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Functional genomic analysis of novel secondary cell wall genes in poplar (Populus trichocarpa) Tran, Lan T. 2018

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FUNCTIONAL GENOMIC ANALYSIS OF NOVEL SECONDARY CELL WALL GENES IN POPLAR (POPULUS TRICHOCARPA)  by  Lan T. Tran  M.Sc., The University of Victoria, 2011  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Botany)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  September 2018   © Lan T. Tran, 2018  ii   The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled:  Functional genomic analysis of novel secondary cell wall genes in poplar (Populus trichocarpa)  submitted by Lan T. Tran in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Botany  Examining Committee: Dr Lacey Samuels Co-supervisor Dr Jürgen Ehlting Co-supervisor   Supervisory Committee Member Dr Robert Guy University Examiner Dr Patrick Martone University Examiner  Additional Supervisory Committee Members: Dr Ljerka Kunst Supervisory Committee Member Dr Shawn Mansfield Supervisory Committee Member iii  Abstract  Secondary cell walls (SCWs) contain a significant amount of fixed carbon that can be harnessed for the production of renewable energy. However, efficient conversion of wood-based biomass for use as an alternative fuel source is constrained by lignin, a phenolic polymer that is recalcitrant to enzymatic degradation. Many aspects of SCW biosynthesis remain enigmatic, including how genes of broad functional classes affect lignin content and composition. A genetic association mapping (AM) study in poplar (Populus trichocarpa) previously identified novel genes genetically associated with lignin trait variation. To test the hypothesis that these genes influence SCW biosynthesis, I screened 27 lignin-associated genes using in silico analyses and transfer-DNA (T-DNA) mutant phenotyping of Arabidopsis (Arabidopsis thaliana) homologs and identified two genes for in-depth functional characterization. First, Coiled-coil Protein of Unknown Function (CPU) was identified to be highly expressed in xylem and co-expressed with well-known SCW-related genes including SND1, a key transcriptional regulator of SCW biosynthesis in fibres. While AM predicted CPU to be significantly associated with total lignin content variation, this was not found in transgenic poplar over-expressing poplar CPU. Instead, transgenic poplars exhibited altered fibre length compared to wild-type. In Arabidopsis, a genetic interaction for CPU and Cellulose-Microtubule Uncoupling (CMU) was identified as the cpucmu1cmu2 triple mutant had decreased SCW thickening in fibre cells compared to wild-type, suggesting CPU to also influence microtubules during SCW deposition. Secondly, P. trichocarpa Nitrate Peptide Family 6.1 (PtNPF6.1), a member of the nitrate1/peptide transporter superfamily was characterized. PtNPF6.1 is expressed in the vascular tissue, as detected from transgenic PtNPF6.1pro:GUS lines. Transgenic poplar suppressed in PtNPF6.1 had elevated levels of total nitrogen corresponding to elevated levels of free glutamic acid and aspartic acid compared to wild-type. Under luxuriant nitrogen conditions, PtNPF6.1-suppressed lines produced wood with less syringyl lignin compared to wild-type. The findings suggest PtNPF6.1 may help regulate the long-distance transport of nitrogen. Altogether, previously unsuspected classes of genes identified through AM has broadened our understanding of genes that impact the cellular and physiological processes that contribute to wood formation which may enable further optimization of woody plants for a diversity of applications including bioethanol production. iv  Lay Summary  A significant portion of wood is cellulose, a structural carbohydrate that has the potential to be converted into bioethanol. However, the extraction process is hindered by lignin, a non-carbohydrate polymer in wood. Wood development is a complex process that requires the function of numerous genes, most of which remain unknown. I characterized the biological role of two previously unknown genes identified from their genetic influences on lignin traits in poplar (Populus trichocarpa). Using transgenic approaches, I found that gain of function of one gene altered wood fibre length, suggesting a role in cell wall structure organization. The second gene, predicted to be a transporter, may regulate internal nitrogen redistribution and thus indirectly affect lignin biosynthesis. The outcomes of this research have advanced our knowledge of the cellular and physiological processes that contribute to wood development and expand the potential gene targets for the optimization of poplar breeding for industrial applications.   v  Preface  All research chapters of this thesis are presented as draft manuscripts in preparation for publication.  Chapter 2: Lan Tran contributed to the experimental design, executed the experiments, and prepared the manuscript draft. Dr Carl Douglas and Dr Jürgen Ehlting conceived the research project, assisted in experimental planning, and provided resources for the research.   Chapter 3: Lan Tran contributed to the research idea and experimental design, executed the experiments, and prepared the manuscript draft. Dr Cuong Le (Ehlting Lab, University of Victoria) conducted the phylogenetic analysis. Dr Oliver Corea (Ehlting Lab, University of Victoria) ran the co-expression analysis for the poplar developing xylem transcriptome data. Dr Misato Ohtani and Nobuhiro Akiyoshi (Demura Lab, NAIST) provided guidance (during an internship at NAIST) and collaborated on the in situ hybridization experiment. Dr Faride Unda provided instrument assistance for the cell wall structural carbohydrate analysis. Yaseen Mottiar performed the crystallinity and microfibril angle measurements. Dr Carl Douglas and Dr Jürgen Ehlting conceived the research project, assisted in the experimental design, and provided resources for the research. Dr Jürgen Ehlting assisted with the manuscript preparation. Dr Lacey Samuels provided experimental input, and editing and critical discussion of the manuscript.  Chapter 4: Lan Tran contributed to the research idea and experimental design, executed the experiments, and prepared the manuscript draft. Samantha Robbins (Hawkins Lab, University of Victoria) provided instrument assistance for the elemental analysis. Andre Bindon (Analytical Chemistry Services Laboratory at the Ministry of Environment & Climate Change Strategy, Victoria, BC) conducted the free amino acid analysis. Tyler Irwin, an undergraduate Honours student under my direct supervision (Ehlting Lab, University of Victoria), assisted with the anthocyanin quantification. Yaseen Mottiar assisted with the instrument and data analysis for the thioacidolysis experiment. Dr Carl Douglas and Dr Jürgen Ehlting conceived the research, assisted in the experimental planning, and provided resources for the research. Dr Jürgen Ehlting assisted with the manuscript preparation. Dr Lacey Samuels provided editing and critical discussion of the manuscript. vi  Table of Contents  Abstract ......................................................................................................................................... iii Lay Summary ............................................................................................................................... iv Preface .............................................................................................................................................v Table of Contents ......................................................................................................................... vi List of Tables ................................................................................................................................ xi List of Figures .............................................................................................................................. xii List of Abbreviations ................................................................................................................. xiv Acknowledgements .................................................................................................................... xvi Dedication ................................................................................................................................. xviii Chapter 1. Introduction ................................................................................................................1 1.1 Xylem and tracheary element formation ........................................................................... 1 1.2 Secondary cell wall biosynthesis ....................................................................................... 2 1.3 Lignin transport, polymerization, and deposition .............................................................. 4 1.4 Transcriptional regulation of secondary cell wall development ........................................ 5 1.5 An alternative approach to forward and reverse genetics to identify new candidate genes .......................................................................................................................................... 6 1.6 Genetic association mapping in poplar .............................................................................. 7 1.7 Poplar and Arabidopsis as model systems ......................................................................... 8 1.8 Research objectives............................................................................................................ 9 Chapter 2. Initial characterization of novel candidate secondary cell wall biosynthesis genes identified through genetic association mapping in poplar .......................................................11 2.1 Introduction ...................................................................................................................... 11 2.2 Materials and Methods..................................................................................................... 15 2.2.1 Sequence analysis .................................................................................................. 15 2.2.2 Gene expression and co-expression analysis ......................................................... 15 2.2.3 Plant growth conditions and analysis .................................................................... 15 2.2.4 Microscopy ............................................................................................................ 16 2.3 Results .............................................................................................................................. 18 2.3.1 Summary of the genetic association mapping study in P. trichocarpa ................. 18 2.3.2 Genes genetically associated with lignin trait variation ........................................ 19 2.3.3 Screening lignin-associated genes for functional characterization ........................ 20 2.3.4 Sequence similarity trees reveal Arabidopsis homologs of the poplar lignin-associated genes .............................................................................................................. 22 2.3.5 Gene expression analysis using in silico and transcriptomic data ......................... 26 2.3.6 Co-expression analysis of lignin-associated Arabidopsis homologs ..................... 29 2.3.7 Reverse genetic screen of Arabidopsis T-DNA insertion mutants ........................ 33 2.4 Discussion ........................................................................................................................ 37 2.4.1 Reverse genetic screen of lignin-associated gene homologs did not reveal impacts on primary stem development in Arabidopsis ................................................................ 37 2.4.2 In silico analyses can identify novel genes involved in secondary cell wall biosynthesis..................................................................................................................... 39 2.4.3 Transporters and their contribution to secondary cell wall biosynthesis ............... 40 2.4.4 Conclusion ............................................................................................................. 42 vii  Chapter 3. Coiled-coil protein of unknown function (CPU) and its role in SCW deposition in fibres .........................................................................................................................................44 3.1 Introduction ...................................................................................................................... 44 3.2 Materials and Methods..................................................................................................... 47 3.2.1 Plant material and growth conditions .................................................................... 47 3.2.2 In silico analyses .................................................................................................... 48 3.2.3 Plasmid construction .............................................................................................. 48 3.2.4 Generation of transgenic plants ............................................................................. 49 3.2.5 Analysis of transgenic plants ................................................................................. 49 3.2.6 GUS staining and microscopy ............................................................................... 50 3.2.7 In situ hybridization ............................................................................................... 51 3.2.8 Yeast two-hybrid analysis...................................................................................... 51 3.2.9 Cell wall analyses .................................................................................................. 52 3.2.10 Wood ultrastructure analyses ............................................................................... 53 3.3 Results .............................................................................................................................. 55 3.3.1 Structural and phylogenetic analysis of CPU ........................................................ 55 3.3.2 PtCPU expression in developing wood ................................................................. 58 3.3.3 Wood phenotyping analysis of transgenic poplar over-expressing PtCPU ........... 63 3.3.4 Fibre length analysis .............................................................................................. 63 3.3.5 Cellulose microfibril angle and crystallinity analysis ........................................... 66 3.3.6 Heterologous over-expression of PtCPU in Arabidopsis ...................................... 67 3.3.7 Yeast two-hybrid screen for potential interactors of AtCPU ................................. 68 3.3.8 Expression of AtCPU and its putative interactors CMU1 and CMU2 ................... 72 3.3.9 cpucmu1cmu2 higher order mutant analysis .......................................................... 75 3.4 Discussion ........................................................................................................................ 80 3.4.1 Co-expression analysis of CPU indicates a potential role in secondary cell wall deposition in fibre cells ................................................................................................... 81 3.4.2 CPU interacts with a microtubule-associated tetratricopeptide repeat protein in vitro ................................................................................................................................. 82 3.4.3 Conclusion ............................................................................................................. 83 Chapter 4. Characterization of a Populus trichocarpa nitrogen-related transporter, PtNPF6.1 .......................................................................................................................................85 4.1 Introduction ...................................................................................................................... 85 4.2 Materials and Methods..................................................................................................... 89 4.2.1 Plant maintenance and stress treatments ................................................................ 89 4.2.2 Generation of transgenic poplar ............................................................................. 90 4.2.3 Expression analysis ................................................................................................ 91 4.2.4 Elemental analysis of carbon and nitrogen ............................................................ 92 4.2.5 Free amino acid analysis ........................................................................................ 93 4.2.6 Phenolic compound extraction and quantification ................................................ 93 4.2.7 Lignin analysis ....................................................................................................... 94 4.3 Results .............................................................................................................................. 94 4.3.1 PtNPF6.1 is evolutionary distinct as it lacks orthologs in Arabidopsis ................. 94 4.3.2 PtNPF6.1 is expressed in vascular tissue .............................................................. 97  viii  4.3.3 Total nitrogen concentration is altered in transgenic poplar down-regulated in PtNPF6.1 ........................................................................................................................ 99 4.3.4 High-light and ultraviolet-B stress causes a reduction in total soluble phenolic compounds in transgenic poplar down-regulated in PtNPF6.1 .................................... 103 4.3.5 Exogenous nitrogen application alters syringyl lignin composition in transgenic poplar down-regulated in PtNPF6.1 ............................................................................. 107 4.4 Discussion ...................................................................................................................... 109 4.4.1 PtNPF6.1 expression in vascular tissue suggests a role in long-distance transport ...................................................................................................................................... 109 4.4.2 PtNPF6.1 impacts total nitrogen and free amino acids in source leaves ............. 110 4.4.3 PtNPF6.1 has an indirect influence on phenylpropanoids including S lignin ..... 112 4.4.4 Conclusion ........................................................................................................... 113 Chapter 5. Discussion ................................................................................................................114 5.1 Main findings ................................................................................................................. 114 5.1.1 Analysis of Arabidopsis thaliana T-DNA insertion mutant stems ...................... 114 5.1.2 Characterization of CPU ...................................................................................... 115 5.1.3 Characterization of PtNPF6.1 ............................................................................. 117 5.2 Perspectives on genetic association mapping in poplar ................................................. 118 5.3 Conclusion ..................................................................................................................... 119 Literature Cited .........................................................................................................................121 Appendices ..................................................................................................................................139 Appendix A. Additional materials for Chapter 2 ........................................................... 139 Appendix A.1 Groups of genes tested for the P. trichocarpa genetic association mapping study conducted by Porth et al. (2013a). ....................................................... 139 Appendix A.2 Genes from the P. trichocarpa genetic association mapping study significantly associated with wood chemistry and ultrastructure traits. ....................... 140 Appendix A.3 Sequence similarity tree for UMAMIT9 (POPTR_0001s06980), a gene associated with insoluble lignin content ....................................................................... 143 Appendix A.4 Sequence similarity tree for CKL6 (POPTR_0002s03730), a gene significantly associated with insoluble lignin content .................................................. 144 Appendix A.5 Sequence similarity tree for MPK20 (POPTR_0002s06080), a gene significantly associated with insoluble lignin content .................................................. 145 Appendix A.6 Sequence similarity tree for CWP (POPTR_0005s07810), a gene significantly associated with insoluble lignin content .................................................. 146 Appendix A.7 Sequence similarity tree for LAX2 (POPTR_0009s13470), a gene significantly associated with insoluble lignin content .................................................. 147 Appendix A.8 Sequence similarity tree for BBEL13 (POPTR_0011s16200), a gene significantly associated with insoluble lignin content .................................................. 148 Appendix A.9 Sequence similarity tree for MAP20 (POPTR_0017s01510), a gene significantly associated with insoluble lignin content .................................................. 149 Appendix A.10 Sequence similarity tree for P4H7 (POPTR_0017s11150), a gene associated with insoluble lignin content ....................................................................... 150 Appendix A.11 Sequence similarity tree for SKS12 (POPTR_0001s03760), a gene significantly associated with soluble lignin content ..................................................... 151 ix  Appendix A.12 Sequence similarity tree for FPP7 (POPTR_0007s10810), a gene significantly associated with soluble lignin content ..................................................... 152 Appendix A.13 Sequence similarity tree for UXS2 (POPTR_0014s12380), a gene significantly associated with soluble lignin content ..................................................... 153 Appendix A.14 Sequence similarity tree for DIR6 (POPTR_0001s10120), a gene significantly associated with syringyl lignin content .................................................... 154 Appendix A.15 Sequence similarity tree for LHT1 (POPTR_0001s36340), a gene significantly associated with syringyl lignin content .................................................... 155 Appendix A.16 Sequence similarity tree for NPF6.1 (POPTR_0002s03070), a gene significantly associated with syringyl lignin content .................................................... 156 Appendix A.17 Sequence similarity tree for GH3.9 (POPTR_0002s20790), a gene significantly associated with syringyl lignin content .................................................... 157 Appendix A.18 Sequence similarity tree for GA2OX2 (POPTR_0004s06380), a gene significantly associated with syringyl lignin content .................................................... 158 Appendix A.19 Sequence similarity tree for UMAMIT12 (POPTR_0006s08270), a gene significantly associated with syringyl lignin content .................................................... 159 Appendix A.20 Sequence similarity tree for HAD (POPTR_0006s08720), a gene significantly associated with syringyl lignin content .................................................... 160 Appendix A.21 Sequence similarity tree for BLH9 (POPTR_0008s06130), a gene significantly associated with syringyl lignin content .................................................... 161 Appendix A.22 Partial sequence similarity tree for SAR2 (POPTR_0010s15180), a gene significantly associated with syringyl lignin content .................................................... 162 Appendix A.23 Sequence similarity tree for TLP3 (POPTR_0014s04020), a gene significantly associated with syringyl lignin content .................................................... 163 Appendix A.24 Sequence similarity tree for PGIP1 (POPTR_0016s05010), a gene significantly associated with syringyl lignin content .................................................... 164 Appendix A.25 Sequence similarity tree for CML3 (POPTR_0018s12720), a gene significantly associated with syringyl lignin content .................................................... 165 Appendix A.26 Sequence similarity tree for NPF6.3 (POPTR_0001s12890), a gene significantly associated with total lignin content .......................................................... 166 Appendix A.27 Sequence similarity tree for CPU (POPTR_0001s28570), a gene significantly associated with total lignin content .......................................................... 167 Appendix A.28 Sequence similarity tree for WRKY32 (POPTR 0006s19850), a gene significantly associated with total lignin content .......................................................... 168 Appendix A.29 Sequence similarity tree for DGP (POPTR_0008s11150), a gene significantly associated with total lignin content .......................................................... 169 Appendix A.30 In silico expression data for UMAMIT9 and CKL6 obtained from the BAR eFP browser ......................................................................................................... 170 Appendix A.31 In silico expression data for MPK20 and CWP obtained from the BAR eFP browser .................................................................................................................. 171 Appendix A.32 In silico expression data for LAX2 and BBEL13 obtained from the BAR eFP browser .................................................................................................................. 172 Appendix A.33 In silico expression data for MAP20 and P4H7 obtained from the BAR eFP browser .................................................................................................................. 173 x  Appendix A.34 In silico expression data for SKS12 obtained from the BAR eFP browser ...................................................................................................................................... 174 Appendix A.35 In silico expression data for FPP7 and UXS2 obtained from the BAR eFP browser .................................................................................................................. 175 Appendix A.36 In silico expression data for DIR6 and LHT1 obtained from the BAR eFP browser .................................................................................................................. 176 Appendix A.37 In silico expression data for NPF6.1 and GH3.9 obtained from the BAR eFP browser .................................................................................................................. 177 Appendix A.38 In silico expression data for GA2OX2 and UMAMIT12 obtained from the BAR eFP browser ................................................................................................... 178 Appendix A.39 In silico expression data for HAD and BLH9 obtained from the BAR eFP browser .................................................................................................................. 179 Appendix A.40 In silico expression data for SAR2 and TLP3 obtained from the BAR eFP browser .................................................................................................................. 180 Appendix A.41 In silico expression data for PGIP1 and CML3 obtained from the BAR eFP browser .................................................................................................................. 181 Appendix A.42 In silico expression data for NPF6.3 and CPU obtained from the BAR eFP browser .................................................................................................................. 182 Appendix A.43 In silico expression data for WRKY32 and DGP obtained from the BAR eFP browser .................................................................................................................. 183 Appendix B. Additional materials for Chapter 3............................................................ 184 Appendix B.1 CPU homologs analyzed in the maximum likelihood phylogeny. ........ 184 Appendix B.2 Amino acid alignment of homologous CPU sequences used for the maximum likelihood phylogenetic analysis ................................................................. 186 Appendix B.3 Entire list of genes co-expressed with PtCPU in developing P. trichocarpa wood transcriptomes at a PCC of at least 0.60 ......................................... 194 Appendix B.4 Wood anatomy for wild-type and transgenic poplar 35S:PtCPU over-expression lines ............................................................................................................. 201 Appendix B.5 Over-expression of 35S:YFP-PtCPU in Arabidopsis produced rounded leaves and shorter siliques compared to wild-type ....................................................... 202 Appendix B.6 Phenotypes of ten-day-old seedlings of wild-type, cpu, cmu1cmu2, and cpucmu1cmu2 ............................................................................................................... 203 Appendix C. Additional materials for Chapter 4 ........................................................... 204 Appendix C.1 Sequence information for PtNPF6.1 and PtNPF6.2 ............................. 204 Appendix C.2 Unidentified anthocyanin peaks detected from transgenic poplar RNAi PtNPF6.1 lines exposed to natural outdoor high-light/UV-B radiation. ...................... 205 Appendix C.3 Phenotypes of wild-type and RNAi PtNPF6.1 lines fertilized with high (10 mM) or low (0.10 mM) ammonium nitrate ............................................................ 206 Appendix C.4 Wild-type and RNAi PtNPF6.1 lines fertilized with high (10 mM) or low (0.10 mM) ammonium nitrate for eight weeks did not exhibit biomass differences .... 207 xi  List of Tables  Table 2.1 Arabidopsis T-DNA insertion lines analyzed for homologs of the lignin-associated genes from Porth et al. (2013a) ...................................................................................... 17 Table 2.2 Complete list of lignin-associated genes analyzed identified from a genetic association mapping experiment in poplar (P. trichocarpa) conducted by Porth et al. (2013a) ...... 21 Table 2.3 Arabidopsis homologs of the poplar lignin-associated genes identified from sequence similarity trees ................................................................................................................ 24 Table 2.4 Expression of lignin-associated genes in P. trichocarpa developing secondary xylem and leaves from Geraldes et al. (2011) ........................................................................... 28 Table 2.5 Lignin-associated genes that were screened for cell wall-specific defects using Arabidopsis T-DNA insertion lines ................................................................................ 35 Table 3.1 Primer sequences used in this study ............................................................................. 54 Table 3.2 Expression of PtCPU and its paralog in developing secondary xylem of P. trichocarpa ........................................................................................................................................ 58 Table 3.3 MapMan annotation abundance for genes co-expressed with PtCPU at a PCC of at least 0.60 ......................................................................................................................... 62 Table 3.4 Analysis of structural cell wall carbohydrate composition and total lignin content in wild-type and transgenic hybrid poplar lines over-expressing PtCPU .......................... 64 Table 3.5 Thioacidolysis analysis of guaiacyl and syringyl lignin in transgenic poplar lines over-expressing PtCPU ........................................................................................................... 65 Table 3.6 AtCPU-interacting proteins identified from a yeast two-hybrid screen ....................... 70 Table 3.7 Lignin analysis of wild-type, cpu and cpucmu1cmu2 mutants .................................... 79 Table 4.1 Number of NPFs from representative species of four major taxonomic groups described by von Wittgenstein et al. (2014) ................................................................... 97 Table 4.2 Total C and N concentration in leaves of RNAi PtNPF6.1 lines............................... 102 Table 4.3 Free amino acid content in transgenic RNAi PtNPF6.1 lines ................................... 103 Table 4.4 Total anthocyanins in RNAi PtNPF6.1 lines exposed to natural outdoor high-light/UV-B radiation ..................................................................................................... 105 Table 4.5 Analysis of lignin content and composition in wild-type and transgenic poplar RNAi PtNPF6.1 lines grown under luxuriant nitrogen (10 mM ammonium nitrate) fertilization ...................................................................................................................................... 108 Table 4.6 Total percentage of structural cell wall carbohydrates in wild-type and transgenic poplar RNAi PtNPF6.1 lines grown under luxurious nitrogen (10 mM ammonium nitrate) fertilization ....................................................................................................... 109  xii  List of Figures  Figure 2.1 Categories of genes significantly associated with wood chemistry and ultrastructure traits identified from a genetic association mapping experiment in poplar (Populus trichocarpa) conducted by Porth et al. (2013a) .............................................................. 19 Figure 2.2 Categories of genes significantly associated with lignin trait variation in poplar (Populus trichocarpa) identified from a genetic association mapping experiment conducted by Porth et al. (2013a). .................................................................................. 20 Figure 2.3 Experimental approach used for the selection of novel LAGs for functional characterization ............................................................................................................... 22 Figure 2.4 Unrooted sequence similarity tree for UMAMITs in land plants ............................... 25 Figure 2.5 In silico expression data for the poplar lignin-associated genes and their Arabidopsis homologs in different tissues and organs ........................................................................ 27 Figure 2.6 Co-expression analysis of lignin-associated genes from Porth et al. (2013a) using the Arabidopsis Legacy Expression Angler tool .................................................................. 30 Figure 2.7 Co-expression analysis for the total lignin-associated gene CPU from Porth et al. (2013a) using the Arabidopsis Legacy Expression Angler tool ..................................... 31 Figure 2.8 Co-expression network analysis of lignin-associated genes from Porth et al. (2013a) using ATTED-II .............................................................................................................. 32 Figure 2.9 Genotyping T-DNA insertion mutants to identify homozygous lines for phenotypic analysis ........................................................................................................................... 34 Figure 2.10 Phenotypic analysis of T-DNA insertion lines for Arabidopsis homologs of the poplar lignin-associated genes ........................................................................................ 36 Figure 3.1 Secondary structure prediction for CPU from Arabidopsis (A. thaliana) and poplar (P. trichocarpa) using SMART ...................................................................................... 56 Figure 3.2 An unrooted maximum likelihood phylogenetic analysis of CPU in land plants ...... 57 Figure 3.3 In silico expression analysis of PtCPU in phloem and developing wood using AspWood ........................................................................................................................ 59 Figure 3.4 In situ hybridization analysis of PtCPU expression in developing secondary xylem of one-month-old P. trichocarpa stems .............................................................................. 60 Figure 3.5 The top 25 co-expressed genes to PtCPU after mean-centering, based on 384 P. trichocarpa developing wood transcriptomes ................................................................ 61 Figure 3.6 Expression analysis of wild-type and transgenic poplar over-expressing PtCPU ..... 64 Figure 3.7 Mean fibre lengths measured for wild-type and transgenic poplar over-expressing PtCPU ............................................................................................................................. 65 Figure 3.8 Microfibril angle and crystallinity analysis for transgenic poplar over-expressing PtCPU ............................................................................................................................. 66 Figure 3.9 Over-expression of 35S:YFP-PtCPU in Arabidopsis Col-0 increases secondary cell wall deposition in interfascicular fibres compared to wild-type .................................... 67 Figure 3.10 Western analysis of LexA-AtCPU expression in the yeast (Saccharomyces cerevisiae) strain NMY51 ............................................................................................... 68 Figure 3.11 Yeast two-hybrid screen of LexA-AtCPU identified an armadillo-repeat protein and Cellulose-Microtubule Uncoupling 1 to be strong interactors ....................................... 69 Figure 3.12 Protein-protein interaction of CPU and CMUs from Arabidopsis (A. thaliana) and poplar (P. trichocarpa) ................................................................................................... 71 xiii  Figure 3.13 AtCPUpro:GUS expression analysis in bolting Arabidopsis inflorescence stems ... 73 Figure 3.14 Expression analysis of CMU1 and CMU2 in bolting Arabidopsis inflorescence stems ............................................................................................................................... 74 Figure 3.15 Reverse-transcription PCR analysis of cpu, cmu1cmu2, and cpucmu1cmu2 mutants relative to actin ............................................................................................................... 76 Figure 3.16 Anatomical analysis of the basal portion of the main inflorescence stem for WT, cpu, cmu1cmu2, and cpucmu1cmu2 mutants stained with toluidine blue-O .................. 77 Figure 3.17 Quantification of secondary cell wall thickness in interfascicular fibre cells of WT, cpu, cmu1cmu2, and cpucmu1cmu2 ............................................................................... 78 Figure 3.18 Holocellulose and alpha-cellulose content in stems of wild-type and cpu, cmu1cmu2, and cpucmu1cmu2 mutants ......................................................................... 80 Figure 4.1 An unrooted maximum likelihood phylogeny of the Nitrate1/Peptide Family transporters from land plants .......................................................................................... 96 Figure 4.2 In silico expression analysis of PtNPF6.1 and its paralog PtNPF6.2. ....................... 98 Figure 4.3 Expression of PtNPF6.1pro:GUS in the vascular tissue of one-month-old greenhouse-grown transgenic poplar ............................................................................ 100 Figure 4.4 Gene expression analysis of RNAi PtNPF6.1 lines ................................................. 101 Figure 4.5 Three-month-old WT and RNAi PtNPF6.1 lines before and at day eight of outdoor high-light/ultraviolet-B exposure.................................................................................. 104 Figure 4.6 Representative HPLC chromatograms (520 nm) of anthocyanins in WT and transgenic poplar RNAi PtNPF6.1 lines before and during high light/UV-B exposure  ...................................................................................................................................... 105 Figure 4.7 Anthocyanins quantified as cyanidin-3-glucoside for wild-type and transgenic poplar RNAi PtNPF6.1 lines exposed to natural outdoor high-light/UV-B stress for four days ...................................................................................................................................... 106 Figure 4.8 Total phenolics quantified as gallic acid equivalents for wild-type and transgenic RNAi PtNPF6.1 poplar lines before and during exposure to high-light/UV-B stress . 107  xiv  List of Abbreviations  ABC ATP-Binding Cassette AM Association Mapping 3-AT 3-amino-1,2,4-triazole BAR Bio-Analytic Resource  BBEL Berberine Bridge Enzyme Like BLAST Basic Local Alignment Tool cDNA complementary DNA CesA Cellulose Synthase CIM Callus Inducing Media CMU Cellulose-Microtubule Uncoupling CORD Cortical Microtubule Disordering CPU Coiled-coil Protein of Unknown Function CSC Cellulose Synthase Complex CTAB hexadecyltrimethylammonium bromide DAB 3,3’-diaminobenzidine DIG digoxigenin DUF Domain of Unknown Function eFP electronic Fluorescent Pictograph FRA Fragile Fiber GFP Green Fluorescent Protein GUS β-glucuronidase GWAS Genome-Wide Association HCT Hydroxycinnamoyl Transferase HPLC High Performance Liquid Chromatography HRP Horseradish Peroxidase IBA Indole Butyric Acid IM Induction Medium 2iP N6-(2-isopentenyl)adenine IQD IQ67 Domain IRX Irregular Xylem LAG Lignin-Associated Gene LPI Leaf Plastochron Index MAP Microtubule-Associated Protein MS Murashige and Skoog NAA 1-Naphthaleneacetic Acid NPF Nitrate Peptide Family NRT1 Nitrate1 Transporter NST NAC Secondary Wall Thickening Promoting Factor PCC Pearson correlation coefficient POF Protein of Obscure Function PUF Protein of Unknown Function PTR Peptide Transporter SCW Secondary Cell Wall xv  SD Synthetic Defined SLC15 Solute Carrier 15 SMART Simple Modular Architecture Research Tool SND Secondary Cell Wall NAC Domain  SNP Single Nucleotide Polymorphism T-DNA Transfer-DNA TE Tracheary Element UMAMIT Usually Multiple Acids Move In and Out Transporter UV ultraviolet VND Vascular-Related NAC-Domain WT Wild-Type X-gluc 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid YFP Yellow Fluorescent Protein Y2H Yeast Two-Hybrid  xvi  Acknowledgements  First, I would like to express my sincere gratitude to my supervisors, Dr Carl Douglas, Dr Jürgen Ehlting, and Dr Lacey Samuels. Thank you to Dr Carl Douglas and Dr Jürgen Ehlting for the opportunity to work together, and to pursue this research project. Thank you to Dr Samuels for the opportunity to be a part of your lab and helping me reach the finish line. Each of you have provided exceptional guidance and support in many ways throughout this challenging experience and I am truly grateful to have been mentored by you.   I would also like to thank my committee members, Dr Ljerka Kunst and Dr Shawn Mansfield, for their support and input into my research. Thank you to the Douglas Lab, the Samuels Lab, and the past and present members of the Ehlting Lab and Constabel Lab for their assistance and support. Many other people have contributed to my research. For cell wall analyses assistance, I would like to thank the Mansfield Lab, in particular, Dr Faride Unda, Yaseen Mottiar, Pablo Chung, Foster Hart, and Francis De Araujo. I would also like to thank Dr Staffan Persson and Dr Marc Somssich for providing the cmu1cmu2 (designated here as cmu1-1cmu2) seeds.  A thank you to Dr Taku Demura for giving me the opportunity to have a foreign scientific experience in the Demura Lab, and Dr Misato Ohtani and Nobuhiro Akiyoshi for taking the time to assist me in learning in situ hybridization and their efforts on the completion of the experiment. I would also like to thank Dr Hitoshi Endo for his hospitality during my exchange, as well as the Demura Lab, for making me feel welcome. I would also like to thank Dr Masa Yamaguchi for insightful research discussions during his sabbatical in the Douglas Lab.  