTPVKKSQP WMNPMKKGQP WMTPIKKSQP WMTPIKKSQP WMTPIKKAQP WMTPMKRKQP WMTPVKKSQP WMTPIKKNQP  ....|....| 205 SSHAVIVGQW SPHEIMIGKW SPHEVMIGKW SAHDVMVGKW SAHEVIVGKW SAHQVMVGKW SAHDVFVGNW SAHQVMVGKW SAHDVLVGNW SAHDVFVGKW SAHDIFVGNW SAHDVFVGKW SAHDAFVGNW SAHEAFLGKW SPHDVFVGNW SPHDVFVGNW SAHDVFVGSW SAHDVFVGNW SAHDIFVGNW SAHDVFVGNW SAHDVFVGNW SAHDAFVGNW SAHDVFVGNW SAHDVFVGTW SAHDAFVGNW SAHDAFVGNW SAHDAFVGNW SAHDAFVGNW SAHQAFVGDW SAHDIFVGNW SAHDAFVGNW SAHDAFVGNW SAHEAFVGTW  ....|....| 215 SPSGADRAAG VPTKNDTLAY VPTKNDTLAN IPTKNDTNSF VPTKNDTKSF VPTKNDTEAL KPTKNDTESY VPTKNDTNSL KPTKNDTASF KPTKNDTAAK KPTKNDTLSK KPTKNDTLAK KPTKNDTMGN KPTKNDTLAK KPTKNDTLSK KPTKNDTLSK KPTKNDTLAK KPTKNDTLAK KPTKNDTEEK KPTKNDTLAK KPTKNDTLSK KPTKKDTLSK KPTKNDTLAK KPTKNDTLAK KPTKNDTLAK KPTKNDTLSK KPTKNDTLSK KPTKNDTLSK KPTKNDTLSK KPTKNDTLSK KPTKKDTLSK KPTKNDTLSK KPTKNDTLSK  ....|....| 225 RVPGFGVITN RKPGFGMTIN RKPGFGMTIN RLPGFGMTIN RLPGFGMTIN RLPGFGMTIN RLPGFGMVIN RHPGFGMTIN RFPGFGMTIN RTPGFGATIN RGPTFGSTMN RVPGFGTTMN RVPGFGATMN RVPGFGTTMN RVPGFGATIN RVPGFGATIN RVPGFGATMN RVPGFGTTMN RGPTFGSTMN RLPGFGATMN RYPGFGATMN RYPGFGATMN RVPGFGTTMN RIPGFGATMN RVHGFGTTMN RGPNFGTTMN RLPGFGTTMN RLPGFGATMN RFPGFGTTMN RGPTFGTTMN RYPGFGVTMN RGPNFGTTMN RLPGFGATMN  ....|....| 235 IINGGIECGH VKASDVECGH IKASDVECGH ILDGDAECGK ILDGDAECGK ILKADAECGT VLNGGLECGK ILKGEAECGA VLNGGLECGQ VLYGDQICNS VLYGEYTCGQ VLYGDQVCGK ILYGEGVCGQ VLYGDQVCGQ VLYGDQVCDQ LLYGDQTCGQ VLYGDQVCGR VLYGDQVCGQ VLYGDYTCGQ VLYGDQICGK ILYGDLICGQ ILYGDAICGK ILYGDQVCGQ VLYGDSVCGQ VLYGDQVCGK ILYGESVCGQ ILYGDAVCGQ ILYGESICGK ILYGESVCGK VLYGEYTCGQ ILYGDAICGK ILYGESVCGQ ILYGESICGK  90  HorvuCHI26 PhypaCTL1 PhypaCTL2 CycruCTL GinBiCTL PintaCTL1 PintaCTL2 PicglCTL1 PicglCTL2 ArathCTL2 ArathCTL1 PoptrCTL1 PissaCTL PoptrCTL2 MedtrCTL GlymaCTL GoshiCTL1 GoshiCTL2 GoshiCTL3 OrisaCTL1 OrisaCTL2 TriaeCTL VinviCTL1 VinviCTL2 CitsiCTL1 CitsiCTL2 SoltuCTL ZeamaCTL MaldoCTL BranaCTL HorvuCTL PontrCTL SacofCTL  ....