I would like to thank the undergraduate students that I have worked with during the course of my studies, in particular Tyler Irwin for his dedication to this research. I am very appreciative of your efforts, as well as the efforts of Ayumi Carrington and Junsu Kwon. I would also like to thank Dr Barbara Hawkins and Samantha Robbins for assistance with the elemental analysis. A thank you to Dr Bob Chow, Dr Louise Page, and Nova Hanson for use of their microscopes. I would also like to thank Andre Bindon for his efforts related to the free amino acid analysis. xvii   Although the RNA-seq experiment was not discussed here, I would like to extend a thank you to Dr Jim Mattson and Juan Aldana for guidance in creating RNA-seq libraries, Dr Mike Rott, Heidi Rast, and Ian Boyes for allowing access and assistance with the NextSeq sequencer, and Kate Donaleshen for assistance with data analysis.  I am very appreciative of my training experience while under the “Working on Walls” group, and would like to thank Dr Brian Ellis, Margaret Ellis, and Martha Kertesz for their efforts in creating a fulfilling graduate student experience. I would like to thank Dr Miki Fujita and Dr Melissa Roach for providing training in areas related to my research while part of this group. I would also like to thank Alice Luo, Veronica Oxtoby, Isabel Ferens, and the Department of Botany office for their assistance over the years. Thank you to the Centre for Forest Biology at the University of Victoria for allowing me to share your space. I would also like to thank Brad Binges for access to the Bev Glover Greenhouse Facility and assistance with plant maintenance. I am thankful to have had the opportunity to conduct my research at two different universities, and to have met great friends with a common interest in plants.   Lastly, I would like to thank my family and friends for their continued love and support, especially the Cheng family for welcoming me into their home. I appreciate the help, support, and friendship of the many people that have made this experience possible and I apologize for not being able to acknowledge everyone. This experience would also not be possible without generous funding from NSERC CREATE, NSERC, the University of British Columbia, and the Department of Botany.    xviii  Dedication    In memory of Professor Carl Douglas    1  Chapter 1. Introduction  Vascular plants include lycophytes, monilophytes, and seed plants, all of which contain xylem and phloem. Xylem enables the long-distance transport of water while phloem is largely responsible for the translocation of organic compounds such as amino acids and carbohydrates. Xylem is impregnated with lignin, a hydrophobic phenolic polymer that facilitates water transport and provides mechanical stability for erect growth. The genetic factors that regulate its development have been studied primarily in seed plants as xylem is the basis for secondary xylem or wood, and thus provides potential opportunities for the improvement of wood-based products.  1.1 Xylem and tracheary element formation Xylem is a complex tissue composed of tracheary elements (TEs), xylary fibres, and parenchyma (Demura and Fukuda, 2007). TEs include tracheids and vessel elements, the lignified cells required for water and solute conduction (Myburg et al., 2013). Fibres are sclerenchymatic cells that impart structural support to the plant body. Xylem is produced during primary and secondary growth and is referred to as primary and secondary xylem, respectively. During primary growth, meristematic cells of the procambium (derived from the shoot apical meristem) divide and differentiate to form primary xylem. In plants that undergo secondary growth, for example trees, secondary xylem or wood develops from periclinal divisions of the vascular cambium, a lateral meristem derived from the procambium (Schuetz et al., 2013).  Xylem TE development has been studied in model systems such as zinnia (Zinnia elegans) mesophyll cells (Fukuda, 1997; Pesquet et al., 2005) and Arabidopsis (Arabidopsis thaliana) suspension cells (Kubo et al., 2005; Derbyshire et al., 2015; Takeuchi et al., 2018). Despite not undergoing extensive secondary growth, Arabidopsis inflorescence stems have also been instrumental in the identification of genes required for secondary cell wall biosynthesis (Ye, 2002; Demura and Fukuda, 2007). Woody perennial plants including eucalyptus (genus Eucalyptus), poplar (genus Populus) and pine (genus Pinus) are also important models for wood development (Jansson and Douglas, 2007; Courtois-Moreau et al., 2009; Jokipii-Lukkari et al., 2018). TE and fibre development require a series of sequential coordinated steps common to both primary and secondary xylem development which includes cell expansion, secondary cell 2  wall deposition, lignification, and programmed cell death (Bollhöner et al., 2012). During cell expansion, vessels undergo radial expansion while fibres undergo longitudinal expansion via intrusive growth (Mellerowicz et al., 2001). In the end, mature TEs and fibres are formed and possess the structural features that facilitate their physiological functions.   The deposition of secondary cell walls (SCWs) in TEs occurs in specialized patterns. For example, protoxylem SCWs form in an annular or helical pattern that permits axial elongation during primary growth. Secondary cell wall deposition in metaxylem, on the other hand, exhibits pitted or reticulate patterns (Schuetz et al., 2014). Cortical microtubules regulate SCW deposition (Oda et al., 2005), likely facilitated by microtubule-associated proteins (MAPs). For example, MAP70-5 identified from Arabidopsis TEs in vitro appears to restrict the area of secondary cell wall deposition (Pesquet et al., 2010). Cortical Microtubule Disordering 1, identified from transcriptomic analysis of MAPs in developing xylem, is another MAP required for pitted secondary cell wall patterning in metaxylem vessels (Sasaki et al., 2017). As the patterned deposition of SCWs in TEs involves MAPs that facilitate cortical microtubule organization, other novel proteins of a related function also likely contribute to secondary cell wall deposition and remain to be identified.  1.2 Secondary cell wall biosynthesis  Secondary cell walls provide mechanical support to specialized cell types and are deposited between the primary cell wall and the plasma membrane after cell expansion has completed, often in multiple layers each containing cellulose, hemicelluloses, and lignin (Samuels et al., 2006). Secondary cell walls are comprised of an S1, S2 and S3 layer, where the cellulose microfibrils are deposited in a parallel orientation and differs in each layer (Plomion et al., 2001; Donaldson, 2008). S2, the thickest layer, is largely responsible for the mechanical strength (Mellerowicz et al., 2001). The biosynthesis of these components has largely been studied using the Arabidopsis inflorescence stem as a model since both primary xylem cells and interfascicular fibres contain lignified secondary cell walls (Liepman et al., 2010; Strabala and MacMillan, 2013). These walls resemble their equivalents found in the secondary xylem of woody plants.  3  Cellulose, the major constituent of both primary and secondary cell walls, is a polymer of β-1,4 glucose molecules arranged in microfibrils (McFarlane et al., 2014). Cellulose microfibrils are synthesized by cellulose synthase (CesA) complexes (CSCs) localized at the plasma membrane. In Arabidopsis, there are ten CesA genes (Endler and Persson, 2011). CesA4, CesA7, and CesA8 encode for the subunits that form the CSCs required for cellulose deposition in SCWs of Arabidopsis (Taylor et al., 2003), which were first identified from genetic screens based on collapsed vessel phenotypes in mutants (Turner and Somerville, 1997; Taylor et al., 1999; Taylor et al., 2000). While ten CesAs have been identified and well characterized in Arabidopsis, up to 18 CesA genes have been identified in poplar (Djerbi et al., 2004). Visualization of fluorescent tagged-CesA7s expressed in epidermal cells of a transgenic tracheary element induction system in Arabidopsis revealed secondary cell wall CesAs to be enriched in the plasma membrane where cortical microtubules bundled (Watanabe et al., 2015), suggesting cellulose deposition during secondary cell wall biosynthesis is reliant on MT organization as previously seen during cellulose deposition in primary cell walls (Paredez et al., 2006).  Hemicelluloses associate with cellulose microfibrils via hydrogen bonds to form part of the cell wall matrix (Scheller and Ulvskov, 2010). These polysaccharides are synthesized at the Golgi and consist of a β-1,4-linked glycan backbone decorated with glycosyl side chains (Liepman et al., 2010; Pauly et al., 2013). Xylans are the most abundant hemicellulose in eudicot SCWs and can be substituted with different side chains such as glucuronic acid. In poplar, glucuronoxylan is abundant (Mellerowicz et al., 2001). Considerable progress has been made towards the identification and characterization of different glycosyltransferase families and proteins involved in hemicellulose biosynthesis. Co-expression analysis of CesA7 has aided the identification of candidate glycosyltransferase genes (Brown et al., 2005; Persson et al., 2005). For example, Brown et al. (2005) identified a suite of putative glycosyltransferases using such an approach. While initial analysis of transfer-DNA (T-DNA) insertion mutants corresponding to several of the identified glycosyltransferases did not find collapsed vessels or an irregular xylem (irx) phenotype, further characterization of one of the genes and its paralog, Irregular Xylem 15 (IRX15) and IRX15L, found decreased xylan content in the irx15 irx15l double mutant (Brown et al., 2011; Jensen et al., 2011). However, the authors suggest IRX15/IRX15L to unlikely be a glycosyltransferase but instead represent an undefined class of biosynthetic proteins required for 4  xylan biosynthesis (Brown et al., 2011). While several xylan biosynthetic enzymes have been characterized, the mechanism of hemicellulose biosynthesis is not entirely understood but appears to require protein-protein interactions via the formation of biosynthetic complexes at the Golgi (Meents et al., 2018).  Lignin, the third major compound found in many secondary cell walls, is a heterogeneous phenolic polymer. Lignin biosynthesis stems from the phenylpropanoid pathway and involves at least ten enzymes (Bonawitz and Chapple, 2010). The first step requires the deamination of phenylalanine via phenylalanine ammonium lyase. Subsequent enzymatic conversions of cinnamic acid by cinnamate 4-hydroxylase and 4-coumarate:CoA ligase form p-coumaroyl CoA, the precursor for monolignol biosynthesis (Bonawitz and Chapple, 2010) and other phenolic compounds including flavonoids (Vogt, 2010). During monolignol biosynthesis, p-coumaroyl CoA is esterified to shikimate and hydroxylated to form caffeoyl-shikimate. Caffeoyl shikimate esterase, a lignin biosynthetic enzyme, hydrolyzes caffeoyl-shikimate into caffeate (Vanholme et al., 2013a); its recent identification in Arabidopsis suggests our understanding of lignin biosynthesis is still incomplete. Downstream of caffeoyl-shikimate, a series of enzymes further catalyze the hydroxylation and O-methylation reactions of the aromatic ring and reduction reactions of the terminal carbonyl group to produce the monolignols coniferyl alcohol and sinapyl alcohol (Boerjan et al., 2003). When incorporated into lignin, these monolignols are referred to as guaiacyl (G) and syringyl (S) units, respectively. G and/or S units are common in lignin from angiosperms such as poplar and Arabidopsis; p-coumaryl alcohol-derived p-hydroxyphenyl (H) units can also be found (Bonawitz and Chapple, 2010). The distribution and abundance of monolignols differs widely from species to species, among cell types, and even cell wall layers which reflects the overall chemical and physical diversity of lignin (Campbell and Sederoff, 1996; Vanholme et al., 2012).  1.3 Lignin transport, polymerization, and deposition  Monolignols are synthesized in the cytoplasm and delivered to the extracellular space for polymerization. However, the process by which they are transported across the plasma membrane is not well understood. ATP-binding cassette (ABC) transporter proteins have been implicated in monolignol transport due to their ability to transport molecules of different classes 5  (Verrier et al., 2008). Global transcript profiling of developing Arabidopsis inflorescence stems identified candidate ABC transporter genes but functional validation did not support the monolignol transport hypothesis (Ehlting et al., 2005; Kaneda et al., 2011). Expression analysis of lignifying TEs in Arabidopsis suspension cells found members from ABCC and ABCG to be coordinately expressed with MYB58, a transcriptional regulator of lignin biosynthesis (Takeuchi et al., 2018). Alejandro et al. (2012) reported ABCG29 from Arabidopsis to transport p-coumaryl alcohol across yeast plasma membranes. While there is experimental support for ABC proteins and monolignol transport, it cannot be excluded that other transporter families may also be potential candidates. For example, recent evidence suggests Nitrate Peptide Family (NPF) transporters to have broader substrate specificities than previously thought. Most characterized NPFs indeed transport nitrate or small peptides (Corratgé-Faillie and Lacombe, 2017) but secondary metabolites including glucosinolates and non-N-containing hormones have also been identified as substrates (Kanno et al., 2012; Nour-Eldin et al., 2012). Once in the apoplastic space, monolignols are polymerized via oxidative coupling by laccases and peroxidases that are spatially distributed in the secondary cell walls of vessel elements and fibres (Schuetz et al., 2014; Chou et al., 2018).   1.4 Transcriptional regulation of secondary cell wall development Different regulatory programs exist to control the deposition of SCWs in different cell types and at specific developmental stages (Ehlting et al., 2005; Zhong and Ye, 2007). This regulation is mediated by a hierarchical network of transcription factors, separated into three tiers. The top tier consists of NAC domain proteins, the master switches of secondary cell wall biosynthesis (Nakano et al., 2015). Vascular-Related NAC-Domain 6 (VND6) and VND7, identified from Arabidopsis cell suspension, are master regulators of metaxylem and protoxylem vessel differentiation, respectively (Kubo et al., 2005). Secondary Wall-Associated NAC Domain Protein 1 (SND1)/NAC Secondary Wall Thickening Promoting Factor1 (NST1) are master regulators of fibre cell formation in Arabidopsis (Zhong et al., 2006; Mitsuda et al., 2007; Zhong et al., 2007). The second tier consists of MYB proteins, MYB46 and MYB83 (Zhong and Ye, 2009). Transcription factors from both tiers can regulate downstream transcription factors, such as the lignin-specific MYB58/63 (Zhou et al., 2009), or secondary cell wall biosynthesis 6  genes directly. As a whole, the transcriptional regulation of secondary cell wall biosynthesis is highly complex and likely regulated by feed-forward loops (Taylor-Teeples et al., 2015).  1.5 An alternative approach to forward and reverse genetics to identify new candidate genes The application of forward and reverse genetics has provided significant contributions to our understanding of SCW biosynthesis, for example, the identification of CesA genes. However, despite the identification and characterization of master transcriptional regulators and the biosynthetic enzymes that act directly on SCW biosynthesis, many factors still remain unidentified including the upstream signalling components, regulatory processes, and the interplay with other physiological conditions that indirectly affect this complex biological process. Environmental cues including nitrogen availability in soil, for example, strongly influence SCW biosynthesis in woody plants. Therefore, additional strategies can be applied in attempt to uncover and dissect processes that influence secondary cell wall deposition.  A genetics-based approach that can be used to identify novel genes involved in SCW development is genetic association mapping (AM). Studies of this type aim to link allelic variations in genetic markers across individuals from a natural population to variations of a phenotypic trait (Rafalski, 2010). The genetic markers tested are often single nucleotide polymorphisms (SNPs) since these are most abundant and suitable for large-scale genotyping (Rafalski, 2002). AM can be applied using genetic markers targeted to selected candidate genes, or that span the entire genome. The use of candidate genes relies on choosing genes that have an influence on the phenotypic trait of interest, identifying allelic variants in these genes, and correlating these variants with the trait. Such genes are often chosen based on experimental evidence such as gene expression or a presumed or characterized function (Ingvarsson and Street, 2011).  A genome-wide association (GWAS) approach is a global perspective that tests most, if not all, allelic variants for correlations with the trait of interest. This method has been employed extensively in human genetics but can also be applied to plant biology. Association mapping in Arabidopsis has been successful in identifying genes that have moderate contributions to phenotypic effects, in contrast to mutant screens and the appearance of strong phenotypes. For 7  example, a GWAS experiment conducted for Arabidopsis accessions exposed to drought identified significant SNPs in novel genes not yet implicated in abscisic acid (ABA) stress and were validated to affect ABA levels through reverse genetic analysis of T-DNA insertion mutants (Kalladan et al., 2017). In trees, the first association mapping experiment was conducted in eucalyptus (Eucalyptus nitens) which analyzed allelic variants in cinnamoyl coA reductase, a lignin biosynthetic gene, and its associations to microfibril angle variation (Thumma et al., 2005). With the current accessibility to single nucleotide polymorphism genotyping arrays and sequencing technologies, it is now feasible to identify genetic markers en masse and assess them in genetic association mapping studies for both model and non-model plants, including trees.   1.6 Genetic association mapping in poplar Black cottonwood (Populus trichocarpa) and other poplars have been developed as model trees, partly because of the numerous genomic resources available including a sequenced genome (Tuskan et al., 2006; Jansson and Douglas, 2007). Phenotypic variations are evident within the natural range of P. trichocarpa, which extends along the west coast of North America from Alaska to Mexico (Brunner et al., 2004). The underlying genetic variation that causes the phenotypic differences can be examined by sequencing individuals from the population. For example, 0.5 million putative SNPs were identified in more than 26,000 genes from 20 xylem transcriptomes of natural P. trichocarpa accessions (Geraldes et al., 2011). This suggests that natural populations are suitable for genetic association mapping studies to identify genetic variants that influence phenotypic differences such as wood-related traits.  Association genetics has been conducted previously in poplar (Wegrzyn et al., 2010; Guerra et al., 2013). More recently, Porth et al. (2013a) conducted a large-scale candidate gene-based association genetics study using clonal replicates of unrelated P. trichocarpa individuals from the Pacific Northwest coast of North America (Xie et al., 2009) planted in a common garden. First, a SNP genotyping array was used to genotype over 300 individuals at multiple SNP sites to identify SNPs in 3,500 genes which potentially contribute to SCW formation (Geraldes et al., 2013). Using the same individuals, 17 different wood traits including cell wall chemistry and ultrastructure were quantified (Porth et al., 2013b). Genetic association of the SNP data with the phenotypic measurements found 141 significant SNPs in numerous genes 8  associated with 16 different wood traits, including multiple lignin-related phenotypes (Porth et al., 2013a). Current work now involves understanding the biological function of these genes and how their genetic variants influence wood trait variation. This work may contribute to our understanding of the underlying mechanisms that influence SCW structure and therefore, will help optimize poplar for commercial uses, including biofuel production.  1.7 Poplar and Arabidopsis as model systems Arabidopsis is the classical model for many fields of plant biology due to its small physical size, short generation time and usefulness for genetics-based studies (Koornneef and Meinke, 2010). Poplar has been developed as a model tree because of comparable traits including rapid growth, ease of propagation, and amenability to molecular manipulations in the laboratory. Although Arabidopsis and poplar exhibit different angiosperm traits, the former being an herbaceous annual and the latter a woody perennial, the two species share a relatively close phylogenetic relationship as members of the Eurosid clade (Jansson and Douglas, 2007). However, there are some physiological traits that are better explored in woody plants such as the phenotypic plasticity of poplar in response to nitrogen as carbon partitioning is influenced by nitrogen content in the environment (Novaes et al., 2009). With the abundance of genomic resources available for both plants including entire sequenced genomes, comparative studies can be undertaken to determine the extent of conserved genes that contribute to wood and secondary cell wall formation.   Many poplar genes have Arabidopsis orthologs (Tuskan et al., 2006) and such genes and their respective gene families can be studied in both systems using common tools and techniques. One advantage of using Arabidopsis as an initial system for investigation, however, is the more rapid pace of reverse genetic approaches. The large collection of T-DNA insertion lines available for many genes in the Arabidopsis genome is a useful resource to study gene function (O’Malley and Ecker, 2010). Orthologous genes with putative roles related to wood and SCW development can then be further investigated in poplar. For example, SND1, a key transcriptional regulator of SCW biosynthesis in fibre cells was first identified in Arabidopsis. Subsequent studies in poplar found orthologs with similar functions (Zhong et al., 2010a; Ohtani et al., 2011). This suggests that aspects of SCW biosynthesis and its regulation are conserved in herbaceous annuals and woody 9  plants. Thus, Arabidopsis is suitable as an initial model to investigate genes identified in poplar. Many secondary cell wall-related genes identified in Arabidopsis have yet to be explored in woody plants, including poplar. By pursuing such comparative studies our knowledge of conserved biological processes will be enhanced.  1.8 Research objectives Trees are long-living perennials that store a significant amount of carbon in secondary cell walls of wood which is composed primarily of a matrix of cellulose, hemicellulose, and lignin. This carbon represents a renewable resource that humans use for various purposes, including lumber, pulp and paper, and fuel. Expanding the sustainable use of wood has the potential to alleviate our dependence on fossil fuels. Lignin is a major component of wood and is one of the most abundant biological polymers on Earth. Its composition and abundance defines many wood properties. For example, lignin causes wood to be resistant to both chemical and enzymatic degradation as it forms tight associations with other cell wall components such as cellulose. Therefore, in order to optimize wood-based SCWs for different applications, we must first understand the genetic and molecular factors that affect their biosynthesis.   Many investigations have focused on lignin biosynthesis, mainly the biosynthetic enzymes and transcriptional regulators of the pathway. However, other classes of genes that have a direct or indirect impact on lignin content and composition variation remain largely enigmatic. I hypothesized the lignin-associated genes from the poplar genetic association mapping experiment (Porth et al., 2013a) to contribute to lignin trait variation in wood and/or secondary cell walls. To test this hypothesis, my first objective was to screen the lignin-associated genes to identify two candidates for functional characterization. In Chapter 2, I analyzed 27 genes significantly associated with lignin traits from Porth et al. (2013a) using in silico analyses (sequence similarity-based trees and gene expression mining) and a reverse genetic screen of Arabidopsis T-DNA insertion mutants to identify homologs that exhibit phenotypes characteristic of cell wall defects. My second objective was to characterize two selected candidate genes, CPU and PtNPF6.1, using a combined functional genomics approach in both poplar and Arabidopsis to determine whether the morphological phenotype(s) caused by mis-expression and/or loss of function of the lignin-associated genes affected lignin traits and to 10  understand their biological functions related to wood and/or secondary cell wall development. In Chapter 3, I characterized a Coiled-coil Protein of Unknown Function (CPU) which did not influence lignin traits but possibly cell wall ultrastructure. In Chapter 4, I characterized a P. trichocarpa Nitrate Peptide Family 6.1 (PtNPF6.1) transporter protein which appears to impact nitrogen redistribution in planta and imparts an indirect effect on lignin composition and soluble phenolics.  11  Chapter 2. Initial characterization of novel candidate secondary cell wall biosynthesis genes identified through genetic association mapping in poplar  2.1 Introduction  Trees are long-lived perennials that accumulate a significant amount of secondary xylem, or wood, during their lifetime. As a renewable resource, trees provide a number of wood-based products including lumber, pulp and paper, and potential raw material for biofuels (Sannigrahi et al., 2010; Porth et al., 2013b). Efficient conversion of lignocellulosic biomass to alternative fuel sources has limitations however, largely because of the complex structure of wood. To overcome this challenge, our current knowledge of wood development needs to be expanded. Further investigation of the genetic factors that influence wood traits will increase our basic understanding of this important developmental process which may assist in the selection of ideal genotypes for various applications, including biofuel production.  Lignified secondary cell walls (SCWs) provide structural reinforcement to the secondary xylem (Groover et al., 2010). Secondary xylem is produced from radial divisions of initial cells located in the vascular cambium, and accumulates on the inner side of the cambium (Myburg et al., 2013; Schuetz et al., 2013). In angiosperms, these cells differentiate into ray parenchyma, xylary fibres, tracheids, and vessel elements, of which the latter three are rich in lignin. While secondary xylem is a distinguishing feature of woody plants, a similar tissue can also form in the stem, hypocotyl and root of the model plant Arabidopsis (Arabidopsis thaliana) (Dolan et al., 1993; Chaffey et al., 2002; Barra-Jiménez and Ragni, 2017). Under short day conditions, an abundance of xylary fibres and vessel elements that resemble those of woody angiosperms such as poplar are deposited in the hypocotyl but ray parenchyma, which facilitate metabolite storage and transport, are absent (Murakami et al., 1999; Chaffey et al., 2002). As this herbaceous annual can undergo secondary growth it can be used as a model for the identification of potential genes involved in wood and SCW development (Ubeda-Tomas et al., 2007) for further investigation in woody plants.  In the SCW, the lignin polymer is embedded in a matrix of cellulose and hemicellulose. Cellulose, a highly structured polymer of β-1,4-linked glucose residues, is synthesized via the activity of cellulose synthase (CesA) complexes at the plasma membrane (McFarlane et al., 12  2014). In Arabidopsis, cellulose biosynthesis in the SCW requires the catalytic activity of CesA4, CesA7, and CesA8 (Taylor et al., 2000; Taylor et al., 2003). Hemicelluloses cross-link the cellulose microfibrils of the SCW. In angiosperms, xylans are the most common hemicelluloses (Pauly et al., 2013) and glycosyltransferases localized at the Golgi are required for their biosynthesis (Wu et al., 2009; Scheller and Ulvskov, 2010). Unlike cellulose and hemicellulose, lignin is a phenolic polymer comprised mainly of three cinnamyl-derived alcohols or monolignols, coniferyl alcohol, sinapyl alcohol, and p-coumaryl alcohol. When incorporated into lignin, guaiacyl (G), syringyl (S), and p-hydroxyphenyl (H) units are produced, respectively (Bonawitz and Chapple, 2010; Vanholme et al., 2012). While in vitro studies have aided the characterization of lignin biosynthetic enzymes (Humphreys et al., 1999; Schoch et al., 2001; Franke et al., 2002), it is largely from genetic screens in Arabidopsis that genes required for SCW biosynthesis have been identified (Turner and Somerville, 1997; Ruegger and Chapple, 2001; Zhong et al., 2005). Genetic mutations that affect the biosynthesis of SCW components can result in morphological abnormalities such as severe dwarfism (Franke et al., 2002; Persson et al., 2007) and pendant stems (Zhong and Ye, 1999; Ratcliffe et al., 2000; Zhong et al., 2006; Mitsuda et al., 2007). Collapsed vessel elements, referred to as irregular xylem (irx), are also characteristic of mutants impaired in SCW biosynthesis; the identification of irx mutants has led to the characterization of numerous SCW-related genes in Arabidopsis (Turner and Somerville, 1997; Brown et al., 2005; Persson et al., 2005; Grienenberger and Douglas, 2014).  In addition to forward genetic approaches, reverse genetic approaches are also utilized for functional genomics-based gene characterization. In many cases, genes of interest are first identified from in silico analyses such as phylogenetic relationships or co-expression networks and then investigated in planta. A well-annotated Arabidopsis genome (The Arabidopsis Genome Initiative, 2000) complemented with a collection of thousands of transfer-DNA (T-DNA) insertion mutants (Alonso et al., 2003) has enabled large-scale reverse genetic screens to identify phenotypes that may infer gene function (Krysan et al., 1999; Sessions et al., 2002; O’Malley and Ecker, 2010). While Arabidopsis does not produce true wood, its inflorescence stem is abundant in lignified cells, including an extensive ring of interfascicular fibres between the vascular bundles (Ehlting et al., 2005; Strabala and MacMillan, 2013) and thus Arabidopsis is a suitable system to investigate genes that contribute to SCW biosynthesis. 13  A significant portion of our current understanding of cellulose, hemicellulose, and lignin biosynthesis is based on experimentation in Arabidopsis. However, studies in woody plants such as eucalyptus (Eucalyptus spp.) and poplar (Populus spp.) have broadened our knowledge of this highly regulated process (Paux et al., 2005; Ohtani et al., 2011; Zhong et al., 2011; Hefer et al., 2015). Lignin biosynthesis, for example, is an attractive target for genetic manipulation because traits such as lignin composition define wood properties and are important considerations during industrial processing. Consequently, orthologs of transcription factors and biosynthetic enzymes known to alter lignin content and composition in Arabidopsis have been characterized in poplar (Coleman et al., 2008; Zhong et al., 2010a). While our knowledge of lignin biosynthesis is advanced, it is far from complete as biosynthetic enzymes such as caffeoyl shikimate esterase (Vanholme et al., 2013a) are still being uncovered. Furthermore, we are only starting to understand how physiological responses to internal and external stimuli influence wood and SCW biosynthesis in trees. Therefore, untargeted approaches that are not reliant upon previous knowledge offer new opportunities for gene discovery.  One such approach is genetic association mapping (AM), which exploits the genetic variation that exists between individuals in order to associate genotypes with phenotypes using correlation analyses (Chan et al., 2011). The identification of genetic variants, mainly single nucleotide polymorphisms (SNPs), that are causal or very closely linked to the causal variant for a specific quantitative trait can be beneficial for breeding, or even for novel gene discovery. AM has been applied to Arabidopsis as well as crop plants to find genetic variants associated, or co-inherited, with complex traits such as disease-resistance and flowering time (Aranzana et al., 2005; Buckler et al., 2009; Ehrenreich et al., 2009). In contrast to classical genetic mapping techniques that utilize segregating crosses, AM can be applied to less-related individuals, or even species-wide populations (Zhao et al., 2007; Chan et al., 2011). However, AM requires a large number of SNPs to be assessed since only SNPs in very close proximity to the causal variant will be co-inherited within a relatively unrelated population (Ingvarsson and Street, 2011).  The genus Populus (aspens, cottonwoods, and poplars) is ideal for studying wood and SCW development through AM to improve lignocellulosic traits for bioethanol and other industrial applications. Aside from favourable wood properties and the availability of an 14  extensive genomic toolbox (Tuskan et al., 2006; Devappa et al., 2015), poplars are fast-growing woody perennials that are wide-spread across the Northern Hemisphere and can thrive on marginal soils. Given the large degree of phenotypic variation within a natural population (Brunner et al., 2004; Porth et al., 2013b) and the abundance of sequence data available including mapped SNPs for multiple P. trichocarpa accessions (Gilchrist et al., 2006; Tuskan et al., 2006; Geraldes et al., 2011), AM studies are feasible in poplar.  The first AM study for cellulose and lignin-related traits in poplar (P. trichocarpa) tested SNPs in 40 candidate genes (Wegrzyn et al., 2010). Porth et al. (2013a) conducted a similar but larger-scale AM experiment and tested SNP associations in c. 3,500 candidate genes with multiple wood-related traits, including lignin content and composition, using 334 natural accessions of P. trichocarpa grown in a common garden. Significant genetic associations to different lignin-related phenotypes were detected for 25 genes of different functional classes, most of which have not yet been implicated in a SCW-related function. The wealth of genomic, phenotypic and genetic association data from Porth et al. (2013a) identified numerous novel candidate genes to be involved in wood or SCW development in poplar. However, this woody perennial species is not amenable to medium- or high-throughput studies for functional validation as loss of function mutants are not available and gene knock-down experiments in poplar are time-consuming. In contrast, Arabidopsis T-DNA insertion mutant collections are publicly available and have been used extensively for reverse genetic characterization. Therefore, my research objective for this chapter was to survey the lignin-associated genes from Porth et al. (2013a) by using in silico and reverse genetic approaches in Arabidopsis to identify a subset of novel genes for in-depth functional characterization in poplar. I hypothesize that at least some of the AM genes have a direct effect on SCW biosynthesis. From this analysis, our understanding of how genes of different functional classes contribute to secondary cell wall-related traits will be enhanced.     15  2.2 Materials and Methods  2.2.1 Sequence analysis  Protein sequences for each of the lignin-associated genes from Porth et al. (2013a) were searched using the P. trichocarpa genome (version 2.2) hosted on Phytozome (https://phytozome.jgi.doe.gov/), the Plant Comparative Genomics portal of the Department of Energy’s Joint Genome Institute (Goodstein et al., 2011; Grigoriev et al., 2012). For each gene, homologous protein sequences from selected embryophyte genomes (Physcomitrella patens, Selaginella moellendorffii, Brachypodium distachyon, Oryza sativa, Sorghum bicolor, Arabidopsis thaliana, Eucalyptus grandis, Medicago truncatula, and Ricinus communis) were also analyzed. Sequences sharing at least 60% similarity were retained and aligned, and sequence similarity trees (Dereeper et al., 2010) were generated using the automated bioinformatics platform JalView available within Phytozome. The output trees were visualized using FigTree (version 1.4.3).   2.2.2 Gene expression and co-expression analysis  In silico expression data was obtained through the Bio-Analytic Resource (BAR) for Plant Biology (bar.utoronto.ca) electronic Fluorescent Pictograph (eFP) browsers (Winter et al., 2007; Wilkins et al., 2009). Absolute expression values were analyzed. Co-expression data was obtained through the Arabidopsis Legacy Expression Angler (Toufighi et al., 2005), also available from the BAR. The top 25 correlated genes from the “AtGen Plus-Extended Tissue Compendium” were analyzed after median centering and normalization. Co-expression data was also obtained through the network drawer function from ATTED-II (atted.jp) (Obayashi et al., 2009). The developing xylem and leaf transcriptome data was obtained from the analysis of 20 P. trichocarpa accessions as described in Geraldes et al. (2011).  2.2.3 Plant growth conditions and analysis T-DNA insertion lines were identified using The Arabidopsis Information Resource (www.arabidopsis.org). Where available, two independent T-DNA lines corresponding to A. thaliana homologs of the P. trichocarpa lignin-associated genes, as identified from the sequence similarity trees, were obtained from the Arabidopsis Biological Resource Centre. The SALK insertion lines were preferred (Alonso et al., 2003) and chosen based on the T-DNA insertion 16  position, where insertions located in the exon were most preferred and homozygosity considered. Seeds were surface-sterilized using 70% ethanol for 1 min and sown on solid half-strength Murashige and Skoog (MS) medium (2.15 g L-1 MS salts (PhytoTechnology Laboratories), 1% (w/v) sucrose, 0.6% (w/v) agar, pH 5.8). Seeds were stored for 48 h at 4°C in the dark and transferred to a plant tissue culture chamber (Caron) maintained at 22°C under long day conditions (16 h light, 8 h dark). Ten-day-old seedlings were transferred to Sunshine Mix #4 (Sun Gro Horticulture) and maintained in a growth chamber (Conviron) at 21°C under long day conditions and 110 to 120 µmol photons m-2 s-1. Genomic DNA was isolated from rosette leaves in 400 µL of extraction buffer (250 mM NaCl, 200 mM Tris-HCl, 25 mM EDTA, 0.5% (v/v) SDS) and 300 µL of 2-propanol, and resuspended in 100 µL of TE (Weigel and Glazebrook, 2002). Primer sequences for genotyping T-DNA insertion lines (Table 2.1) were obtained using the iSect Primers function from the SIGnAL SALK T-DNA Express: Arabidopsis Gene Mapping Tool (http://signal.salk.edu/cgi-bin/tdnaexpress). For the SALK lines, the left border primer LBb1.3 (5’-attttgccgatttcggaac-3’) was used. For the SAIL lines, one of three left border primers (LB1: 5’-gccttttcagaaatggataaatagccttgcttcc-3’; LB2: 5’-gcttcctattatatcttcccaaattaccaataca-3’; LB3: 5’-tagcatctgaatttcataaccaatctcgatacac-3’) was used. T-DNA insertion lines were genotyped as described by O’Malley and Ecker (2010).  2.2.4 Microscopy Manual cross-sections of the basal portion (bottom 1 cm) of the main inflorescence stem (at least 15 cm in height) of five to six-week-old Arabidopsis plants were stained with 0.25% (w/v) toluidine blue-O (Sigma-Aldrich) for histochemical analysis. Sections were mounted in water, and viewed and imaged with bright-field settings using a Nikon Eclipse TE2000-U equipped with a Nikon Digital Sight DS-U1 camera and ACT-2U control software. For the T-DNA insertion mutant screens, confirmed homozygous lines were analyzed and compared with wild-type (Columbia-0 (Col-0) for SALK lines and Col-3 for SAIL lines). In cases where homozygous lines were not identified, T-DNA insertion mutants were re-analyzed in the next generation. Mutant lines with putative phenotypes observed during the initial screen were re-grown and re-analyzed in the subsequent generation.   17         Table 2.1 Arabidopsis T-DNA insertion lines analyzed for homologs of the lignin-associated genes from Porth et al. (2013a)  Lignin Trait  Associated Gene ATG ID T-DNA Linea Position Left Primer (5’ to 3’) Right Primer (5’ to 3’)  Insoluble UMAMIT9 POPTR_0001s06980 At5g07050 SALK_022640 intron ggggattcgaatatggttttc gtttgtgatggaacacaaccc     SALK_095303 promoter accgtttctactcccatcctg gttattttcggctgagccttc  CWP POPTR_0005s07810* At4g39840 SALK_028622 exon ctacggaagctatggttggtg agagaatctgcaacatgacgg     SALK_093172 5’ UTR gcagatctgtccaaatcaagc aaaagtttccaaccatccacc  LAX2 POPTR_0009s13470* At2g21050 CS808596 exon aacctgaatgacatggtttctg   atttgctcgagaaacgaaagc  MAP20 POPTR_0017s01510* At5g37478 SALK_021552 5’ UTR tttggggatgtaacagctctg gaaggaaatggttccaaaagc  P4H7 POPTR_0017s11150 At3g28480 SALK_004428 exon tgggtccaaaattcctctacc ttcctcatcagaaagaaaccc     SALK_039733 promoter gtgtgtgtggacgatcatgag aaatccgagaatccattttcg    At3g28490 SALK_067682 exon tgccacacaatatttttctgaag taccctcaggaagaaaggtcc     SALK_101949 promoter aaaatgccctgaagaaaccac ggtataccgcgggttaaaatc Soluble SKS12 POPTR_0001s03760* At1g55560 SALK_017243 exon cgtttaaagtgataccattgcg gaaaaccctttgccttgaatc    At1g55570 SALK_061973 exon cgtcattaatcttccaagccc catcttcagggtcagcgtaag    At3g13390 SALK_096049 exon tgggactccaggaactatgtg gcaaagaaggagtaaccgtcc    At3g13400 SALK_005755 exon gtgtcaacgtgtgatacaccg gtcttcaacaacctcgacgag  FPP7 POPTR_0007s10810* At2g23360 SALK_026499 exon ttgtcctcaatcttgggtttg ctcctcacgaacaaaacgaag     SALK_045879 exon aaaccgttgttgaaagcaatg cccgtaacctttgacactctg Syringyl LHT1 POPTR_0001s36340* At5g40780 SALK_034566 exon ctgtacatccccaaaaatcatg acctgagagacataacggcag     SALK_083700 intron cagctcataactcttgtgccc   tgtcagtgggctaaaaatgtg  NPF6.1 POPTR_0002s03070* At3g54140 SALK_103927 intron ggatgattaacacgtccttcg atggatagatccaatcagggc     CS872049 exon cgctcctttctatttccttgg tagtgcccatgccatagtagg    At5g01180 SALK_022619 exon gagtgacaacaaaggagcagc ctagcggaatctcagcacaac       CS872776 exon ctagcggaatctcagcacaac tgataaggcagcagtggaaac  GH3.9 POPTR_0002s20790* At2g47750 SALK_025354 promoter tccaagatcccacgtaaaatg ttgatgctgtgctgttgaaac  GA2OX2 POPTR_0004s06380* At1g30040 SALK_011239 exon tggagatgtgcaattcaactg ctaacgggaggttcaagagtg    At2g34555 SALK_002824 promoter atggcaactctgatttcatgg tggtgatcatcatctcttgaag  UMAMIT12 POPTR_0006s08270* At2g37450 SALK_151855 promoter gtgcagagggtcctttagtcc attcaatgtttggtttgcgag    At2g37460 SALK_101986 exon gaaaccatgatgcttttgacg agagctcagcagggtaagtcc     CS814465 3’ UTR gctcagcattggcttactacg aagtacgtcaacaaccaaccg  SAR2 POPTR_0010s15180* At1g56330 SALK_013096 promoter aacaggttgcaaacttgaaag tggtttcgtgatttcgtcttc    At3g62560 SALK_020919 intron cctagatgataacgcagctcg aaaaccaccttgcttcacatg    At4g02080 SALK_087289 exon aatcgaacatggttgaattgg atggggagaaaaatttgatgg     CS811207 promoter tgtgctatttgtgggaatttttc ggctgatgctgtaccaatctc  TLP3 POPTR_0014s04020* At1g75030 SALK_068601 exon gccagagattcatcatcatcc aaagcaacattgcaaatttgc     SALK_089145 promoter atttgcaatttccgaatcctc agagtaagaaggccgttgctc    At1g75050 SALK_087483 promoter tatttctgaacccattcgtgg cggtgtaaggacaactgttctg     SALK_141355 intron agattggaagaccatcacacg ttacatttaagaacaccgcc Total NPF6.3 POPTR_0001s12890* At5g13400 SALK_007230 promoter cgatgactttctctgtgagcc cgaaatacgccattctctctg     SALK_080802 promoter aagcctccaagaacagaggag aggaaccaagggctcatagac  WRKY32 POPTR_0006s19850* At4g30935 SALK_062133 promoter ccttgtaccctatctcctccg ggtgaaaggaaatcctcatcc     SALK_091352 5’ UTR gctatcagtctcctgccaatg ttggaagaacgcaaaacaaac           *denotes significant lignin-associated genes from Porth et al. (2013a). aT-DNA lines labelled CS are SAIL lines.18  2.3 Results  2.3.1 Summary of the genetic association mapping study in P. trichocarpa To further understand the genetic factors that contribute to wood and SCW biosynthesis, Porth et al. (2013a) tested genetic associations for 3,543 candidate genes (Geraldes et al., 2013) predicted to influence wood properties (Appendix A.1). The candidate genes were largely based on literature-supported evidence and gene expression profiles in developing xylem (Geraldes et al., 2011). However, other genes included ecophysiology-related genes as environmental conditions also impact wood formation (Berta et al., 2010; Groover et al., 2010; Plavcová et al., 2013). A natural population of 334 unrelated P. trichocarpa individuals grown in a common garden were genotyped for 29,233 SNPs in (and near) the candidate genes and phenotyped for 17 wood chemistry and ultrastructure traits (Porth et al., 2013a); the final analysis found 141 SNPs in 105 different genes to be significantly associated with 16 of the 17 traits (Appendix A.2).  The 105 significantly associated genes represented 11 different categories defined by Porth et al. (2013a). Most of the significantly associated genes were grouped in a category designated as “other,” which included genes that possess distinct functions as well as unknown functions (Figure 2.1). For example, the soluble lignin-associated gene SKU5 Similar 12 (SKS12) was categorized as “other” and is the only associated gene of its kind (Appendix A.2). SKS12 is related to SKU5, a glycoprotein that is structurally-related to the multiple-copper oxidase proteins (Sedbrook et al., 2002). Next, 16% of the significantly associated genes were categorized as “expression” or “transcription.” Transcription factors involved in the regulation of SCW biosynthesis have been well-studied, most notably the Vascular-Related NAC-Domain (VND) and Secondary Wall-Associated NAC Domain (SND) proteins (Kubo et al., 2005; Zhong et al., 2006; Hussey et al., 2011; Endo et al., 2015). Lastly, 2% of the significantly associated genes had functions related to cell wall structural proteins or signalling (Appendix A.2). It should be noted that while some categories such as “cytoskeleton-related” (Appendix A.1) did not appear to be represented by the significantly associated genes (Appendix A.2) it is possible that related genes were grouped in categories such as “expression” or “other.” Thus, genes of broad classes were found to have significant genetic associations with wood and/or SCW biosynthesis. 19   Figure 2.1 Categories of genes significantly associated with wood chemistry and ultrastructure traits identified from a genetic association mapping experiment in poplar (Populus trichocarpa) conducted by Porth et al. (2013a).                          2.3.2 Genes genetically associated with lignin trait variation Wood is recalcitrant to enzymatic degradation during industrial processing due to the presence of lignin, and thus lignin biosynthesis is a target for wood improvement. Porth et al. (2013a) identified genetic associations to insoluble, soluble, and total lignin content variation. Associations to syringyl lignin were also identified, as poplar wood has a relatively high amount of S lignin and increasing the S:G ratio is favourable for chemical processing (Studer et al., 2011; Mansfield et al., 2012). Because of the importance of lignin in defining wood properties, the AM genes associated with lignin trait variation (i.e. insoluble, soluble, total, and S lignin) were chosen as the main focus for functional characterization.  Porth et al. (2013a) reported 25 genes to be significantly associated with lignin content and composition variation (Appendix A.2). Here, these genes will be referred to as the lignin-associated genes (LAGs). Of the 25 LAGs, 12 were associated with S lignin composition and six were associated with insoluble lignin content including BBEL13, a gene also associated with 20  total lignin content (refer to Appendix A.2 for full gene names). The categorical distribution for the LAGs was similar to the complete set of 105 genes significantly associated to all the wood traits tested as most of the genes were categorized as “other” (Figure 2.2). LAGs categorized as “expression,” i.e. genes that were input into the AM experiment based on their expression profiles in developing xylem, and “transport” were also highly represented. Five transporter genes (LAX2, LHT1, NPF6.1, NPF6.3, and UMAMIT12) were significantly associated with lignin content and composition variation even though UMAMIT12 was not categorized as “transport” (Appendix A.2). Overall, only one cell wall carbohydrate metabolism-related gene was significantly associated with lignin content variation (Figure 2.2).                            2.3.3 Screening lignin-associated genes for functional characterization While most of the LAGs reported by Porth et al. (2013a) have not been implicated in wood or SCW development, some have been previously characterized such as Microtubule-Figure 2.2 Categories of genes significantly associated with lignin trait variation in poplar (Populus trichocarpa) identified from a genetic association mapping experiment conducted by Porth et al. (2013a). 21  Associated Protein 20 (MAP20) (Rajangam et al., 2008). Such genes were still examined but the intent was to focus on the novel LAGs. Therefore, I included two additional candidate genes (UMAMIT9 and P4H7) in my analysis that were identified by Ilga Porth et al. during preliminary AM studies (personal communication). However, these genes did not pass the threshold (α ≤ 0.05) in the final analysis (Porth et al., 2013a). A complete list of the LAGs analyzed here is presented in Table 2.2.   Table 2.2 Complete list of lignin-associated genes analyzed identified from a genetic association mapping experiment in poplar (P. trichocarpa) conducted by Porth et al. (2013a)  Poplar Gene ID (v2.2) Category ATG ID A. thaliana Annotationa  Insoluble Lignin POPTR_0001s06980 NA At5g07050 Usually Multiple Acids Move In and Out Transporter 9 (UMAMIT9)b POPTR_0002s03730* Protein Kinase At4g28540 Casein Kinase I-Like 6 (CKL6) POPTR_0002s06080* Protein Kinase At2g42880 Mitogen-Activated Protein Kinase 20 (MPK20) POPTR_0005s07810* Other At4g39840 Cell Wall Integrity/Stress Response Component-Like Protein (CWP) POPTR_0009s13470* Transport At2g21050 Like Auxin Resistant 2 (LAX2) POPTR_0011s16200* Expression  At1g30760 Berberine Bridge Enzyme-Like 13 (BBEL13) POPTR_0017s01510* Expression  At5g37478 Microtubule-Associated Protein 20 (MAP20) POPTR_0017s11150 NA At3g28480 Prolyl 4-Hydroxylase 7 (P4H7)  Soluble Lignin POPTR_0001s03760* Other At1g55570 SKU5 Similar 12 (SKS12) POPTR_0007s10810* Other At2g23360 Filament-Like Plant Protein (FPP7) POPTR_0014s12380* CW Carbohydrate At3g62830 UDP-Glucuronic Acid Decarboxylase 2 (UXS2)  Syringyl Lignin POPTR_0001s10120* Expression  At4g23690 Dirigent Protein 6 (DIR6) POPTR_0001s36340* Transport At5g40780 Lysine Histidine Transporter 1 (LHT1) POPTR_0002s03070* Transport At3g54140 Nitrate1/Peptide Family 6.1 (NPF6.1)c POPTR_0002s20790* Phytohormone At2g47750 Indole-3-Acetic Acid-Amido Synthetase (GH3.9) POPTR_0004s06380* Phytohormone At1g30040 Gibberellin 2-Oxidase 2 (GA2OX2) POPTR_0006s08270* Other At2g37460 Usually Multiple Acids Move In and Out Transporter 12 (UMAMIT12)b POPTR_0006s08720* Other At5g02230 Haloacid Dehalogenase-Like Hydrolase Family Protein (HAD) POPTR_0008s06130* Transcription At5g02030 BEL1-Like Homeodomain 9 (BLH9) POPTR_0010s15180* Other At4g02080 Secretion-Associated RAS Super Family 2 (SAR2) POPTR_0014s04020* Other At1g75030 Thaumatin-Like Protein 3 (TLP3) POPTR_0016s05010* Other At5g06860 Polygalacturonase Inhibiting Protein 1 (PGIP1) POPTR_0018s12720* Expression  At3g07490 Calmodulin-Like 3 (CML3)  Total Lignin POPTR_0001s12890* Transport At5g13400 Nitrate1/Peptide Family 6.3 (NPF6.3)c POPTR_0001s28570* Other At1g07120 Coiled-coil Protein of Unknown Function (CPU) POPTR_0006s19850* Expression  At4g30935 WRKY DNA-Binding Protein 32 (WRKY32) POPTR_0008s11150* Expression  At1g13635 DNA Glycosylase Superfamily Protein (DGP) POPTR_0011s16200* Expression  At1g30760 Berberine Bridge Enzyme-Like 13 (BBEL13)     aA. thaliana annotations are based on Araport (www.araport.org), TAIR (www.arabidopsis.org), and published literature. bUMAMIT proteins are also known as nodulin MtN21/EamA-like transporters (Denancé et al., 2014). cNitrate1/Peptide Family (NPF) proteins cited here are as designated by Léran et al. (2014). *denotes significantly associated genes from Porth et al. (2013a).    22  In order to select novel LAGs for functional characterization, each gene (Table 2.2) was examined using an experimental approach that consisted of in silico and in planta-based analyses (Figure 2.3). First, sequence similarity trees were constructed to determine the number of poplar (P. trichocarpa) and Arabidopsis (A. thaliana) homologs. Next, I used publicly available in silico tools to mine gene expression and co-expression data. Finally, a reverse genetic screen of Arabidopsis T-DNA insertion mutants was carried out to examine the effect(s) of knockout mutations on lignified cells of the vascular tissue. Note, for each experiment, specific criteria were implemented to aid the final selection of two genes for in-depth functional characterization.                            2.3.4 Sequence similarity trees reveal Arabidopsis homologs of the poplar lignin-associated genes Poplar is related to the herbaceous annual Arabidopsis (A. thaliana) as both are members of the Eurosid clade within the angiosperms (Jansson and Douglas, 2007). Since extensive genomic resources are available for Arabidopsis, the first analysis was to determine the number  in silico expression mining xylem-specific expression reverse genetics screen xylem phenotype lignin-associated genes BLAST analysis sequence-based trees P. trichocarpa paralogs A. thaliana homologs Figure 2.3 Experimental approach used in the selection of novel LAGs for functional characterization.   Figure 2.3 Experimental approach used for the selection of novel LAGs for functional characterization. (i) In silico data was analyzed (left), and (ii) T-DNA insertion mutants of Arabidopsis (A. thaliana) homologs for the LAGs identified from sequence similarity trees were analyzed (right). 23  of poplar LAGs that have an Arabidopsis homolog using NCBI protein BLAST (Figure 2.3, Table 2.3). Next, poplar (P. trichocarpa) and Arabidopsis (A. thaliana) genomes as well as other land plant genomes representing major lineages, including moss (Physcomitrella patens) and a lycopod (Selaginella moellendorffii), were mined for homologous LAGs using Phytozome (Goodstein et al., 2011). Homologous sequences from eucalyptus (Eucalyptus grandis) were also analyzed as this woody perennial, like poplar, is also targeted as a potential biofuel crop (Camargo et al., 2014). For each LAG, sequence similarity trees (Dereeper et al., 2010) were constructed and assessed for homologs in poplar and Arabidopsis (Appendix A.3 to A.29).  Most genes in the P. trichocarpa genome contain at least one paralog as the Salicaceae lineage experienced a whole genome duplication event approximately 65 million years ago (Tuskan et al., 2006). From the tree-based analyses, 17 LAGs had at least one paralog (Table 2.3). Here, a paralog was defined as a related poplar sequence that is most similar to the LAG than a homolog from a different species. For example, in the UMAMIT tree (Figure 2.4A) one poplar sequence (POPTR_0003s19170) is immediately adjacent to the insoluble lignin-associated UMAMIT9 (Figure 2.4B, clade II) and was defined as a paralog. By contrast, the closest UMAMIT12 homolog is from a different species and thus no poplar paralog is present (Figure 2.4B, clade I). More distantly related poplar homologs may exist in the other clades. Interestingly, nine LAGs were single copy in poplar, or only had distantly related homologs (Table 2.3). Further analysis of the sequence similarity trees revealed that 14 LAGs have putative Arabidopsis orthologs, most of which have a maximum of two orthologs (Table 2.3). In the end, 23 LAGs were found to have a maximum of two homologs in both poplar and Arabidopsis. Reverse genetic approaches in poplar have been successful but are limited by experimental constraints such as the time required to complete multiple generations. Therefore, the presence of orthologous LAGs facilitated reverse genetic studies in the model plant Arabidopsis (see below).     24        Table 2.3 Arabidopsis homologs of the poplar lignin-associated genes identified from sequence similarity trees  Associated Gene Poplar Paralog(s) Arabidopsis Additional Commentsa Blast Hit; % Ortholog(s)       Insoluble Lignin UMAMIT9 POPTR_0001s06980* POPTR_0003s19170 At5g07050; 81.8 At5g07050  CKL6 POPTR_0002s03730* POPTR_0005s24860 At4g28540; 87.2 At3g23340    POPTR_0008s16790  At4g14340    POPTR_0010s08110  At4g28540    POPTR_0013s04430    MPK20 POPTR_0002s06080* POPTR_0005s22350 At2g42880; 83.6  Under investigation (BE Ellis Lab) during initial analysis CWP POPTR_0005s07810* POPTR_0007s05560 At4g39840; 46.0 At4g39840  LAX2 POPTR_0009s13470* POPTR_0004s17860 At2g21050; 82.9 At2g21050  BBEL13 POPTR_0011s16200* POPTR_0001s46710 At5g44400; 76.9  Additional poplar homologs in sister clades;      At1g30760, At2g34790 annotated as co-orthologs** MAP20 POPTR_0017s01510*  At5g37478; 53.4 At5g37478 Characterized by Rajangam et al. (2008) P4H7 POPTR_0017s11150*  At3g28480; 83.8  At3g28490 a possible co-homolog Soluble Lignin SKS12 POPTR_0001s03760* POPTR_0001s03770 At1g55570; 83.8  At1g55560, At1g55570, At3g13390, and At3g13400   POPTR_0003s20670   annotated as co-orthologs**   POPTR_0003s20680    FPP7 POPTR_0007s10810** POPTR_0005s18350 At2g23360; 48.3 At2g23360  UXS2 POPTR_0014s12380*  At3g62830; 85.7  At2g47650 and At3g62830 annotated as co-orthologs** Syringyl Lignin DIR6 POPTR_0001s10120*  At1g64160; 62.4  Six co-orthologs**  LHT1 POPTR_0001s36340*  At5g40780; 88.0   NPF6.1 POPTR_0002s03070* POPTR_0005s25490 At3g54140; 61.4  At5g01180 a co-homolog GH3.9 POPTR_0002s20790*  At2g47750; 82.6 At2g47750 At2g47750 annotated as an ortholog in Phytozome GA2OX2 POPTR_0004s06380* POPTR_0001s38760 At1g30040; 81.0  At1g30040, At2g34555 annotated as co-orthologs**   POPTR_0011s09770    UMAMIT12 POPTR_0006s08270*  At2g37460; 68.6 At2g37450      At2g37460  HAD POPTR_0006s08720**  At5g02230; 73.4 At5g02230  BLH9 POPTR_0008s06130* POPTR_0010s20480 At4g34610; 38.8 At2g27990 Under investigation (CJ Douglas Lab) during initial analysis SAR2 POPTR_0010s15180* POPTR_0008s10720 At3g62560; 87.6  At3g62560, At4g02080 annotated as co-orthologs** TLP3 POPTR_0014s04020** POPTR_0002s13430 At1g75030; 70.0 At1g75030 Possible Arabidopsis homologs in sister clade PGIP1 POPTR_0016s05010* POPTR_0006s05740 At5g06860; 67.8  Possible poplar and Arabidopsis homologs in sister clades CML3 POPTR_0018s12720* POPTR_0006s06480 At1g05990; 45.5  Possible poplar and Arabidopsis homologs in sister clades Total Lignin NPF6.3 POPTR_0001s12890*  At5g13400; 80.2 At5g13400  CPU POPTR_0001s28570* POPTR_0009s07770 At1g07120; 67.0 At1g07120  WRKY32 POPTR_0006s19850*  POPTR_0018s11630 At4g30935; 50.7  Potri.006G184800 is the closest gene model to genome v2.2 DGP POPTR_0008s11150* POPTR_0010s14710 At1g13635; 78.1 At1g13635              *denotes significant lignin-associated genes identified by Porth et al. (2013a). WRKY32 gene model (POPTR_0006s19850) has changed in poplar genome v3.0.       aco-orthologs** denotes as annotated from Phytozome (phytozome.jgi.doe.gov).            25             A B UMAMIT12  At2g37450 At2g37460  ii i                                           6.0  Figure 2.4 Unrooted sequence similarity tree for UMAMITs in land plants. (A) UMAMIT sequences from angiosperms (black), including P. trichocarpa (blue) and A. thaliana (orange). Sequences from moss (Physcomitrella patens) and a lycopod (Selaginella moellendorffii) were also analyzed but homologs were not identified. (B) UMAMIT12 (clade I) is significantly associated with S lignin (Porth et al., 2013a). While only UMAMIT9 (clade II) has a poplar paralog both UMAMIT sequences have putative Arabidopsis orthologs (orange). UMAMIT9 and UMAMIT12 were analyzed as part of the lignin-associated genes.  A B   I   I II III26  2.3.5 Gene expression analysis using in silico and transcriptomic data  Gene expression resources are integral for mining data related to the spatial and temporal expression patterns for a gene of interest. Therefore, expression data was analyzed for each LAG to determine their expression levels throughout the plant. First, in silico data from poplar and Arabidopsis electronic Fluorescent Pictograph (eFP) browsers (Winter et al., 2007; Wilkins et al., 2009) available through the BAR for Plant Biology was examined (Appendix A.30 to A.43). Since SCW biosynthesis occurs primarily in xylem cells, LAGs displaying xylem-specific expression in both poplar and the Arabidopsis homologs were of primary interest. Based on the available data, LAX2, MAP20, DIR6, CPU, and DGP were found to be the most highly expressed in poplar xylem (Figure 2.5). BBEL13 was also highly expressed in poplar xylem but to a slightly lesser extent. Five LAG homologs (BBEL13, FPP7, BLH9, SAR2, and CPU) appeared to be the most highly expressed in the second internode of the Arabidopsis stem (Figure 2.5). Interestingly, comparison of the in silico data found only BBEL13 and CPU to exhibit similar expression profiles in both poplar and Arabidopsis. While BBEL13 is highly expressed in the xylem/stem and root, CPU is expressed exclusively in the xylem/stem (Figure 2.5).               27   Figure 2.5 In silico expression data for the poplar lignin-associated genes and their Arabidopsis homologs in different tissues and organs. Electronic fluorescent pictograph (eFP) data was obtained for poplar (left) and Arabidopsis (right) from the Bio-Analytic Resource for Plant Biology (Winter et al., 2007; Wilkins et al., 2009). Grey regions represent data that was not available.            To assess the relative expression levels of the LAGs in developing secondary xylem, transcriptomic data for 20 different P. trichocarpa accessions was also examined (Geraldes et al., 2011). Eight of the 27 LAGs were preferentially expressed in xylem and of these, MAP20 exhibited the highest level of expression (Table 2.4). DIR6 and DGP also had significant UMAMIT9 At5g07050CKL6 At4g28540MPK20 At2g42880CWP At4g39840LAX2 At2g21050BBEL13 At1g30760MAP20 At5g37478P4H7 At3g28480SKS12 At1g55570FPP7 At2g23360UXS2 At3g62830DIR6 At4g23690LHT1 At5g40780NPF6.1 At3g54140GH3.9 At2g47750GA2OX2 At1g30040UMAMIT12 At2g37460HAD At5g02230BLH9 At5g02030SAR2 At4g02080TLP3 At1g75030PGIP1 At5g06860CML3 At3g07490NPF6.3 At5g13400CPU At1g07120WRKY32 At4g30935DGP At1g13635Insoluble Soluble Total Syringyl Female Catkin Male Catkin Xylem Young Leaf Mature Leaf Root Mature Pollen Flower (stage 15) Seeds (stage 5) w/ Silique Stem (2nd internode) Mature Rosette Root Poplar Arabidopsis min                                               max 28  expression levels that were at least 100 times greater in xylem compared to leaves, whereas CPU exhibited 50 times higher expression in xylem. P4H7, an insoluble lignin-associated gene that was included in the analysis here but was not significantly associated in Porth et al. (2013a) also displayed differential expression in xylem compared to leaves (Table 2.4). Taken together, the transcriptomic data was similar to the in silico data (Figure 2.5) for the poplar LAGs BBEL13, MAP20, DIR6, CPU, and DGP. However, while the in silico data suggested LAX2 to be highly expressed in poplar xylem, this was not evident from the transcriptomic data.   Table 2.4 Expression of lignin-associated genes in P. trichocarpa developing secondary xylem and leaves from Geraldes et al. (2011)  Traita Lignin-Associated Gene FPKM DX DL X L        Insol UMAMIT9 POPTR_0001s06980 2.7 3.1   Insol CKL6 POPTR_0002s03730 56.2 51.0   Insol MPK20 POPTR_0002s06080 38.0 29.0   Insol CWP POPTR_0005s07810 20.3 28.1   Insol LAX2 POPTR_0009s13470 75.3 42.7   Insol/Total BBEL13 POPTR_0011s16200 78.2 4.9 Y   Insol MAP20 POPTR_0017s01510 1024.7 0.06 Y   Insol P4H7 POPTR_0017s11150 50.4 16.7 Y  Sol SKS12 POPTR_0001s03760 NA NA   Sol FPP7 POPTR_0007s10810 18.0 1.1 Y  Sol UXS2 POPTR_0014s12380 185.5 68.2   Syrl DIR6 POPTR_0001s10120 685.7 5.4 Y  Syrl LHT1 POPTR_0001s36340 0.0 0.2   Syrl NPF6.1 POPTR_0002s03070 0.2 0.7   Syrl GH3.9 POPTR_0002s20790 0.5 1.4   Syrl GA2OX2 POPTR_0004s06380 0.1 0.3   Syrl UMAMIT12 POPTR_0006s08270 3.4 2.9   Syrl HAD POPTR_0006s08720 6.6 235.6  Y Syrl BLH9 POPTR_0008s06130 14.7 8.3   Syrl SAR2 POPTR_0010s15180 212.9 53.6 Y  Syrl TLP3 POPTR_0014s04020 6.3 1.3   Syrl PGIP1 POPTR_0016s05010 0.1 0.2   Syrl CML3 POPTR_0018s12720 10.3 3.2   Total NPF6.3 POPTR_0001s12890 0.1 6.0  Y Total CPU POPTR_0001s28570 31.7 0.6 Y  Total WRKY32 POPTR_0006s19850 2.2 1.9   Total DGP POPTR_0008s11150 78.0 0.3 Y   atrait abbreviations: Insol: insoluble lignin; Sol: soluble lignin; Syrl: syringyl lignin; Total: total lignin. X: xylem; DX: significantly higher transcript abundance in xylem compared to leaves. L: leaf; DL: significantly higher transcript abundance in leaves compared to xylem. FPKM: fragments per kilobase million. NA: not available.  29  2.3.6 Co-expression analysis of lignin-associated Arabidopsis homologs   Co-expression analysis has been a successful approach for the identification and functional characterization of SCW biosynthesis genes (Brown et al., 2005; Persson et al., 2005). Therefore, Arabidopsis LAG homologs were analyzed for correlated expression with known SCW-related genes. Using the Legacy Expression Angler tool (Toufighi et al., 2005), the top 25 co-expressed genes were analyzed. Here, 14 homologs were found to have reliable co-expression correlation coefficients (r ≥ 0.70), including the stem-expressed BBEL13, FPP7, BLH9, SAR2, and CPU (Figure 2.6). Three of the 14 LAG homologs, SKS12, SAR2 and TLP3, were significantly enriched in correlated genes related to “cell organization and biogenesis” (Figure 2.6). However, none of the three genes appeared to be expressed with SCW-related genes. A closer examination of the remaining LAG homologs revealed that CPU and FPP7 were in fact coordinately expressed with characterized SCW biosynthesis genes, though this was not obvious based on their enrichment in genes related to “other cellular processes” and “other metabolic processes” (Figure 2.6). CPU is co-expressed with CesA7 (Figure 2.7), a gene required for cellulose biosynthesis in SCWs of Arabidopsis (Taylor et al., 1999). While CPU and FPP7 had correlated expression with similar SCW biosynthesis genes, the overall correlation coefficient for the top 25 co-expressed genes was higher for CPU than FPP7 (Figure 2.6).   The ATTED-II co-expression tool (Obayashi et al., 2009) was also employed to assess genes coordinately expressed with the Arabidopsis LAG homologs. Here, homologs that exhibited co-expression with genes enriched in cell wall biogenesis, modification, and organization were of primary interest; CPU, SKS12 and MPK20 were enriched in the latter categories, respectively (Figure 2.8). While the correlations for MPK20 and SKS12 did not indicate a role related to SCW biosynthesis, CPU was co-expressed with similar SCW biosynthesis genes as seen from the Expression Angler analysis (Figure 2.7). The Expression Angler also indicated FPP7 to have correlated expression with SCW biosynthesis genes. However, the ATTED-II analysis indicated an enrichment for genes involved in cell plate formation processes for FPP7 (Figure 2.8). It is noteworthy that none of the Arabidopsis LAG homologs were co-expressed with lignin biosynthetic enzymes.   30  Figure 2.6 Co-expression analysis of lignin-associated genes from Porth et al. (2013a) using the Arabidopsis Legacy Expression Angler tool (Toufighi et al., 2005). The top 25 co-expressed genes were analyzed for each lignin-associated gene homolog, where data was available. The r-value represents the range for the top 25 co-expressed genes. Numbers 1 to 26 denote functional classification categories represented in the Expression Angler. Significantly enriched functional classification categories for each lignin-associated gene are indicated by P < 0.05 to 0.001. Lignin-associated genes in bold indicate strong co-expression in the stem.                        UMAMIT9 At5g07050 0.903-0.864CKL6 At4g28540 0.769-0.678MPK20 At2g42880 0.786-0.676CWP At4g39840 0.681-0.519LAX2 At2g21050 0.656-0.575BBEL13 At1g30760 0.935-0.819P4H7 At3g28480 0.873-0.801SKS12 At1g55570 0.978-0.991FPP7 At2g23360 0.823-0.760UXS2 At3g62830 0.740-0.684DIR6 At4g23690 0.732-0.584LHT1 At5g40780 0.716-0.649NPF6.1 At3g54140 0.833-0.722GH3.9 At2g47750 0.928-0.845GA2OX2 At1g30040 0.770-0.570UMAMIT12 At2g37460 0.813-0.748HAD At5g02230 0.566-0.508BLH9 At5g02030 0.911-0.877SAR2 At4g02080 0.819-0.778TLP3 At1g75030 0.922-0.822PGIP1 At5g06860 0.895-0.729CML3 At3g07490 0.954-0.861NPF6.3 At5g13400 0.692-0.597CPU At1g07120 0.970-0.916WRKY32 At4g30935 0.828-0.669                                                 Functional Classification/                                               Gene           r-value   1     2    3    4     5    6     7    8    9    10  11  12   13  14   15  16  17   18  19  20  21   22  23  24   25   26 1. DNA or RNA Metabolism 2. Cell Organization and Biogenesis  3. Developmental Processes 4. Electron Transport or Energy Pathways 5. Hydrolase Activity 6. Kinase Activity 7. Nucleotide Binding 8. Other Binding 9. Other Biological Processes 10. Other Cellular Processes 11. Other Enzymatic Activity 12. Other Metabolic Processes 13. Other Molecular Functions 14. Protein Binding 15. Protein Metabolism 16. Response to Abiotic or Biotic Stimulus 17. Response to Stress 18. Signal Transduction 19. Structural Molecule Activity 20. Transcription, DNA-Dependent 21. Transcription Factor Activity 22. Transferase Activity 23. Transport 24. Transporter Activity 25. Unknown Biological Process 26. Unknown Molecular Process  0.05                 0.01             0.001                 Insoluble Soluble Syringyl Total 31      Tissue/          Gene      r-value   1.  At1g07120 1.000 CPU   2.  At1g07120 1.000 CPU   3.  At5g17420 0.970 CesA7   4.  At3g50220 0.969 IRX15   5.  At5g15630 0.964 COBL4   6.  At5g54690 0.961 IRX8    7.  At1g27380 0.958 RIC2   8.  At5g03170 0.956 FLA11    9.  At3g16920 0.956 CTL2  10. At3g18660 0.955 GUX1  11. At5g44030 0.954 CesA4  12. At4g27435 0.954 DUF1218  13. At1g32770 0.953 SND1  14. At1g22480 0.951 cupredoxin   15. At4g18780 0.951 CesA8  16. At2g31930 0.943 unknown  17. At1g08340 0.940 Rho GTPase  18. At1g63910 0.939 MYB103  19. At3g15050 0.938 IQD10  20. At4g28500 0.938 SND2  21. At2g38080 0.938 LAC4  22. At2g41610 0.937 unknown  23. At5g67210 0.927 IRXL15  24. At3g62020 0.924 GLP10  25. At4g28380 0.923 LRR protein  26. At2g29130 0.917 LAC2   27. At1g27440 0.916 IRX10   0                                        0.6  Figure 2.7 Co-expression analysis for the total lignin-associated gene CPU from Porth et al. (2013a) using the Arabidopsis Legacy Expression Angler tool (Toufighi et al., 2005). The top 25 co-expressed genes were analyzed (r ≥ 0.90) and strong co-expression with secondary cell wall biosynthesis genes was seen in the stem (red). 32   Figure 2.8 Co-expression network analysis of lignin-associated genes from Porth et al. (2013a) using ATTED-II (Obayashi et al., 2009), where data was available for the Arabidopsis homologs. Numbers in parentheses (left) indicate the number of genes out of the total number of co-expressed genes that suggest enrichment in a gene ontology (GO) category (top) at P < 0.05 to 0.001. Black squares indicate the enriched GO category for the lignin-associated gene.                           GO Enrichment/                                Gene               p value UMAMIT9 At5g07050 (2/15) < 0.05CLK6 At4g28540 (1/9) < 0.05MPK20 At2g42880 (9/15) < 0.001CWP At4g39840 (9/11) < 0.001LAX2 At2g21050 (4/11) < 0.01BBEL13 At1g30760 (5/9) < 0.001P4H7 At3g28480 (1/6) < 0.05SKS12 At1g55570 (7/20) < 0.001FPP7 At2g23360 (6/11) < 0.001DIR6 At4g23690 (4/13) < 0.001LHT1 At5g40780 (6/11) < 0.001NPF6.1 At3g54140 (2/9) < 0.05GA2OX2 At1g30040 (7/11) < 0.001UMAMIT12 At2g37460 (2/15) < 0.05HAD At5g02230 (5/15) < 0.001BLH9 At5g02030 (7/8) < 0.001SAR2 At4g02080 (5/10) < 0.01TLP3 At1g75030 (5/17) < 0.001PGIP1 At5g06860 (5/10) < 0.001NPF6.3 At5g13400 (7/16) < 0.05CPU At1g07120 (7/17) < 0.001KL  Insoluble Soluble Syringyl Total 33  2.3.7 Reverse genetic screen of Arabidopsis T-DNA insertion mutants  SCW biosynthesis can be investigated in Arabidopsis due to the presence of lignified cells in the mature inflorescence stem. To complement the in silico analyses for the poplar LAGs in planta, Arabidopsis T-DNA insertion mutants were screened to determine whether a gene knockout caused an obvious SCW-related phenotype. Using the SiGnAL T-DNA Express database (http://signal.salk.edu) two independent T-DNA insertion lines were obtained for each LAG homolog, where possible (Table 2.1). In some instances, orthologs were not obvious based on the sequence similarity trees and therefore the closest homolog was examined. In total, 40 insertional mutants were analyzed, corresponding to 16 of the 27 LAGs.    A population of 10 to 15 plants for each insertion mutant was grown with wild-type (WT) under long day conditions (16 h light, 8 h dark) and genotyped to validate homozygous individuals (Figure 2.9). Homozygous individuals were desired for experimental analysis as both alleles should be interrupted by the T-DNA and allow for recessive phenotypes to be realized (O’Malley and Ecker, 2010). If a homozygous individual was not identified during the first generation then the plants were permitted to self-fertilize and the progeny from the next generation were re-genotyped. A total of 33 out of 40 insertion lines were validated to be homozygous.  During development, the morphology of WT and T-DNA insertion lines were compared but no obvious differences were apparent for any of the mutants. After six weeks of growth, the base of the mature inflorescence stem was dissected for analysis. Cross-sections were stained with the metachromatic dye toluidine blue-O and observed for changes in tissue organization, or obvious cell wall defects in SCW-containing cells. Most of the mutants did not show obvious cellular phenotypes. However, in 11 T-DNA insertion lines corresponding to seven lignin-associated genes, collapsed vessels also known as irregular xylem, or increased interfascicular fibres were observed (Table 2.5). One T-DNA insertion line for UMAMIT9 had a very mild irx phenotype compared to WT (Figure 2.10). The T-DNA insertion lines corresponding to Arabidopsis PTR1 and PTR5, homologs of poplar NPF6.1 which is significantly associated with S lignin (Porth et al., 2013a), were observed to have a slight qualitative increase in fibre deposition compared to WT (Table 2.5, Figure 2.10). The phenotypes observed for the mutants 34  Figure 2.9 Genotyping T-DNA insertion mutants to identify homozygous lines for phenotypic analysis. (A) Schematic diagram for PCR-based analysis of T-DNA insertion mutants. Two primer sets were used to confirm the genotype of the mutants. First, the gene-specific left primer (LP) and right primer (RP) were used to detect the presence of the gene. Second, the left border primer (LBb) and RP were used to detect the presence of the inserted T-DNA. Adapted from rarge-v2.psc.riken.jp/chloroplast. (B) Genotyping of a T-DNA insertion mutant for the syringyl lignin-associated CML3 (Porth et al., 2013a; not studied in detail here). Two wild-type (WT) and insertion mutant individuals (1, 2) from a population are shown. The absence of a band in the LP/RP panel for the two mutant individuals and the presence of bands for the same plants in the LBb/RP panel indicates homozygous individuals. Bands in WT in the LP/RP panel indicate the presence of the endogenous gene examined.  corresponding to the LAGs were indicative of vascular or fibre developmental defects during the initial screen. Therefore, the same T-DNA insertion mutants were re-screened in the subsequent generation to determine if the previously observed phenotypes could be confirmed. However, repeated analysis of the same mutant lines did not result in reproducible phenotypes across generations.                                     LP RP LBb T-DNA A B WT1 WT2  1 2 - control LP/RP  LBb/RP 35          Table 2.5 Lignin-associated genes that were screened for cell wall-specific defects using Arabidopsis T-DNA insertion lines  Trait   Associated Gene  ATG ID T-DNA Line Position Phenotype (T1)a  Insoluble UMAMIT9 POPTR_0001s06980 At5g07050 SALK_022640 intron mild irx  CWP POPTR_0005s07810* At4g39840 SALK_093172 5’ UTR none  LAX2 POPTR_0009s13470* At2g21050 CS808596  exon none  MAP20 POPTR_0017s01510* At5g37478 SALK_021552 5’ UTR none   P4H7 POPTR_0017s11150 At3g28480 SALK_004428 exon increased fibres     SALK_039733 promoter none    At3g28490 SALK_067682 exon increased fibres     SALK_101949 promoter none  Soluble SKS12 POPTR_0001s03760* At1g55560 SALK_017243 exon increased fibres    At1g55570 SALK_061973 exon none    At3g13390 SALK_096049 exon none    At3g13400 SALK_005755 exon none  FPP7 POPTR_0007s10810* At2g23360 SALK_045879 exon none  Syringyl LHT1 POPTR_0001s36340* At5g40780 SALK_034566 exon none     SALK_083700 intron none  NPF6.1 POPTR_0002s03070* At3g54140 CS872049 exon increased fibres    At5g01180 SALK_022619 exon increased fibres     CS872776 exon increased fibres  GH3.9 POPTR_0002s20790* At2g47750 SALK_025354 promoter none  GA2OX2 POPTR_0004s06380* At1g30040 SALK_011239 exon increased fibres    At2g34555 SALK_002824 promoter none  UMAMIT12 POPTR_0006s08270* At2g37460 SALK_101986 exon mild irx     CS814465 3’ UTR mild irx  SAR2 POPTR_0010s15180* At1g56330 SALK_013096 promoter none    At3g62560 SALK_020919 intron none    At4g02080 SALK_087289 promoter none  TLP3 POPTR_0014s04020* At1g75030 SALK_089145 promoter none    At1g75050 SALK_087483 promoter none     SALK_141355 intron none        Total NPF6.3 POPTR_0001s12890* At5g13400 SALK_007230 promoter none     SALK_080802 promoter none  WRKY32 POPTR_0006s19850* At4g30935 SALK_062133 promoter increased fibres     SALK_091352 5’ UTR None  *denotes significant associations in Porth et al. (2013a).  adenotes phenotypes observed during the first generation (T1) screen.      36                    Altogether, 27 LAGs were analyzed of which eight genes exhibited significant differential expression in developing secondary xylem of poplar. Co-expression analysis of the LAG homologs found only one gene, CPU, to have co-expression with well-known secondary cell wall biosynthetic genes. While the analysis of the LAGs extended to a reverse genetic screen of Arabidopsis T-DNA mutants and cell wall-related phenotypes were observed, they were not reproducible. WT WT ptr5 umamit9  Figure 2.10 Phenotypic analysis of T-DNA insertion lines for Arabidopsis homologs of the poplar lignin-associated genes. Stem cross-sections from wild-type (WT, left) and T-DNA insertion lines (right) were stained with toluidine blue-O. A very mild irx phenotype in the xylem (x) was observed for umamit9 (top right, denoted by arrows). A slight increase in interfascicular fibres (iff) was observed for ptr5 (bottom right) compared to WT (bottom left). Scale bar represents 50 µm. x iff iff x iff x iff x 37  2.4 Discussion  SCW biosynthesis is a highly-coordinated process that is regulated and realized by a large suite of gene products. Although reproducible phenotypes were not obtained from the reverse genetic screen of Arabidopsis T-DNA insertion mutants, some novel genes associated with lignin trait variation from the P. trichocarpa AM experiment were identified as suitable candidates for in-depth functional characterization based on the in silico analyses. However, from my analyses I found little supporting evidence for roles in wood or secondary cell wall formation for most of the LAGs, highlighting the challenges in linking genes identified from AM to their associated phenotype.  2.4.1 Reverse genetic screen of lignin-associated gene homologs did not reveal impacts on primary stem development in Arabidopsis  The identification and validation of genetic variants and their phenotypic effect is a definite challenge in genetics (Geraldes et al., 2013; Korte and Farlow, 2013). Many phenotypic traits including wood properties are complex and are thus influenced by multiple genes that contribute either a direct or indirect effect (Mizrachi and Myburg, 2016). A single gene will exert a relatively minor effect on complex phenotypic traits, which explains why Porth and colleagues reported only three to seven percent of the measured phenotypic variation from the individual associations to be attributed to a given locus (Porth et al., 2013a). Mutant screens typically result in a low percentage of phenotypes since single gene knockouts frequently do not show differences (Hanada et al., 2009; Ransbotyn et al., 2015; Rutter et al., 2017). Although secondary xylem development can be investigated in the model plant Arabidopsis, it remains possible that at least some of the lignin-associated genes identified from AM in poplar contribute to wood development processes that are unique to poplar, or trees in general (Nieminen et al., 2004; Li et al., 2008).   Wood development is an example of a complex trait at any developmental stage, but some wood properties become more apparent as the tree matures; trees are also highly responsive to environmental factors such as seasonal transitions and stresses (Groover et al., 2010; Wegrzyn et al., 2010). Since nine-year-old trees exposed to a natural environment were tested for wood traits by Porth et al. (2013a), phenotypic comparisons to the primary vascular system of 38  Arabidopsis maintained under a controlled setting cannot mimic all the factors that may influence lignin deposition during wood formation. The genetic regulation of reaction wood, for example, cannot be directly studied in an herbaceous annual since it forms in trees under mechanical bending stress. Therefore, while common mechanisms exist to regulate SCW biosynthesis in herbaceous annuals and woody perennial plants, it is likely that the latter has more complex impacts from indirect effects on wood formation and SCW biosynthesis, such as environmental cues and whole-plant developmental stages. Given that well-characterized lignin biosynthetic enzymes were not associated with lignin traits in Porth et al. (2013a), for example caffeoyl CoA O-methyltransferase which was associated with holocellulose content, it is possible that many of the lignin-associated genes have an indirect contribution to lignin content and/or composition variation.   AM studies targeting wood and biomass properties have been conducted for several poplar species, including P. deltoides (Fahrenkrog et al., 2017), P. nigra (Guerra et al., 2013; Allwright et al., 2016), and P. trichocarpa (Wegrzyn et al., 2010; Porth et al., 2013a). Yet very few genes with significant associations were found to overlap between experiments which could partly be explained by differences in population size and age, as well as the number and type of candidate genes used. While Porth et al. (2013a) based their experiment on c. 3,500 diverse candidate genes, Wegrzyn et al. (2010) and Guerra et al. (2013) tested associations using only 40 cellulose and lignin biosynthetic enzymes. However, both AM studies in P. trichocarpa found significant associations in SCW CesA genes. Wegrzyn et al. (2010) detected associations in poplar orthologs of Arabidopsis CesA4, CesA7, and CesA8 to lignin content variation, whereas Porth et al. (2013a) found CesA7 to be associated with glucose content variation. Therefore, the findings suggest that changes in cellulose biosynthesis can impact cellulose in addition to other wood traits. This is not unexpected as correlation studies have observed an inverse relationship between alpha-cellulose and total lignin content (González-Martínez et al., 2007; Porth et al., 2013b).  It is apparent that the experimental design of an AM study can influence its outcome. AM in plants for example, is often based on smaller sample sizes compared to typical studies in humans, which consist of thousands of individuals (Atwell et al., 2010; Korte and Farlow, 2013). 39  Smaller sample sizes impact the statistical power and as a result, the associations that are deemed significant after multiple testing corrections (Wangler et al., 2017). As AM strongly relies on statistical analyses, the risk of detection of false negatives or false positives over true associations has to be taken into account (Zhao et al., 2007; Mitchell-Olds, 2010; Platt et al., 2010). Another consideration is the type of association study conducted. A candidate gene approach requires the selection of genes with a priori knowledge for a given biological process. However, this approach is restricted by the genes input into the analysis as causal mutations in excluded genes will not be detected (Ingvarsson and Street, 2011). Since the selected candidate genes are often based on experimental evidence, it is possible that the phenotypes identified from clonal mutants in a laboratory setting may not be reflective of the phenotypic variations that exist within a natural population (Ingvarsson and Street, 2011).  2.4.2 In silico analyses can identify novel genes involved in secondary cell wall biosynthesis As thousands of genes are required for wood formation (Mizrachi and Myburg, 2016) many studies have profiled xylem transcriptomes in order to identify the core genes that are essential for its development (Sterky et al., 1998; Hertzberg et al., 2001; Ko et al., 2006; Pavy et al., 2008; Hefer et al., 2015). Comparative analysis of xylem transcriptomes from poplar and Arabidopsis suggest that some genes may be unique to woody plants (Sterky et al., 1998). However, the sequence similarity trees suggest the lignin-associated genes from Porth et al. (2013a) to all have homologs in Arabidopsis, and most have putative orthologs in Arabidopsis. This finding suggests most of the lignin-associated genes to be functionally conserved in poplar and Arabidopsis.   Eight of the 25 lignin-associated genes have preferential expression in P. trichocarpa xylem (Geraldes et al., 2011). Of these, MAP20, DGP, and DIR6 are expressed more than 100 times higher in xylem when compared to leaves. While DIR6 was identified as a putative direct target of Arabidopsis VND7 (Yamaguchi et al., 2011), co-expression-based evidence for a SCW-related role was not found here. Analysis of expression levels alone however, can lead to the discovery of novel genes involved in SCW biosynthesis. For example, the highest P. trichocarpa xylem-expressed lignin-associated gene, MAP20, was first identified based on its preferential 40  expression in poplar wood (Rajangam et al., 2008). MAP20 localizes to cortical microtubules and has a possible role related to cellulose biosynthesis.  FPP7 is one of only two lignin-associated genes to have both significant expression in xylem, and co-expression with characterized SCW biosynthetic genes in Arabidopsis. Using the Arabidopsis Expression Angler tool (Toufighi et al., 2005), FPP7 was found to be the most coordinately expressed with a polygalacturonase gene (At1g80170), whereas ATTED-II (Obayashi et al., 2009) predicted correlations with microtubule-related genes. However, promoter-GUS lines for At1g80170 showed expression in mature anthers (González-Carranza et al., 2007). While the results here do not indicate a clear function for FPP7, FPP7 has been defined as a long coiled-coil protein of unknown function (Gindullis et al., 2002). As some coiled-coil proteins are cytoskeletal proteins (Gardiner et al., 2011), FPP7 may interact with actin or microtubules and contribute to SCW biosynthesis.  The most compelling results from the gene expression analyses were found for CPU, a gene encoding an unknown protein that has homologs in land plants. First, CPU has approximately 50 times higher expression in P. trichocarpa xylem compared to leaves and exhibits the fourth-highest xylem expression of all the lignin-associated genes. Second, its single Arabidopsis ortholog (At1g07120) is also expressed in the stem and is tightly co-expressed (r ≥ 0.90) with well-characterized SCW transcription factors and biosynthetic genes which highly suggests a function related to SCW biosynthesis. For example, CPU is co-expressed with IRX15 and the SCW CesAs, CesA4, CesA7, and CesA8. IRX15, a xylan biosynthetic gene, was identified based on its in silico expression pattern in the Arabidopsis root and co-expression with CesA4, CesA7, and CesA8 (Brown et al., 2005; Brown et al., 2011). Lastly, identification of unknown cell wall proteins by Mewalal et al. (2014) found CPU to be present in various SCW-related datasets, thereby giving the strongest support for this gene to have a role related to SCW biosynthesis. For these reasons, CPU was chosen for further investigation (Chapter 3).  