|....| 245 GQDS-RVADR G-EDPRMQSR G-DDPRMLSR G-DVEKMDNR G-DIEKMSNR DSDDKQMNTR G-DIDAMNNR GSDDKQMNKR G-DIEAMSNR GfDNDEMNNI GS-IDPMNNI G-D-ESMNNI G-DVDSMNNI G-DIDAMNNF GSDNEAMSNI GSDNEAMNNI G-DVDTMNNI G-DSDSMNNM G-DIDPMNII GY-IDDMNVI GS-IDKMNVI GS-IDNMNGI G-DVDSMNNI G-DVDSMNNI G-DDESMNNM G-DIDAMNNI G-DVDSMNNI GY-VDAMNVI G-DIDAMNNI G-DIEPMSNI GT-TESMNAI G-DIDAMNNI GF-IDAMNTI  ....|....| 255 IGFYKRYCDI ISHYLTFLRD ISHYLDFLQN ISHYLYFLDL ISHYLYFLDL IAHYLDFLDH ISHYLYFLDL IAHYLYFLDQ VSHYLHFLDL VSHYLYYLDL ISHYLYFLDL VSHYLYYLDL ASHFLFYLDL ISHYLYYLDL ISHYLYYLDL ISHYLYYLDL ISHYLSYLDL ISHYLYYLDL ISHYLHYLDL ISHYQYYLDL VSHYQHYLDL ISHYQHYLDL ISHYQYYLDL VSHYQYYLDL ISHYLYYLDL VSHYLYYLDL ISHYQYYLDL ISHYQYYLDL VSHYQYYLDL VSHYLYFLDL ISHYQHYLDL VSHYLYYLDL ISHYQYYLDL  ....|....| 265 LGVG---YGN TFQLDDP-GS KFQVQDP-GA MGVGRQYSGD MGVGRQFAGV MDVGRENAGD LGVGREQAGD LDVGRDNAGD MGVGRELAGD IGVGREEAGP MGIGREDAGP MGVGREEAGS LGVGRDKGGT LGLNREDAGP LGVGREEAGP LGVGREEAGP MGVGREEAGP LGVGREEAGP LGVGREEAGP MGVGREHSGD MGVGSDKAGD MGVGAQHSGD LGVGREQAGP MGVGREEAGP MGVGREEAGP LGVGREQAGP MGVGREEAGP MGVGREHSGD MGVNRDEAGP LGIGREDAGP MGVGVQHSGD LGVGREQAGP MGVGREHSGD  ....|....| 275 N--LDCYSQNLD--CGLQG NLD--CGLQG NLD--CGQQNLD--CGQQNVD--CSEQNLD--CGQQNLD--CSDQHLD--CGEQHEKLSCADQNDELSCAEQHDVLSCAEQHDVLTCAEQHEYLTCAEQNEILSCAEQNEVLSCAEQHEVLTCEEQHDMLTCEEQHEELSCAEQNRD--CAEQNLD--CADQNLD--CADQHENVTCAEQHEVLTCAEQNEVLSCGEQNEELSCAEQHEVLNCAEQNRD--CAEQHEVLTCAEQNEELSCAEQNLD--CADQNEELSCAEQNRD--CAEQ-  ....|... 285 -RPF---VIPLAY-VVPLAY--VALNPAS -VPLNPSA -KVLNPSS -VPLNPVS -KVLNPSS -VALNPVS -EPFSSSS -KPFNPST -LPFNQAS -RPFNPNT -VAFNPST -AAFKPTG -AAFKPSG -KPFTVSP -EPFTVSP -KAFNPTP -AAFNPSY -VAFNPSS -VPFNPSS -IAFNPSY -EAFNPSS -EPFNPSS -KAFNPNT -KPFNPTA -APFNPSS -VAFNPIq -KAFNPAT -VPFNPSS -KAFNPNT -LPFNPSS  91  


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