2.4.3 Transporters and their contribution to secondary cell wall biosynthesis The AM study identified four transporter genes to be significantly associated with lignin content and composition variation which suggests a direct or indirect role in lignin deposition. Transporters have long been speculated as a possible mechanism for monolignol translocation 41  across the plasma membrane and into the extracellular matrix. Expression profiling of the Arabidopsis inflorescence stem by Ehlting et al. (2005) identified several ATP-binding cassette (ABC) proteins as possible candidates for monolignol transport based on their coordinated expression profiles to known lignin biosynthetic genes. Subsequent investigation of the latter by Kaneda et al. (2011) found ABCB14 to be involved in auxin transport in the stem while another candidate, ABCG29, has since been reported as a p-coumaryl alcohol transporter (Alejandro et al., 2012). Porth et al. (2013a) did not find ABC transporters to be significantly associated with any of the wood traits. Instead, their results indicate that other types of transporters may influence aspects of wood and/or SCW biosynthesis. For example, UMAMIT12 is significantly associated with S lignin composition variation. UMAMIT or MtN21/EamA-like nodulin proteins (Denancé et al., 2014) represent a family of transporters that are not well-studied but individual members appear to have broad substrates including amino acids as in the case of Siliques are Red 1 (Ladwig et al., 2012). Walls are Thin 1 (WAT1), an auxin transporter, is the only characterized UMAMIT to have a SCW-related phenotype as wat1 mutants have reduced SCW deposition in the interfascicular fibres (Ranocha et al., 2010; Ranocha et al., 2013). The mild but non-reproducible irx phenotype seen for the UMAMIT T-DNA insertion mutants suggest these transporters may impact vascular development and thus have an influence on SCW biosynthesis. However, since the UMAMIT genes analyzed here have homologs, some degree of functional redundancy is expected. This presents these genes as being less ideal for functional characterization particularly in poplar as longer generation times are required and multi-locus manipulations are not simple.  Nitrogen (N) is an essential element for plant development. Its acquisition from the environment and translocation throughout the plant is in part, mediated by the Nitrate1/Peptide (NRT1/PTR) Family (NPF), also known as Solute Carrier 15 (SLC15) transporters (Léran et al., 2014; von Wittgenstein et al., 2014). These proteins mainly transport nitrate and di- and tripeptides but hormones and secondary metabolites have also been identified as substrates. The peptide transporters have been far less studied than their nitrate counterparts. Porth et al. (2013a) found NPF6.1 and NPF6.3 to be significantly associated with syringyl and total lignin variation, respectively. While nitrogen homeostasis affects wood formation (Plavcová et al., 2013; Euring et al., 2014), NPFs have yet to be implicated in SCW biosynthesis. Indeed, NPFs have not yet 42  been characterized in detail in woody plants. A comparative analysis of monocot and eudicot transcriptomes to identify conserved genes involved in vascular development found an enrichment in genes related to peptide transport (Xu et al., 2013). It is therefore conceivable that NPF proteins contribute a direct or indirect influence on vascular tissue development via the regulation of nitrogen distribution.  NPF6.1, a putative peptide transporter, is significantly associated with syringyl lignin composition. Unlike most genes in the poplar genome, NPF6.1 does not have an Arabidopsis ortholog as seen from the phylogeny of SLC15 proteins from various land plants constructed by von Wittgenstein et al. (2014). Arabidopsis PTR1, the most similar homolog of NPF6.1, is expressed in vascular tissue (Dietrich et al., 2004). The broad in silico expression pattern seen for NPF6.1 in poplar also suggests this gene to be expressed in the vascular tissue. Since ptr1 and ptr5 T-DNA insertion lines appeared to have slightly increased interfascicular fibres compared to WT, the association to syringyl lignin is plausible as angiosperm fibres are high in syringyl lignin (Weng et al., 2008; Bonawitz and Chapple, 2010; Zhou et al., 2011). Therefore, the evolutionary relationship of NPF6.1 and its single poplar paralog combined with its likely expression in vascular tissue presents it as an interesting and novel candidate to investigate the role of NPF transporters in SCW biosynthesis in woody plants.  2.4.4 Conclusion Our knowledge of SCW biosynthesis is currently well-established in terms of the biosynthetic enzymes and transcriptional proteins that directly regulate the biosynthesis of cellulose, hemicellulose, and lignin. However, the impact of other developmental processes and whole-plant physiology on wood and SCW formation are much less understood. Most of the significantly associated genes from Porth et al. (2013a) have not yet been described to influence SCW biosynthesis. Here, I employed in silico and reverse genetic analyses to further test a role in wood and SCW formation for the lignin-associated genes. Challenges were apparent in linking these genes to their associated phenotypes and for a majority of the genes, my analysis did not provide further evidence for a role in SCW biosynthesis. However, the negative results do not exclude such a role for these genes, rather they may reflect the fact that AM identifies alleles that contribute minor phenotypic variation. The abundance of genomic, transcriptomic and 43  phenotypic data generated from the large-scale association mapping study in poplar will facilitate the application of functional genomics to characterize two new genes associated with lignin trait variation, CPU and NPF6.1. Their possible functions in wood and SCW biosynthesis will be discussed in detail in the subsequent chapters. CPU will be presented in Chapter 3, and NPF6.1 will be presented in Chapter 4.   44  Chapter 3. Coiled-coil protein of unknown function (CPU) and its role in SCW deposition in fibres  3.1 Introduction  Wood is a composite tissue that contains fibres, tracheary elements (tracheids and vessel elements), and parenchyma. Fibre and tracheary element (TE) development occurs through a progression of steps, starting with differentiation at the vascular cambium followed by cell expansion and elongation (Bollhöner et al., 2012). After cell expansion/elongation has ceased, the secondary cell wall components cellulose, hemicellulose, and lignin are deposited in a spatially and temporally controlled manner. For example, cellulose and hemicellulose deposition occurs during the early stages of secondary cell wall deposition, followed by lignification (Meents et al., 2018). In the final stages, organellar contents including the vacuole are degraded. While the development of TEs and fibres have overlapping processes they also exhibit differences. For example, in woody plants, programmed fibre cell death occurs at much slower rates than for TEs to facilitate increased cell wall thickening and lignification (Schuetz et al., 2013). Despite being a significant constituent of wood, the molecular processes related to fibre development are not as well-understood compared to vessel elements.   Our understanding of secondary cell wall biosynthesis has largely been facilitated through experimentation in the model herbaceous annual Arabidopsis (Arabidopsis thaliana). As there are practical limitations to working with trees such as their sizes and long generation times, Arabidopsis is an ideal system to investigate secondary cell wall biosynthesis as a large portion of the inflorescence stem is comprised of interfascicular fibres which contribute mechanical strength. In comparison to the biosynthesis genes that have been identified, our knowledge of the cytoskeletal-related genes involved in secondary cell wall biosynthesis is fragmented. The role of cortical microtubules in cell wall biosynthesis, for example, has been best characterized in relation to cellulose biosynthesis as these proteins guide cellulose synthase (CesA) complexes in both the primary and secondary cell wall (Paredez et al., 2006; Watanabe et al., 2015). This suggests an interconnected relationship between cortical microtubule organization and cellulose microfibril orientation (Smith and Oppenheimer, 2005). In Arabidopsis, the fragile fiber 1 (fra1) mutant exhibits reduced mechanical strength in the stem as a consequence of disorganized 45  cellulose microfibril arrangement in fibre cells compared to wild-type (WT) (Zhong et al., 2002). However, as microtubule organization was not affected, this suggests other proteins may influence cortical microtubules during secondary cell wall biosynthesis such as microtubule-associated proteins (MAPs).  It is estimated that up to 15% of the 27,000 protein-coding genes in the Arabidopsis (A. thaliana) genome contribute to cell wall biosynthesis (Mewalal et al., 2014). Most of these genes remain uncharacterized as experimental evidence is limited for cell wall-related genes (Yang et al., 2011). Proteins of unknown function (PUF) have been implicated in cell wall biology due to their co-expression with known cell wall proteins. For example, Horan et al. (2008) tested whether PUF genes from publicly available expression data for Arabidopsis were co-regulated with known biological processes. One of the most significant gene expression clusters identified was a subset of PUFs enriched in cell wall-related gene annotations, including cellulose synthases, which suggests the suitability of using such an approach to identify PUF genes and their potential biological functions (Horan et al., 2008). As secondary cell wall biosynthesis requires CesA4, CesA7, and CesA8 to form a functional cellulose synthase complex (Taylor et al., 2003; McFarlane et al., 2014), these genes are expected to be tightly co-regulated.  Co-expression analysis using CesA7 as a bait has been highly successful in the identification of novel genes involved in secondary cell wall biosynthesis. Brown et al. (2005) and Persson et al. (2005), for example, both identified glycosyltransferases involved in hemicellulose biosynthesis through co-expression of different expression data sets based on CesA7. In subsequent studies, Persson et al. (2007) characterized Irregular Xylem 8 (IRX8) while Brown et al. (2011) characterized IRX15/IRX15L. Both genes are required for xylan biosynthesis in Arabidopsis as irx8 and irx15/irx15l have decreased xylan content. The CesA7 co-expressed genes identified by Brown and Persson et al. have also been a resource for the identification and characterization of novel genes involved in secondary cell wall biosynthesis. For example, the double mutant of a lignin-related gene containing a domain of unknown function (DUF1218), first identified by Persson et al. (2005), was characterized to have changes in lignin content compared to wild-type (Mewalal et al., 2016) suggesting that co-expression using CesA7 as a bait can find genes involved in cellulose, hemicellulose, or lignin biosynthesis. However, apart 46  from PUFs there are proteins of obscure function (POF) which do not have domains or motifs that resemble known sequences (Mewalal et al., 2014). POFs can also be identified based on co-expression analysis. POFs identified from a comparative co-expression analysis based on CesA8 include Vascular Protein Unknown 1 (VUP1). Over-expression of VUP1 in Arabidopsis resulted in an irregular xylem phenotype and may affect hormone signaling during secondary cell wall biosynthesis (Grienenberger and Douglas, 2014). Taken together, PUFs and POFs represent understudied proteins that have potential impacts on secondary cell wall biosynthesis.   POFs tend to engage in protein-protein interactions (Mewalal et al., 2014). However, there are only a few reports of protein-protein interactions for secondary cell wall-related proteins. In vitro systems such as zinnia (Zinnia elegans) cell culture have led to the identification of a suite of genes required for secondary cell wall thickening, including genes of unknown function. For example, screening of zinnia cells undergoing TE differentiation found an intriguing protein identified as TE Differentiation-Related 6 (TED6) and TED7 (Endo et al., 2009), a transmembrane protein lacking functional domains. The former was identified to form a protein-protein interaction with cellulose synthase complexes in the secondary cell wall. Interestingly, several other proteins have been proposed to interact with the cellulose synthase complex, at least in the primary cell wall, including the β-1,4 endoglucanase Korrigan (KOR) (Vain et al., 2014). However, a physical interaction has not been detected in the secondary cell wall (Szyjanowicz et al., 2004). A recent protein-interaction screen of the poplar (Populus trichocarpa) xylem proteome suggests a myriad of protein interactions to facilitate secondary cell wall biosynthesis (Petzold et al., 2018).   Here, I describe the characterization of a POF named Coiled-coil Protein of Unknown Function (CPU), which was originally identified from its genetic association to total lignin content variation in P. trichocarpa (Porth et al., 2013a). Analyses of P. trichocarpa xylem transcriptome data sets found CPU to be highly expressed in developing secondary xylem. Here, I demonstrate that over-expression of PtCPU in transgenic poplar wood led to decreased fibre length and increased cellulose microfibril angle, while total lignin content was not altered, compared to wild-type. Further analysis in Arabidopsis of the putative ortholog AtCPU found Cellulose-Microtubule Uncoupling l (CMU1) to be a protein interaction partner through a yeast 47  two-hybrid screen. CMU1 is a microtubule-associated protein that is proposed to anchor microtubules to the plasma membrane to maintain them as guides during cellulose synthase interactions (Liu et al., 2016). Analysis of cpu and cpucmu1cmu2 found decreased interfascicular fibre secondary cell wall thickness in the triple mutant compared to WT. Taken together, the findings suggest a role for CPU in influencing cellulose ultrastructure, perhaps through its interaction with microtubule-associated proteins and the microtubules that control cellulose deposition during secondary cell wall formation in poplar and Arabidopsis.   3.2 Materials and Methods  3.2.1 Plant material and growth conditions Hybrid poplar (Populus tremula x Populus alba) clone INRA 717-1B4 was maintained in vitro on solid half-strength Murashige and Skoog (MS) basal medium (PhytoTechnology Laboratories) supplemented with 0.5 μg mL-1 indole butyric acid (IBA) and grown in a plant tissue culture chamber (Caron) at 22°C under long day conditions (16 h light, 8 h dark). For experimentation in the greenhouse, in vitro plantlets were transferred into soil and acclimated in a mist chamber for three weeks. Plantlets were transplanted into one-gallon pots containing Sunshine Mix #4 (Sun Gro Horticulture) supplemented with slow-release fertilizer (21.4 g L-1 Acer 21-7-14, 11.4 g L-1 dolomite lime, 2.9 g L-1 Micromax Micronutrients, and 1.1 g L-1 superphosphate) and grown under long-day conditions (16 h light, 8 h dark) at 22°C with automated irrigation. Four-month-old stems were harvested approximately 5 cm above the base of the root collar. For all Arabidopsis (Arabidopsis thaliana) experiments, the wild-type ecotype Columbia-0 (Col-0) was used. T-DNA insertion lines (cpu: SALK_208580; cmu1: SALK_121703; cmu2: SALK_148296) were obtained from the Arabidopsis Biological Research Centre (Ohio, USA). Surface-sterilized seeds were sown on solid half-strength MS medium and stored at 4°C for 48 h under darkness and transferred to a plant tissue culture chamber (Caron) at 22°C under long day conditions. Ten-day-old seedlings were transferred to Sunshine Mix #4 and maintained in growth chambers (Conviron) at 21°C under long day conditions at a photon flux density of 120 ± 10 μmol m-2 s-1. Basal sections of the main stem (bottom 1 cm) of six to eight-week-old plants were analyzed, unless stated otherwise. All plants were maintained at the Bev Glover Greenhouse Research Facility at the University of Victoria (Victoria, Canada). 48  3.2.2 In silico analyses Sequences obtained from Phytozome (https://phytozome.jgi.doe.gov) and the 1000 plants initiative (www.onekp.com) were aligned using Clustal Omega v1.2.4 (Sievers et al., 2011) and manually trimmed; positions in the alignment that contained a gap in more than 95% of the sequences were removed. A maximum likelihood phylogeny with the Jones Taylor Thornton amino substitution model (Jones et al., 1992) and 1,024 bootstrap replicates was generated using ProtTest3 (Darriba et al., 2011) and PhyML 3.3 (Guindon and Gascuel, 2003). The protein motif analysis was performed using SMART (smart.embl-heidelberg.de) (Letunic et al., 2015) and conducted under normal mode using the entire protein sequence. CONAN (Corea et al., unpublished) was used to identify pairwise gene co-expression relationships from mean-centered normalized, variably-expressed genes from 384 P. trichocarpa xylem transcriptomes. Gene expression values were log2(n+1)-transformed and Pearson correlation coefficients were calculated between each gene, with a minimum cut-off of r = 0.60. The in silico resource Aspwood (aspwood.popgenie.org) was used to examine the PtCPU expression during wood development in naturally growing 45-year-old P. tremula clones (Sundell et al., 2017).  3.2.3 Plasmid construction Primers were generated to amplify coding sequences as annotated from the poplar (P. trichocarpa) genome (v2.2) and the Arabidopsis (A. thaliana) genome, both available through Phytozome (phytozome.jgi.doe.gov) (Goodstein et al., 2011). All poplar coding sequences were amplified from cDNA isolated from young P. trichocarpa (Nisqually-1) stems. Arabidopsis promoters were isolated from genomic DNA while coding sequences were amplified from cDNA isolated from mature Col-0 inflorescence stems. Plant transformation constructs were cloned into the Gateway pCR8/TOPO donor vector (Thermo Fisher Scientific) and recombined into the appropriate destination vector using LR Clonase II as recommended by the manufacturer. The double 35S:PtCPU over-expression construct introduced into poplar was generated using the destination vector pMDC32 while the promoter-GUS constructs were produced from the destination vector pMDC163 (Curtis and Grossniklaus, 2003). 35S:YFP-PtCPU constructs expressed in Arabidopsis were produced via recombination into the pEarleyGate 104 destination vector (Earley et al., 2006). All plant transformation constructs were transformed into Agrobacterium tumefaciens C58 pMP90 (for poplar) or GV3101 (for Arabidopsis) via 49  electroporation. The yeast two hybrid screen LexA-AtCPU bait vector was constructed by ligating the full-length coding sequence of AtCPU into pLexA-N via the restriction sites EcoRI and PstI. For other yeast two-hybrid assay constructs, DNA fragments containing restriction sites were ligated into the bait vector pGBKT7-BD or the prey vector pGADT7-AD (Clontech). All cloned DNA fragments were amplified using Q5 High-Fidelity DNA Polymerase (New England Biolabs). All primer sequences used are listed in Table 3.1.  3.2.4 Generation of transgenic plants  Transgenic poplars were generated from hybrid aspen (P. tremula x P. alba) clone INRA 717-1B4 as described by Ma et al. (2004). Agrobacterium were grown at 28°C for 48 h and resuspended in MS induction medium (IM) to OD600 0.5 to 0.6. Explants were incubated in the Agrobacterium suspension for 1 h and transferred onto solid callus-induction medium (CIM) supplemented with 5 μM 2iP and 10 μM NAA for 48 h at 22°C, in darkness. Explants were transferred to CIM containing carbenicillin and cefotaxime (or timentin) and maintained for 21 d at 22oC in the dark. Callus cultures were moved onto shoot-induction medium supplemented with 0.2 μM thidiazuron and maintained at 22oC under subdued light until shoots started to regenerate from callus. Regenerated shoots were excised and micropropagated onto solid half-strength MS medium supplemented with 0.5 μM IBA and 10 µg mL-1 hygromycin, and further tested to confirm insertion of the transgene. Arabidopsis over-expression lines were created using the floral dip method (Clough and Bent, 1998). Transgenic seedlings were sowed on solid half-strength MS medium (2.5 g L-1 MS salts, 1% (w/v) sucrose, 3.5 g L-1 phytagar, pH 5.8) supplemented with either 50 µg mL-1 Basta (pEarleyGate 104) or 25 µg mL-1 hygromycin (pMDC163) for selection.  3.2.5 Analysis of transgenic plants  Total RNA from poplar xylem scrapings was isolated using the CTAB method as described by Haruta et al. (2001). In brief, 0.5 g of tissue in 5 mL of extraction buffer (2% hexadecyltrimethylammonium bromide (CTAB), 1% (v/v) β-mercaptoethanol, 1.4 M NaCl, 0.1 M Tris-HCl (pH 9.5), 20 mM EDTA) was incubated at 65°C for 10 min and extracted using chloroform:isoamyl alcohol (24:1) and chloroform:phenol (1:1). Total RNA was precipitated at  50  −80°C for 10 min using 65 μL of 3 M sodium acetate and 850 μL of 100% ethanol. To remove contaminating genomic DNA, 10 μg of total RNA was treated with DNase I (Ambion) according to the manufacturer’s recommendations. One microgram of DNase-treated RNA was used for cDNA synthesis via Superscript III reverse-transcriptase (Thermo Fisher Scientific). PtCPU over-expression in transgenic poplar was analyzed using quantitative real-time PCR run on a Bio-Rad CFX96. Reactions (15 μL) were prepared (in triplicate) using the SsoFast EvaGreen mix (Bio-Rad) according to the manufacturer’s recommendations with 300 nM of primer (Table 3.1) and 2 μL of diluted cDNA template. Amplification conditions consisted of 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 55°C for 5 s. Gene expression (delta Ct) was quantified relative to elongation factor 1β (Coleman et al., 2008). For the analysis of Arabidopsis T-DNA insertion mutants, plants were genotyped (Table 3.1) as described by O’Malley and Ecker (2010) to confirm homozygosity and analyzed by reverse-transcription PCR to confirm the absence of transcripts using gene-specific primers (Table 3.1). Total RNA was extracted from 100 mg of ground Arabidopsis stems (isolated at 5 cm from the base) and 0.5 mL of Purelink Plant RNA Reagent (Thermo Fisher Scientific) according to the manufacturer’s recommendations for small scale RNA isolation. Isolated total RNA free of genomic DNA contamination was reverse-transcribed into cDNA. Semi-quantitative reverse transcription PCR was run for 28 cycles for the target genes and 34 cycles for the no reverse transcriptase control.   3.2.6 GUS staining and microscopy For GUS staining, hand-cut cross-sections of the primary stem were vacuum infiltrated in cold 90% acetone for 5 min at 25 psi and maintained in cold 90% acetone for 25 min. Sections were rinsed three times with 0.1 M NaPO4 (pH 7.0) and incubated in X-gluc solution (1 mM 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid (X-gluc), 0.5 mM K3[Fe(CN)6], 0.5 mM K4[Fe(CN)6], 0.1 M NaPO4 (pH 7.0), 0.01% (v/v) Triton X-100) at 37°C until the appearance of blue precipitate. Sections were cleared with 75% ethanol as required to remove chlorophyll. For anatomical analysis of stems, hand cut cross-sections were stained with 0.25% (w/v) toluidine blue-O (Sigma-Aldrich). Sections were mounted in water and visualized and imaged with Nikon Eclipse TE2000-U equipped with a Digital Sight DS-U1 camera and ACT-2U control software. Cell wall thickness measurements were carried out using ImageJ (imagej.nih.gov).  51  3.2.7 In situ hybridization  In situ hybridization was performed as described by Ohtani et al. (2011). One-month-old stem segments (internode six) from greenhouse-grown P. trichocarpa were fixed in 4% (v/v) paraformaldehyde in PBS and dehydrated in an increasing ethanol series (20% to 95%) for 60 min each at 4°C. Paraffin sections cut at 8 μm using a microtome were hybridized with digoxigenin (DIG)-labelled probes (synthesized from the full-length coding sequence of PtCPU) at 50°C overnight (Roche). Probes were detected with anti-DIG-alkaline phosphatase and Western blue reagent (Promega).  3.2.8 Yeast two-hybrid analysis  The yeast two-hybrid screen was carried out as recommended by the manufacturer (Dualsystems Biotech). Expression of the bait construct in the yeast (Saccharomyces cerevisiae) strain NMY51 was analyzed via immunoblotting detected with mouse anti-LexA (Santa Cruz) at a 1:200 dilution and goat anti-mouse-HRP (Bio-Rad) at a 1:3000 dilution. Blot detection was achieved through incubation with DAB (Sigma-Aldrich). A single colony of NMY51 was inoculated in 2xYPAD at 30°C for 16 h. Cells were diluted to an initial OD546 of 0.2. At an OD546 of 0.6 to 0.8, cells were pelleted at 2,500 g for 5 min and resuspended in water. The bait and a prey cDNA mixed tissue (stem and leaf) library were added to 100 µL of cells and 300 µL of PEG/LiOAc and incubated at 42°C for 45 min. Cells were pelleted at 700 g for 5 min and resuspended in 150 µL of 0.9% NaCl and plated on SD/-Leu/-Trp, SD/-Leu/-Trp/-His, and SD/-Leu/-Trp/-His/-Ade, and incubated at 30°C for four days. To validate interactions, prey plasmids were isolated from formed colonies using the Zymoprep yeast plasmid miniprep kit (Zymo Research) and independently transformed into yeast as described by the manufacturer. The small scale yeast two-hybrid transformation was carried out as recommended by the manufacturer (Clontech). A single colony of the yeast strain Y2H Gold was inoculated in YPAD and grown at 30°C for 16 h. Cells were diluted to an initial OD600 of 0.2 and grown to a final OD600 of 0.6. Bait and prey plasmid DNA (0.1 μg) was added to 100 µL of cells and transformed using the PEG/LiOAc method at 30°C for 30 min, 42°C for 15 min, and plated on SD/-Leu/-Trp and SD/-Leu/-Trp/-His. Cells were incubated at 30°C for three to four days until colonies appeared. 52  3.2.9 Cell wall analyses For all cell wall analyses, four-month-old poplar stems with the periderm and pith removed, or dried stems (10 cm from the base) from six to eight-week-old Arabidopsis plants, were ground using a Wiley mill through a 40-mesh screen and acetone-extracted at 70°C for 16 h to remove extractives; all samples were dried overnight at 50°C before experimentation. Holocellulose and alpha-cellulose measurements were conducted as described by Porth et al. (2013b). To extract holocellulose, 3.5 mL of buffer (60 mL glacial acetic acid (GAA) and 1.3 g L-1 NaOH) and 1.5 mL of 20% (w/v) sodium chlorite was added to 100 mg of ground tissue and gently agitated at 50°C for 16 h. The reaction was quenched on ice and the extraction solution was removed. The extraction was repeated. The reacted stem material was washed twice with 50 mL of 1% GAA into a pre-weighed coarse sintered crucible, followed by a wash with 10 mL of acetone. Crucibles were dried at 50°C overnight. The yield was determined gravimetrically after 60 min in a dessicator at room temperature. To extract alpha-cellulose, 30 mg of holocellulose was reacted with 2.5 mL of 17.5% NaOH for 30 min at room temperature. A 2.5 mL aliquot of water was added and the reaction was stirred for 1 min and allowed to react for 29 min. The reaction solution was washed with distilled water (3 x 30 mL) into a pre-weighed course sintered crucible. The retentate in the crucible was soaked with 10 mL of 1.0 M acetic acid for 5 min and dried at 50°C overnight. The yield was determined gravimetrically after 60 min in a dessicator at room temperature. Lignin analysis was conducted as described by Skyba et al. (2013) and Mewalal et al. (2016). For Klason lignin analysis, 200 mg of ground tissue was treated with 3 mL of 72% sulfuric acid and consistently macerated for 2 h. Water was added to a final volume of 115 mL and autoclaved at 121°C for 1 h. Samples were filtered to isolate insoluble and soluble lignin in a glass crucible. Soluble lignin was quantified by measuring the absorbance at 205 nm. Structural cell wall carbohydrates were quantified from the soluble lignin fraction using a Dionex DX-600 equipped with a PA1 column. Monolignol composition was analyzed using the thioacidolysis method described by Robinson and Mansfield (2009). In brief, 1 mL of reaction mixture (2.5% (v/v) boron trifluoride etherate and 10% (v/v) ethanethiol in distilled dioxane) was added to 10 mg of tissue under nitrogen gas and heated at 100°C for 4 h, with manual agitation at 1 h intervals. The aqueous phase was extracted using 1 mL of methylene chloride and 2 mL of water, and vacuum-dried. Samples were resuspended in 1 mL of methylene chloride; 20 µL of 53  sample was derivatized with 20 µL of pyridine and 100 µL of N,O-bis(trimethylsilyl)acetamide (Sigma-Aldrich). Reactions were incubated at room temperature for 2 h and analyzed on a Thermo Fisher Scientific TRACE 1310 gas chromatograph.  3.2.10 Wood ultrastructure analyses Fibre length analysis was conducted as described by Porth et al. (2013b). Wood chips cut from a one centimeter diameter cookie were delignified in Franklin solution (1 volume of GAA to 1 volume of 30% H2O2) at 70°C for 48 h. Wood fibres were rinsed with water until neutralized and blended until a heterogeneous suspension was obtained. The suspension was diluted in water and analyzed on a Fibre Quality Analyzer (OpTest Equipment) where an average of 5,000 fibres was scanned to determine the length weighted average. For the crystallinity and microfibril angle measurements, X-ray diffraction patterns were recorded in transmission mode using a Bruker D8 Discover X-ray diffractometer with CuKα radiation generated at 40 kV and 40 mA. The source was equipped with a 0.5 mm collimator. Radial stem cross-sections analyzed were cut with a precision pneumatic saw to a thickness of 1.6 mm. For crystallinity, the source was set to an angle of 17 degrees while the detector was held at 0 degrees. Integrated density plots were generated between 4 to 40 degrees 2θ and -180 to 0 degrees chi. The crystallinity index was evaluated as a ratio of the intensity of the 002 diffraction peak to the intensity at the minimum between the 002 and 101 peaks (Segal et al., 1959), and calibrated with a bacterial cellulose sample of known crystallinity. For microfibril angle, both source and detector were maintained at 0 degrees. Diffraction patterns were analyzed between 20 and 24 degrees 2θ and -180 to 180 degrees chi. The average T-values were evaluated from the angular separation of the zero-intensity intercepts for lines drawn tangent to the curve at the inflection points on integrated intensity plots of the 002 diffraction arc. The microfibril angle was calculated using a poplar calibration curve (MFA = 0.9583*(T-value) − 3.8364) generated previously with measurements made using the differential interference contrast microscopy methodology as described by Wang et al. (2001).    54  Table 3.1 Primer sequences used in this study  Primer  Sequence (5’ to 3’)  Plant transformation   PtCPU F  ggaaaggacgagagagagca PtCPU R  ccccgtgttcagtaaccatt PtCPU FL-F  atgcgattccaatctcct PtCPU FL-R  caattgtttgagactacc AtCPU FL-F  atgttacctaatggagaa AtCPU FL-R  taatcctctggtttcacc AtCPU pro F  cgcttcctacggtggattgtt  AtCPU pro R  cttcaccagacgcaggagat  CMU1 pro F  ggcaccactcaaaatcgtct  CMU1 pro R  tgctggcatgaatgtgtct  CMU2 pro F  tatgcagctgtagtccacgt  CMU2 pro R  ggcctccaaaactcacaac    T-DNA mutant analysis   cpu LP  acctgtccaatcagtcctgtg cpu RP  agaagttgcccgtctaagagc cmu1 LP  ccccttcctctacaccatctc cmu1 RP  atcctcaacgcattctcacag cmu2 LP  gttcctggagtcggttttagg cmu2 RP  gtccagggcgagttttatttc CPU F  atgttacctaatggagaa CPU R  tgccctttcatctaccaagg CMU1 F1  aagattccccaaccgtatcc CMU1 R1  tcctcaacgcattctcacag CMU2 F1  aaagacgattctgcccttca CMU2 R1  gcgtgttttcccaatcttgt actin F  gcgacaatggaactggaat actin R  ggatagcatgtggaagtgcatacc   Y2H analysis   EcoRI pLexA AtCPU F  gaattcatgttacctaatggaggag PstI pLexA AtCPU R  ctgcagttatcctctggtttcacc EcoRI pGAD-CMU1 F  gaattcatgccagcaatgccaggtct BamHI pGAD-CMU1 R  ggatcctcagaacttgaaaccgaggct EcoRI pGAD-CMU2 F  gaattcatggacgtaggagagagcaatgag BamHI pGAD-CMU2 R  ggatcctcaataaaccggtctctgtccatttg EcoRI pGAD-CMU3 F  gaattcatggaaggagggtctgttaatg BamHI pGAD-CMU3 R  ggatccttaacgaagagctgaagaagaagtgag NdeI PtCPU F  catatgatgcgattccaatctcct EcoRI PtCPU R  gaattcctattgtttgagactacc NdeI pGAD-PtCMU1.1  catatgatgcctggtcttgtctctgt EcoRI pGAD-PtCMU1.1  gaattctcaaattctaaaaccccacttctt NdeI pGAD-PtCMU1.2 F  catatgatgccaggtcttgtctctgtta EcoRI pGAD-PtCMU1.2 R  gaattctcaaattctaaaaccccacttcttc ClaI pGAD-PtCMU2.1 F  atcgatatgccgggactagccatggatgtttta BamHI pGAD-PtCMU2.1 R  ggatccttacgagacctcaagatcatcgtc NdeI pGAD PtCMU4.1 F  catatgatgttgggcctagtctctgcaaa EcoRI pGAD PtCMU4.1 R  gaattcctaggagctggaatccaggaga ClaI GAD PtCMU4.2 F  atcgatctacgagctagaatctaggaga EcoRI GAD PtCMU4.2 R  gaattcatgttgggcctagtctct       55  3.3 Results   3.3.1 Structural and phylogenetic analysis of CPU PtCPU was found to have genetic association to total lignin content variation in poplar (Porth et al., 2013a) and encodes a POF (Mewalal et al., 2014). A BLAST search of the P. trichocarpa genome identified one paralog (POPTR_0009s07770/Potri.009G073600) that is 78% identical to PtCPU at the amino acid level. The Arabidopsis homolog of PtCPU, AtCPU (identification described in Chapter 2), shares 55% identity at the amino acid level. While sequence similarity is evenly distributed across the Arabidopsis and poplar homologs, differences in length are apparent, mainly at the N-terminus and an indel starting at position 105 of the alignment (Figure 3.1). As PtCPU and AtCPU are POFs without obvious protein domains, the in silico Simple Modular Architecture Research Tool (SMART) (Letunic et al., 2015) was used to identify structural motifs and to predict the secondary structure. This POF is predicted to possess coiled-coil domains at homologous amino acid positions at the N- and C-termini in poplar and Arabidopsis (Figure 3.1). A proline-rich region in between the coiled-coil domains was also evident in both CPU sequences. Together, these protein structures suggest CPU may engage in protein-protein interactions (Mewalal et al., 2014).  Previous sequence similarity trees (described in Chapter 2) indicated that CPU homologs are present in diverse land plants, including the non-vascular plant Physcomitrella patens. A more detailed phylogenetic reconstruction was carried out to examine the evolutionary history of PtCPU (Figure 3.2). Homologous CPU sequences from land plant representatives including moss, a lycopod, ferns, conifers, and angiosperms were analyzed (Appendix B.1 and Appendix B.2). The extent of homologs analyzed included a single characterized protein, Chloroplast Unusual Positioning 1 (CHUP1), an Arabidopsis actin-binding protein (Oikawa et al., 2003) that shares 43% similarity with PtCPU. A maximum likelihood phylogenetic analysis clustered the sequences into four distinct clades (I to IV). Poplar CPU homologs were present in each clade, while Arabidopsis representatives were found only in clades I to III. In clade I, seed plant CPU homologs clustered with proteins from all land plant representatives, including moss and lycopods. This clade also contained CHUP1. While AtCPU has been identified as a homolog of    56                  10        20        30        40        50        60       70                     ....|....|....|....|....|....|....|....|....|....|....|....|....|....|  AtCPU  -----------------MLPNGEDDSDLLRLVKELQAYLVRNDKLEKENHELRQEVARLRAQVSNLKSHE  PtCPU  MRFQSPVIAPMKAEQERKMRKEEDESLIIYLKKEVEAALLRTDSLEKENQDLRQEVVRLKAQICSLKAHD                   80        90       100       110       120       130      140                ....|....|....|....|....|....|....|....|....|....|....|....|....|....|  AtCPU  NERKSMLWKKLQSSYDGSNTDGSNLKAPESVKSN--------------------TKGQEVRNPNPKPTIQ  PtCPU  NERKSMLWKKLQNPFDSSKTEVFLQKQSDFVKVSERSVEHSSPRPSIQELAAIKEKHAKVPNPPPRPTYV                  150       160       170       180       190       200      210              ....|....|....|....|....|....|....|....|....|....|....|....|....|....|  AtCPU  GQS----------TATKPPPPPPLPSKRTLGKRSVRRAPEVVEFYRALTKRESHMGNKINQNGVLSPAFN  PtCPU  APPSLKEANDNKLPLTSAPPPPPPPPNMCAGSKAVRRVPEVVEFYRLLTRRDAHMENRTNSAAIPVVAFT                  220       230       240       250       260       270      280              ....|....|....|....|....|....|....|....|....|....|....|....|....|....|  AtCPU  RNMIGEIENRSKYLSDIKSDTDRHRDHIHILISKVEAATFTDISEVETFVKWIDEELSSLVDERAVLKHF  PtCPU  PNMIGEIENRSSYLSAIKSDVEKQKEFINFLIKEVESSAFKDISDVKAFVKWLDDELSSLVDERAVLKHF                  290       300       310       320       330       340      350              ....|....|....|....|....|....|....|....|....|....|....|....|....|....|  AtCPU  PKWPERKVDSLREAACNYKRPKNLGNEILSFKDNPKDSLTQALQRIQSLQDRLEESVNNTEKMRDSTGKR  PtCPU  PQWPERKADALREAAFNYRDLMNLESEVSSFQDNPKDLLTLALGRMQALQDRLERSIDNMERTRESMIKR                  360       370       380       390       400       410      420              ....|....|....|....|....|....|....|....|....|....|....|....|....|....|  AtCPU  YKDFQIPWEWMLDTGLIGQLKYSSLRLAQEYMKRIAKELESNGSGKEGNLMLQGVRFAYTIHQFAGGFDG  PtCPU  YRDFQIPWEWLLNTGLIGEMKLSSLRLAKVYLKRITKELQLNECSGEDNLLLQGARFAYRVHQFAGGFDA                  430              ....|....|....|....  AtCPU  ETLSIFHELKKITTGETRG*  PtCPU  ETIRAFQELKKIGMGSLKQ*   Figure 3.1 Secondary structure prediction for CPU from Arabidopsis (A. thaliana) and poplar (P. trichocarpa) using SMART (Letunic et al., 2015). Coiled-coil domains in AtCPU (dashed boxes) are predicted at amino acid positions 9 to 55 and 269 to 299, separated by a low complexity region enriched in proline residues at position 111 to 118 (solid box). For PtCPU, coiled-coil domains (underlined) are predicted at positions 29 to 65 and 320 to 354; a proline-rich region is also seen for PtCPU (solid box) but a low complexity region was not predicted by SMART. Amino acids shaded in grey and black represent similar or identical amino acids, respectively.   CHUP1 alongside two other Arabidopsis sequences (At1g48280 and At4g18570) (Oikawa et al., 2003), the phylogeny indicates AtCPU, CHUP1, and At4g18570 to be evolutionary distinct. At1g48280 was included in the initial phylogenetic analysis but was removed as it did not appear to resolve into any of the clades. Based on the alignment by Oikawa et al. (2003), At4g18570 is clearly divergent at the C-terminus compared to the other three homologs. In clades II, III, and      57     Spu03  83  Figure 3.2 An unrooted maximum likelihood phylogenetic analysis of CPU in land plants. Sequences from moss (Physcomitrella patens; dark green), a lycopod (Selaginella moellendorffi; light green), ferns (black), conifers (Pinus spp.; rose), monocots (red), and eudicots (light blue) resolve into four clades (I to IV). PtCPU and its homolog AtCPU group in clade III (also shown in the inset as Ptr01 and Ath01, respectively). Poplar (P. trichocarpa) sequences are indicated in dark blue and Arabidopsis (A. thaliana) sequences are in orange. The characterized CHUP1 (At3g25690) is in clade I. A complete list of species is listed in Appendix B.1. Bootstrap values over 90% are indicated by the black dots. The inset shows bootstrap values for clade III.                                              58  IV, only sequences from angiosperms were observed and for each clade, a clear split was evident between monocots and eudicots. Thus seed plant CPU homologs have been separated multiple times, at least prior to the monocot and eudicot split. Clade III contains PtCPU and its single poplar paralog, both of which contain putative orthologs in willow (Salix purpurea), a close relative of poplar within the Salicaceae. In clade III, a single Arabidopsis member is present which is AtCPU, the closest Arabidopsis CPU homolog to poplar PtCPU (Figure 3.2).  3.3.2 PtCPU expression in developing wood Analysis of developing wood transcriptomes of P. trichocarpa accessions (Geraldes et al., 2011) found PtCPU and its paralog (POPTR_0009s07770/Potri.009G073600) to both be expressed in developing secondary xylem. However, the mRNA abundance of PtCPU is approximately two and a half times higher than its paralog (Table 3.2). The in silico resource Aspwood (Sundell et al., 2017) was also used to examine PtCPU expression throughout wood development. RNA-seq analysis of sample clusters of phloem, vascular cambium, expanding xylem, and lignified xylem indicate PtCPU to be expressed at increasing levels in the vascular cambium, and remain at high levels in expanding and lignified xylem (Figure 3.3). The paralog is expressed in a similar manner but with decreased levels during xylem lignification.   Table 3.2 Expression of PtCPU and its paralog in developing secondary xylem of P. trichocarpa  CPU FPKM*    PtCPU  31.7 POPTR_0009s07770 13.4   *from Geraldes et al. (2011). FPKM: fragments per kilobase million.   To assess the cell-specific expression of PtCPU in planta, in situ mRNA hybridization was performed on one-month-old P. trichocarpa stems (Figure 3.4). PtCPU expression was detected via an anti-sense probe in cells of developing wood, including xylary fibres and vessel elements (Figure 3.4C), which is consistent with cells undergoing lignification as seen from cells stained red with safranin-astra blue (Srebotnik and Messner, 1994). Lignifying phloem fibres also expressed PtCPU. The signals detected for PtCPU were specific as the sense probe did not yield any detectable signal. 59                         PtCPU expression in developing wood suggested coordinated expression with secondary cell wall-related genes. A co-expression analysis based on almost 400 P. trichocarpa developing wood transcriptomes from nearly 200 accessions also used in the genetic association mapping study (Porth et al., 2013a), was performed. Using PtCPU as the bait, 300 genes of different functional classes were positively correlated at a Pearson correlation coefficient (PCC) of at least 0.60 (Appendix B.3). Of the top 25 co-expressed genes (PCC ≥ 0.69) the most highly co-expressed gene with PtCPU was an ortholog of SND1, a fibre SCW-related transcription factor (Zhong et al., 2006) (Figure 3.5). Korrigan, a β-1,4-endoglucanase, was also among the most highly co-regulated genes with PtCPU. Some lignin-related genes were also co-expressed with PtCPU. For example, an ortholog of Laccase 17 (LAC17), an enzyme active in lignin polymerization (Berthet et al., 2011), was highly co-expressed. ABC transporters have been implicated in monolignol transport. Here, ABC transporter AtABCB9 (PGP9), identified as a direct target of SND1 and VND7 (Zhong et al., 2010b), was also co-expressed with PtCPU. However, monolignol biosynthetic genes, most notably ones involved in sinapyl alcohol  Figure 3.3 In silico expression analysis of PtCPU in phloem and developing wood using AspWood (Sundell et al., 2017). Four different sample clusters from poplar were analyzed: phloem, the vascular cambium, expanding xylem, and lignified xylem.  phloem vascular cambium expanding xylem lignified xylem 60   Figure 3.4 In situ hybridization analysis of PtCPU expression in developing secondary xylem of one-month-old P. trichocarpa stems (internode 6). (A) Control cross-section hybridized with a sense probe for PtCPU. (B) Positive hybridization signal (purple) detected via an anti-sense probe is apparent in phloem fibres, xylary fibres, and vessel elements. (C) Close-up of positive hybridization signal seen in B. (D) Safranin-astra blue-stained stem cross-section indicating lignified cells (red). Pf: phloem fibres; pi: pith; sx: secondary xylem; ve: vessel elements; xf: xylary fibres. Scale is 100 μm in A and B, and 50 μm in C and D.   biosynthesis (HCT, C3H, and F5H) were not among the top 25 co-expressed genes but co-expressed with PtCPU at lower correlation coefficients (Appendix B.3).                                           50 µm 50 µm 100 µm 100 µm A B C D pi sx pf ve xf pi pf ve xf pf sx pi 61   Figure 3.5 The top 25 co-expressed genes to PtCPU after mean-centering, based on 384 P. trichocarpa developing wood transcriptomes. Yellow indicates high expression and blue indicates low expression. The entire co-expression list is presented in Appendix B.3.               PCC Poplar Gene ID (v2.2/v3.0) Arabidopsis Hit Gene Annotation  1.00 POPTR_0001s28570/Potri.001G279000 At1g07120 CPU 0.76 POPTR_0001s45250/Potri.001G448400 At2g46770 SND1  0.74 POPTR_0009s06540/Potri.009G060600 At5g12840 NF-YA1 transcription factor 0.73 POPTR_0003s15140/Potri.003G151700 At5g49720 KOR1  0.73 POPTR_0001s34120/Potri.001G333600 At5g14230 ankyrin repeat protein 0.73 POPTR_0008s14520/Potri.008G145900 At1g24030 protein kinase family protein 0.73 POPTR_0006s09370/Potri.006G092600 At5g02010 ROPGEF7 0.72 POPTR_0001s34280/Potri.001G354900 At4g18050 ABCB9  0.72 POPTR_0010s00550Potri.010G003100 At1g48280 hydroxyproline-rich glycoprotein family protein 0.71 POPTR_0001s41170/Potri.001G401300 At5g60020 LAC17  0.71 POPTR_0006s22780/Potri.006G211200 At5g22400 rac GTPase activating protein (putative) 0.71 POPTR_0001s44200/Potri.001G416300 At5g54670 ATK3 0.71 POPTR_0019s10720/Potri.019G078400 At1g72180 leucine-rich repeat transmembrane protein kinase 0.71 NA/Potri.008G144900 At1g10850 serine/threonine protein kinase 0.71 POPTR_0005s25250/Potri.005G230900 At1g27620 transferase family protein 0.70 POPTR_0003s06780/Potri.003G070200 At3g14170 CORD1 0.70 POPTR_0006s22110/Potri.006G204700 At3g52500 aspartyl protease family protein 0.70 POPTR_0010s19140/Potri.010G184000 At2g40320 trichome birefringence 33  0.70 POPTR_0011s07381/Potri.011G066100 At1g29170 WAVE2 0.70 POPTR_0003s14180/Potri.003G141900 At5g42050 asparagine-rich protein  0.70 POPTR_0016s05240/Potri.016G051900 At3g20860 NEK5  0.70 POPTR_0013s11220/Potri.013G107700 At1g22610 MCTP6  0.70 NA/Potri.010G184100 At5g05450 RH18  0.69 POPTR_0017s00520/Potri.017G000300 At2g44160 MTHFR2 0.69 POPTR_0018s05760/Potri.T137300 At1g15260 ATP-dependent RNA helicase-like protein 0.69 POPTR_0010s25110/Potri.010G244900 At5g06390 FLA17           While functional categories related to unknown genes and transcriptional regulators constituted the largest portion of PtCPU co-expressed genes, genes related to cell organization were also highly abundant (Table 3.3). ROPGEF7, for example, appeared to be the most highly co-expressed cell organization-related gene (Figure 3.5). ROPGEFs are Rho GTPase proteins 384 P. trichocarpa developing xylem transcriptomes  62  implicated in signalling processes required for plant development and environmental sensing (Mucha et al., 2011). Arabidopsis ROPs have been implicated in patterning of the secondary cell wall via interaction with IQ67 Domain 13 (IQD13) (Sugiyama et al., 2017) and Kinesin-13A (Oda and Fukuda, 2013). Furthermore, Cortical Microtubule Disordering 1 (CORD1), previously known as Domain of Unknown Function 936 (DUF936), was also coordinately expressed with PtCPU. Recent characterization of CORD1 in Arabidopsis found a role related to the formation of the pitted patterning of secondary cell walls in metaxylem vessel elements, suggesting CORD1 to be a MAP (Sasaki et al., 2017).                Table 3.3 MapMan annotation abundance for genes co-expressed with PtCPU at a PCC of at least 0.60  MapMan Annotation Gene Count Functional Classificationa Term ID    Not Assigned (Unknown) 35.2 52 RNA Regulation of Transcription 27.3 29 Protein Degradation 29.5 24 Cell Organization 31.1 22 Protein Post-Translational Modification 29.4 17 Development (Unspecified) 33.99 14 Misc Transport 34 13 Misc UDP Glucosyl and Glucoronyl Transferases 26.2 10 Amino Acid Metabolism (Synthesis) 13.1 9 Secondary Metabolism (Phenylpropanoids) 16.2 8 Signalling (Receptor Kinases) 30.2 8 Cell Wall (Degradation) 10.6 8 Abiotic Stress  20.2 8 Cell Wall (Cellulose Synthesis) 10.2 7 Secondary Metabolism (Simple Phenols) 16.1 5 Signalling (Calcium) 30.3 5 Cell Wall (Cell Wall Proteins - AGP) 10.5 5 Hormone Metabolism (Auxin) 17.2 4 DNA Synthesis (Chromatin Structure) 28.1 4 Misc Cytochrome P450 26.1 4 Misc Signalling 30 4 Cell Wall (Hemicellulose Synthesis) 10.3 3 Misc Lipid Metabolism 11 3 Misc Acid and Other Phosphatases 26.13 3 Protein Glycosylation 29.7 3 Protein Targeting (Secretory Pathway) 29.3 3 Amino Acid Metabolism (Degradation) 13.2 2 Cell Vesicle Transport 31.4 2 C1 Metabolism 25 2                                       afunctional classifications represented by at least two genes are listed.        Refer to Appendix B.3 for the entire gene list.  63  3.3.3 Wood phenotyping analysis of transgenic poplar over-expressing PtCPU To investigate the biological role of CPU in wood development, the entire coding sequence of PtCPU was over-expressed in hybrid poplar clone 717 (P. tremula x P. alba) under the constitutive control of a double 35S cauliflower mosaic virus (CaMV) promoter. Transgenic levels of PtCPU compared to WT were analyzed using quantitative real-time PCR. Transgenic lines 34 and 32 exhibited significant over-expression levels compared to WT, whereas line 14 was not statistically significant due to an outlier and the limitation of having analyzed only three biological replicates per line despite the greater than ten-fold increase in expression (Figure 3.6). Stem sections of over-expression lines stained with toluidine blue-O did not indicate cellular defects compared to WT (Appendix B.4). Since PtCPU had a genetic association to total lignin content variation in P. trichocarpa (Porth et al., 2013a), Klason lignin was measured. Total lignin content in WT and PtCPU over-expression lines ranged from 22.8 to 23.3 % DW (Table 3.4), but no significant changes in insoluble, soluble, or total lignin content were found. However, quantification of structural cell wall carbohydrates revealed a minor but significant decrease in glucose content in over-expression lines 14 and 34 compared to WT. Monolignol composition was also analyzed in WT and two PtCPU over-expression lines through thioacidolysis. Changes in guaiacyl and syringyl lignin were also not detected (Table 3.5).  3.3.4 Fibre length analysis In poplar wood, nearly half of the cells are fibres (Mellerowicz et al., 2001). To test whether over-expression of PtCPU influenced fibre morphology in poplar, wood fibre length was measured. Using a Fibre Quality Analyzer, 5,000 fibres were scanned to determine the length weighted average fibre length for WT and the three PtCPU over-expression lines. Wood from all three PtCPU over-expression lines had shorter fibres relative to WT; the phenotype was significant (P < 0.01) for lines 14 and 32 (Figure 3.7). Transgenic lines 14 and 32 which exhibited the highest over-expression levels had nearly a 10 to 15% reduction in fibre length compared to WT, whereas the moderate over-expression line 34 had close to a 5% reduction in fibre length.    64   Figure 3.6 Expression analysis of wild-type (WT) and transgenic poplar over-expressing PtCPU. Expression was measured relative to elongation factor 1β. Mean and standard error of the mean for three clonal replicates is shown in grey. Individual data points in black represent the mean of three technical replicates measured for each clonal replicate. Asterisks represent significant differences from WT based on a Student’s t-test (P < 0.05).                             Table 3.4 Analysis of structural cell wall carbohydrate composition and total lignin content in wild-type (WT) and transgenic hybrid poplar lines over-expressing PtCPU  Line Carbohydrates (% DW)  Klason Lignin (% DW) Arabinose Rhamnose Galactose Glucose Xylose Mannose  Insoluble Soluble Total  WT 0.32 (0.01) 0.43 (0.02) 0.65 (0.01) 45.29 (0.14) 16.73 (0.28) 1.92 (0.10)  20.59 (0.51) 2.72 (0.06) 23.32 (0.46) 14 0.38 (0.01) 0.46 (0.01) 0.69 (0.01) 42.63 (0.27) 16.09 (0.17) 1.90 (0.11)  20.49 (0.56) 2.63 (0.09) 23.12 (0.50) 32 0.39 (0.01) 0.44 (0.02) 0.71 (0.03) 44.67 (1.89) 15.76 (0.92) 1.34 (0.14)  20.01 (0.10) 2.77 (0.17) 22.78 (0.23) 34 0.33 (0.01) 0.45 (0.01) 0.66 (0.01) 43.35 (0.12) 16.25 (0.16) 1.87 (0.10)  20.46 (0.32) 2.66 (0.06) 23.12 (0.37)         Mean and standard error of the mean (in parentheses) for three clonal replicates.         Bold values indicate significant differences from WT based on a Student’s t-test (P < 0.01).        DW: dry weight. * * 65   Figure 3.7 Mean fibre lengths measured for wild-type (WT) and transgenic poplar over-expressing PtCPU. Mean and standard error of the mean for three clonal replicates is shown in grey. Individual data points in black represent the length weighted average of 5000 fibres measured for each clonal replicate. Asterisks represent significant differences from WT based on a Student’s t-test (*P < 0.05, **P < 0.01).    Table 3.5 Thioacidolysis analysis of guaiacyl and syringyl lignin                                         in transgenic poplar lines over-expressing PtCPU      Line      Lignin S:G Guaiacyl (% G)  Syringyl (% S)      WT 29.92 (0.19)  70.08 (0.19) 2.34 (0.02) 14 29.77 (0.45)  70.23 (0.45) 2.36 (0.05) 34 29.66 (0.58)  70.34 (0.58) 2.37 (0.06)      Mean and standard error of the mean (in parentheses) for three clonal replicates. No significant differences from WT based on a Student’s t-test (P < 0.05).                                 WT 34 14 32 * ** ** 66   Figure 3.8 Microfibril angle and crystallinity analysis for transgenic poplar over-expressing PtCPU. (A) Microfibril angle for transgenic lines 14 and 32 are significantly different from wild-type (WT) based on a Student’s t-test (P < 0.05), indicated by an asterisk. (B) Crystallinity did not differ between WT and the poplar transgenics. Mean and standard error of the mean for three clonal replicates is shown in grey. Individual data points in black represent the average of three technical measurements for each clonal replicate.  A         B      3.3.5 Cellulose microfibril angle and crystallinity analysis To test whether mis-regulation of PtCPU influenced ultrastructural wood properties, WT and the PtCPU over-expression lines were analyzed for crystallinity and microfibril angle (MFA). The MFA is the angle of the cellulose microfibril relative to the long axis of the cell (Maloney and Mansfield, 2010). Here, transgenic lines 14 and 32 exhibited a significant increase in MFA, which was nearly a 50% increase compared to WT (Figure 3.8A). The MFA in the moderate over-expression line 34 had approximately a 10% increase in MFA. Changes in cellulose crystallinity between WT and the transgenic poplars were not observed (Figure 3.8B).   * * 67  3.3.6 Heterologous over-expression of PtCPU in Arabidopsis To expand the analysis for CPU, fluorescent protein-tagged PtCPU was over-expressed in Arabidopsis ecotype Columbia-0 (Col-0). Stem cross-section analysis of transgenic over-expression lines of 35S:YPF-PtCPU were found to have increased secondary cell wall deposition in the interfascicular fibres compared to WT (Figure 3.9). 35S:YPF-PtCPU over-expression (OX) line 4 had nearly a 50% increase in cell wall thickness compared to WT. Stem cross-section analysis of the same transgenic lines did not indicate an irregular xylem phenotype in the vascular bundles (Appendix B.5). However, the rosette leaves and silique morphology differed from WT as these organs appeared to be more rounded (Appendix B.5).                              plant 1 plant 2  Figure 3.9 Over-expression of 35S:YFP-PtCPU in Arabidopsis Col-0 increases secondary cell wall deposition in the interfascicular fibres compared to wild-type (WT). Cross-sections of basal stems from six-week-old T2 plants were stained with toluidine blue-O. Interfascicular fibres of WT (A) and 35S:YFP-PtCPU (B). Scale bar represents 50 µm. (C) Quantification of secondary cell wall thickness in interfascicular fibre cells. For each genotype two plants were analyzed. Black dots are individual measurements, where 80 cells were measured in total. The boxplot represents a 95% confidence interval and the black bar in the box represents the median. The blue dashed line represents the mean.  B A C 68  Figure 3.10 Western analysis of LexA-AtCPU expression in the yeast (Saccharomyces cerevisiae) strain NMY51. The expected size of LexA-AtCPU is approximately 60 kDa.  The blot was detected using anti-LexA from mouse (1:200 dilution) and DAB. 3.3.7 Yeast two-hybrid screen for potential interactors of AtCPU  CPU does not have annotated functional domains but contains structural motifs that could facilitate protein-protein interactions. To investigate whether AtCPU has a protein-interaction partner, a yeast two-hybrid (Y2H) screen was conducted using a LexA DNA-binding domain fused to AtCPU (LexA-AtCPU) as the bait and an Arabidopsis mixed tissue (leaf and stem) cDNA library as the prey. Western analysis confirmed the expression of the LexA-AtCPU construct in yeast, at an expected molecular weight of approximately 60 kDa (Figure 3.10, second lane). Transformation of LexA-AtCPU with the prey library resulted in approximately 300 colonies after four days of growth on SD/-Leu/-Trp/-His in the absence of 3-AT.                         Due to the high number of colonies obtained on SD/-Leu/-Trp/-His, all 300 colonies were replated under high stringency selection conditions (SD/-Leu/-Trp/-His/-Ade) to identify the LexA-AtCPU LexA-p53 (+) MW 58  MW 80 kDa 69  Figure 3.11 Yeast two-hybrid screen of LexA-AtCPU identified an armadillo-repeat protein (ARM) and Cellulose-Microtubule Uncoupling 1 (CMU1) to be strong interactors under stringent selection conditions. Left: Control conditions on SD/-Leu/-Trp. Right: Stringent selection conditions on SD/-Leu/-Trp/-His/-Ade. (a) KNAT3. (b) CMU1. (c) CMU1 (full-length cDNA clone). (d) ARM. (e) p53/ACT positive control. (f) SEC3A. (g) PAKRP1. (h) MAP65-7. strongest interactors for AtCPU. Of the 300 colonies, 45 colonies formed on SD/-Leu/-Trp/-His/-Ade. To validate the interactions detected under the stringent conditions, plasmids were extracted from each of the colonies and retransformed into yeast with the bait. From interactions where at least 100 colonies formed on a single plate, the corresponding prey plasmid was subjected to DNA sequencing. BLAST analysis revealed two cDNAs, an armadillo-repeat protein (ARM) and Cellulose-Microtubule Uncoupling 1 (CMU1), to be present more than once out of ten plasmids  sequenced including a full-length cDNA clone for the latter (Figure 3.11). PCR screening identified additional clones for ARM and CMU1. To further examine the suite of putative interactors for AtCPU, formed colonies from the less stringent selection conditions (SD/-Leu/-Trp/-His) were also analyzed. DNA sequencing of 40 additional prey plasmids identified five other partial cDNA sequences with at least two clones, including MAP65-7 (Table 3.6). While MAP65-7 has not been characterized, over-expression of MAP65-1 from zinnia (Zinnia elegans) in Arabidopsis cell suspension results in cortical microtubule bundling, indicating a potential role in tracheary element formation (Mao et al., 2006). Taken together, ARM and CMU1 were the most prominent among the interacting proteins and interacted with AtCPU under stringent selection conditions with comparable growth to the positive control (Figure 3.11). CMU1 was chosen for further experimentation as recent evidence from literature and in silico expression data suggested a cell wall-related role (Liu et al., 2016).                    SD/-Leu/-Trp/-His/-Ade SD/-Leu/-Trp a b c d e f g hb c d e 70  Table 3.6 AtCPU-interacting proteins identified from a yeast two-hybrid screen using LexA-AtCPU as the bait and a mixed tissue (leaf and stem) cDNA library as the prey  Interactor  % clones Arabidopsis thaliana TAIR annotation -A -H    At5g10200 28 ARM armadillo-repeat protein + + At4g10840 26 CMU1 cellulose-microtubule uncoupling 1  + + At5g65495 18 NA unknown protein - + At5g25220* 6 KNAT3 class II knotted1-like homeobox transcription factor - + At4g35550  6 WOX13 WUSCHEL-related homeobox transcription factor - + At1g47550* 6 SEC3A member of the exocyst complex gene family - + At1g14690* 6 MAP65-7 microtubule-associated protein 65-7 - + At5g39590 4 NA TLD-domain containing nucleolar protein - + At4g14150  4 PAKRP1 phragmoplast-associated kinesin-related protein 1 - + At1g31780  4 COG7 conserved oligomeric Golgi complex 7 - +       -A: growth on SD/-Leu/-Trp/-His/-Ade; -H: growth on SD/-Leu/-Trp/-His.   The interaction between AtCPU and CMU1 was found under the most stringent conditions of the Y2H screen. CMU1 is a member of a small gene family in Arabidopsis containing three members (Bürstenbinder et al., 2013). BLAST searches of the poplar genome found seven CMU-like sequences that group into four clades; three of the clades contain an Arabidopsis CMU ortholog while PtCMU4.1 and PtCMU4.2 in the CMU-IV clade appear to lack an Arabidopsis counterpart (Figure 3.12A). To confirm the original AtCPU interaction with CMU1 and to extend the analysis to other CMU proteins, an independent Y2H system was used. AtCPU and its potential interaction with all three Arabidopsis CMUs (CMU1, CMU2, and CMU3) were tested (Figure 3.12B). AtCPU fused to a Gal4 DNA-binding domain was transformed into yeast with a CMU prey fused to a Gal4 activation domain. When selected on SD/-Leu/-Trp/-His, CMU2 was the only sequence detected to have a positive interaction, while the original interaction with CMU1 could not be confirmed in this system (Figure 3.12B). Next, I tested whether PtCPU can interact with poplar CMU members of the CMU-I, CMU-II and CMU-IV clades. Interestingly, PtCPU was found only to interact with PtCMU4.2, a member of the CMU-IV clade (Figure 3.12A, B). The latter can also can interact with AtCPU in yeast. PtCMU4.1 and PtCMU2.1 (CMU-II clade) are co-expressed with PtCPU (Appendix B.3, PCC = 0.63). Taken together, the findings suggest CPU to interact with distinct members of the CMU family in both poplar and Arabidopsis.71   Figure 3.12 Protein-protein interaction of CPU and CMUs from Arabidopsis (A. thaliana) and poplar (P. trichocarpa). (A) Unrooted neighbour-joining tree of CMUs from Arabidopsis (orange) and poplar (blue), which separate into four clades: CMU-I, CMU-II, CMU-III, and CMU-IV. (B) Protein-interaction for CPU (bait) and CMU (prey) in yeast. Colony growth was compared to the appropriate controls after three days at 30°C. GBKT7-BD: DNA-binding domain bait vector. GADT7-AD: activation domain prey vector. Control on SD/-Leu/-Trp. Selection on SD/-Leu/-Trp/-His (0 mM 3-AT). PtCMU1.1 (POPTR_0001s10980); PtCMU1.2 (POPTR_0003s14320); PtCMU2.1 (POPTR_0008s09430); PtCMU3.1 (POPTR_0012s08160); PtCMU3.2 (POPTR_0015s08660); PtCMU4.1 (no alias for poplar genome v2.2/Potri.002G173400 from v3.0); PtCMU4.2 (POPTR_0014s09590).   I IV III II                              0.08 AtCPU/CMU2 AtCPU/PtCMU4.2 AtCPU/CMU3 AtCPU/CMU1 GBKT7-BD/GADT7-AD AtCPU/GADT7-AD PtCPU/PtCMU4.2 PtCPU/PtCMU4.1 PtCPU/GADT7-AD PtCPU/PtCMU1.1 PtCPU/PtCMU1.2 PtCPU/PtCMU2.1 A  B 72  3.3.8 Expression of AtCPU and its putative interactors CMU1 and CMU2 Analysis of the Arabidopsis eFP browser (Winter et al., 2007) indicated AtCPU to be expressed in the second internode of the mature stem. To analyze AtCPU expression in vivo, a 1100 bp promoter region was fused to GUS. The bolting main inflorescence stem was examined at the shoot apex, mid-stem, and at the base as xylem and interfascicular fibre cells develop at different stages (Ehlting et al., 2005). Transgenic Arabidopsis expressing AtCPUpro:GUS showed localized expression in regions of developing xylem tissue, including at the tip and mid-stem (Figure 3.13B, C). AtCPUpro:GUS expression was also observed in the interfascicular fibre region, consistent with developing interfascicular fibres. At the basal stem, AtCPUpro:GUS expression localized to the phloem (Figure 3.13D). As interacting proteins need to be expressed in overlapping spatial and temporal patterns, the Arabidopsis eFP browser was also analyzed for in silico expression patterns of CMU1 and CMU2. Like AtCPU, CMU1 and CMU2 are also highly expressed in the second internode of the stem (Figure 3.14A, B). Again, to examine their expression in vivo, transgenic Arabidopsis CMU promoter-GUS lines were generated. In bolting inflorescence stems, CMU1pro:GUS and CMU2pro:GUS expression was evident in the cortex and developing protoxylem strands in the shoot tip (Figure 3.14C, D). In the basal stem where secondary cell wall biosynthesis has ceased in both the xylem and interfascicular region, GUS expression in CMU1 and CMU2 promoter-GUS lines remained localized in the cortex but was also evident in the phloem (Figure 3.14E, F). Taken together, AtCPU and its protein interaction partner(s) CMU1 and CMU2 exhibit overlapping expression profiles in the Arabidopsis inflorescence stem in regions of fibre development, consistent with the eFP expression data.               73                                       Figure 3.13 AtCPUpro:GUS expression analysis in bolting Arabidopsis inflorescence stems. Cross-sections of: (B) shoot apex (1 cm from the tip), (C) mid-stem (5 cm from the base), and (D) basal stem (1 cm segment at 10 cm) stained in X-gluc. (A) control (unstained) stem section. Iff: interfascicular fibres; ph: phloem; pi: pith; x: xylem. Scale bar is 50 µm.   A B C D pi x x ph iff iff x ph pi pi pi x ph ph 74          at CMU1pro:GUS CMU2pro:GUS  Figure 3.11 Expression analysis of CMU1 and CMU2 in bolting Arabidopsis inflorescence stems. (A, B) Arabidopsis eFP expression for CMU1 (A) and CMU2 (B). High expression is seen in the second internode of the stem. (C to F) CMU1 (C, E) and CMU2 (D, F) promoter∷GUS analysis in x-gluc. (C, D) Cross-section of the shoot apex (1 cm from the tip). (E, F) Cross-section of the basal stem (10 cm). Scale bar is 50 µm. A B C E D F                                           Figure 3.14 Expression analysis of CMU1 and CMU2 in bolting Arabidopsis inflorescence stems. (A, B) Arabidopsis eFP expression for CMU1 (A) and CMU2 (B). High expression is seen in the second internode of the stem ( ircled). (C to F) CMU1 (C, E) and CMU2 (D, F) promoter-GUS analysis in X-gluc. (C, D) Cross-section of the shoot apex (1 cm from the tip). (E, F) Cross-section of the basal stem (1 cm segment at 10 cm). Co: cortex; iff: interfascicular fibres; ph: phloem; pi: pith; x: xylem. Scale bar represents 50 µm.  x pi ph pi x ph iff x pi ph pi iff x ph co co 75  3.3.9 cpucmu1cmu2 higher order mutant analysis To investigate the role of AtCPU and its interaction with CMU1 and CMU2 in vivo, T-DNA insertion mutants were obtained for cpu, cmu1, and cmu2. As functional redundancy is possible for CMU1 and CMU2, the double mutant cmu1cmu2 was created. Further to this, a higher order cpucmu1cmu2 triple mutant was generated to assess the biological effect of their interaction. The T-DNA insertion mutants appeared to be true knockouts as seen through reverse-transcription PCR analysis (Figure 3.15). Ten days after germination, cpu did not exhibit obvious developmental phenotypes compared to WT. However, a root skewing phenotype was apparent for the cmu1cmu2 double mutant and the cpucmu1cmu2 triple mutant (Appendix B.6); a cell twisting phenotype in the hypocotyl was previously reported for cmu1cmu2 (Liu et al., 2016), designated here as cmu1-1cmu2 since a different cmu1 allele was used by Liu et al. (2016). In six-week-old plants, developmental differences were not apparent. Cross-sections of the main inflorescence stem for cpu, cmu1cmu2, and cpucmu1cmu2 mutants were analyzed for anatomical defects. A mild irregular xylem phenotype was seen in the vascular bundles of the cpucmu1cmu2 triple mutant but not in the cpu single mutant, the cmu1cmu2 double mutant, or WT (Figure 3.16). Within the interfascicular region, secondary cell wall thickness in the fibres appeared to decrease in the triple mutant compared to WT, but not in the cpu and cmu1cmu2 mutants (Figure 3.17). The phenotype of the triple loss of function mutant is opposite to the increased cell wall thickness phenotype observed for the PtCPU over-expression mutants in WT Arabidopsis.               76                                             CPU  actin  no RT  WT cpu A B WT cmu1cmu2 cpucmu1cmu2 CPU  CMU1 CMU2  actin  no RT  Figure 3.15 Reverse-transcription PCR analysis of cpu, cmu1cmu2, and cpucmu1cmu2 mutants relative to actin. (A) Analysis of CPU in cpu. (B) Analysis of CPU, CMU1, and CMU2 in the cmu1cmu2 double mutant and the cpucmu1cmu2 triple mutant. CPU, CMU1, and CMU2 transcripts appear to be absent in the relevant T-DNA mutants and present in wild-type (WT). All genes were analyzed for 28 cycles. The no reverse-transcriptase (no RT) control was run for 34 cycles.   77                                               WT cmu1cmu2 cpu cpucmu1cmu2  Figure 3.16 Anatomical analysis of the basal portion of the main inflorescence stem for wild-type (WT), cpu, cmu1cmu2, and cpucmu1cmu2 mutants stained with toluidine blue-O. Left: vascular bundles of WT and mutants. Right: interfascicular fibre region of WT and mutants. The triple mutant cpucmu1cmu2 appears to have a mild irx phenotype (denoted by the arrow), relative to WT, cpu, and cmu1cmu2. Scale bar is 100 μm.  78                                The basal portion of mature inflorescence stems from WT and the higher order mutants were subjected to chemical analyses. As stated above, over-expression of PtCPU in transgenic poplar did not have a measurable effect on total lignin content. Likewise, a significant difference in total lignin content (or S:G ratio) relative to WT was not found for any of the Arabidopsis mutants analyzed (Table 3.7). Since the co-expression analysis suggested PtCPU be coordinately expressed with cellulose biosynthesis genes, holocellulose and alpha-cellulose was extracted and  Figure 3.17 Quantification of secondary cell wall thickness in interfascicular fibre cells of wild-type (WT), cpu, cmu1cmu2, and cpucmu1cmu2. For each genotype, two plants were analyzed where 80 cells were measured in total (represented by the black dots). The boxplot represents a 95% confidence interval and the bar in the box is the median. The blue dashed line is the mean.   WT cpu cmu1cmu2 cpucmu1cmu2 plant 1 plant 2 79  quantified for WT, cpu, cmu1cmu2, and cpucmu1cmu2. Mean holocellulose content was significantly reduced in all three mutants compared to WT (Figure 3.18A). The cpu single mutant showed the least reduction, while its combination with cmu1cmu2 had the greatest reduction; cpucmu1cmu2 contained 41% DW holocellulose content compared to WT at 44.5% DW. While there appeared to be an additive effect on holocellulose reduction in the triple mutant, the within sample variation was high and therefore, differences in reduction between mutants were not significant. Differences in alpha-cellulose content for WT and the three mutants were not found, which may also be attributed to the within sample variation and the limited number of replicates (n = 3) available for the analysis (Figure 3.18B).    Table 3.7 Lignin analysis of wild-type (WT), cpu and cpucmu1cmu2 mutants  Line Klason Lignin (% DW)  Thioacidolysis Insoluble Soluble Total  % G % S S:G         WT 15.6 (0.14) 2.2 (0.30) 18.08 (0.24)  54.59 (0.71) 27.00 (0.47) 0.49 cpu* 16.1 (0.08) 3.0 (0.42) 19.17 (0.35)  55.83 (0.06) 28.99 (0.20) 0.54 cmu1cmu2 - - -  55.66 (0.58) 27.93 (0.29) 0.50 cpucmu1cmu2 - - -  55.94 (0.11) 26.19 (0.30) 0.48         Mean and standard error of the mean (in parentheses) for three aliquots of pooled Arabidopsis stems, where applicable. Klason lignin not analyzed for cmu1cmu2 and cpucmu1cmu2 due to limited sample. *thioacidolysis performed in duplicate for this sample. No significant differences from WT based on a Student’s t-test (P < 0.05). DW: dry weight.            80                               Figure 3.18 Holocellulose and alpha-cellulose content in stems of wild-type (WT) and cpu, cmu1cmu2, and cpucmu1cmu2 mutants. (A) Holocellulose. (B) Alpha-cellulose. Grey dots represent the mean and standard error of the mean for three replicates. Black dots represent individual measurements from an aliquot of pooled Arabidopsis stems (bottom 10 cm). Significant differences in holocellulose content in mutants compared to WT were detected based on a Student’s t-test, indicated by an asterisk (P < 0.05). DW: dry weight.   3.4 Discussion                                                                                                                                                                                                                                                                                                 Studies related to wood development in trees are limited, relative to our knowledge of secondary cell wall biosynthesis in Arabidopsis. The identification of CPU from a genetic association mapping experiment in poplar (P. trichocarpa) provided an opportunity to investigate a POF hypothesized to influence secondary cell wall deposition. While a connection cpu cmu1cmu2 cpucmu1cmu2 WT cpu cmu1cmu2 cpucmu1cmu2 WT       A                                                    B * * * 81  to total lignin content variation was not found through chemical analysis of transgenic poplar over-expressing PtCPU, the latter exhibited altered fibre length and cellulose microfibril angle compared to WT wood. In Arabidopsis, over-expression of PtCPU also yielded a fibre phenotype, where secondary cell wall thickness appeared to increase compared to WT. Analysis of CPU in Arabidopsis found AtCPU interacted with CMU1 and CMU2 in vitro; the cpucmu1cmu2 triple mutant had reduced secondary cell wall thickness in interfascicular fibres and an irregular xylem phenotype, both of which are typical of mutants with altered secondary cell walls. Thus, the underlying connection to the cortical microtubules seen here suggests an indirect role for CPU in cellulose ultrastructure during secondary cell wall deposition in both poplar and Arabidopsis.  3.4.1 Co-expression analysis of CPU indicates a potential role in secondary cell wall deposition in fibre cells While genes encoding for lignin biosynthetic enzymes such as C3H and F5H were co-expressed with PtCPU to some level, the highest coordinated gene expression with PtCPU was to an ortholog of SND1/NST3, a key transcriptional regulator of cellulose, xylan, and lignin biosynthetic genes in fibres of Arabidopsis (Zhong et al., 2006; Mitsuda et al., 2007). Orthologs of Arabidopsis NAC domain proteins have been identified in poplar (P. trichocarpa) (Zhong et al., 2010a; Ohtani et al., 2011), suggesting these proteins to have similar regulatory functions in wood. Transcriptomic analysis of Arabidopsis SND1 over-expression lines found a five-fold increase in AtCPU and other secondary cell wall-related genes including the transcriptional regulator SND2 (Hussey et al., 2011), relative to WT (Ko et al., 2007). SND2 over-expression in Arabidopsis increased secondary cell wall thickening in the interfascicular fibres (Zhong et al., 2008). Transgenic Arabidopsis over-expressing PtCPU also exhibited enhanced secondary cell wall deposition in the interfascicular fibres compared to WT. While SND2 is not a direct target of SND1, SND2 can activate cellulose biosynthetic genes (Zhong et al., 2008). Therefore, it is plausible that over-expression of PtCPU has an indirect effect on the deposition of secondary cell wall components such as cellulose, regulated through a mechanism mediated by SND1 and/or SND2.  82  PtCPU over-expression in transgenic poplar resulted in shorter fibres and increased cellulose microfibril angle relative to WT. Fibres develop via intrusive tip growth, i.e. as the cell elongates it penetrates through the middle lamella of neighbouring cells (Snegireva et al., 2010). It is largely accepted that after cell expansion has ceased, the secondary cell wall is deposited and lignification persists until programmed cell death, the last developmental stage (Samuels et al., 2006; Bollhöner et al., 2012). However, it has been proposed that wood fibre cells continue to elongate at the tips as the secondary cell wall is deposited in the main cell body which leads to delayed secondary cell wall deposition at the tips (Gorshkova et al., 2012). Presumably, this is mediated by proteins involved in cortical microtubule re-orientation. For example, fibre length and cellulose and hemicellulose deposition was altered in interfascicular fibres of fra2 mutants, consistent with a role for Fragile Fiber 2/Katanin 1 as a putative microtubule-severing protein required for cell elongation and cell wall deposition (Burk et al., 2001). In the same fra2 mutants, Burk and Ye (2002) found cellulose microfibril orientation to be distorted in the secondary cell walls of fibre cells, correlated with disorganized cortical microtubules. The β-1,4-endoglucanase KOR is another protein that has a role in cell elongation and cellulose biosynthesis and modification (Sato et al., 2001; Glass et al., 2015). In poplar, suppression of KOR altered cellulose ultrastructure as transgenic poplar had a smaller microfibril angle compared to WT (Maloney and Mansfield, 2010), suggesting an indirect role for KOR during cellulose biosynthesis. Interestingly, KOR was the third most highly co-expressed gene with PtCPU. Taken together, it is plausible that the phenotypes for the transgenic poplar PtCPU over-expression lines are a consequence of altered cellulose microfibril deposition.   3.4.2 CPU interacts with a microtubule-associated tetratricopeptide repeat protein in vitro The enrichment in “cell organization” genes from the PtCPU co-expression analysis is consistent with the CPU-CMU protein interaction in vitro. While I identified seven putative CMUs in poplar, only PtCMU2.1 and PtCMU4.2 were co-expressed with PtCPU and the latter was found to have a protein interaction with PtCPU in yeast. AtCPU and CMUs also appear to have a genetic interaction in vivo as suggested by the appearance of decreased secondary cell wall thickness in the interfascicular fibres of the cpucmu1cmu2 triple mutant compared to WT, which was not evident in the cpu and cmu1cmu2 mutants. Cortical microtubules help to organize secondary cell wall formation (Oda et al., 2005) such as for the guidance of cellulose synthase 83  complexes during cellulose deposition (Watanabe et al., 2015). However, microtubule-associated proteins are also required. In Arabidopsis, CMUs stabilize cortical microtubules as cellulose synthases traverse the plasma membrane during cellulose biosynthesis in primary cell walls; in the cmu1cmu2 mutant, microtubule-based guidance of cellulose synthase complexes was not seen due to lateral microtubule displacement (Liu et al., 2016). MAPs, including CMUs, have been detected in Arabidopsis suspension cells undergoing TE differentiation (Derbyshire et al., 2015). Therefore, it is plausible that CMUs exhibit similar roles during cellulose biosynthesis in secondary cell walls. While cpu, cmu1cmu2, and cpucmu1cmu2 higher order mutants did not have altered alpha-cellulose content as a whole, it is tempting to speculate that CPU alongside the CMUs may affect the directionality of the cellulose synthase complexes and as a consequence affect cellulose MFA. Therefore, an analysis of cellulose ultrastructure for the cpucmu1cmu2 mutant is warranted as both CMU and KOR appear to be co-expressed with primary cell wall CesAs (Yang et al., 2011).  Bürstenbinder et al. (2013) reported CMU1 to interact with the calmodulin (CaM) binding protein IQD1, identified through a Y2H screen. Furthermore, a pull-down assay also indicated IQD1 to bind CaM2. Since GFP-tagged IQD1 localizes to microtubules in vivo, the authors hypothesize IQD1 to act as a scaffold protein for both CaM2 and CMU1, where a kinesin motor protein bound through CMU1 facilitates the directional transport of cargo via the microtubules (Bürstenbinder et al., 2013). While IQD1 was not co-expressed with PtCPU, other IQDs including an ortholog of IQD13 were. IQD13 is a xylem-specific microtubule-associated protein that functions with Rho of Plant GTPase proteins to delineate patterned secondary cell wall deposition in tracheary elements (Sugiyama et al., 2017). Interestingly, a ROP guanine nucleotide exchange factor (GEF), which activate Rho of Plant GTPases, is also co-expressed with PtCPU. Therefore, it is plausible that CPU acts as a scaffold protein that binds CMU as part of a larger protein complex that includes IQD and a kinesin such as the PtCPU co-expressed kinesin 3 (ATK3) to facilitate directionality of cellulose deposition in secondary cell walls.   3.4.3 Conclusion While the molecular function of CPU remains speculative, the in silico and reverse genetic evidence clearly indicate a role related to secondary cell wall deposition. While the 84  predicted association for PtCPU to total lignin content variation was not confirmed here in four-month-old trees, it cannot be excluded that PtCPU has an indirect effect on lignin as the ultimate cellulose-hemicellulose network deposited in secondary cell walls of mature trees could affect the overall lignin content and/or composition. A proposed role for CPU in defining cellulose ultrastructure is consistent with the gene expression pattern of CPU, which appears to be the most prominent during cellulose deposition. In general, it is challenging to assign functional roles to proteins of unknown function, in particular those proteins that appear to have an accessory or structural-based function in protein complexes, such as in the case of CPU, which are unlikely to result in strong phenotypes when mis-regulated. As CPU is speculated to engage in a protein-protein interaction with CMUs in vivo, further investigation into this relationship will help determine how CPU influences cellulose ultrastructure during secondary cell wall deposition.    85  Chapter 4. Characterization of a Populus trichocarpa nitrogen-related transporter, PtNPF6.1  4.1 Introduction  Nitrogen (N) is an essential macronutrient for plant growth and development, most notably as a component of chlorophyll, amino acids, and proteins (Cantón et al., 2005; Parker and Newstead, 2014). Plant secondary metabolites, such as the non-N-containing phenylpropanoids, also require N-containing precursors in the form of phenylalanine (Phe) (Razal et al., 1996). N can be acquired from the soil as inorganic nitrate (NO3-) or ammonium (NH4+), or as organic N in the form of amino acids and peptides (Paungfoo-Lonhienne et al., 2008; Wang et al., 2012). However, N is often limited in the environment and its availability can impact plant biomass production (Tegeder and Rentsch, 2010). While N transport has been well-studied in the model system Arabidopsis (Arabidopsis thaliana) and in several crop plants due to its direct impact on agricultural output, less research has been conducted with woody perennial plants, despite observations that forest ecosystems are commonly N-limited (Näsholm et al., 1998; Cooke et al., 2005). Hence, investigating the molecular mechanisms that influence N distribution in trees is of significance as it may provide a genetic basis for either the improvement of woody biomass or manipulation of precursors into phenolic metabolism.   As N has a direct effect on biomass, the physiological and anatomical responses of trees to N fertilization have been studied (Cooke et al., 2005; Novaes et al., 2009; Euring et al., 2014). The influence of ammonium nitrate (NH4NO3) on wood development in poplar (Populus spp.), for example, is interesting, as the application of N has been proposed as a means to improve shoot biomass for commercial products such as lumber, pulp and paper, and biofuel feedstocks (Gallardo et al., 2003; Cooke et al., 2005). Under luxuriant N conditions, trees develop larger leaves and accumulate more shoot than root biomass (Cooke et al., 2005; Li et al., 2012). The shoot diameter also increases but the wood quality is affected; thinner-walled fibres, wider vessel elements, and decreased syringyl (S) lignin composition have all been reported (Luo et al., 2005; Plavcová et al., 2013; Euring et al., 2014). Under limited N conditions, the shoot to root ratio decreases as the production of lateral roots is stimulated (Novaes et al., 2009; Wei et al., 2013). 86  Therefore, trees are highly responsive to N levels in the environment as evidenced by their development under different N conditions (Cooke et al., 2003).  The metabolic relationship between carbon (C) and N influences plant growth and development (Nunes-Nesi et al., 2010). Mature leaves are the source of photosynthetic carbon which can be assimilated into sucrose, the main transport form of carbon in most plants via the phloem (Coleman et al., 2009; Roach et al., 2012). Sucrose is translocated to sink organs and is required for the biosynthesis of structural carbohydrates, including those that constitute wood (Coleman et al., 2009; Roach et al., 2017). Trees sequester vast amounts of carbon in the secondary cell walls of wood as the carbohydrate-based polymers cellulose and hemicellulose. Lignin, the second most abundant biopolymer on Earth after cellulose (Suárez et al., 2002), is a phenolic polymer that also requires a significant input of fixed carbon through the shikimate pathway as well as N, due to its amino acid precursor Phe. During lignin biosynthesis, the recycling of the ammonium ion from the deamination of Phe is necessary to maintain N homeostasis in planta (Razal et al., 1996; Cantón et al., 2005). While the transport of sucrose delivers carbon skeletons for important anabolic reactions, N-containing amino acids, or perhaps even the catabolism of oligopeptides (Hildebrandt et al., 2015), are also required for wood development.  In addition to the secondary cell wall structural polymer lignin, the levels of related phenolic compounds for defense and pigmentation can also be modulated in response to N availability, which demonstrates the intricate physiological relationship between C and N (Dixon and Paiva, 1995; Randriamanana et al., 2014). Woody plants such as poplar (Populus spp.) accumulate anthocyanins and proanthocyanidins (or condensed tannins) as major end products of the flavonoid biosynthetic pathway (Tsai et al., 2006). Condensed tannins represent a significant phenolic carbon sink (Kao et al., 2002) as these compounds can constitute up to 20% leaf dry weight in some species, such as quaking aspen (P. tremuloides) (Barbehenn et al., 2009; Barbehenn and Constabel, 2011). While the mechanisms that underlie the relationship between C and N in phenolic metabolism are not well understood, the protein competition model (PCM) proposes that the competition for Phe, a common precursor for both protein and phenylpropanoid biosynthesis, is one of the factors that affects photosynthate allocation, towards either protein 87  biosynthesis (for growth) or phenolic compound biosynthesis (for defence) (Jones and Hartley, 1999; Donaldson et al., 2006; Bandau et al., 2015). In addition, it is also possible that the C and N balance is partly regulated via N transport. Co-expression analysis of different N transporters from Arabidopsis found significant enrichment in gene ontology categories related to phenolic metabolism, including phenylpropanoid biosynthesis (He et al., 2016).  N transport in plants includes uptake of both inorganic (ammonium and nitrate) as well as organic forms, as plant roots can take up N as amino acids and oligopeptides (Näsholm et al., 1998; Paungfoo-Lonhienne et al., 2008). While organic N is mobile from root to shoot via the xylem, it can also undergo xylem to phloem transfer for sink tissue unloading/loading (Rentsch et al., 2007). In roots, inorganic nitrate can be reduced to ammonium for assimilation into amino acids, or transported into the shoot and reduced in mature leaves (Tegeder, 2014). Ammonium, the preferred source of inorganic N in woody plants (Suárez et al., 2002), is converted into glutamine (Gln) and glutamate (Glu) via the glutamine synthetase (GS)/glutamate synthase (GOGAT) cycle (Cantón et al., 2005). Gln and Glu are important N donors for the biosynthesis of chlorophyll and other amino acids, including asparagine (Asn) and aspartate (Asp) (Cantón et al., 2005; Hildebrandt et al., 2015). Gln and Asn are the main amino acids that move through the vascular system to supply N from source to sink organs (Suárez et al., 2002; Cooke et al., 2003; Millard and Grelet, 2010), likely via transport proteins. Exactly how this movement throughout the plant is carried out is not known as organic N transporters have not been well-studied in woody plants (Castro-Rodríguez et al., 2017). Amino Acid Permease 11 (AAP11), a proline transporter expressed during xylem differentiation in poplar, is the only organic N transporter characterized to date (Couturier et al., 2010).   From work in non-woody species, it is clear that transporters of different families can facilitate the uptake, translocation, storage, and remobilization of inorganic and organic N (Masclaux-Daubresse et al., 2010; Xu et al., 2012), including the nitrate1/peptide (NRT1/PTR) transporters. NRT1/PTR, recently renamed as the NRT1/PTR Family (NPF) (Léran et al., 2014), consist of nitrate and peptide transporters that are homologous to the Solute Carrier 15 (SLC15) di- and tripeptide transporters in animals (Steiner et al., 1995; Daniel and Kottra, 2004; von Wittgenstein et al., 2014; Corratgé-Faillie and Lacombe, 2017). In plants, NRT1 and PTR 88  transporters are phylogenetically related, although they may have diverse functions (Parker and Newstead, 2014; von Wittgenstein et al., 2014). Some NPFs prefer to mobilize nitrate, whereas others mainly utilize di- and tripeptides as substrates (Tsay et al., 2007). Recent studies indicate NPFs also transport secondary metabolites and hormones. Nour-Eldin et al. (2012) reported that Glucosinolate Transporter 1 (GTR1) has a role in the long-distance transport of glucosinolates from source leaves to seeds in Arabidopsis. ABA-Importing Transporter 1 (AIT1), previously characterized as nitrate transporter NRT1.2 (Huang et al., 1999), has been implicated in abscisic acid (ABA) uptake based on its protein interaction with an ABA receptor (Kanno et al., 2012). Therefore, NPFs appear to have evolved to transport N and non-N-derived substrates.   Genome-wide searches for NPFs have identified large families that contain dozens of members in a number of herbaceous and perennial species (Ouyang et al., 2010; Criscuolo et al., 2012; Bai et al., 2013; Léran et al., 2014; von Wittgenstein et al., 2014; Pellizzaro et al., 2017; Santos et al., 2017). The rice genome, for example, contains 80 members (Tsay et al., 2007). However, the total number of characterized genes in planta remains limited. The A. thaliana genome contains 53 NPF genes, of which 32 have been studied (Tsay et al., 2007; Corratgé-Faillie and Lacombe, 2017). Most of the characterized NPFs are NRT1s from Arabidopsis. Their expression patterns suggest distinct N-related functions for at least some members, including nitrate sensing, N uptake, vascular tissue-related loading/unloading, and maintaining N homeostasis in the leaves (Tsay et al., 1993; Chiu et al., 2004; Lin et al., 2008; Ho et al., 2009; Masclaux-Daubresse et al., 2010). By contrast, only six Arabidopsis PTRs have been investigated (Frommer et al., 1994; Steiner et al., 1994; Song et al., 1996; Dietrich et al., 2004; Karim et al., 2005; Komarova et al., 2008; Weichert et al., 2012). Like the NRT1s, PTRs also have distinct expression patterns. For example, Arabidopsis PTR1 is expressed in the phloem and facilitates the long distance transport of di- and tripeptides (Dietrich et al., 2004), while its paralog PTR5 is expressed in germinating pollen (Komarova et al., 2008). PTR homologs characterized from crop plants including barley (Hordeum vulgare), rice (Oryza sativa), and broad bean (Vicia faba) have provided insight into di- and tripeptide transport in developing seeds (West et al., 1998; Miranda et al., 2003; Fang et al., 2013) and its significance during plant development. Although it is evident that PTRs utilize short oligopeptides as substrates, in vitro 89  studies suggest that some of the Arabidopsis PTRs are also capable of hormone transport (Chiba et al., 2015).  In Populus trichocarpa, 68 to 70 NPF genes are predicted yet none have been functionally characterized (Bai et al., 2013; von Wittgenstein et al., 2014). Poplar is a model for tree biology, partly because of its favourable wood chemistry, rapid growth, and ability to thrive on marginal lands. Furthermore, as poplar can acclimate to different N forms available in its native environment (Min et al., 2000), it is an ideal woody species to investigate N-related transport mechanisms. As previously described in Chapter 2, a large-scale genetic association study in poplar (P. trichocarpa) conducted by Porth and colleagues identified a novel NPF6.1 gene to be associated with variation in S lignin (Porth et al., 2013a). In this chapter, the functional characterization of PtNPF6.1 using a reverse genetics approach in poplar is presented. Analysis of PtNPF6.1pro:GUS lines revealed PtNPF6.1 to be expressed in vascular tissue throughout the plant. Transgenic poplar down-regulated in PtNPF6.1 had an increase in total foliar N concentration and a concomitant decrease in foliar total soluble phenolics, compared to wild-type (WT) trees. The same transgenic PtNPF6.1 lines grown under luxuriant N conditions were found to have decreased S lignin in developing wood, compared to WT. Based on these findings, I propose that PtNPF6.1 encodes an NPF transporter involved in vascular tissue-related (un)loading of N-containing compounds to maintain N homeostasis in developing tissues, which could indirectly influence wood development. This work investigates how nutrient control at the cellular level may have an impact on whole-plant physiology.  4.2 Materials and Methods  4.2.1 Plant maintenance and stress treatments Hybrid aspen (Populus tremula x P. alba) clone INRA 717-1B4 was maintained in vitro on solid half-strength Murashige and Skoog (MS) basal medium (PhytoTechnology Laboratories) supplemented with 0.5 μg mL-1 indole butyric acid (IBA) and grown in a plant tissue culture chamber (Caron) under long day conditions (16 h light, 8 h dark) at 22°C. For experiments conducted in the greenhouse, in vitro plantlets were transferred into soil and maintained in a mist chamber for three weeks. Greenhouse-acclimatized plantlets were then transplanted into one-gallon pots containing Sunshine Mix #4 (Sun Gro Horticulture) 90  supplemented with slow-release fertilizer (21.4 g L-1 Acer 21-7-14, 11.4 g L-1 dolomite lime, 2.9 g L-1 Micromax Micronutrients, and 1.1 g L-1 superphosphate) and grown under long-day conditions (16 h light, 8 h dark) at 22°C with automated irrigation, unless otherwise stated. For high-light/ultraviolet-B (UV-B) exposure, three-month-old trees were moved from the greenhouse [average maximum photosynthetically active radiation: 253 μmol m-2 s-1 (LI-250A light meter, LI-COR Biosciences); average UV-B irradiance: 0.002 mW cm-2 (PMA 2200 Single-Input Radiometer, Solar Light)] and placed outdoors (south-east exposure) for eight days during early August 2016 (maximum photosynthetically active radiation: 1088 μmol m-2 s-1; average UV-B irradiance: 0.09 mW cm-2). Leaves were indexed according to Larson and Isebrands (1971), starting at the first developing leaf with a maximum lamina length of 20 mm designated as leaf plastochron index (LPI) zero. For luxuriant nitrogen fertilization, all genotypes were grown in one-gallon pots containing Sunshine Mix #2 and manually irrigated with modified Long Ashton solution (Ehlting et al., 2007), where NH4NO3 was the main nitrogen source (10 mM NH4NO3, 0.5 mM KNO3, 0.9 mM CaCl2, 0.3 mM MgSO4, 0.6 mM KH2PO4, 42 μM K2HPO4, 10 μM Fe-EDTA, 2 μM MnSO4, 10 μM H3BO3, 7 μM Na2MoO4, 0.05 μM CoSO4, 0.2 μM ZnSO4, 0.2 μM CuSO4). All trees were watered with fixed volumes that adequately saturated the soil two to three times per week, for eight weeks. All experiments were conducted at the Bev Glover Greenhouse Research Facility at the University of Victoria (Victoria, Canada).  4.2.2 Generation of transgenic poplar  PtNPF6.1 promoter and hairpin RNA sequences were amplified from P. trichocarpa (Nisqually-1) using Q5 High-Fidelity DNA Polymerase (New England Biolabs) as recommended by the manufacturer. For the PtNPF6.1pro:GUS construct, a 1558 bp region upstream of PtNPF6.1 was amplified from genomic DNA using the following primer pair (forward: 5’-aggattttaccgacggatga-3’; reverse: 5’-actcgttggcatgctttctt-3’). Adenine residues were added to the 3’ termini of the amplicon via Taq DNA polymerase to facilitate TA cloning into the Gateway donor vector pCR8/GW/TOPO (Thermo Fisher Scientific). The donor insert was recombined into the destination vector pMDC163 (Curtis and Grossniklaus, 2003) to produce the hygromycin-resistant plant transformation construct using an LR Clonase II reaction. To construct the PtNPF6.1 hairpin RNA cassette, a 305 bp fragment corresponding to the 5’ coding sequence of PtNPF6.1 (POPTR_0002s03070/Potri.002G029200) in the P. trichocarpa genome 91  v2.2 (position 182 to 486) was amplified from cDNA isolated from young shoots using the following primer pair (forward: 5’-gaggatggaaagctgctcct-3’; reverse: 5’-gcattttggtggacgtaagc-3’). This region was selected to suppress both PtNPF6.1 and its paralog PtNPF6.2 (POPTR_0005s25490/Potri.005G23350) as their sequences are at least 90% identical at the nucleotide level and homologous to NPF6.1 in P. tremula x P. alba (Appendix C.1). cDNA fragments were cloned sequentially into pKannibal (Wesley et al., 2001) via restriction sites introduced through amplification, as described by Coleman et al. (2008). In brief, the sense fragment was ligated via 5’-EcoRI and 3’-KpnI, while the anti-sense fragment was ligated via 5’-BamHI and 3’-HindIII. The entire hairpin RNA cassette including the 35S CaMV promoter and the OCS terminator was excised via NotI and subcloned into the kanamycin-resistant binary vector pArt27 (Gleave, 1992). Plant transformation constructs were verified by DNA sequencing and introduced into Agrobacterium tumefaciens C58 pMP90 via electroporation.  Transgenic poplars were generated from hybrid aspen clone INRA 717-1B4 as described by Ma et al. (2004). Agrobacterium were grown in YEB medium supplemented with appropriate antibiotics at 28°C for 48 h and resuspended in MS induction medium (IM) to a final OD600 of 0.5 to 0.6. Explants obtained from in vitro cultures were incubated in the IM suspension for 1 h and transferred to solid callus-induction medium (CIM) containing 5 μM 2iP and 10 μM NAA, for 48 h at 22°C in the dark. Explants were transferred to CIM supplemented with carbenicillin and cefotaxime (or timentin) and cultured for 21 d at 22oC in the dark. Callus cultures were transferred to shoot-induction medium containing 0.2 μM thidiazuron and maintained at 22oC under subdued light until regenerated shoots appeared. Shoots were micropropagated on half-strength MS medium supplemented with 0.5 μM IBA and the appropriate selection antibiotic and further tested to confirm insertion of the transgene.  4.2.3 Expression analysis Total RNA was isolated using the CTAB method described by Haruta et al. (2001). In brief, 0.5 g of leaf tissue in 5 mL of extraction buffer (2% hexadecyltrimethylammonium bromide (CTAB), 1% (v/v) β-mercaptoethanol, 1.4 M NaCl, 0.1 M Tris-HCl (pH 9.5), 20 mM EDTA) was incubated at 65°C for 10 min, and extracted using chloroform:isoamyl alcohol (24:1) and chloroform:phenol (1:1). Total RNA was precipitated at −80°C for 10 min with 65 μL 92  of 3 M sodium acetate and 850 μL of 100% ethanol. To remove contaminating genomic DNA, 10 μg of total RNA was treated with DNase I (Ambion) according to the manufacturer’s recommendations. One microgram of DNase-treated RNA was used for cDNA synthesis via Superscript III reverse-transcriptase (Thermo Fisher Scientific). PtNPF6.1 suppression was analyzed using quantitative real-time PCR run on a Bio-Rad CFX96. Reactions (15 μL) were prepared (in triplicate) using the SsoFast EvaGreen mix (Bio-Rad) according to the manufacturer’s recommendations with 300 nM of primer (forward: 5’-catgccaacgagtttggtaag-3’; reverse: 5’-ctttccatcctccagtagttcg-3’) and 2 μL of cDNA template. Amplification conditions consisted of 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 55°C for 5 s. Gene expression (delta Ct) was quantified relative to elongation factor 1β (Coleman et al., 2008). Mean-centred values based on log2-transformed expression values across multiple experiments from publicly available Affymetrix microarray data (Guo et al., 2014) were used for co-expression analysis. Pearson correlation coefficients were determined using Excel and genes co-expressed with the bait (r ≥ 0.50) were retained. For in situ GUS staining, hand-cut sections were vacuum infiltrated in cold 90% acetone for 5 min at 25 psi and maintained in cold 90% acetone for 25 min. Sections were rinsed three times with 0.1 M NaPO4 (pH 7.0) and incubated in X-gluc solution (1 mM 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid (X-gluc), 0.5 mM K3[Fe(CN)6], 0.5 mM K4[Fe(CN)6], 0.1 M NaPO4 (pH 7.0), 0.01% (v/v) Triton X-100) at 37°C until the appearance of blue precipitate. Sections were cleared with 75% ethanol as required to remove chlorophyll. Cross-sections were mounted in water and viewed and imaged under bright-field settings using a Nikon Eclipse TE2000-U equipped with a Digital Sight DS-U1 camera and ACT-2U control software. Whole mounts were viewed and imaged using an Olympus SZX9 stereo microscope equipped with a DP72 camera and DP2-BSW software.  4.2.4 Elemental analysis of carbon and nitrogen Ground lyophilized leaves were oven-dried overnight at 60°C. Tin capsules (Isomass Scientific) were packaged with approximately 5 mg of tissue and analyzed on a FlashEA 1112 NC Analyzer (Thermo Fisher Scientific). Total carbon and nitrogen concentration was quantified using Douglas-fir (Pseudotsuga menziesii) needles as a standard.  93  4.2.5 Free amino acid analysis Free amino acids were extracted from 100 mg of ground lyophilized leaves in 5 mL of 80% (v/v) methanol at 80°C. The extraction was repeated two more times and pooled for a total volume of 15 mL. Amino Acid Standard H (Thermo Fisher Scientific) was added to 200 µL of extract, vacuum-dried, and resuspended in 20 µL of ethanol:water:TEA (2:2:1). Samples were redried and derivatized with 40 µL of ethanol:water:TEA:PITC (7:1:1:1) and incubated at room temperature for 20 min. Samples evaporated to dryness under vacuum were resuspended in 200 µL of phosphate buffer (pH 7.4) and separated on a Waters 2960 equipped with a Kinetex 2.6 µm C18 (100Å) column (150 x 4.6 mm) and a photodiode array detector (254 nm). Eluent A consisted of 1.8% sodium acetate and 0.05% TEA in water (pH 6.8). Eluent B was acetonitrile. The gradient consisted of 93% eluent A at 0 min, changing to 50% eluent A by 15 min. The column was flushed with 80% acetonitrile and equilibrated for 5 min between injections.  4.2.6 Phenolic compound extraction and quantification For the extraction of soluble phenolic compounds, 25 mg of ground lyophilized leaves was homogenized in 1.5 mL of 100% (v/v) methanol using a Precellys 24 tissue homogenizer (Bertin Instruments) at 3,500 rpm for 45 s. Extracts were sonicated for 10 min and centrifuged at 15,000 rpm for 10 min. The extraction procedure was repeated two more times using 1 mL of 100% methanol and pooled for a total volume of 3.5 mL. Quantification of total soluble phenolics was carried out using 20 μL of extract, 100 μL of Folin and Ciocalteu’s phenol reagent (Sigma-Aldrich), and 500 μL of 20% Na2CO3 in a final volume of 1 mL. Reactions were incubated at room temperature for 45 min, centrifuged at 13,000 rpm for 1 min, and measured at 735 nm on a Victor X5 multilabel plate reader (PerkinElmer). Soluble phenolics were quantified using gallic acid (Sigma-Aldrich) as a standard. Anthocyanin extraction was performed as described by Yoshida et al. (2015). Ground lyophilized leaves (50 mg) were extracted in 500 μL of 1% (v/v) HCl in methanol at room temperature for 16 h with gentle agitation. Water was added to the extract (1:1), followed by 1 mL of chloroform and centrifuged at 13,000 rpm for 15 min. The aqueous phase (20 μL) was separated on a Dionex UltiMate 3000 equipped with a Kinetex 2.6 µm C18 (100Å) column (150 x 4.6 mm) and a photodiode array detector (520 nm) using a linear gradient (A: 4% formic acid in water; B: 4% formic acid, 50% acetonitrile in 94  water; 15% B, 7.5 min; 45% B, 20 min; to 100% B, 24 min). Cyanidin-3-glucoside was quantified using cyanidin-3-O-glucoside chloride (Carbosynth) as a standard.  4.2.7 Lignin analysis Stems harvested approximately 2.5 cm above the soil were stripped of periderm and air-dried. Segments of the basal stem with the pith removed were ground using a Wiley mill and passed through a 40-mesh. The wood flour was acetone-extracted at 70°C for 16 h and oven-dried overnight at 50°C before use. The lignin analysis conducted was as described by Skyba et al. (2013) and Mewalal et al. (2016). For Klason lignin analysis, 200 mg of wood flour was treated with 3 mL of 72% sulfuric acid and consistently macerated for 2 h. Water was added to a final volume of 115 mL and autoclaved at 121°C for 1 h. Samples were filtered through sintered glass crucibles to isolate insoluble and soluble lignin. Insoluble lignin was quantified gravimetrically and soluble lignin was quantified by UV absorbance (205 nm). Structural cell wall carbohydrates were separated on a Dionex DX-600 equipped with a PA1 column. Monolignol composition was determined using the thioacidolysis method described by Robinson and Mansfield (2009). In brief, 1 mL of reaction mixture (2.5% (v/v) boron trifluoride etherate and 10% (v/v) ethanethiol in distilled dioxane) was added to 10 mg of wood flour under nitrogen gas and heated at 100°C for 4 h, with manual agitation at hourly intervals. The aqueous phase was extracted using 1 mL of methylene chloride and 2 mL of water, and evaporated to dryness at 45°C under vacuum. Samples were resuspended in 1 mL of methylene chloride; 20 µL of sample was derivatized with 20 µL of pyridine and 100 µL of N,O-bis(trimethylsilyl)acetamide (Sigma-Aldrich). Reactions were incubated at room temperature for 2 h and 1 µL was separated on a Thermo Fisher Scientific TRACE 1310 gas chromatograph.   4.3 Results  4.3.1 PtNPF6.1 is evolutionary distinct as it lacks orthologs in Arabidopsis  The genetic association of PtNPF6.1 to the variation in S lignin composition in a natural population of poplar (Porth et al., 2013a) provided a unique opportunity to investigate NPFs in trees and their potential role in wood, secondary cell wall formation, and/or phenolic metabolism. A BLAST search based on the amino acid sequence of PtNPF6.1 found PTR1 95  (Dietrich et al., 2004) and PTR5 (Komarova et al., 2008) from Arabidopsis (A. thaliana) as the most similar homologs, suggesting PtNPF6.1 to be a peptide transporter rather than a nitrate transporter. AtPTR1 shared slightly greater sequence similarity to PtNPF6.1 (61.4%) than its paralog AtPTR5 (59.8%). However, a phylogenetic reconstruction of NPFs from representative land plants revealed that PtNPF6.1 is in fact evolutionary distinct from AtPTR1 and AtPTR5 (Léran et al., 2014; von Wittgenstein et al., 2014).   To assess the evolutionary relationship of PtNPF6.1, a phylogenetic reconstruction of NPFs described by von Wittgenstein et al. (2014) was explored. Their analysis of 30 land plants including poplar (P. trichocarpa) indicated that NPF sequences cluster into ten distinct supergroups (A to J) (Figure 4.1). Most clades contained NPFs from four major taxonomic groups (i.e. moss, lycopods, monocots, and eudicots), except supergroups D and J. Supergroup D is an angiosperm-specific clade that is comprised of both monocot and eudicot sequences and contained the largest number of poplar NPFs with 19 sequences (Table 4.1). In poplar, up to 70 NPFs have been identified (Bai et al., 2013; von Wittgenstein et al., 2014). Arabidopsis glucosinolate transporters GTR1 and GTR2 (Nour-Eldin et al., 2012) are also members of supergroup D. Like supergroup D, supergroup J is an angiosperm-specific clade, and it is comprised of NPF sequences from only select eudicot species. PtNPF6.1 (and its single paralog PtNPF6.2) is a member of the latter, a small clade of nine including putative orthologs from Carica papaya, Manihot esculenta, Mimulus guttatus, Prunus persica, Ricinus communis, and Vitis vinifera (Figure 4.1, inset). Arabidopsis NPFs were notably absent from supergroup J (Table 4.1). AtPTR1 and AtPTR5 cluster in supergroup F (Figure 4.1). Interestingly, supergroup E contains only one poplar NPF sequence (Table 4.1) which also had a significant genetic association to lignin content variation (Porth et al., 2013a). While poplar NPF sequences were represented in all ten clades, the phylogenetic position of PtNPF6.1 in supergroup J suggests a unique function.         96        0.3  Figure 4.1 An unrooted maximum likelihood phylogeny of the Nitrate1/Peptide Family (NPF) transporters from land plants including eudicots (blue), monocots (red), a lycophyte (orange), and a moss (yellow). Related sequences from chlorophytes (green) and non-plants (brown) are also included. PtNPF8.1 is a member of supergroup J and contains homologs from Carica papaya, Manihot esculenta, Mimulus guttatus, Prunus persica, Ricinus communis, and Vitis vinifera (see inset). Arabidopsis thaliana PTR1 and PTR5, the most similar homologs to PtNPF8.1, are members of supergroup F. Black dots represent characterized NPFs that transport secondary metabolites; supergroup A contains the ABA-Importing Transporters (AITs) (Kanno et al., 2012) and supergroup D contains the glucosinolate transporters (GTRs) (Nour-Eldin et al., 2012). Modified from von Wittgenstein et al. (2014) BMC Evol Biol 14:11 with permission from authors. B   Figure 4.1 An unrooted maximum likelihood phylogeny of the Nitrate1/Peptide Family (NPF) transporters from land plants including eudicots (blue), monocots (red), a lycopod (orange), and moss (yellow). Related sequences from chlorophytes (green) and non- lants (brown) are also included. PtNPF6.1 (inset, star) is a member of supergroup J and contains homologs from Carica papaya, Manihot esculenta, Mimulus guttatus, Prunus persica, Ricinus communis, and Vitis vinifera (see inset). Arabidopsis thaliana PTR1 and PTR5, the most similar homologs to PtNPF6.1, are members of sup rgroup F. Black ots represent characterized NPFs that transport secondary metabolites; supergroup A contains the ABA-Importing Transporters (AITs) (Kanno et al., 2012) and supergroup D contains the glucosinolate transporters (GTRs) (Nour-Eldin et al., 2012). Modified from von Wittgenstein et al. (2014) with permission from authors.  97  Table 4.1 Number of NPFs from representative species of four major taxonomic groups described by  von Wittgenstein et al. (2014)  Supergroup  Number of Genes  P. trichocarpa A. thaliana S. moellendorffiia P. patensb       A  11 7 4 3 B  2 3 3 2 C  4 1 4 1 D  19 16 2 1 E  1 1 2 1 F  7 5 6 1 G  5 3 5 3 H  14 12 7 5 I  5 3 0 2 J  2 0 0 0       Total  70 51 31 18                a two of the four sequences in supergroup C also root supergroup D.                b the sequence in supergroup C is also the root of supergroup D.   4.3.2 PtNPF6.1 is expressed in vascular tissue  Analysis of the in silico expression data from the poplar eFP browser (Wilkins et al., 2009) indicated that PtNPF6.1 is moderately expressed in most organs and tissues including xylem, but most pronounced in male catkins and young leaves (Figure 4.2A). PtNPF6.2, the paralog of PtNPF6.1, exhibited a slightly different expression profile than PtNPF6.1 as it lacked expression in the xylem. While both paralogs displayed high expression in male catkins, only PtNPF6.2 appeared to be expressed at low levels in female catkins. The expression profiles for PtNPF6.1 and PtNPF6.2 were further investigated using publicly available microarray expression data (Figure 4.2B). As seen from the expression profile, PtNPF6.1 is expressed throughout the plant, mainly in floral organs. PtNPF6.1 expression is also high in the bark which was not represented in the poplar eFP browser. The microarray data for PtNPF6.2 suggests slight differences in expression compared to its paralog, as PtNPF6.2 appears to be mainly expressed in young and mature leaves. Co-expression analysis of the microarray data found five genes to be co-expressed with PtNPF6.1 and only gene to be co-expressed with PtNPF6.2 at a Pearson correlation coefficient (PCC) of at least 0.60 (Figure 4.2B). Interestingly, an Arabidopsis BAHD acyltransferase in the same phylogenetic clade as the hydroxycinnamoyl transferase (HCT) involved in lignin biosynthesis (Hoffmann et al., 2003) was co-regulated with PtNPF6.1 at a  98   Figure 4.2 In silico expression analysis of PtNPF6.1 and its paralog PtNPF6.2. (A) Expression profile for PtNPF6.1 and PtNPF6.2 based on poplar eFP browser data (Wilkins et al., 2009). (B) Co-expression analysis of PtNPF6.1 (top) and PtNPF6.2 (bottom) using publicly available Affymetrix microarray data for poplar at a Pearson correlation coefficient of at least 0.50.      PtNPF6.1 PtNPF6.2 PtNPF6.1 PtNPF6.3                   log2 (signal/mean) PTR1  - - - DCR  - - - - CLE26  - - - - BLH11  HCT  - PA2  - THI1 - APRR2  Peptide Transporter 1  - DUF247/DUF862 DUF247 BAHD acyl-transferase protein  unknown protein - - DUF2431 Clavata3/ESR-Related 26  tetraspanin family protein  - tetraspanin family protein  DUF594 BEL1-like homeodomain 11  Hydroxycinnamoyl Transferase DUF594 Peroxidase 2  RING/U-box superfamily protein  thiazole biosynthetic enzyme - Pseduo Response Regulator 2 POPTR_0002s03070 POPTR_0001s39530 POPTR_0001s05530 POPTR_0403s00200 POPTR_0012s12890 POPTR_0004s15700 NA POPTR_0006s07110 POPTR_0006s00460 POPTR_0008s19570 POPTR_0004s04160 POPTR_0014s18750 POPTR_0011s05050 POPTR_0015s12510 POPTR_0002s03230 POPTR_0005s02810 POPTR_0015s12600 POPTR_0003s21640 POPTR_0003s09190 POPTR_0011s13820 NA POPTR_0003s08600  1.00 0.70 0.66 0.62 0.61 0.61 0.60 0.60 0.59 0.59 0.59 0.58 0.58 0.58 0.58 0.57 0.57 0.57 0.56 0.56 0.56 0.56  Poplar ID                r-value Best Arabidopsis Hit PTR1  - ACA4  CHX19  - FUC1  - ACA4  - RLP33  - - - - Peptide Transporter 1  - Alpha Carbonic Anhydrase 4  Cation/H+ Exchanger 19  leucine-rich repeat protein kinase  Alpha-L-Fucosidase 1  carbohydrate-binding protein  Alpha Carbonic Anhydrase 4  - Receptor-Like Protein 33  - unknown protein - - POPTR_0005s25490 POPTR_0004s22310 POPTR_0006s04600 POPTR_0271s00200 NA POPTR_0015s14140 POPTR_0010s18070 POPTR_0006s04600 NA POPTR_0046s00250 NA POPTR_0006s01860 NA POPTR_0001s43370 1.00 0.60 0.58 0.53 0.53 0.53 0.53 0.52 0.51 0.51 0.51 0.50 0.50 0.50 A B cvcv PtNPF6.1 PtNPF6.2 PtNPF .  PtNPF6.2  99  at a PCC of 0.57. However, due to the limited number of co-expressed genes for both PtNPF6.1 and PtNPF6.2 at a PCC of at least 0.60 and the lack of characterized functions for these co-expressed genes, inferences to support a possible biological function could not be confidently made.  To expand the cell- and tissue-specific expression of PtNPF6.1 in planta, a 1558 bp fragment upstream of PtNPF6.1 was fused to the reporter gene β-glucuronidase (GUS) and introduced into hybrid poplar clone 717 (P. tremula x P. alba). Analysis of whole in vitro plantlets expressing PtNPF6.1pro:GUS found GUS expression to be most pronounced around the vascular system (data not shown). In one-month-old greenhouse-grown transgenic poplar, the vascular tissue-related expression pattern remained consistent. PtNPF6.1 promoter-driven expression was apparent in the leaf lamina vasculature (Figure 4.3A). Furthermore, petioles from LPI one displayed clear PtNPF6.1pro:GUS expression in the xylem (Figure 4.3B). PtNPF6.1 expression was also analyzed in the stem. At internode two, PtNPF6.1pro:GUS expression was evident in the developing secondary xylem. However, later in stem development when the xylem cells have become lignified and undergone programmed cell death, most of the PtNPF6.1pro:GUS expression appeared to localize to the vascular cambium and the phloem (Figure 4.3D), consistent with the in silico expression of PtNPF6.1 in the bark (Figure 4.2B). PtNPF6.1 expression was also seen in the young root stele and tip (Figure 4.3G). However, not all root segments analyzed had clear expression in young tissues (Figure 4.3H), suggesting PtNPF6.1 is not consistently expressed in all roots.   4.3.3 Total nitrogen concentration is altered in transgenic poplar down-regulated in PtNPF6.1  To characterize the function of PtNPF6.1, RNA interference-mediated suppression (RNAi) of PtNPF6.1 in transgenic poplar was used to target both PtNPF6.1 and PtNPF6.2 (Appendix C.1). Expression analysis indicated a significant reduction in PtNPF6.1 transcript abundance in three of the four transgenic lines compared to WT (Figure 4.4). Lines A4 and D5 had the most reduced levels of PtNPF6.1 expression, while in line B2 PtNPF6.1 expression was suppressed only by 20% compared to WT.   100   Figure 4.3 Expression of PtNPF6.1pro:GUS in the vascular tissue of one-month-old greenhouse-grown transgenic poplar. (A) Leaf lamina epidermis. (B) Cross-section of petiole from LPI one. (C, D, E) Cross section of stem internodes 2 (C), 6 (D), and 10 (E). (F) Cross-section of a mature root. (G, H) Whole mounts of young roots. (G) Young root showing expression in the stele and tip. (H) Young roots showing PtNPF6.1pro:GUS expression in only one (denoted by the arrow) of the two lateral roots. Ph: phloem; pi: pith; sx: secondary xylem; x: xylem. Scale bar represents 10 μm in B-F, 100 μm in A, 1 mm in G, and 2 mm in H.                                               10 µm sx ph F D sx ph 10 µm x ph B 100 µm 10 µm sx ph pi 10 µm 10 µm sx ph 1 mm 2 mm A E H G 101                            Most characterized NPF transporters utilize nitrate or di- and tripeptides as substrates. To test whether PtNPF6.1 is a nitrogen-related transporter, total foliar nitrogen concentration was quantified in mature leaves of WT and RNAi PtNPF6.1 lines via elemental analysis. Three of the four transgenic lines were found to have a significant increase in total nitrogen concentration relative to WT. In RNAi PtNPF6.1 line A4, total nitrogen concentration increased significantly by nearly 50% compared to WT (Table 4.2). Total carbon concentration was also quantified. However, significant differences were not found as both WT and transgenic poplar RNAi  Figure 4.4 Gene expression analysis of RNAi PtNPF6.1 lines relative to elongation factor 1β. The mean (except line B4 which had an extreme outlier) and standard error of the mean for three clonal replicates are represented in grey. Individual measurements (black dots) are the means of three technical replicates measured for each clonal replicate. Significant differences from wild-type (WT) are based on a Student’s t-test (P < 0.05), indicated by an asterisk.   * * 102  PtNPF6.1 lines contained 48 to 49% total carbon. Therefore, the significant change in total nitrogen concentration alone is consistent with PtNPF6.1 as a nitrogen-related transporter.     Table 4.2 Total C and N concentration in leaves of RNAi PtNPF6.1 lines  Line % DW C:N Carbon (C) Nitrogen (N)     WT 48.01 (0.15) 2.49 (0.20) 19.58 (1.36) A4 48.03 (0.20) 3.50 (0.06) 13.75 (0.24) B2 48.53 (0.22) 3.37 (0.33) 14.87 (1.66) B4 48.46 (0.18) 3.24 (0.12) 15.03 (0.65) D5 48.36 (0.19) 3.26 (0.15) 14.92 (0.69)        LPI 9 was measured.       Mean and standard error of the mean (in parentheses) for four clonal replicates.        Bold values indicate significant differences from WT based on a Student’s t-test (P < 0.05).    To test potential peptide transport, total free amino acid content was quantified in transgenic RNAi PtNPF6.1 poplar lines. This approach was chosen over di- and tripeptide analysis as peptide transporter substrates can be broad. Of the 20 standard amino acids, 14 were successfully quantified from the same leaves analyzed for total nitrogen concentration in WT and two independent transgenic lines. Glutamic acid (Glu) and aspartic acid (Asp) accumulated to significantly higher levels in RNAi PtNPF6.1 lines B4 and D5 compared to WT (Table 4.3). In particular, the concentration of Glu was nearly 2.5 times higher in the transgenic lines compared to WT. Asp levels in the transgenic lines were approximately double the amount of WT. Interestingly, leucine also accumulated to higher levels in the transgenic lines compared to WT. Phe, an amino acid that is required for both primary and secondary metabolism, was also analyzed. Its concentrations in the two transgenic lines were 1.6 and 5.5-fold lower than WT. However, because of the low Phe concentration and limited sample size, only line B4 passed the significance threshold (P < 0.05), whereas line D5 did not (P = 0.051). Thus, it remains open as to whether Phe levels are significantly affected by the mis-regulation of PtNPF6.1. Significant changes were not detected for the other amino acids in both transgenic poplar lines suppressed in PtNPF6.1 when compared to WT. 103            Table 4.3 Free amino acid content in transgenic RNAi PtNPF6.1 lines  Amino Acid (nmol mg-1 DW)a RNAi Line WT B4 D5     Arginine 0.084 (0.012) 0.091 (0.003) 0.082 (0.009) Aspartic Acid 0.123 (0.009) 0.193 (0.005) 0.245 (0.020) Glutamic Acid 0.123 (0.023) 0.300 (0.006) 0.349 (0.016) Glycine 0.299 (0.014) 0.223 (0.018) 0.229 (0.024) Histidine 0.054 (0.003) 0.043 (0.003) 0.039 (0.006) Isoleucine 0.101 (0.011) 0.104 (0.005) 0.100 (0.008) Leucine 0.028 (0.011) 0.087 (0.012) 0.055 (0.004) Lysine 0.026 (0.001) 0.023 (0.000) 0.021 (0.002) Phenylalanine 0.030 (0.003) 0.006 (0.002) 0.018 (0.002) Proline 0.388 (0.008) 0.398 (0.038) 0.368 (0.023) Serine 0.071 (0.015) 0.078 (0.010) 0.074 (0.010) Threonine 0.084 (0.008) 0.065 (0.003) 0.062 (0.004) Tyrosine 0.933 (0.063) 0.869 (0.187) 0.711 (0.028) Valine 0.019 (0.001) 0.027 (0.003) 0.023 (0.003) Total 2.364 (0.126) 2.516 (0.284) 2.380 (0.054)     aalanine, cysteine and methionine were not detectable; asparagine, glutamine and tryptophan were not measured from this assay. Mean and the standard error of the mean (in parentheses) for three clonal replicates. Bold values indicate significant differences from WT based on a Student’s t-test (P < 0.05). P value for Phe in line D5 = 0.051. LPI 9 was analyzed. DW: dry weight.   4.3.4 High-light and ultraviolet-B stress causes a reduction in total soluble phenolic compounds in transgenic poplar down-regulated in PtNPF6.1  Phenolic compounds, including anthocyanins, can be induced by abiotic stresses such as ultraviolet radiation and N deficient conditions (Dixon and Paiva, 1995). Greenhouse-grown WT and RNAi PtNPF6.1 lines were placed outdoors for natural exposure to high-light/UV-B radiation. After eight days of abiotic stress, the upper canopy of the transgenic RNAi lines clearly accumulated less anthocyanins compared to WT (Figure 4.5). Leaves before and during exposure to high-light/UV-B radiation at day four were collected for phytochemical analysis in the midst of anthocyanin accumulation phase, before the stress response started to plateau. While under greenhouse conditions, only minor amounts of anthocyanins were detected (Table 4.4). At day four of high-light/UV-B exposure outdoors, it was evident that anthocyanins accumulated in both WT and RNAi PtNPF6.1 lines, but the latter accumulated only half the anthocyanin content as WT. For example, RNAi PtNPF6.1 line A4 had an anthocyanin content of 32 ± 3 A530 g-1 DW compared to WT at 63 ± 1 A530 g-1 DW. To further examine the change in anthocyanin content, 104   Figure 4.5 Three-month-old wild-type (WT) and RNAi PtNPF6.1 lines before and at day eight of outdoor high-light/ultraviolet-B exposure. Top panel: Before (0 h) outdoor high-light/UV-B exposure. Bottom: Day eight (8 d) of outdoor high-light/UV-B exposure. WT (left) and A4, a representative RNAi PtNPF6.1 line (right).  the same anthocyanin extracts for WT and RNAi PtNPF6.1 lines were separated using high performance liquid chromatography (HPLC). While anthocyanins were not detected before high-light/UV-B exposure, peak detection at 520 nm for day four WT and RNAi PtNPF6.1 lines revealed three peaks, one major peak at eight minutes and two minor peaks close to 10 minutes (Figure 4.6). The most prominent peak corresponded to cyanidin-3-glucoside (Cy3G) based on comparision of its retention time and UV absorption spectra with a Cy3G standard. As Cy3G is a common anthocyanidin in poplar (Cho et al., 2016), the concentration of Cy3G in RNAi PtNPF6.1 lines was quantified. All four RNAi PtNPF6.1 lines had significantly decreased Cy3G content compared to WT, where nearly a two-fold difference was observed (Figure 4.7), corroborating the total anthocyanin results. However, down-regulation of PtNPF6.1 did not signficiantly affect the concentrations (based on peak area) of the two other peaks, which remain unidentied (Appendix C.2).                            WT 0 d 8 d A4 RNAi 105   Figure 4.6 Representative HPLC chromatograms (520 nm) of anthocyanins in wild-type (WT) and transgenic poplar RNAi PtNPF6.1 lines before (0 d) and during high light/UV-B exposure at day four (4 d). The major anthocyanin peak (peak 1) is cyanidin-3-glucoside (Cy3G), present at approximately eight minutes followed by two unknown peaks (peak 2 and 3) at approximately 10 minutes. The inset shows a representative UV-visible spectrum for Cy3G.          Table 4.4 Total anthocyanins in RNAi PtNPF6.1 lines exposed to natural outdoor high-light/UV-B radiation  Line Anthocyanin (A530 g-1 DW) 0 d 4 d    WT 5.56 (0.45) 62.57 (1.35) A4 2.64 (0.44) 32.00 (2.81) B2 3.46 (0.47) 29.18 (5.29) B4 2.93 (0.57) 40.05 (5.24) D5 3.38 (0.26) 36.77 (6.66)    Mean and standard error of the mean (in parentheses) for three to four clonal replicates.  Bold values indicate significant differences from WT based on a Student’s t-test (P < 0.05).    0 d: before outdoor high-light/UV-B exposure; 4 d: day four of outdoor high-light/UV-B exposure. DW: dry weight.              0 d 4 d Time (min) WT A4 B4 B2  D5 1 2 3106                               As the anthocyanins were altered in the RNAi PtNPF6.1 lines, soluble phenolic compounds were tested for differences in total phenolic content. RNAi PtNPF6.1 lines before outdoor exposure had a 10% decrease in soluble phenolic content (measured as gallic acid equivalents) relative to WT. Four days after natural high-light/UV-B exposure, it was evident that phenolic compounds accumulated in WT and RNAi PtNPF6.1; however, the latter accumulated significantly less phenolics than WT (Figure 4.8). Altogether, the change in total phenolics is consistent with the altered total anthocyanin content. b  Figure 4.7 Anthocyanins quantified as cyanidin-3-glucoside for wild-type (WT) and transgenic poplar RNAi PtNPF6.1 lines exposed to natural outdoor high-light/UV-B stress for four days. Anthocyanins were not detected before outdoor exposure. Closed grey circles represent the mean of three to four clonal replicates. Open grey circles are the individual measured values for each clonal replicate based on the corresponding HPLC peak at 520 nm. The boxplot represents a 95% confidence interval and the black bar in the box is the median. Significant differences from WT are based on a Student’s t-test (P < 0.01), indicated with an asterisk. DW: dry weight.   * * * * 107                                       4.3.5 Exogenous nitrogen application alters syringyl lignin composition in transgenic poplar down-regulated in PtNPF6.1  PtNPF6.1 has been genetically associated with syringyl lignin composition (Porth et al., 2013a), and lignin content and composition can be affected by exogenous nitrogen availability (Pitre et al., 2007a). Therefore, WT and two RNAi PtNPF6.1 lines were fertilized with either luxuriant (10 mM) or low (0.10 mM) ammonium nitrate (NH4NO3) regimes for eight weeks. After six weeks of fertilization, it was apparent that the low nitrogen trees started to show  Figure 4.8 Total phenolics quantified as gallic acid equivalents for wild-type (WT) and transgenic RNAi PtNPF6.1 poplar lines before (0 d) and during exposure (4 d) to high-light/UV-B stress. Closed grey circles are the means of three to four clonal replicates. Open grey circles are the individual measured data points for each clonal replicate. The boxplot represents a 95% confidence interval and the black bar in the box is the median. Significant differences were observed from WT based on a Student’s t-test (P < 0.05), indicated by an asterisk. DW: dry weight.  * * * * * * * * 108  nitrogen deficiency symptoms with the onset of leaf chlorosis, while the luxuriant nitrogen-supplemented trees retained large, dark green foliage in both WT and transgenic RNAi PtNPF6.1 lines (Appendix C.3). In general, the visible changes in tree morphology indicated that the nitrogen fertilization regime was effective. After eight weeks of growth, the trees were dissected for biomass measurements. However, significant differences were not detected in stem height and diameter (Appendix C.4).  As high nitrogen conditions have been shown to affect lignification (Pitre et al., 2007a), guaiacyl (G) and syringyl (S) lignin composition was analyzed using thioacidolysis for the luxuriant nitrogen-fertilized trees. A minor but significant decrease in S lignin was observed in RNAi PtNPF6.1 lines compared to WT (Table 4.5). In general, RNAi PtNPF6.1 lines had a slight increase in G units and a slight decrease in S units. As a consequence, RNAi PtNPF6.1 lines had significantly reduced S:G ratios (2.2) compared to WT (2.4). Since the slight alterations in S lignin was apparent in RNAi PtNPF6.1 lines, insoluble and soluble lignin contents were quantified for the same nitrogen-fertilized WT and transgenic poplar RNAi lines. Insoluble, soluble and total lignin content was not impacted in these trees as WT and RNAi PtNPF6.1 lines had an average total lignin content of 21% (Table 4.5). Structural cell wall carbohydrates were also unaltered (Table 4.6).   Table 4.5 Analysis of lignin content and composition in wild-type (WT) and transgenic poplar RNAi PtNPF6.1 lines grown under luxuriant nitrogen (10 mM ammonium nitrate) fertilization. Total lignin measured using Klasons and monolignol composition measured using thioacidolysis  Line Thioacidolysis  Klason Lignin (% DW) % G % S S:G  Insoluble Soluble Total         WT 29.4 (0.4) 69.7 (0.4) 2.37 (0.04)  18.0 (0.13) 3.3 (0.27) 21.2 (0.20) A4 30.8 (0.2) 68.6 (0.2) 2.23 (0.02)  17.9 (0.17) 3.4 (0.19) 21.3 (0.32) B4 31.3 (0.2) 68.1 (0.2) 2.17 (0.02)  17.9 (0.33) 3.2 (0.20) 21.1 (0.35)             Mean and standard error of the mean (in parentheses) of four clonal replicates.      Bold values indicate significant differences from WT based on a Student’s t-test (P < 0.05).     DW: dry weight.     109  Table 4.6 Total percentage of structural cell wall carbohydrates in wild-type (WT) and transgenic poplar RNAi PtNPF6.1 lines grown under luxurious nitrogen (10 mM ammonium nitrate) fertilization  Line Carbohydrates (% DW) Arabinose Rhamnose Galactose Glucose Xylose Mannose         WT 0.30 (0.03) 0.46 (0.03) 0.76 (0.05) 37.0 (2.86) 16.6 (1.73) 1.1 (0.08)  A4 0.32 (0.01) 0.47 (0.02) 0.81 (0.05) 38.0 (1.21) 17.5 (0.20) 1.3 (0.03)  B2 0.30 (0.02) 0.45 (0.03) 0.73 (0.06) 35.0 (1.87) 16.2 (0.92) 1.2 (0.07)                     Mean and standard error of the mean (in parentheses) of four clonal replicates.            Significant differences from WT were not detected based on a Student’s t-test (P < 0.05).             DW: dry weight.   4.4 Discussion  RNAi-mediated suppression of PtNPF6.1 in transgenic poplar resulted in increased total nitrogen concentration and decreased total soluble phenolics compared to WT. Under luxuriant nitrogen conditions, S lignin composition also decreased in transgenic poplar compared to WT. The inverse relationship between phenolic compounds and N seen here demonstrates the phenotypic plasticity of poplar in response to nitrogen and suggests an indirect role for PtNPF6.1 on phenylpropanoid accumulation through maintaining nitrogen homeostasis within the plant.   4.4.1 PtNPF6.1 expression in vascular tissue suggests a role in long-distance transport  PtNPF6.1pro:GUS expression in transgenic poplar was detected in the vasculature of the leaf, stem, and roots. In the young portion of the stem, PtNPF6.1 expression localized mainly in developing xylem. However, at the onset of secondary xylem development, PtNPF6.1 expression was localized primarily in the vascular cambium and phloem. While this is likely the first NPF promoter-GUS expression analysis in woody plants, studies in Arabidopsis have also found NPF expression in the vascular tissue. In situ hybridization of Arabidopsis NRT1.5 revealed its expression in the pericycle, supporting its role in xylem loading of nitrate for transport from root to shoot (Lin et al., 2008). Arabidopsis PTR1, the closest homolog to PtNPF6.1, is expressed throughout the plant (root to shoot), and in reproductive organs and siliques; promoter-GUS analysis found PTR1 to be expressed in the phloem and is hypothesized to be a phloem-loading, long-distance transporter of di- and tripeptides (Dietrich et al., 2004). Although PtNPF6.1 is expressed in vascular tissue, it is unlikely to have a role in nitrogen uptake since its expression is largely restricted to the stem and leaves. By contrast, NRT1.1 (CHL1), a 110  nitrate uptake transporter in Arabidopsis, is expressed in epidermal cells at the root tip and in the root cortex (Huang et al., 1996). The cell-specific expression of characterized Arabidopsis NPFs suggests discrete functions for these proteins. Thus, the expression of PtNPF6.1 is consistent with a role in long-distance transport. Consistent with the high expression seen in the anthocyanin-rich male catkins and young leaves from the in silico data and increased Glu and Asp content in mis-regulated PtNPF6.1 transgenic poplar, it is plausible that PtNPF6.1 facilitates nitrogen-related transport to developing sinks via the xylem or phloem. In long-lived woody perennials, nitrogen is maintained internally (Rennenberg et al., 2010) and the long-distance remobilization of nitrogen is required to supplement organs and tissues during adequate and changing nitrogen levels. Examples include developmental changes that occur in the spring, such as flower development, bud flush, leaf expansion, and vascular cambium initiation (Jansson and Douglas, 2007). Amino acid measurements in xylem sap of Salix during seasonal transitions found an increase in Glu during male catkin development and bud flush (Sauter, 1981) which parallels the general expression pattern of PtNPF6.1 and the amino acids that accumulate when PtNPF6.1 is mis-regulated. It would be interesting to test whether PtNPF6.1 expression increases in the spring to facilitate nitrogen-related transport to sink tissues.   4.4.2 PtNPF6.1 impacts total nitrogen and free amino acids in source leaves  Free amino acid profiling in leaves of transgenic poplar down-regulated in PtNPF6.1 found an increase in Glu and Asp relative to WT in mature leaves, suggesting a direct role for PtNPF6.1 in nitrogen reallocation from source tissues to presumably developing sink tissues. Although phylogenetically distinct, PtNPF6.1 is most similar to peptide transporters within the NPF superfamily. NPF peptide transporters mainly transport di- and tripeptides, but free amino acids have also been identified as a substrate. For example, the first characterized peptide transporter Arabidopsis PTR2 is capable of histidine (His) transport in vitro (Frommer et al., 1994; Steiner et al., 1994). While His was not affected in transgenic RNAi PtNPF6.1 lines, it appears plausible that PtNPF6.1 can transport free amino acids such as Glu and Asp, which accumulated to high levels in the transgenic lines, as well as di- and tripeptides. Arabidopsis loss of function mutants of the nodulin/MtN21/UMAMIT transporter Siliques Are Red 1 (SIAR1) have reduced Gln and Glu content in the siliques, suggesting this protein to be an amino acid exporter (Ladwig et al., 2012). Amino acid depletion in the siar1 mutant coincided with 111  anthocyanin accumulation, a symptom commonly induced under nitrogen-deficient conditions. PtNPF6.1 down-regulated lines had decreased anthocyanins concomitant with an increase in total nitrogen concentration compared to WT, consistent with a role for PtNPF6.1 in nitrogenous compound transport. However, this does not exclude the possibility that PtNPF6.1 may instead transport phenolic compounds or their precursors.   PtNPF6.1 is part of the phylogenetic supergroup J, and no NPFs in this clade have been characterized. In general, it is difficult to predict the biochemical functions of NPF transporters based on their phylogenetic classification (von Wittgenstein et al., 2014). An amino acid complementation screen of 26 Arabidopsis NPFs in a peptide transport-deficient strain of yeast (Saccharomyces cerevisiae) failed to identify new peptide transporters (Léran et al., 2015). Using the same set of Arabidopsis NPFs in a screen of Xenopus laevis oocytes for nitrate accumulation, only one uncharacterized nitrate transporter was identified, suggesting that several of the NPFs tested may transport alternative substrates. In recent years, an increasing number of NPFs have been identified to transport nitrogen and non-nitrogen derived substrates. Arabidopsis GTR1, a possible evolutionary descendent of the phloem nitrate transporter NRT1.9 (Wang and Tsay, 2011), facilitates the long-distance transport of glucosinolates (Nour-Eldin et al., 2012). The vacuolar transport of a monoterpene indole alkaloid intermediate in Catharanthus roseus also suggests a specialized role for NPF proteins in the transport of secondary metabolites (Payne et al., 2017). Aside from novel secondary metabolite transporters, previously characterized NPFs known to transport nitrate have been identified to have dual transport capability. Arabidopsis NRT1.2, originally characterized as a low affinity nitrate transporter also acts as an abscisic acid-importing transporter (AIT). When AIT is over-expressed, plants become hypersensitive to ABA, suggesting a role in ABA mobilization (Kanno et al., 2012). A screen for Arabidopsis mutants compromised in gibberellin transport identified NPF3, a plasma membrane localized gibberellin transporter in root endodermal cells (Tal et al., 2016). Interestingly, AtPTR1 also has transport capability for jasmonoyl-isoleucine in yeast, based on a large-scale in vitro screen for NPF hormone transport activity (Chiba et al., 2015). Leucine content increased in transgenic poplar suppressed in PtNPF6.1 suggesting that, like its Arabidopsis homolog, it may have some degree of jasmonoyl-leucine transport activity. Thus, while amino acid/peptide transport activity 112  remains the most plausible biochemical activity for PtNPF6.1, a role in hormone or secondary metabolite transport cannot be excluded.  4.4.3 PtNPF6.1 has an indirect influence on phenylpropanoids including S lignin PtNPF6.1 mis-regulated lines showed reduced levels of total phenolics and an attenuated response in anthocyanin production under high-light/UV-B exposure compared to WT. Consistent with this, a minor but significant decrease in S lignin was detected in transgenic RNAi PtNPF6.1 lines compared to WT under luxuriant nitrogen conditions. The negative impact on soluble phenolics and S lignin contrasts the elevated nitrogen concentration seen in leaves. The protein competition model suggests that nitrogen availability influences photosynthate allocation, towards either protein for growth or phenolic compound biosynthesis for defense and mechanical stability; competition for Phe, an amino acid that links primary metabolism via the shikimate pathway and secondary metabolism via the phenylpropanoid pathway, is one of the factors affecting this inverse relationship (Jones and Hartley, 1999; Bandau et al., 2015). Therefore, the increased nitrogen concentration and free amino acids in the RNAi PtNPF6.1 lines may be channelled to enhance protein biosynthesis at the expense of phenolic compound biosynthesis. Previous studies examining the effect of nitrogen on wood development showed that under high nitrogen fertilization, S lignin decreases (Pitre et al., 2007a). This could be a consequence of a transcriptional response to regulate carbon and nitrogen balance (Novaes et al., 2009; Plavcová et al., 2013; Euring et al., 2014). The change in S lignin composition in transgenic PtNPF6.1 poplar also supports the genetic association of PtNPF6.1 to variation in S lignin detected by Porth et al. (2013a). Individuals with the homozygous CC allele had slightly lower levels of S lignin than the alternative TT allele (Porth et al., 2013a). As for most of the lignin trait associations, the association detected in nine-year-old trees only accounted for 4.6% difference of the observed variation in total S lignin composition (Porth et al., 2013a). It is possible that individuals with the non-synonymous genetic variant could have impaired substrate transport activity due to an amino acid change in the protein structure. In general, the findings here and from Porth et al. (2013a) suggest PtNPF6.1 to have a minor influence on S lignin deposition which can most easily be explained as an indirect effect of internal nitrogen availability due to environmental constraints. 113  4.4.4 Conclusion The identification of PtNPF6.1 based on its genetic association to S lignin variation suggested an influence on wood development (Porth et al., 2013a). My results suggest this effect is indirectly caused by a role for PtNPF6.1 in nitrogen redistribution within the plant. PtNPF6.1 knock-down lines over-accumulated total nitrogen and amino acids in source leaves, which correlated with decreased phenylpropanoid production both in leaves (soluble phenolics including anthocyanins) and to a lesser extent in stems (S lignin). However, the impact of PtNPF6.1 on nitrogen homeostasis is possibly embedded in a more complex and partly redundant manner given that PtNPF6.1-related transporters (belonging to supergroup J) are not present in all plants, and also because suppression phenotypes only become apparent under inductive conditions. Transgenic mis-regulated PtNPF6.1 lines can now be used as tool to further understand the relationship between carbon-based phenolic compounds and nitrogen redistribution on a whole-plant level. As a whole, the increased total nitrogen concentration paralleled with increased amino acid content in mature source leaves in transgenic poplar suggest in that WT, the role of PtNPF6.1 is to translocate nitrogen from source leaves to developing sinks experiencing active growth. The knowledge gained here may be used to fine tune internal nitrogen redistribution and suggests a feasible approach to augment plant performance under nitrogen-limited conditions frequently found in forest ecosystems.      114  Chapter 5. Discussion  5.1 Main findings  Genetic association mapping (AM) in poplar (Populus trichocarpa) previously identified novel genes significantly associated with lignin trait variation (Porth et al., 2013a). I hypothesized the latter have potential roles related to secondary cell wall (SCW) biosynthesis. After screening in silico data and T-DNA insertion mutants for Arabidopsis homologs of the lignin-associated genes (LAGs), I identified CPU and PtNPF6.1 for in-depth functional characterization in poplar, described in Chapter 2. In Chapter 3, I found CPU to have a function in SCW deposition in fibre cells, possibly through protein-protein interactions with microtubule-associated proteins that help guide cellulose synthase complexes in the plasma membrane. In Chapter 4, I characterized PtNPF6.1, a putative long-distance N-related transporter impacting nitrogen (N) homeostasis that resulted in a concomitant decrease in phenylpropanoids including syringyl (S) lignin under high-light/UV-B exposure and luxuriant N conditions, respectively. Altogether, these findings highlight two novel cell wall-related genes identified through AM and their distinct roles in the cellular and physiological processes required for secondary cell wall biosynthesis in poplar.   5.1.1 Analysis of Arabidopsis thaliana T-DNA insertion mutant stems Of the 27 LAGs analyzed, eight exhibited high expression in poplar xylem, consistent with a cell wall-related function. However, irreproducible stem-related phenotypes for the Arabidopsis T-DNA insertion mutants analyzed suggest the LAGs to contribute either an indirect or minor effect on wood and/or secondary cell wall biosynthesis. Associations identified from AM typically detect multiple single nucleotide polymorphisms (SNPs) that each explain only a small fraction of the variation observed in complex phenotypic traits (Korte and Farlow, 2013). Wood and secondary cell wall development are indeed complex traits and thus it is expected that multiple mild-effect loci contribute to the phenotypic variation, as found by Porth et al. (2013a). This could explain the phenotypes observed in Arabidopsis despite having studied loss of function alleles for the associated genes. Natural alleles of the AM candidate genes may only express phenotypes under certain genetic backgrounds or environmental conditions (Kalladan et al., 2017). Variation in genes that exert large phenotypic effects may be selected against within a natural population because these alleles likely cause detrimental effects, which may explain why 115  such genes are not captured through AM. The latter would be expected for near or complete loss of function alleles as these alleles are maintained at low frequencies in a natural population and are difficult to detect (Vanholme et al., 2013b). As a rare example, a natural mutant allele of the gene encoding the lignin biosynthetic enzyme HCT in P. nigra was identified (Vanholme et al., 2013b). Unlike its loss of function allele in Arabidopsis which is dwarf and contains primarily H lignin (Besseau et al., 2007), homozygous poplar hct mutants only have slightly increased H lignin compared to the heterozygous individuals (Vanholme et al., 2013b). This could be attributed to the presence of HCT paralogs with redundant or partial overlapping functions, which has allowed the homozygous hct allele to remain in the population. As Porth et al. (2013a) indicate the phenotypic variance explained by individual SNPs to range between four to seven percent, it is plausible that the SNPs identified from the AM experiment are the more common alleles in genes that contribute mild-effects to wood development within a population.  While significant differences in cellular composition and cell wall morphology were not apparent from histochemical analysis of stem sections performed here, lignin traits for the T-DNA insertion mutants were not quantified. Thus it remains open as to whether minor but significant changes in lignin content and/or composition are present in mutants of the Arabidopsis LAG homologs. Recombinant in vitro expression of one of the LAGs, BBEL13, suggested a role in monolignol oxidation (Daniel et al., 2015). While BBEL13 was one of the most highly expressed LAGs in poplar developing secondary xylem, it is a member of a large gene family in both poplar and Arabidopsis which presents challenges for reverse genetics. However, it would be worthwhile to analyze higher order mutants for a connection to its association to insoluble and total lignin content variation (Porth et al., 2013a). If validated, this oxidase could represent a new class of enzymes involved in monolignol metabolism.  5.1.2 Characterization of CPU  While several lines of evidence support a SCW-related role for CPU including preferential expression in xylem and co-expression with well-known secondary cell wall-related genes, a role affecting total lignin content variation was not found in transgenic poplar over-expressing PtCPU. Instead, the mutants had significantly shorter fibres and increased cellulose microfibril angle compared to wild-type (WT). Through a protein-interaction screen, AtCPU was 116  identified to interact with Cellulose-Microtubule Uncoupling 1 (CMU1) (Liu et al., 2016). In the higher order cpucmu1cmu2 triple mutant, a genetic interaction was apparent as secondary cell well deposition in the interfascicular fibres was negatively impacted compared to the individual mutants and WT. Over-expression of PtCPU alone in WT Arabidopsis had the opposite effect on cell wall thickness. Together, this suggests a possible protein-interaction localized at the cortical microtubules that affects secondary cell wall formation. This interaction should be further validated in vivo, for example, through co-localization analysis of CPU and CMU1/CMU2, or co-immunoprecipitation. Interestingly, AM in poplar found PtCMU3.1 (POPTR_0012s08160/Potri.012G080100), a putative ortholog of Arabidopsis CMU3, to be significantly associated with fibre length (Porth et al., 2013a). While AtCPU and CMU3 did not interact in vitro, it cannot be ruled out that some degree of functional redundancy exists for the xylem-expressed CMUs. For example, CMU3-related transcripts have been detected in poplar tension wood (Andersson-Gunnerås et al., 2006). In a transcriptomic analysis of elongating poplar (P. trichocarpa) stems under high nitrogen conditions PtCMU2.1 (POPTR_0008s09430/Potri.008G094700), a homolog of CMU2, was also upregulated (Euring et al., 2014). Fittingly, under high nitrogen conditions, P. trichocarpa x P. deltoides trees were found to have shorter xylary fibres possessing thicker cell walls compared to trees grown under regular nitrogen regimes (Pitre et al., 2007b). This suggests that CMU and its interaction with CPU influences fibre cell elongation through an indirect effect of cellulose deposition, especially under rapid growth or particular environmental conditions. Therefore, it would be important to test whether the higher order quadruple mutant cpucmu1cmu2cmu3 has an additional anatomical or chemical phenotypes in the SCWs of fibre cells.   The functional analysis of proteins of unknown function remains one of the biggest challenges in the post-genomic era (Mewalal et al., 2014). The characterization of CPU in this thesis is a good example of these challenges. The tight co-expression with secondary cell wall-related genes strongly suggested a role in this biological process and given the genetic association with total lignin content variation, a direct role in lignin biosynthesis was hypothesized. The absence of detectable catalytic domains in CPU argued against an enzymatic function. Likewise, the absence of domains typically found in transcriptional regulatory proteins such as DNA-binding domains argued against an immediate regulatory role. The presence of 117  coiled-coil domains and tight co-expression with transcriptional regulators suggested a regulatory function through protein-protein interactions, but a role as a co-regulatory protein acting in concert to secondary cell wall transcriptional regulators was rejected as Y2H experiments did not reveal any transcription factor binding. The cDNA clones isolated from the Y2H screen represented different classes of proteins, mainly microtubule-associated proteins. Aside from acting as scaffold proteins, coiled-coil domains have a broad range of functions including cytoskeletal organization and motor protein-based transport (Gardiner et al., 2011). From this perspective, it may not be surprising that my experimental evidence eventually found a possible indirect role for CPU in the assembly or guidance of a large protein complex mediated by microtubules. However, this suggests that the genetic association to total lignin content is likely indirect and may be incurred through structural changes in the secondary cell wall.   5.1.3 Characterization of PtNPF6.1  While the in silico analysis highly supported a secondary cell wall-related role for CPU, it did not for PtNPF6.1. Nevertheless, my experiments revealed PtNPF6.1 expression in the vascular tissue, suggesting a potential function in long-distance transport. Transgenic poplar suppressed in PtNPF6.1 had increased total nitrogen concentration concomitant with decreased total soluble phenolics in mature source leaves compared to wild-type. Under luxuriant nitrogen conditions, the same transgenic lines produced wood with less S lignin units compared to wild-type. Therefore, the changes in total phenolics seen here is most parsimoniously explained with the use of increased nitrogen for biomass accumulation at the expense of the biosynthesis of carbon-based phenylpropanoid compounds such as flavonoids and lignin. This suggests the S lignin association to be an indirect effect of total nitrogen redistribution to different tissues mediated by PtNPF6.1. It is possible that this inverse relationship is mediated by the competition for Phe, an amino acid that links primary metabolism via the shikimate pathway and secondary metabolism via the phenylpropanoid pathway (Jones and Hartley, 1999; Bandau et al., 2015).   As plant nitrogen homeostasis is likely to be more complex in woody perennials compared to herbaceous annuals, it is surprising that only a few studies exist for nitrogen-related transport in poplar and other woody plants. Moreover, peptide transporters within the NPF family have not been well-studied in general. Non-nitrogen compounds are also known to be 118  substrates of NPFs (Wang et al., 2018a). Thus, the actual substrates for PtNPF6.1 remain to be identified. Transporter substrates are conventionally tested using Xenopus laevis oocytes or yeast complementation assays. However, without any knowledge of likely substrates, expressing PtNPF6.1 in poplar protoplasts could be an alternative and a more effective approach to test substrate preferences in a native background. One interesting approach that has been used to monitor organic nitrogen uptake in fungi and their associated plants was through the use of fluorescent nanoscale semiconductors called quantum dots, which can be conjugated to amino or carboxyl groups of organic compounds (Whiteside et al., 2009; Whiteside et al., 2012). Quantum dots bound to γ-aminobutyric acid (GABA) have been used to detect GABA receptors on pollen protoplasts (Yu et al., 2006). Thus, quantum dots bound to amino acids or di- and tripeptides infiltrated into transgenic RNAi PtNPF6.1 lines may be a potential approach to monitor substrate translocation in planta. Aside from transporter substrates, further analysis into the effect of high nitrogen on wood quality in PtNPF6.1-suppressed lines is warranted. Poplar wood is highly responsive to environmental nitrogen; high nitrogen can influence the production of shorter and wider xylary fibres, and the incorporation of non-monolignol phenolics into lignin (Pitre et al., 2007a, b). As wood-related traits were not extensively analyzed for the RNAi PtNPF6.1 lines, it would be interesting to determine whether fibre length and cell wall thickening were affected in these plants under the high nitrogen regime. An analysis of the minor phenolic constituents in poplar lignin such as p-hydroxybenzoic acid (Pitre et al., 2007a; Wang et al., 2018b) would also be justified due to the influence of PtNPF6.1 on phenolic compounds content.   5.2 Perspectives on genetic association mapping in poplar AM can reveal genes that contribute even minor influences on a phenotypic trait of interest (Kalladan et al., 2017). Candidate gene-based approaches have enabled studies related to complex traits in woody plants including wood formation (Wegrzyn et al., 2010; Guerra et al., 2013; Porth et al., 2013a) as current genomic resources enable the selection of a large suite of candidate genes. However, candidate gene AM is based on a hypothesis and is biased towards the genes input into the analysis. Furthermore, as the candidate genes are often chosen based on experimental evidence from loss of function mutations (Hall et al., 2010) the findings may not be reflective of the extent of phenotypic variation caused by allelic variations of the gene acting in a natural environment. Therefore, employing true a genome-wide association study (GWAS) 119  instead of a candidate gene AM approach could improve gene discovery without the initial bias of candidate gene selection and identify a broad range of novel genes not yet implicated in secondary cell wall biosynthesis. However, the inclusion of all genetic variants across the genome demands a much larger population size than is practically feasible, at least for non-model organisms of comparably little economic or societal value. Increasing test population sizes will enable the determination of significance when applying multiple testing corrections for millions of genetic variants across the genome. In addition to multiple testing corrections, genetic association studies are also based on complex statistical analyses that, for example, must account for the population structure which in particular for natural populations such as the P. trichocarpa population used by Porth et al. (2013a), may be difficult to detect. As with any statistical analysis, the pre-defined threshold for significance does influence acceptance of associations (Verslues et al., 2014). While Porth and colleagues chose a threshold of alpha 0.05, it is possible that SNPs not deemed significant could in fact have a significant biological association. A GWAS analysis of proline accumulation in Arabidopsis under drought found this to be the case where genes below the significance threshold exhibited phenotypic differences based on reverse genetic analysis (Verslues et al., 2014). Therefore, the experimental parameters of an AM experiment have to balance the risk of rejecting true positives with minimizing the detection of false positives. As a whole, there are limitations from AM and the results need to be interpreted with caution. Hall et al. (2010) suggest the replication and validation of allelic effects in order to distinguish true positives from false positives through experimentation. Here, I included other evidence as an alternative, in particular reverse genetic profiling of loss of function mutants and gene expression analyses, to further narrow down candidate genes for functional characterization. The findings from this study demonstrate some of the challenges in interpreting AM data, given that only for a comparably small fraction of the LAGs additional evidence further supported their roles in secondary cell wall biosynthesis.  5.3 Conclusion While the SNPs in CPU and PtNPF6.1 did not exhibit a large effect on phenotypic variance in P. trichocarpa (Porth et al., 2013a), the significant genetic association for PtNPF6.1 to S lignin was validated by reverse genetic analysis in poplar. Here AM exposed novel genes, some of which were absent in the model herbaceous annual plant Arabidopsis, including 120  PtNPF6.1, and could therefore be beneficial for the identification of genes that have significant contributions to wood and/or secondary cell wall biosynthesis in woody plants. My findings for CPU exposed coiled-coil microtubule-associated proteins as contributors to secondary cell wall deposition that remain to be characterized in more detail. Due to the experimental challenges and lengthy generation time required to generate transgenic poplar, it was not feasible to undertake a reverse genetics approach to characterize all of the highly xylem-expressed LAGs but my findings suggest that characterizing orthologs in the model plant Arabidopsis presents other challenges and that candidate gene characterization in the native host poplar should be pursued in the future despite the additional experimental work required. Furthermore, my characterization of PtNPF6.1 highlights the importance of including whole-plant physiology when studying secondary cell wall formation as processes seemingly unrelated to its biosynthesis can influence wood properties, such as nitrogen availability in the environment or nitrogen redistribution in the plant. CPU and PtNPF6.1, identified from AM combined with reverse genetic analysis, are novel genes that affect cellular organization and whole-plant physiology, respectively, and therefore have an indirect influence on secondary cell wall biosynthesis. This research demonstrates the potential but also the challenges of AM combined with reverse genetics to uncover how previously unsuspected classes of genes contribute to wood formation and furthers our understanding of the complexity of this developmental process.      121  Literature Cited  Alejandro S, Lee Y, Tohge T, Sudre D, Osorio S, Park J, Bovet L, Lee Y, Geldner N, Fernie AR, Martinoia E (2012) AtABCG29 is a monolignol transporter involved in lignin biosynthesis. 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Plant Cell 21: 248–266   139   Appendices  Appendix A. Additional materials for Chapter 2    129%21%35%45%529%67%70%88%97%106%110%123% Appendix A.1 Groups of genes tested for the P. trichocarpa genetic association mapping study conducted by Porth et al. (2013a).  140  Appendix A.2 Genes from the P. trichocarpa genetic association mapping study significantly associated with wood chemistry and ultrastructure traits  Traita P. trichocarpa Gene ID (v2.2/v3.0)b Categoryc ATG ID A. thaliana Annotationd       Glu POPTR_0002s14750/Potri.002G146500 CW Carbohydrate At4g00110 UDP-D-Glucuronate 4-Epimerase 3 (GAE3) Ara POPTR_0005s16650/Potri.005G168400 CW Carbohydrate At1g78060 glycosyl hydrolase family protein Fibre POPTR_0009s01200/Potri.009G006500 CW Carbohydrate At2g28110 Fragile Fiber 8 (FRA8) AC POPTR_0010s21420/Potri.010G207200 CW Carbohydrate At2g28760 UDP-Xyl Synthase 6 (UXS6) Sol POPTR_0014s12380/Potri.014G129200 CW Carbohydrate At3g62830 UDP-Glucuronic Acid Decarboxylase 2 (UXS2) HC POPTR_0015s05540/Potri.015G029100 CW Carbohydrate At1g73370 Sucrose Synthase 6 (SUS6) HC POPTR_0016s08130/Potri.016G080500 CW Carbohydrate At3g53520 UDP-Glucuronic Acid Decarboxylase 1 (UXS1) AC POPTR_0017s01390/Potri.017G144700 CW Carbohydrate At3g03250 UDP-Glucose Pyrophosphorylase 1 (UGP1) Glu POPTR_0018s11290/Potri.018G103900 CW Carbohydrate At5g17420 Cellulose Synthase Catalytic Subunit 7 (CESA7) Crys POPTR_0002s22020/Potri.002G223300 CW Structural At2g04780 Fasciclin-Like Arabinogalactan 7 (FLA7) Man POPTR_0006s13120/Potri.006G129200 CW Structural At5g03170 Fasciclin-Like Arabinogalactan 11 (FLA11) Syrl POPTR_0001s10120/Potri.001G096900 Expression  At4g23690 Dirigent Protein 6 (DIR6) Glu POPTR_0001s36700/Potri.001G356900 Expression  At1g11910 Aspartic Proteinase 1 (APA1) MFA2 POPTR_0002s22090/Potri.002G222700 Expression  At5g35735 auxin-responsive family protein Aver POPTR_0003s07130/Potri.003G073700 Expression  At1g53730 Strubbelig-Receptor Family 6 (SRF6) Aver POPTR_0004s03720/Potri.004G037000 Expression  At4g03390 Strubbelig-Receptor Family 3 (SRF3) MFA1 POPTR_0004s19470/Potri.004G184000 Expression  At5g25940 early nodulin-like protein Fibre POPTR_0005s25910/Potri.005G237900 Expression  At5g42560 abscisic acid-responsive family protein HC POPTR_0006s00780/Potri.006G006000 Expression  At4g16370 Oligopeptide Transporter 3 (OPT3) Total POPTR_0006s19850/NA Expression  At4g30935 WRKY DNA-Binding Protein 32 (WRKY32) Total POPTR_0008s11150/Potri.008G112300 Expression  At1g13635 DNA glycosylase superfamily protein (DGP) MFA1 POPTR_0009s09910/Potri.009G096200 Expression  At3g19460 reticulon family protein Insol/Total POPTR_0011s16200/Potri.011G158700 Expression  At1g30760 Berberine Bridge Enzyme-Like 13 (BBEL13) Fibre POPTR_0012s08160/Potri.012G080100 Expression  At1g27500 Cellulose-Microtubule Uncoupling 3 (CMU3) Insol POPTR_0017s01510/Potri.T059900 Expression  At5g37478 Microtubule-Associated Protein 20 (MAP20) MFA2 POPTR_0017s14280/Potri.017G108800 Expression  At5g15950 S-Adenosylmethionine Decarboxylase 2 (SAMDC2) Syrl POPTR_0018s12720/Potri.018G127100 Expression  At3g07490 Calmodulin-Like 3 (CML3) AC POPTR_0018s13970/Potri.018G142800 Expression  At5g57200 epsin N-terminal homology superfamily protein Man POPTR_0001s21120/Potri.001G204200 Light-Related At1g66340 Ethylene Response 1 (ETR1) MFA2 POPTR_0007s09720/Potri.007G056400 Light-Related At2g17820 Histidine Kinase 1 (HK1) MFA2 POPTR_0008s07650/Potri.008G076800 Light-Related At3g59470 Far1-Related Sequences-Related Factor 1 (FRF1) Fibre POPTR_0010s04740/Potri.010G037800 Light-Related At1g59940 Response Regulator 3 (ARR3) Man POPTR_0010s08200/Potri.010G071200 Light-Related At1g04400 Cryptochrome 2 (CRY2) Aver POPTR_0012s00600/Potri.012G005900 Light-Related At5g24470 Pseudo-Response Regulator 5 (PRR5) Crys POPTR_0015s00440/Potri.015G002300 Light-Related At5g24470 Pseudo-Response Regulator 5 (PRR5) MFA2 POPTR_0019s08690/Potri.019G058900 Light-Related At3g56380 Response Regulator 17 (ARR17) Aver POPTR_0001s01540/Potri.001G133200 Other At1g32100 Pinoresinol Reductase 1 (PRR1)      atrait abbreviations: AC: alpha-cellulose, Ara: arabinose, Aver: average wood density, Crys: crystallinity, Fibre: fibre length, Glu: glucose, Hemi: hemicellulose, HC: holocellulose, Insol: insoluble lignin, Man: mannose, MFA1/2: microfibril angle 1/2, Sol: soluble lignin, Syrl: syringyl lignin, Total: total lignin, Xyl: xylose. bP. trichocarpa IDs based on genome versions 2.2 and 3.0 (phytozome.jgi.doe.gov). cCategory defined by Porth et al. (2013a). dA. thaliana annotations based on Araport (www.araport.org), TAIR (www.arabidopsis.org) and published literature. Additional details can be found in Porth et al. (2013a).  141  Appendix A.2 (continued) Genes from the P. trichocarpa genetic association mapping study significantly associated with wood chemistry and ultrastructure traits  Traita P. trichocarpa Gene ID (v2.2/v3.0)b Categoryc ATG ID A. thaliana Annotationd      Sol POPTR_0001s03760/Potri.001G000500 Other At1g55570 SKU5 Similar 12 (SKS12) Hemi POPTR_0001s10990/Potri.001G087700 Other NA NA HC POPTR_0001s11720/Potri.001G080400 Other At1g02130 Responsive to Abscisic Acid 1B RA-5 (ARA5) MFA2 POPTR_0001s12220/Potri.001G075600 Other At1g30900 Vacuolar Sorting Receptor 6 (VSR6) Total POPTR_0001s28570/Potri.001G279000 Other At1g07120 Coiled-coil Protein of Unknown Function (CPU) AC POPTR_0002s17740/Potri.002G176800 Other At2g46710 Rho GTPase-activating protein Crys POPTR_0005s04170/NA Other At1g08930 Early Response to Dehydration 6 (ERD6) Insol POPTR_0005s07810/Potri.005G076500 Other At4g39840 cell wall integrity/stress response component-like protein (CWP) Hemi POPTR_0005s21770/Potri.005G195900 Other At5g50860 protein kinase superfamily protein HC POPTR_0006s00740/Potri.006G005600 Other At5g47635 pollen Ole e 1 allergen family protein Syrl POPTR_0006s08270/Potri.006G082700 Other At2g37460 Usually Multiple Acids Move In and Out Transporter 12 (UMAMIT12) Syrl POPTR_0006s08720/Potri.006G086900 Other At5g02230 haloacid dehalogenase-like hydrolase (HAD)  Sol POPTR_0007s10810/Potri.007G046100 Other At2g23360 Filament-Like Plant Protein 7 (FPP7) Crys POPTR_0008s08060/Potri.008G081100 Other At1g79340 Metacaspase 4 (MC4) Aver POPTR_0009s05620/Potri.009G051500 Other At3g21070 NAD Kinase 1 (NADK1) AC/HC POPTR_0010s05940/Potri.010G049600 Other At5g14420 RING Domain Ligase 2 (RGLG2) Ara POPTR_0010s12220/Potri.010G112000 Other At1g30110 Nudix Hydrolase Homolog 25 (NUDX25) Syrl POPTR_0010s15180/Potri.010G141900 Other At4g02080 Secretion-Associated RAS Super Family 2 (SAR2) HC POPTR_0010s19240/Potri.010G185000 Other At2g40280 S-adenosyl-L-methionine-dependent methyltransferase Man POPTR_0011s16690/Potri.011G164000 Other At1g30580 GTP-binding protein Syrl POPTR_0014s04020/Potri.014G040700 Other At1g75030 Thaumatin-Like Protein 3 (TLP3) MFA2 POPTR_0014s09010/Potri.014G094400 Other At2g46300 NDR1/HIN1-LIKE 36 (NHL36) Fibre POPTR_0014s09560/Potri.014G100100 Other NA NA Fibre POPTR_0014s12450/Potri.014G130000 Other At4g02440 Empfindlicher Im Dunkelroten Licht 1 (EID1) Aver POPTR_0014s14480/Potri.014G147600 Other At1g10800 voltage-gated hydrogen channel-like protein Syrl POPTR_0016s05010/Potri.016G049600 Other At5g06860 Polygalacturonase Inhibiting Protein 1 (PGIP1) Aver POPTR_0019s03660/Potri.019G019700 Other At3g04470 ankyrin repeat family protein Fiber POPTR_0002s02610/Potri.002G024500 Phytohormone At1g75590 Small Auxin Upregulated RNA 52 (SAUR52) MFA2 POPTR_0002s02800/Potri.002G026500 Phytohormone At4g31500 Cytochrome P450 Family 83, Subfamily B (CYP83B1) Syrl POPTR_0002s20790/Potri.002G206400 Phytohormone At2g47750 putative Indole-3-Acetic Acid-Amido Synthetase (GH3.9) Man POPTR_0003s04120/Potri.003G044200 Phytohormone At1g72770 Hypersensitive to ABA 1 (HAB1) Syrl POPTR_0004s06380/Potri.004G065000 Phytohormone At1g30040 Gibberellin 2-Oxidase 2 (GA2OX2) Xyl POPTR_0010s02280/Potri.010G022300 Phytohormone At5g18580 Embryo Defective 40 (EMB40) HC POPTR_0011s09770/Potri.011G095600 Phytohormone At1g78440 Gibberellin 2-Oxidase 1 (GA2OX1) MFA1 POPTR_0018s01170/Potri.018G033600 Phytohormone At1g15550 Gibberellin 3-Oxidase 1 (GA3OX1) Insol POPTR_0002s03730/Potri.002G036000 Protein Kinase At4g28540 Casein Kinase I-Like 6 (CKL6) Insol POPTR_0002s06080/Potri.002G059900 Protein Kinase At2g42880 Mitogen-Activated Protein Kinase 20 (MPK20)      atrait abbreviations: AC: alpha-cellulose, Ara: arabinose, Aver: average wood density, Crys: crystallinity, Fibre: fibre length, Glu: glucose, Hemi: hemicellulose, HC: holocellulose, Insol: insoluble lignin, Man: mannose, MFA1/2: microfibril angle 1/2, Sol: soluble lignin, Syrl: syringyl lignin, Total: total lignin, Xyl: xylose. bP. trichocarpa IDs based on genome versions 2.2 and 3.0 (phytozome.jgi.doe.gov). cCategory defined by Porth et al. (2013a). † PPD denotes phenylpropanoid pathway. dA. thaliana annotations based on Araport (www.araport.org), TAIR (www.arabidopsis.org) and published literature. Additional details can be found in Porth et al. (2013a). 142  Appendix A.2 (continued) Genes from the P. trichocarpa genetic association mapping study significantly associated with wood chemistry and ultrastructure traits  Traita P. trichocarpa Gene ID (v2.2/v3.0)b Categoryc ATG ID A. thaliana Annotationd  Glu POPTR_0004s11500/Potri.004G115900 Protein Kinase At3g01090 SNF1-Kinase Homolog 10 (AKIN10) Glu POPTR_0009s07050/Potri.009G066100 Protein Kinase At3g45640 Mitogen-Activated Protein Kinase 3 (MPK3) HC POPTR_0011s12550/Potri.011G128000 Protein Kinase At5g54380 Theseus 1 (THE1) MFA1 POPTR_0012s08890/Potri.012G087000 Protein Kinase At5g40380 Cysteine-Rich Receptor-Like Protein Kinase 42 (CRK42) MFA2 POPTR_0003s09520/Potri.003G096600 Shikimate/PPD† At2g35500 Shikimate Kinase-Like 2 (SKL2) HC POPTR_0008s09210/Potri.008G092600 Shikimate/PPD† At1g69370 Chorismate Mutase 3 (CM3) HC POPTR_0009s10270/Potri.009G099800 Shikimate/PPD† At4g34050 Caffeoyl Coenzyme A O-Methyltransferase 1 (CCoAOMT1) MFA2 POPTR_0013s12510/Potri.013G120800 Shikimate/PPD† At4g35160 N-Acetylserotonin O-Methyltransferase (ASMT) Man POPTR_0001s10750/Potri.001G090200 Signalling  At1g64480 Calcineurin B-Like Protein 8 (CBL8) Xyl POPTR_0015s01480/Potri.015G012500 Signalling  At3g49260 IQ-Domain 21 (IQD21) Man POPTR_0001s15490/Potri.001G155100 Transcription At4g16780 Homeobox Protein 2 (HB2) AC POPTR_0001s22790/Potri.001G220700 Transcription At3g56850 ABA-Responsive Element Binding Protein 3 (AREB3) Hemi POPTR_0001s33080/Potri.001G323500 Transcription At2g33500 B-Box Domain Protein 12 (BBX12) Hemi POPTR_0002s25920/Potri.002G257400 Transcription At5g43990 Set Domain Protein 18 (SUVR2) Ara POPTR_0003s12460/Potri.003G124500 Transcription At1g54830 Nuclear Factor Y, Subunit C3 (NF-YC3) Crys POPTR_0003s17700/Potri.003G178600 Transcription At2g32700 Mucilage-Modified 1 (MUM1) Hemi POPTR_0005s18090/Potri.005G137600 Transcription At3g19184 AP2/B3-like family protein Man POPTR_0007s13910/Potri.007G014400 Transcription At2g18060 Vascular Related NAC-Domain Protein 1 (VND1) Glu POPTR_0008s02580/Potri.008G025600 Transcription At5g03680 Petal Loss (PTL) Syrl POPTR_0008s06130/Potri.008G061000 Transcription At5g02030 BEL1-Like Homeodomain 9 (BLH9) AC POPTR_0009s01990/Potri.009G014500 Transcription At5g60690 Revoluta (REV) Aver POPTR_0009s09250/Potri.009G089400 Transcription At3g19580 Zinc-Finger Protein 2 (ZF2) Crys POPTR_0011s05740/Potri.011G058400 Transcription At4g28500 Secondary Wall-Associated NAC Domain 2 (SND2) AC POPTR_0011s09030/Potri.011G087900 Transcription At4g26640 WRKY DNA-Binding Protein 20 (WRKY20) HC POPTR_0014s04460/Potri.014G045100 Transcription At3g60390 Homeobox-Leucine Zipper Protein 3 (HAT3) Man POPTR_0014s06150/Potri.014G066100 Transcription At1g02030 C2H2-like zinc finger protein Man POPTR_0015s06980/Potri.015G058800 Transcription At5g60970 teosinte branched 1, cycloidea and PCF 5 (TCP5) Total POPTR_0001s12890/Potri.001G068600 Transport At5g13400 Nitrate1/Peptide (NRT1/PTR) Family 6.3 (NPF6.3) Syrl POPTR_0001s36340/Potri.001G335200 Transport At5g40780 Lysine Histidine Transporter 1 (LHT1) Syrl POPTR_0002s03070/Potri.002G029200 Transport At3g54140 Nitrate1/Peptide (NRT1/PTR) Family 6.1 (NPF6.1) Insol POPTR_0009s13470/Potri.009G132100 Transport At2g21050 Like Auxin Resistant 2 (LAX2)      atrait abbreviations: AC: alpha-cellulose, Ara: arabinose, Aver: average wood density, Crys: crystallinity, Fibre: fibre length, Glu: glucose, Hemi: hemicellulose, HC: holocellulose, Insol: insoluble lignin, Man: mannose, MFA1/2: microfibril angle 1/2, Sol: soluble lignin, Syrl: syringyl lignin, Total: total lignin, Xyl: xylose. bP. trichocarpa IDs based on genome versions 2.2 and 3.0 (phytozome.jgi.doe.gov). cCategory defined by Porth et al. (2013a). † PPD denotes phenylpropanoid pathway. dA. thaliana annotations based on Araport (www.araport.org), TAIR (www.arabidopsis.org) and published literature. Additional details can be found in Porth et al. (2013a).   143                         Pp: Physcomitrella patens smo: Selaginella moellendorffii Bradi: Brachypodium distachyon LOC_Os: Oryza sativa zma: Zea mays AT: Arabidopsis thaliana Cucsa: Cucumis sativus Eucgr: Eucalpytus grandis POPTR: Populus trichocarpa rco: Ricinus communius   Appendix A.3 Sequence similarity tree for UMAMIT9 (POPTR_0001s06980), a gene associated with insoluble lignin content. The tree was generated using an automated platform in Phytozome and is based on protein percent identity. The bracket indicates the clade containing the lignin-associated gene (black dot). P. trichocarpa sequences are in blue and A. thaliana sequences are in orange.  yps o: elaginella moellendorffii LOC_Os: Oryza sativa AT: Arabidopsis thaliana Eucgr: Eucalyptus grandis POPTR: Populus trichocarpa  144    Appendix A.4 Sequence similarity tree for CKL6 (POPTR_0002s03730), a gene significantly associated with insoluble lignin content. The tree was generated using an automated platform in Phytozome and is based on protein percent identity. The bracket indicates the clade containing the lignin-associated gene (black dot). P. trichocarpa sequences are in blue and A. thaliana sequences are in orange.  Ppa: Physcomitrella patens smo: Selaginella moellendorffii LOC_Os: Oryza sativa AT: Arabidopsis thaliana POPTR: Populus trichocarpa  145       Appendix A.5 Sequence similarity tree for MPK20 (POPTR_0002s06080), a gene significantly associated with insoluble lignin content. The tree was generated using an automated platform in Phytozome and is based on protein percent identity. The bracket indicates the clade containing the lignin-associated gene. The P. trichocarpa sequence is in blue and the A. thaliana sequences are in orange.  Ppa: Physcomitrella patens smo: Selaginella moellendorffii LOC_Os: Oryza sativa AT: Arabidopsis thaliana Eucgr: Eucalyptus grandis Potri: Populus trichocarpa  146   Appendix A.6 Sequence similarity tree for CWP (POPTR_0005s07810), a gene significantly associated with insoluble lignin content. The tree was generated using an automated platform in Phytozome and is based on protein percent identity. The bracket indicates the clade containing the lignin-associated gene (black dot). P. trichocarpa sequences are in blue and A. thaliana sequences are in orange.    Ppa: Physcomitrella patens smo: Selaginella moellendorffii LOC_Os: Oryza sativa zma: Zea mays AT: Arabidopsis thaliana Eucgr: Eucalyptus grandis rco: Ricinus communis POPTR: Populus trichocarpa  147      Appendix A.7 Sequence similarity tree for LAX2 (POPTR_0009s13470), a gene significantly associated with insoluble lignin content. The tree was generated using an automated platform in Phytozome and is based on protein percent identity. The bracket indicates the clade containing the lignin-associated gene (black dot). P. trichocarpa sequences are in blue and A. thaliana sequences are in orange.  Ppa: Physcomitrella patens smo: Selaginella moellendorffii LOC_Os: Oryza sativa AT: Arabidopsis thaliana Eucgr: Eucalyptus grandis POPTR: Populus trichocarpa  148    Appendix A.8 Sequence similarity tree for BBEL13 (POPTR_0011s16200), a gene significantly associated with insoluble (and total) lignin content. The tree was generated using an automated platform in Phytozome and is based on protein percent identity. The bracket indicates the clade containing the lignin-associated gene (black dot). P. trichocarpa sequences are in blue and A. thaliana sequences are in orange.  Ppa: Physcomitrella patens smo: Selaginella moellendorffii Bradi: Brachypodium distachyon sbi: Sorghum bicolor zma: Zea mays AT: Arabidopsis thaliana Eucgr: Eucalyptus grandis Medtr: Medicago truncatula rco: Ricinus communis POPTR: Populus trichocarpa  149     Appendix A.9 Sequence similarity tree for MAP20 (POPTR_0017s01510), a gene significantly associated with insoluble lignin content. The tree was generated using an automated platform in Phytozome and is based on protein percent identity. The bracket indicates the clade containing the lignin-associated gene. P. trichocarpa sequences are in blue and A. thaliana sequences are in orange.  Ppa: Physcomitrella patens LOC_Os: Oryza sativa sbi: Sorghum bicolor zma: Zea mays AT: Arabidopsis thaliana Eucgr: Eucalyptus grandis POPTR: Populus trichocarpa  150     Appendix A.10 Sequence similarity tree for P4H7 (POPTR_0017s11150), a gene associated with insoluble lignin content. The tree was generated using an automated platform in Phytozome and is based on protein percent identity. The bracket indicates the clade containing the lignin-associated gene. P. trichocarpa sequences are in blue and A. thaliana sequences are in orange.  Ppa: Physcomitrella patens smo: Selaginella moellendorffii LOC_Os: Oryza sativa AT: Arabidopsis thaliana Eucgr: Eucalyptus grandis POPTR: Populus trichocarpa  151     Appendix A.11 Sequence similarity tree for SKS12 (POPTR_0001s03760), a gene significantly associated with soluble lignin content. The tree was generated using an automated platform in Phytozome and is based on protein percent identity. The bracket indicates the clade containing the lignin-associated gene. P. trichocarpa sequences are in blue and A. thaliana sequences are in orange.  Ppa: Physcomitrella patens smo: Selaginella moellendorffii LOC_Os: Oryza sativa AT: Arabidopsis thaliana Eucgr: Eucalyptus grandis Potri: Populus trichocarpa  152       Appendix A.12 Sequence similarity tree for FPP7 (POPTR_0007s10810), a gene significantly associated with soluble lignin content. The tree was generated using an automated platform in Phytozome and is based on protein percent identity. The bracket indicates the clade containing the lignin-associated gene (black dot). P. trichocarpa sequences are in blue and A. thaliana sequences are in orange.  Ppa: Physcomitrella patens smo: Selaginella moellendorffii LOC_Os: Oryza sativa AT: Arabidopsis thaliana Eucgr: Eucalyptus grandis POPTR: Populus trichocarpa  153      Appendix A.13 Sequence similarity tree for UXS2 (POPTR_0014s12380), a gene significantly associated with soluble lignin content. The tree was generated using an automated platform in Phytozome and is based on protein percent identity. The bracket indicates the clade containing the lignin-associated gene. P. trichocarpa sequences are in blue and A. thaliana sequences are in orange.  Ppa: Physcomitrella patens smo: Selaginella moellendorffii LOC_Os: Oryza sativa AT: Arabidopsis thaliana Eucgr: Eucalyptus grandis POPTR: Populus trichocarpa  154      Appendix A.14 Sequence similarity tree for DIR6 (POPTR_0001s10120), a gene significantly associated with syringyl lignin. The tree was generated using an automated platform in Phytozome and is based on protein percent identity. The bracket indicates the clade containing the lignin-associated gene (black dot). P. trichocarpa sequences are in blue and A. thaliana sequences are in orange.  Ppa: Physcomitrella patens smo: Selaginella moellendorffii LOC_Os: Oryza sativa AT: Arabidopsis thaliana Eucgr: Eucalyptus grandis POPTR: Populus trichocarpa  155       Appendix A.15 Sequence similarity tree for LHT1 (POPTR_0001s36340), a gene significantly associated with syringyl lignin. The tree was generated using an automated platform in Phytozome and is based on protein percent identity. The bracket indicates the clade containing the lignin-associated gene. P. trichocarpa sequences are in blue and A. thaliana sequences are in orange.  Ppa: Physcomitrella patens smo: Selaginella moellendorffii Bra: Brachypodium distachyon LOC_Os: Oryza sativa sbi: Sorghum bicolor zma: Zea mays AT: Arabidopsis thaliana Eucgr: Eucalyptus grandis Glyma: Glycine max cassava: Manihot esculenta POPTR: Populus trichocarpa rco: Ricinus communis GSVIVT: Vitis vinifera  156      Appendix A.16 Sequence similarity tree for NPF6.1 (POPTR_0002s03070), a gene significantly associated with syringyl lignin. The tree was generated using an automated platform in Phytozome and is based on protein percent identity. The bracket indicates the clade containing the lignin-associated gene (black dot). P. trichocarpa sequences are in blue and A. thaliana sequences are in orange.  . Ppa: Physcomitrella patens smo: Selaginella moellendorffii Bradi: Brachypodium distachyon LOC_Os: Oryza sativa AT: Arabidopsis thaliana Eucgr: Eucalyptus grandis POPTR: Populus trichocarpa  157     Appendix A.17 Sequence similarity tree for GH3.9 (POPTR_0002s20790), a gene significantly associated with syringyl lignin. The tree was generated using an automated platform in Phytozome and is based on protein percent identity. The bracket indicates the clade containing the lignin-associated gene. P. trichocarpa sequences are in blue and A. thaliana sequences are in orange.  Ppa: Physcomitrella patens smo: Selaginella moellendorffii Bra: Brachypodium distachyon LOC_Os: Oryza sativa sbi: Sorghum bicolor zma: Zea mays AT: Arabidopsis thaliana Eucgr: Eucalyptus grandis Medtr: Medicago truncatula POPTR: Populus trichocarpa rco: Ricinus communis  158      Appendix A.18 Sequence similarity tree for GA2OX2 (POPTR_0004s06380), a gene significantly associated with syringyl lignin. The tree was generated using an automated platform in Phytozome and is based on protein percent identity. The bracket indicates the clade containing the lignin-associated gene (black dot). P. trichocarpa sequences are in blue and A. thaliana sequences are in orange.  Ppa: Physcomitrella patens smo: Selaginella moellendorffii LOC_Os: Oryza sativa AT: Arabidopsis thaliana Eucgr: Eucalyptus grandis POPTR: Populus trichocarpa  159      Appendix A.19 Sequence similarity tree for UMAMIT12 (POPTR_0006s08270), a gene significantly associated with syringyl lignin. The tree was generated using an automated platform in Phytozome and is based on protein percent identity. The bracket indicates the clade containing the lignin-associated gene. P. trichocarpa sequences are in blue and A. thaliana sequences are in orange.  Ppa: Physcomitrella patens smo: Selaginella moellendorffii LOC_Os: Oryza sativa AT: Arabidopsis thaliana Eucgr: Eucalyptus grandis POPTR: Populus trichocarpa  160      Appendix A.20 Sequence similarity tree for HAD (POPTR_0006s08720), a gene significantly associated with syringyl lignin. The tree was generated using an automated platform in Phytozome and is based on protein percent identity. The bracket indicates the clade containing the lignin-associated gene. P. trichocarpa sequences are in blue and A. thaliana sequences are in orange.  Ppa: Physcomitrella patens smo: Selaginella moellendorffii LOC_Os: Oryza sativa AT: Arabidopsis thaliana Eucgr: Eucalyptus grandis POPTR: Populus trichocarpa rco: Ricinus communis  161      Appendix A.21 Sequence similarity tree for BLH9 (POPTR_0008s06130), a gene significantly associated with syringyl lignin. The tree was generated using an automated platform in Phytozome and is based on protein percent identity. The bracket indicates the clade containing the lignin-associated gene (black dot). P. trichocarpa sequences are in blue and A. thaliana sequences are in orange.  Ppa: Physcomitrella patens smo: Selaginella moellendorffii Bra: Brachypodium distachyon LOC_Os: Oryza sativa sbi: Sorghum bicolor zma: Zea mays AT: Arabidopsis thaliana POPTR: Populus trichocarpa rco: Ricinus communis  162     Appendix A.22 Partial sequence similarity tree for SAR2 (POPTR_0010s15180), a gene significantly associated with syringyl lignin. The tree was generated using an automated platform in Phytozome and is based on protein percent identity. The bracket indicates the clade containing the lignin-associated gene (black dot). P. trichocarpa sequences are in blue and A. thaliana sequences are in orange.  Bra: Brachypodium distachyon AT: Arabidopsis thaliana POPTR: Populus trichocarpa  163        Appendix A.23 Sequence similarity tree for TLP3 (POPTR_0014s04020), a gene significantly associated with syringyl lignin. The tree was generated using an automated platform in Phytozome and is based on protein percent identity. The bracket indicates the clade containing the lignin-associated gene. P. trichocarpa sequences are in blue and A. thaliana sequences are in orange.  Ppa: Physcomitrella patens smo: Selaginella moellendorffii LOC_Os: Oryza sativa AT: Arabidopsis thaliana Eucgr: Eucalyptus grandis POPTR: Populus trichocarpa  164     Appendix A.24 Sequence similarity tree for PGIP1 (POPTR_0016s05010), a gene significantly associated with syringyl lignin. The tree was generated using an automated platform in Phytozome and is based on protein percent identity. The bracket indicates the clade containing the lignin-associated gene (black dot). P. trichocarpa sequences are in blue and A. thaliana sequences are in orange.  Ppa: Physcomitrella patens smo: Selaginella moellendorffii LOC_Os: Oryza sativa AT: Arabidopsis thaliana Eucgr: Eucalyptus grandis POPTR: Populus trichocarpa  165      Appendix A.25 Sequence similarity tree for CML3 (POPTR_0018s12720), a gene significantly associated with syringyl lignin. The tree was generated using an automated platform in Phytozome and is based on protein percent identity. The bracket indicates the clade containing the lignin-associated gene (black dot). P. trichocarpa sequences are in blue and A. thaliana sequences are in orange.  Ppa: Physcomitrella patens smo: Selaginella moellendorffii LOC_Os: Oryza sativa AT: Arabidopsis thaliana Eucgr: Eucalyptus grandis POPTR: Populus trichocarpa  166      Appendix A.26 Sequence similarity tree for NPF6.3 (POPTR_0001s12890), a gene significantly associated with total lignin content. The tree was generated using an automated platform in Phytozome and is based on protein percent identity. The bracket indicates the clade containing the lignin-associated gene. P. trichocarpa sequences are in blue and A. thaliana sequences are in orange. Ppa: Physcomitrella patens smo: Selaginella moellendorffii LOC_Os: Oryza sativa AT: Arabidopsis thaliana Eucgr: Eucalyptus grandis POPTR: Populus trichocarpa  167  Ppa: Physcomitrella patens smo: Selaginella moellendorffii Bradi: Brachypodium distachyon LOC_Os: Oryza sativa sbi: Sorghum bicolor zma: Zea mays AT: Arabidopsis thaliana Medtr: Medicago truncatula rco: Ricinus communis POPTR: Populus trichocarpa Eucgr: Eucalyptus grandis At1g07120 POPTR_0009s07770 POPTR_0001s28570 \    Appendix A.27 Sequence similarity tree for CPU (POPTR_0001s28570), a gene significantly associated with total lignin content. The tree was generated using an automated platform in Phytozome and is based on protein percent identity.  \\ 168      Appendix A.28 Sequence similarity tree for WRKY32 (POPTR 0006s19850), a gene significantly associated with total lignin content. The tree was generated using an automated platform in Phytozome and is based on protein percent identity. The bracket indicates the clade containing the lignin-associated gene (black dot). P. trichocarpa sequences are in blue and A. thaliana sequences are in orange.  Ppa: Physcomitrella patens smo: Selaginella moellendorffii LOC_Os: Oryza sativa AT: Arabidopsis thaliana Eucgr: Eucalyptus grandis POPTR: Populus trichocarpa  169      Appendix A.29 Sequence similarity tree for DGP (POPTR_0008s11150), a gene significantly associated with total lignin content. The tree was generated using an automated platform in Phytozome and is based on protein percent identity. The bracket indicates the clade containing the lignin-associated gene (black dot). P. trichocarpa sequences are in blue and A. thaliana sequences are in orange.  Ppa: Physcomitrella patens smo: Selaginella moellendorffii LOC_Os: Oryza sativa AT: Arabidopsis thaliana Eucgr: Eucalyptus grandis POPTR: Populus trichocarpa  170          UMAMIT9               CKL6  Appendix A.30 In silico expression data for UMAMIT9 and CKL6 obtained from the BAR eFP browser. UMAMIT9 (left) is associated with insoluble lignin content, while CKL6 (right) is significantly associated with insoluble lignin content (Porth et al., 2013a). Top: poplar browser results (Wilkinson et al., 2009). Bottom: Arabidopsis browser results (Winter et al., 2007). 171                                Appendix A.31 In silico expression data for MPK20 and CWP obtained from the BAR eFP browser. MPK20 (left) and CWP (right) are significantly associated with insoluble lignin content (Porth et al., 2013a). Top: poplar browser results (Wilkinson et al., 2009). Bottom: Arabidopsis browser results (Winter et al., 2007).  MPK20               CWP 172      Appendix A.32 In silico expression data for LAX2 and BBEL13 obtained from the BAR eFP browser. LAX2 (left) is significantly associated with insoluble lignin content (Porth et al., 2013a), while BBEL13 (right) is significantly associated with insoluble and total lignin content. Top: poplar browser results (Wilkinson et al., 2009). Bottom: Arabidopsis browser results (Winter et al., 2007).         LAX2               BBEL13 173      Appendix A.33 In silico expression data for MAP20 and P4H7 obtained from the BAR eFP browser. MAP20 (left) is significantly associated with insoluble lignin content, while P4H7 (right) is associated with insoluble lignin content (Porth et al., 2013a). Top: poplar browser results (Wilkinson et al., 2009). Bottom: Arabidopsis browser results for P4H7 (Winter et al., 2007). Expression data unavailable for Arabidopsis MAP20.      MAP20               P4H7 174  SKS12   Appendix A.34 In silico expression data for SKS12 obtained from the BAR eFP browser. SKS12 is significantly associated with soluble lignin content (Porth et al., 2013a). Left: poplar browser results (Wilkinson et al., 2009). Right: Arabidopsis browser results (Winter et al., 2007).     175   Appendix A.35 In silico expression data for FPP7 and UXS2 obtained from the BAR eFP browser. Both FPP7 (left) and USX2 (right) are significantly associated with soluble lignin content (Porth et al., 2013a). Top: poplar browser results (Wilkinson et al., 2009). Bottom: Arabidopsis browser results (Winter et al., 2007).             FPP7                UXS2 176     Appendix A.36 In silico expression data for DIR6 and LHT1 obtained from the BAR eFP browser. Both DIR6 (left) and LHT1 (right) are significantly associated with syringyl lignin (Porth et al., 2013a). Top: poplar browser results (Wilkinson et al., 2009). Bottom: Arabidopsis browser results (Winter et al., 2007).       DIR6                LHT1 177      Appendix A.37 In silico expression data for NPF6.1 and GH3.9 obtained from the BAR eFP browser. Both NPF6.1 (left) and GH3.9 (right) are significantly associated with syringyl lignin (Porth et al., 2013a). Top: poplar browser results (Wilkinson et al., 2009). Bottom: Arabidopsis browser results (Winter et al., 2007).       NPF6.1                GH3.9 178      Appendix A.38 In silico expression data for GA2OX2 and UMAMIT12 obtained from the BAR eFP browser. Both GA2OX2 (left) and UMAMIT12 (right) are significantly associated with syringyl lignin (Porth et al., 2013a). Top: poplar browser results (Wilkinson et al., 2009). Bottom: Arabidopsis browser results (Winter et al., 2007).       GA2OX2               UMAMIT12 179           HAD               BLH9  Appendix A.39 In silico expression data for HAD and BLH9 obtained from the BAR eFP browser. Both HAD (left) and BLH9 (right) are significantly associated with syringyl lignin (Porth et al., 2013a). Top: poplar browser results (Wilkinson et al., 2009). Bottom: Arabidopsis browser results (Winter et al., 2007). 180      Appendix A.40 In silico expression data for SAR2 and TLP3 obtained from the BAR eFP browser. Both SAR2 (left) and TLP3 (right) are significantly associated with syringyl lignin (Porth et al., 2013a). Top: poplar browser results for SAR2 (Wilkinson et al., 2009). Expression data unavailable for poplar TLP3. Bottom: Arabidopsis browser results (Winter et al., 2007).             SAR2                        TLP3 181          PGIP1                 CML3  Appendix A.41 In silico expression data for PGIP1 and CML3 obtained from the BAR eFP browser. Both PGIP1 (left) and CML3 (right) are significantly associated with syringyl lignin (Porth et al., 2013a). Top: poplar browser results for PGIP1 (Wilkinson et al., 2009). Expression data unavailable for poplar CML3. Bottom: Arabidopsis browser results (Winter et al., 2007). 182          NPF6.3                 CPU  Appendix A.42 In silico expression data for NPF6.3 and CPU obtained from the BAR eFP browser. Both NPF6.3 (left) and CPU (right) are significantly associated with total lignin content (Porth et al., 2013a). Top: poplar browser results (Wilkinson et al., 2009). Bottom: Arabidopsis browser results (Winter et al., 2007). 183           WRKY32                 DGP  Appendix A.43 In silico expression data for WRKY32 and DGP obtained from the BAR eFP browser. Both WRKY32 (left) and DGP (right) are significantly associated with total lignin content (Porth et al., 2013a). Top: poplar browser results (Wilkinson et al., 2009). Bottom: Arabidopsis browser results for WRKY32 (Winter et al., 2007). Expression data unavailable for Arabidopsis DGP. 184  Appendix B. Additional materials for Chapter 3  Appendix B.1 CPU homologs analyzed in the maximum likelihood phylogeny  Phylum Represented Species ID Gene Model Sourcea  Bryophyta P. patens Ppa02 Pp3c6_26200 Phytozome   Ppa03 Pp3c7_17580    Ppa01 Pp3c15_19960  Lycopodiophyta S. moellendorffii Smo03 85268 Phytozome   Smo01 438117    Smo02 438203  Monilophyta A. aleuticum Aal03 WCLG-2009943 1000 Plants  P. acrostichoides Pac02 FQGQ-2005003 1000 Plants   Pac03 FQGQ-2005004    Pac04 FQGQ-2012287  Coniferophyta P. engelmannii PEn01 AWQB-2054760 1000 Plants   PEn05 AWQB-2056860    PEn06 AWQB-2057111   P. jeffreyi Pje03 MFTM-2000373 1000 Plants   Pje04 MFTM-2000374    Pje05 MFTM-2000383    Pje07 MFTM-2015100    Pje08 MFTM-2083584   P. parviflora PiPa01 IIOL-2006293 1000 Plants   PiPa03 IIOL-2079974    PiPa06 IIOL-2075996   P. ponderosa PPo03 JBND-2002707 1000 Plants   PPo02 JBND-2016898    PPo04 JBND-2011749    PPo01 JBND-2012595  Anthophyta  B. distachyon  Bra01 Bradi1g65430 Phytozome (monocots)  Bra02 Bradi2g15630    Bra03 Bradi2g15767    Bra04 Bradi2g45310   O. sativa Osa01 LOC_Os01g46340 Phytozome   Osa02 LOC_Os03g18300    Osa03 LOC_Os05g49820    Osa04 LOC_Os08g03560    Osa05 LOC_Os11g01439    Osa06 LOC_Os12g01449   S. bicolor Sbi01 Sobic.001G405400 Phytozome   Sbi02 Sobic.003G239600    Sbi03 Sobic.007G027900    Sbi04 Sobic.009G240300    Sbi05 Sobic.009G242100   Z. mays Zma01 GRMZM2G019225 Phytozome   Zma02 GRMZM2G047255    Zma03 GRMZM5G850640    Zma04 AC206223.3_FG001  Anthophya  A. trichopoda Atr01 evm_27.TU.AmTr_v1.0_scaffold00021.78 Phytozome (eudicots)  Atr02 evm_27.TU.AmTr_v1.0_scaffold00147.32    Atr03 evm_27.TU.AmTr_v1.0_scaffold00155.9   aPhytozome (https://phytozome.jgi.doe.gov); 1000 plants (www.onekp.com). 185  Appendix B.1 (continued) CPU homologs analyzed in the maximum likelihood phylogeny  Phylum Represented Species ID Gene Model Sourcea  Anthophya   Ath01 At1g07120 Phytozome (eudicots)  Ath02 At3g25690    Ath03 At4g18570   E. grandis Egr01 Eucgr.B03060 Phytozome   Egr02 Eucgr.D01021    Egr03 Eucgr.E02711    Egr05 Eucgr.J01897   M. truncatula Mtr01 Medtr1g016290 Phytozome   Mtr02 Medtr1g093850    Mtr03 Medtr4g087780    Mtr04 Medtr5g053270    Mtr05 Medtr5g071817    Mtr06 Medtr5g071840   P. trichocarpa Ptr01 Potri.001G279000 Phytozome   Ptr02 Potri.001G460300    Ptr03 Potri.004G054900    Ptr04 Potri.009G073600    Ptr05 Potri.010G130800    Ptr06 Potri.011G064500    Ptr07 Potri.011G156500   S. purpurea Spu01 SapurV1A.0042s0400    Spu02 SapurV1A.0079s0580 Phytozome   Spu03 SapurV1A.0106s0120    Spu04 SapurV1A.0131s0370    Spu05 SapurV1A.0270s0050    Spu06 SapurV1A.0304s0130    Spu07 SapurV1A.0602s0070    Spu08 SapurV1A.1185s0120       aPhytozome (https://phytozome.jgi.doe.gov); 1000 plants (www.onekp.com).    186                       10        20        30        40        50        60        70        80                            ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| Atr01       ----VASLLRFIDQLQENQSRLQTELLEFKLLRESV---KVFEREVASKNAVLKSLAEENERFRC------EAL------  PEn05       -------LEQLLNELQAREAELQRELVDYKMLKERASRVSELEKELEMKNVQAEGFIKRINLLESEKNQMYEETARVSIL  PJe08       ---DVNDLEQLLNELQAREAELQRELVDYKVLKERANRVSELEKELEMKNVQAEALIKRINLMESEKNQMYEEIARVSIL  PPo04       ---DVNDLEQLLNELQAREAELQRELVDYKVLKERANRVSELEKELEMKNVQAEALIKRINLMESEKNQMYEEIARVSIL  Osa04       LFLELDHLREQLRESKERELALQSELRQCR---ENP-RVSELEKDLDSRKNEIDRLVRLKTSLEVEKTSLSEQLSALSCM  SBi03       LFLELDHLREQLRESKERELALQSELRQCR---ENP-KVSELEKELDSMRDEVDRLARLKTSLEAEKTNISEQLSALSSM  ZMa01       LFLELDHLREQLRESKERELTLQSELRQCR---ENP-KVSELEKELDSMRDEVDRLARLKISLEAEKTSISEQLSALSSM  Egr02       LFVELDHLRGLLQESKEREFKLQGELTECK---RNP-RVLDLEREVEVKKSEIDELRRRVELLECEKGSLSGQLPSVPVE  Ptr02       LFVELDQLRSLLQESKEREFKLQAELSELK---RNG-RVVDIERELEARKNEVDELCKRIGVLESEKSGLCEQVNELCLI  Spu07       LFVELDQLRSLLQESKEREFKLQAELSELK---RNG-RVVDLERELEARTNEVDELCKRIGVLESEKSGLCEQVNELCLI  Ptr07       LFVELDQLRSLLQESKEREFKLQAELSEVK---RNG-RVVDLERELEARRNEVDELCKRIGVLESEKSGLCEQVNELCLI  Spu04       LIVELDQLRSLLQESKEREFKLQAELSEIK---RNG-RVVDLERELEAKRNEVDELCKRIGVLESEKSGLCEQVNELCLI  Mtr01       LFTELDHMRSLLQESKEREAKLNAELVECR---KNQSEVDE--------------LVKKVALLEEEKSGLSEQLVALS--  Atr03       LFFELEHLRTSLQESKERETKLHSEIQEFK---GLKSKILELERELELKKTEAETFSQRVCMLEDEKEKMSEQLASLSTV  PEn01       --------------------------------------------------------------------------------  PiPa06      --------------------------------------------------------------------------------  PJe03       --------------------------------------------------------------------------------  PJe04       --------------------------------------------------------------------------------  PPo02       --------------------------------------------------------------------------------  SBi05       VASEIERLRSLVREMEEREAKLEGELLEYYGMKEMETDVTELQKQLKIKAVEINMLNDTINSLQEERKKLQDDVARGEVA  Bra02       -------LRGLVRELEEREVKLEGELLEYYGLKEQETDVTELQKQLKIKTVEVDMLNITISSLQAERKKLQDDVVRGAAT  Osa05       -------LRGLVRELEEREVKLEGELLEYYGLKEQETDVVELHRQLKIKMVEIDMLKMTINSLQEERKKLQDDVARGTGA  Osa06       -------LRGLVRELEEREVKLEGELLEYYGLKEQETDVVELHRQLKIKMVEIDMLKMTINSLQEERKKLQDDVARGTGA  Mtr02       SDSEIEWLRNVVEELEEREMKLQSELLEYYSLKEQVPVIEEFQRQLRIKSVEIDMLHMTIKSLQEENNKLQEELIHEASA  Ath02       NDGELERLKQLVKELEEREVKLEGELLEYYGLKEQESDIVELQRQLKIKTVEIDMLNITINSLQAERKKLQEELSQNGIV  Mtr04       NDSELERLRQLVKELEEREVKLEGELLEYYGLKEQESDIVELQRQLKIKTVEIDMLNITINSLQAERKKLQEELTNGASA  Egr01       NASELEGLRRLVKELEEREMKLEGELLEYYGLKEQESDVIELQRQLKIKTVEIDMLKITINSLQAERKKLQDEIAHGASV  Spu02       TVSELKCLRNLVKELEEREVKLEGELLEYYALKEQEPDIVELQRQLKIKTVEIDMLNITINSLQAERKKLQEEILLGASA  Ptr05       NASELECLRNLVRELEEREVKLEGELLEYYGLKEQESDVVELQRQLKIKTVEIDMLNITINSLQAERKKLQEEISHGASS  Spu06       NASELERLRYLVKELEEREVKLEGELLEYYGLKEQESDIVELQRQLKIKTVEIDMLNITINSLQAERRKLQEEISHGASS  Smo03       --------------------------------------------------------------------------------  Ppa02       DAAELHALRETVKVLKQKESRMEMELMEYYALEDQEYERQKLEGEVVLKTNQIARLKERIGALEERSMQVADEAASVTIL  Ppa03       NETIIEMLRITVAELKEREIRLEGELLEYYGLQEREIECFEKKRILEEQAKTIETLKLHIENLEVHSNGLSSMIIQDNIV  Pac02       DSTELLQLKALVADLQQKEMKLQEELLEYYGLREQEETHSDLERQLRKKSAEIEKLNGRLNALEEQKKVLSEELAEKENL  Pac03       DSTELLQLKALVADLQQKEMKLQEELLEYYGLREQEETHSDLERQLRKKSAEIEKLNGRLNALEEQKKVLSEELAEKENL  Aal03       DSAELVKLKALVTELQQKEVKLEAELLEYYGLKEQERDHLELERQLRRKSTEIEKLNGKIKALEEQKKNLSEELMGKENL  Pac04       --------------------------------------------------------------------------------  Ppa01       ATAELRALRETVKVLKNKEARMEAELLEYYDLEDQEAELVKLEEEMEEKNSQIAKLKERIGILEARSTKLADEAASVTGL  PEn06       NSAELDRLRNLVKELEEREVTLEGELLEYYGMKEQETSIGELQRQLQIKGVEIDMLKIKINSMETQKKKMEEEVAKAMIM  PiPa01      NSAELNRLRNLVKELEEREVTLEGELLEYYGMKEQETSIGELQRQLQIKVVEIDMLNIKVNSLETQKKKMEEEVAKAMIM  PJe07       NSAELNRLRNLVKELEEREVTLEGELLEYYGMKEQETSIGELQRQLQIKVVEIDMLKIKVNSLETQKKKMEEEVAKTMIM  PPo03       NSAELNRLRNLVKELEEREVTLEGELLEYYGMKEQETSIGELQRQLQIKVVEIDMLKIKVNSLETQKKKMEEEVAKTMIM  Smo01       PVDEIGDLKARLQQLQEKERKLNAELLDYYGLKEREKGVKELEAQLLVKDEQITSLTASIRKLEDEKKKMADDIKAASKS  Smo02       LVDEIGDLKARLQQLQEKERKLNAELLDYYGLKEREKGVKELEAQLLVKDEQITSLTASIRKLEDEKKKMADDIKAASKS  Atr02       NATELERLRNLVKELEEREVKLEGELLEYYGLKEQEANIAELQRQLKIKAVEIDMLNITINSLQAERKKLQEEVTIGVAA  PiPa03      --------------------------------------------------------------------------------  PJe05       --------------------------------------------------------------------------------  PPo01       NAAEIERLKNLVEELQKSEENLQGELLEYYGLTEQETNIVELQKQLQLNTVEIEMLNLKLNSLETQKKKLEEEVEESSLV  Bra01       -------------------------------------MM-EGDPCVALLRSKLHGLIERNHTLEEENKQLRHQVSRLKGQ  Osa02       -------------------------------------MM-EGDACVALLRSKLHGLVERNRSLEEENKQLRHQVSRLKGQ  ZMa03       -------------------------------------MM-EGDARVTLLRSKLHGLVERNRDLEEENKQLRHQVSQLKGQ  SBi01       -------------------------------------MM-EGDACVTLLRSKLHGLVERNRALEEENKQLRHQVSRLKGQ  ZMa04       -------------------------------------MM-EGDACVTLLRSKLHGLVERNRALEEENKQLRHQVSRLKGQ  Mtr05       -------------------------------------MLQNESEITTSFKKRLEIHMARNELLQKENQELKEEVSRLKSQ  Mtr06       -------------------------------------MLQNESEITTSFKKRLEIHMARNELLQKENQELKEEVSRLKSQ  Ath01       -------------------------------------MLPEDDSDLLRLVKELQAYLVRNDKLEKENHELRQEVARLRAQ  Egr05       --------------------------------------MPGDDSEIALLRIQLEACLARNTSLEKENQELRHEAARLKSQ  Ptr04       --------------------------------------MREDESLIIYLKKEVEAALLRTDSLEKENQELQQEVVRLKAQ  Spu01       ------------------------------MKPEQERKMREDESLIIYLKKEVEAALLRTDSLEKENQELQQEVVRLKAQ  Ptr01       -------------------MRFQSPVIAP-MKAEQERKMREDESLIIYLKKEVEAALLRTDSLEKENQDLRQEVVRLKAQ  Spu03       ------------------------------MKAQQERKMREDESLIFYLKKEVEAALLRTDSLEKENRELRQEVVRLKAQ  SBi02       APPQVAELLRAIEQLQERESRLRVELLEQKILKETVAIVPFLEAELAAKRSELQRCRDTADRLEAENARLCAELDAAA--  ZMa02       APPQVAELLRAIEQLQERESRLRVELLEQKILKETVAIVPFLEAELAAKKAELQRCRDTAERLEAENGRLCAELDAAA--  Bra04       APPEVAGLLRAIEQLQERESRLRVELLEQKILKETVAIVPFLEAELAAKSGELEKCKEAAARLESENMRLCAELDAAV--  Osa01       APPEVADLLRAIEQLQERESRLRVELLEQKILKETVAIVPFLEAELAAKSSELEKCKDTAARLESENMRLCAELDAAV--  Bra03       AMAEAGELARVVEELRERESRLRTELLEQKILRETVAIVPFLETELAAKSSELGRCRDAMSKLESENARLREQLAAAM--  Osa03       --PEVGELVRLVEELQERESRLRTELLEHKILKETVAIVPFLENELAAKSSELGRCRDALTRLESENARLRAALDAAA--  SBi04       GPAAVADLARLVEELRERESRLRTELLEHKILKETVAIVPFLETELAAKSSDLGRCRDALARLQAENARLRAELDVAV--  Ath03       HNGVVSELRRQVEELREREALLKTENLEVKLLRESVSVIPLLESQIADKNGEIDELRKETARLAEDNERLRREFDR----  Egr03       ---DISELVRLVEDLRERESRLKTELLEHKLIRETASLVPVLEGVICSKNAEIAKSVRRIEELEASNERLRRELEASWGR  Mtr03       ---DGTELLRLVEELRESESRLKTELLEHKLLKESIAIVPVLENELTVRETEIQRNRKRAEEAEEQNEKLKKELEELKQL  Ptr03       ---DVTELLKLVEELRERESLLKTELLEFKLLKESVAIVPVLETVISNKNMEIEKAVKEVESLERENESLKAELSEVRLR  Ptr06       ---DVTEVLKLVEELRERESLLKTELLEYKLLKESVAIIPVLETEITNKILEIEKAVKKIESLELENECLKADLSEVRGR Spu05       ---DVTEVLKLVEELRERESLLKTELLEFKLLKESVAIVPVLETEISNKNLELERAVKKVESLELENECLKADLSEVRGR  Spu08       ---DVTEVLKLVEELRERESLLKTELLEFKLLNESVAIVPVLETEISNKNLELERAVKKVESLELENECLKADLSEVRGR   Appendix B.2 Amino acid alignment of homologous CPU sequences used for the maximum likelihood phylogenetic analysis. Black regions represent identical amino acids. Grey regions represent similar amino acids.   187                       90       100       110       120       130       140       150       160                     ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| Atr01       --EREKASENT------M---QMTLEEENERLRNEAQDLSSEVARLKAKL------------------SCSNQFQGLI--  PEn05       KRELEEHYSRTENEIQELETELMVLRQRNAELQQQKSEMSRKLATAESQVDLVSEAGSAVSMLRHANEDLRNQVEALQIN  PJe08       KGELEQHCSRTENEIHELETELMELRQRNSELELQKSEMSRKLATAESQVDLVSKAETAVSFLRHTNEDLRHQVEGLQTN  PPo04       KGELEQHCSRTENEILELETELMELRQRNSELEQQKSEMSRKLATAESQVDLVSKAETAVSFLRHTNEDLRHQVEGLQTN  Osa04       VEQHESMNGGNASSSENLEIEVVELRRLNKELQFQKRNLAIKLSSAESKLEIVAKVQAEASLLRHTNANLSKQVEGLQMS  SBi03       VEHHESVNGDNT-PSENLEFEVVELRRLNKELQFQKRNLAIKLSSAESKLDIVAKVQAEASLLRHTNANLSKQVEGLQMS  ZMa01       EEHHETVDGDNA-PSENLEFEVVELRRLNKELQFQKRNLAIKLSSAESKLDIVAKVQAEASLLRHTNANLSKQVEGLQMS  Egr02       EALRRRENSATLSCSRDLGMEVLELRRLNKELQLQKRNLACRLSTVESQLDIVAKIKEEASLLRHTNEDLCKQVEGLQMS  Ptr02       SQKRNLKREGHESSLGNLEMEVVELRRLNKELQMDKRNLACKLSSMESQKDIVAKIKAEASLLRHTNEDLCKQVEGLQMS  Spu07       SQKRNLKREGHESSLGNLEMEVLELRRLNKELQMDKRNLACKLSCMESRLDIVAKIKAEASLLRHTNEDLCKQVEGLQMS  Ptr07       SEKRSLKREGNESSVGNLEMEVVELRRLNKELQMDKRNLACKLSSLESQLDVVAKIKAETSLLRHTNEDLCKQVEGLQMS  Spu04       SEKRSLKREENESSVGNLEMEVVELRRLNKELQMDKRSLACKLSSLESQLDVVAKIKAEASLLRHTNEDLCKQVEGLQMS  Mtr01       -----RQEEDKDGSTQNLELEVVELRRLNKELHMQKRNLTCRLSSMESQLDIVAKFKAEASLLRLTNEDLSKQVEGLQTS  Atr03       RESKEQDSERTMKSIRKLEMEAVELRRVNMELQFQKRDLACRLSSSESQLDKVAKVEAEASVLRHTNENLCKQVEGLKMS  PEn01       --------------------------------------------------------------------------------  PiPa06      --------------------------------------------------------------------------------  PJe03       --------------------------------------------------------------------------------  PJe04       --------------------------------------------------------------------------------  PPo02       --------------------------------------------------------------------------------  SBi05       KKELEVEVERKLKKLKEFEVEVLELRRKNKELLYEKRDLIVKLDAAEGKIDVVANAREEINKLRHTNEDLTKQVEGLQMN  Bra02       KKELEAEIEQKLKKLKELEVEVLELRRKNKELLYEKRDLIVKLDAAQGKIDVVAHAREEISNLRHTNEDLTKQVEGLQMN  Osa05       KRELEAEVQRKLKKLKELEVEVVELRRKNKELLYEKRDLIVKLDAAQGKIDVVSHAREEINKLRHVNEDLTKQVEGLQMN  Osa06       KRELEAEVQRKLKKLKELEVEVVELRRKNKELLYEKRDLIVKLDAAQGKIDVVSHAREEINKLRHANEDLTKQVEGLQMN  Mtr02       KRELEAEIEKKLKTVNDLEIEAVGLKRRNKELQHEKRELTVKLNAAESRIEMIADAKSETGRLRHANEDLQKQVEGLQMN  Ath02       RKELETEVERKLKAVQDLEVQVMELKRKNRELQHEKRELSIKLDSAEARIDKVAKVREEVNNLKHNNEDLLKQVEGLQMN  Mtr04       KRDLEAEIDKKLKAVNDLEVAVVELKRKNKELQYEKRELTVKLNAAESRVEMVAKAKEEVSNLRHANEDLSKQVEGLQMN  Egr01       KKDLEADVERKLKAMKKLET------------------------------ERVASVREEVNNLRHINEDLSKQVEGLQMN  Spu02       KEELEADAEKRLKAAKELEVEVVELKRKNKELQHEKRELTIKLGAAEAKVEIVAKVRTEVNDLRRTKEDLLKQVEGLQMN  Ptr05       KKELEAEVEKRLKAVKELEVEVVELKRKNKELQHEKRELIIKLGAAEAKLEMVAKVREEVNNLKHANEDLLKQVEGLQMN  Spu06       NKELEADVEKRLKAVKELEVEVVELKRKNKELQHEKRELIIKLGAAEAKVEMVAKVREEVNNLKHSNEDLLKQVEGLQMN  Smo03       --------------------------------------------------------------------------------  Ppa02       TKDLEFETEKKLQSLREMEVEVLELRRTNKDLQFQKRELTVQLDAADMDIYRLAEADADNASLRHTNEDLARQVEGLQND  Ppa03       QKELAIELEKKLESLRELEVEVVELRRTSKDIQHQRRDLIIKLSAAESQIALVTQAEEKADALRKANEDLCRQVEKLLNS  Pac02       KKELEFALEKKLQTLKELEVEVVELRRTSKDLQHQKRELTVQLAAAEVKIELVARAQAEASALKLVNDDLSKQVEGLQIN  Pac03       KKELEFALEKKLQTLKELEVEVVELRRTSKDLQHQKRELTVQLAAAEVKIELVARAQAEASALKLVNDDLSKQVEGLQIN  Aal03       KKELEFDMEKKLQTLKELEVEVVELRRTSKELQHQKRELTVQLAAAEAKIDVVARVQSEASALKQANDDLSKQVEGLQMN  Pac04       ---------------------------------------------------------------------------GLQMN  Ppa01       RKDLELETEKKLQALREMEVEIVELRRTNKDLQYQKRELTVKLDAAEMDIDILAEADEELAALRHANEDLARQVEGLQND  PEn06       KKELEFETEKKLQNLKELDVEVVELRRTNKELHHEKRELTVKLDVAEAQIEMVVKAREEANTLRHANADLLKQVEGLQMN  PiPa01      KKELEFETEKKLQNLKELDVEVVELRRTNRELQHEKRELTVKLDAAEAQIEMVMKEREEANTLRHVNADLLKQVEGLQMN  PJe07       KKELEFETEKKLQNLKELDVEVVELRRTNKELQHQKRELTVKLDAAEAQIEMLVKEREEANALRHANADLLKQVEGLQMN  PPo03       KKELEFETEKKLQNLKELDVEVVELRRTNKELQHEKRELTVKLDAAEAQIEMLVKEREEANALRHANADLLKQVEGLQMN  Smo01       RGELSFEIEKKMQTLKELEIEVVELRRTCRELQHQKRDLTVKLSAAEAQVELVARANNESQILRHANDDLMRQVEGLQNN  Smo02       RGELSFEIEKKMQTLKELEIEVVELRRTCRELQHQKRDLTVKLSAAEAQVELVARANNESQILRHANDDLMRQVEGLQNN  Atr02       RKELELEVEKKLKTIKELEVEAVELKRRNKELQHEKRELTVKLDAAEARVDMVAQVRQEVNSLKHINEDLLKQVEGLQMN  PiPa03      --------------------------------------------------------------------------------  PJe05       --------------------------------------------------------------------------------  PPo01       KKELEFEIEKKLQTLKYLEIEVVELRRANKELQHEKRELMLKLDNAETQIETMQKAREELKTLRHVNEDLLKQVEGLQNN  Bra01       VSSF---------EGQDTERK-IMWKKLENSA------------------------------------------------  Osa02       VSSL---------EGQDTDRK-MLWKKL-DNSS-----------------------------------------------  ZMa03       ISSL---------EGQETNKR-MLWKKL-ENSA-----------------------------------------------  SBi01       ISSL---------EGQDTDKK-MLWKKL-ENSA-----------------------------------------------  ZMa04       ISSL---------EGQDTDKR-MLWKKLLESSA-----------------------------------------------  Mtr05       IISL---------KAHNMERKTILWKKIQKSID-----------------------------------------------  Mtr06       IISL---------KAHNMERKTILWKKIQKSID-----------------------------------------------  Ath01       VSNL---------KSHENERKSMLWKKLQSSYD-----------------------------------------------  Egr05       ISSL---------KAHDNERKSLLWKKIQNSLE-----------------------------------------------  Ptr04       ISSL---------KAHDNERKSMLWKKLQNPID-----------------------------------------------  Spu01       ISSL---------KAHDNERKSMLWKKLQNPFE-----------------------------------------------  Ptr01       ICSL---------KAHDNERKSMLWKKLQNPFD-----------------------------------------------  Spu03       ITSL---------KAHDNERKSMLWKKLQNPFD-----------------------------------------------  SBi02       --LEV---TSRKQRIVELEKEMAELLRKQQE-----AADA----------------------------------------  ZMa02       --LEV---TSRKQRIVELEKEMAELRTRQDA-----AADA----------------------------------------  Bra04       --LEV---TSRKQRIVQMENEMTELRKQQQEAL---AADA----------------------------------------  Osa01       --LEV---TSRKQRIVHMEKEMAELKKQQE-AA---AADA----------------------------------------  Bra03       --AGD---KSKAQRIGQLEKEVAELRTPRPPVQ-----VV----------------------------------------  Osa03       --ASS---RDNEQRILEMERQMTELRKRRQRDV---ATGP----------------------------------------  SBi04       --DAA---RSSQQRVVELEKEVAEVKRRRGPVPDREPDHD----------------------------------------  Ath03       SEEMR---RECETREKEMEAEIVELRKLVSS-------------------------------------------------  Egr03       FEEER---KASEERAREMEAEISELKAAASASASAAATPA----------------------------------------  Mtr03       MEDER---IDSERKLKALEDEVTVLKKTASL-------------------------------------------------  Ptr03       FGEER---KEGAKKVKELEAEVVELKKAVSD-------------------------------------------------  Ptr06       FEEER---KEGERKVKELEAEIQELKKAMSD-------------------------------------------------  Spu05       FEEER---KESERKVKELEAEIQEFKKALSD-------------------------------------------------  Spu08       FEEER---KECERKVKELEAEIQEFKKALSD------------------------------------------------- Appendix B.2 (continued) Amino acid alignment of homologous CPU sequences used for the maximum likelihood phylogenetic analysis. Black regions represent identical amino acids. Grey regions represent similar amino acids.   188                      170       180       190       200       210       220       230       240                    ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| Atr01       -------------DVSARSSLMK-----------NGESSSV---SQEGPRVD-----PLETGQSLK--NAEHLE------  PEn05       FSDIEELANLRWVNASLRQKLNTSKVPSGKRSAESPKSSHSSSGSEAGEPDNSFFF-PGLLGFSKRSILKKHREEWSFSQ  PJe08       FSDIEELAHLRRINASLRQELNSSKLPCGKRSAESPKSSHSSSGSEAGESDSSFFF-PGLLGFSKRSILKKHREEWSFLQ  PPo04       FSDIEELAHLRWINASLRQELNSSKLPCGKRSAESPKSSHSSSGSEAGESDSSFFF-PGLLGFSKRSILKKHREEWSFLQ  Osa04       LTEVEELAYLRWINSCLRHELSNSDQAARAMTDAACH---VDD-CDGD-------RLDQNSSDHKKFSIAERIKQWSQND  SBi03       LTEVEELAYLRWINSCLRHELCNSDQAARAMTDIAFN---EGD-GGEG-------AKNAEDSSDIKFSIAERIKQWSQND  ZMa01       LTEVEELAYLRWINSCLRHELCNSDQAARATTDIAFN---EDD-NSEG-------AKTSEDSSDIKFSIAERIKQWSQND  Egr02       LNEVEELAYLRWVNSCLRNELRNTGGEINSDRASSPD-------NDSISSL-SCQE-SIEYSNARRASLIKKLKKWPLTS  Ptr02       LNEVEELAYLRWVNSCLRDELRNSCSTMNSDKSSSPK-------DESAGLM-SCQD-CLEYNSKRRLNLIKKLKKWPITD  Spu07       LNEVEELAYLRWVNSCLRDELSNSSSTMDSEKAASPK-------NESAGSM-SFQD-CLEYSSKRRLNLIKKLKKWPITA  Ptr07       LNEVEELAYLRWVNSCLRDELRNSCSTMNSDKASSPK-------NESAGSI-SCQD-YLESNSKMRLDFIKKLKKWPITD  Spu04       LNEVEELAYLRWVNSCLRDELRNSCST-NSDKASSPK-------NESAGSM-SCQD-YLESNSKMRLNLIKKLKKWPLTD  Mtr01       LNEVEELAYLRWVNSCLRTELKNTCSTLDSDKLSSPQ-SSS------GDSI-SSF---DQCGSANSFNLVKKPKKWPITS  Atr03       FTEVEELVYLRWVNSCLRHELRNVDSQLKKPSALSPNLINTNR-DEDSENL-SCLTSDSSIHSSKKVGLIQKLRKWSKNN  PEn01       --------------------------------------------------------------------------------  PiPa06      --------------------------------------------------------------------------------  PJe03       --------------------------------------------------------------------------------  PJe04       --------------------------------------------------------------------------------  PPo02       --------------------------------------------------------------------------------  SBi05       FSEVEELVYLRWVNACLRFELRNYQTPSGKVSARSPKSSMP-SSPGSEDFDNISISSSRYSFLSKRSNLMQKIKKWGRSK  Bra02       FSEVEELVYLRWVNACLRFELRNYQTPSGKISARSPKSSAP-SSPRSEDFDTVSISSSRYSFLSKRPNLMQKLKKWGRSK  Osa05       FSEVEELVYLRWVNACLRYELRNYQAPSEKISARSPKSSAP-SSPRSEDLDNVSVSSSRYSFFGKRPNLMQKLKKWGRGK  Osa06       FSEVEELVYLRWVNACLRYELRNYQAPSEKISARSPKSSAP-SSPRSEDFDNVSVSSSRYSFFGKRPNLMQKLKKWGRGK  Mtr02       FSEVEELVYLRWVNACLRYELRNYKAPSGKSLARNPKFSHDHSSPGSEDLDNAYIPTYKYSNLSKKTSLIQKLKKWNKNN  Ath02       FSEVEELVYLRWVNACLRYELRNYQTPAGKISARSPKYSQP-SSPGSDDFDNASMSTSRFSSFSKKPGLIQKLKKWGKSK  Mtr04       FSEVEELVYLRWVNACLRYELKNHQAPSGRLSARSPKFSHP-SSPGSEDFDNASIFSSKYSSVSKKTSLIQKLKKWGKTK  Egr01       FSEVEELVYLRWVNACLRYELRNYQTPPGKVSARSPKFSHP-SSPGSEDFDNASISTSRYSSLSRKPGLLQKLKKWGRSK  Spu02       FSEVEELVYLRWVNACLRYELRYYQTPSGKVSARSPKYSHL-SSPGSEDFDNTSISFSRYS-FSKKHGLIQKLKKWGRIK  Ptr05       FSEVEELVYLRWVNACLRYELRNYQTPSGKVSARSPKYSHP-SSPGSEDFDNTSISSSRYS-FSKKPNLIQKLKKWGRSK  Spu06       FSEVEELVYLRWVNACLRYELRNYQTPSGKVSARSPKYSHP-SSPGSEDFDNTSISSSRYS-FSKKPNLIQKLKKWGRGK  Smo03       --------------------------------------------------------------------------------  Ppa02       FTDVEELVYLRWVNACLRFELRSHLAPDGRFSAISPRESSENS-NFIEEYSDITSGSV-SGRFSKKSSLIKRLKNWRGKK  Ppa03       FCEVEELVYLRWVNACLRYELRNLQAPSKKHTALSPKSSESSVPHEPDGVGDLSDSELRLGRVSKKPSLIRRLKKWTGRK  Pac02       FSEVEELVYLRWVNACLRYELRNYQAPPGKFTALSPRSSAPSTPS-EYDPDEMSFQSA-RHSTTKKSGLIKKLKKWGRSK  Pac03       FSEVEELVYLRWVNACLRYELRNYQAPPGKFTALSPRSSAPSTPS-EYDPDEMSFQSA-RHSTTKKSGLIKKLKKWGRSK  Aal03       FSEVEELVYLRWVNACLRYELRNYQAPPGKFTALSPRSSVPSTPS-EYDFDEASFQSA-RHSTSKKSGLMKKLKKWGRSK  Pac04       FSEVEELVYLRWVNACLRYELRNYQAPPGKFTAMSPRSSVPSTPS-EYDFDEAAFHSA-RHSSSKKSGLIKKLKKWGRNK  Ppa01       FTEVEELVYLRWVNACLRYELRNYQAPEGQVSAMSPRSSETSSP--SEDYSDISSGSV-SGHLSKKNSLIKRLKSWTGRK  PEn06       FSEVEEVVYLRWVNACLRYELRNYQVPSGKVTARSPKSSQPSTPSESDDFDDVSVTSS-RQSSFKKSSLIRKLKRWGRSK  PiPa01      FSEVEELVYLRWVNACLRYELRNYQAPPGKVTARSPKSSQPSTPSESDDFDDASITSS-RQSSSKKSSLIRKLKRWGRSK  PJe07       FSEVEELVYLRWVNACLRYELRNYQAPPGKITARSPKSSQPSTPSESDDFDNASVTSS-RQSSFKKSSLIRKLKSWGRTK  PPo03       FSEVEELVYLRWVNACLRYELRNYQAPPGKITARSPKSSQPSTPSESDDFDNASVTSS-RQSSSKKSSLIRKLKSWGRTK  Smo01       FSEVEELVYLRWVNACLRYELRNFKAADGKFTALSPRFSR--TSSDSIDMDD---YGS-EESSSKKPGLIKRLKKWGRSK  Smo02       FSEVEELVYLRWVNACLRYELRNFKAADGKFTALSPRFSR--TSSDSIDMDDSQ-YGS-EESSSKKPGLIKRLKKWGRSK  Atr02       FSEVEELVYLRWVNACLRYEIRNYKIPEGKITARSPRSSH-PSTPGSEDFDNTSIFSS-RYSMTKKPSLIQKLKKWGRSK  PiPa03      --------------------------------------------------------------------------------  PJe05       --------------------------------------------------------------------------------  PPo01       FSEVEELVYLRWVNACLRYELRDYQPPPGKLTARSPRSSHTSTPPESCDFDESSLTST-RLTRLKKPSLIHKLKRWGRSK  Bra01       -------------------------------------------TG-N---FSKEKQFVHNNDDV----------------  Osa02       -----------------------------------------T-GN-S---YLKEKQFVPNN-DA----------------  ZMa03       -----------------------------------------T-GI-S---YSKEKQFVQSNDGA----------------  SBi01       -----------------------------------------T-SI-S---YSKEKQFVQSNDDA----------------  ZMa04       -----------------------------------------T-SI-S---YSKEKQFVQSNDDA----------------  Mtr05       -----------------------------------------D-NN-SDSHHTLKPALQAIM-------------------  Mtr06       -----------------------------------------D-NN-SDSHHTLKPALQAIM-------------------  Ath01       -----------------------------------------G-SN-TDGSNLKAPESVKSN-------------------  Egr05       -----------------------------------------S-ES-ASL--------QKPTAKI----------------  Ptr04       -----------------------------------------S-SK-TDVFLQKQSDFVKVT-------------------  Spu01       -----------------------------------------S-SK-TDVFLQKQSDFVKVSERS----------------  Ptr01       -----------------------------------------S-SK-TEVFLQKQSDFVKVSERS----------------  Spu03       -----------------------------------------S-SK-TDVFQQKQSDFVKVSERS----------------  SBi02       -------------DDC---------------------SSSASAPN--NEHLESSS--------TAAPNRA----------  ZMa02       -------------DDC---------------------SSSASAPS--ERLESSSA--------APKPNRA----------  Bra04       -------------DDC---------------------CSSSASAS--V--GSSG----------AAANPA----------  Osa01       -------------DDC---------------------SSTASVSH--EQPESAS----------SAANPA----------  Bra03       -------------ADC---------------------SSSGHSSD--SA------------------KPA----------  Osa03       -------------DDC---------------------SSSASSDN--SESSNAAT------NSAKSAKVA----------  SBi04       -------------DDC---------------------SSSVSSDN--SDRSNAAT------N----AIVA----------  Ath03       --------------------------------------------ESDDHALSVSQRFQGLMDVSAKSNLIRSLKRVGSLR  Egr03       -------------SSSAAAT--------------TP-SSSASSDRMAGDELCSSQRFQAVAAAEATSRPASTAKISKKGA  Mtr03       ---------------------------------------------HSEEHFSTSQRFQGIGEVSVKSNLMK---TLKKTM  Ptr03       --------------------------------------------RENEIELSSSQRFQGLMEATTKSTLIK---SLKKGV  Ptr06       --------------------------------------------RENEIEFSSSQRFQGLMEVTTKSNLIR---SLKKGV  Spu05       --------------------------------------------RENEMEFSSSQRFQGLMEVTTKSNLIR---NLKKGV  Spu08       --------------------------------------------RENEMEFSSSQRFQGLMEVTTKSNLIR---NLKKGV   Appendix B.2 (continued) Amino acid alignment of homologous CPU sequences used for the maximum likelihood phylogenetic analysis. Black regions represent identical amino acids. Grey regions represent similar amino acids.   189                      250       260       270       280       290       300       310       320                    ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| Atr01       ------ACDEELPENTA---------------------------SVAEKLKPAVSNIPKPPPLPPFPSSSSSISSLERES  PEn05       -NRHSHDSLQNVKQQR-RQNIFLQKEEQTAERQITVTKSSEVAKLTPAEVGKRASRIPKPPPKPSSGSAATNGPAGPRSM  PJe08       -IRHSHDSLQNVKQQKAKQNIFLQKEEQTVERQVTVTKSSEVAKLTPAEVRKRASRIPKPPPKPSSGSAATNRPAGPRSM  PPo04       -NRHSHDSLQNVKQQKAKQNIFLQKEEQTVERQVTVTKSSEVAKLTPAEVRKRASRIPKPPPKPSSGSAATNRPAGPRSM  Osa04       KKTRRHGCAQEFSIVKNKRELLVDKYDFGSESSRFLLGKSEVCKSQSMDVEKRALRIPNPPPRPSVSVPHSGPSN---GS  SBi03       KKTRRHGCAQDFNIIKNKRDLLVDKYDFGSESSRFVLSKPEVCKSQCLDVEKRALRIPNPPPRPSVSVSNSGPSN---GS  ZMa01       KKTRRHGCAQDFNIIKNKRDLLVDKYDFGSESSRFVLSKPEVCKSQCLDVEKRALRIPNPPPRPSVSVSNSGPSN---GS  Egr02       EDR-RHGSAEDIVLNK-MEPLMPTICEWGPQRPQLYTNCQETSKLVPFEVEKRTLRIPNPPPRPSCTFHTESKKGSTV--  Ptr02       EDRRRHGCIEELVPNR-VELVSSEKYEFDIQRPQILAYCQETKKIGPLDVEKRALRVPNPPPRPSCSVS-GPKEEGSAQV  Spu07       EDRRRHGCIEELVPNR-VELVSSEKYEFDIQSPQITANFQETKKIGPLDVEKRALRVPNPPPRPSCSVSAGPKDEDSARV  Ptr07       EDRRRHGCLEDLAPNR-VELVSSEKYELDMQRPQILANCQETNKIGPLDVEKRTLRVPNPPPRPSCSVSTGPKEEVQAQV  Spu04       EDRRRHG-LEDSISNG-IELVNSEKYELDMQRPRILATCQEINKVGPLDVEKRTLRVPKPPPRPSCSVSSGPKEEVPPQV  Mtr01       SNRRRHGSEEEVSVLK-----CLKEIEKEVPMPLF---------VQQCALEKRALRIPNPPPRPSCSISSKTKQE---CS  Atr03       DHRRRHGYAENSTLCK-VNENFPPKYELGVQSPRFLVNKQDLIRIAPSEIEKRALRVPNPPPRPSNLAQNEHKNG---GI  PEn01       --------------------------------------------------------------------------------  PiPa06      -------------------------------------------KFVSSEDGNKPLNLHKPPPKPLSGTTASLTIAPPKSA  PJe03       ----------------------------------------------SLEDGNKPLNLHKPPPKPLSGTTAGLTIASPKSD  PJe04       ----------------------------------------------SLEDGNKPLNLHKPPPKPLSGTTAGLTIASPKSD  PPo02       ----------------------------------------------SLEDGNKPLNSHKPPPKPLSGTTAGLTIASPKSD  SBi05       DS-SPLKPLEALMLKAERHKLATEREKVISSD--NQNNTLVVSQMKLANIEKRATRVPRPPPPRSTTASGATNTA-----  Bra02       DQKR-SKPLESLMLRAERHKLATEREKAISSD--NQNNPLAVTQLKLAQIEKRAPRVPRPPPTASAIASGPTNTA-----  Osa05       DQKSASKPLEALMLRADRHKLATEREKVIPSD--NQNNPLVVTQLKLANIEKRAPRVPRPPPAPSATANT---AS-----  Osa06       DQKSASKPLEALMLRADRHKLATEREKAIPSD--NQNNPLVVTQLKLANIEKRAPRVPRPPPAPSATANT---AS-----  Mtr02       -RCRVSRTLESLMLRAELHKLALAREKQLQSEDVKDFDNQTITEMKLFKIEKRPPRLPRPPPKPSDGAPVSNSLNEIPYA  Ath02       DRGRLSKPLESLMIRAERHKLAVEREKHISNEGKASENAATVTKMKLVDIEKRPPRVPRPPPRSAGGGKSTNLPSARPPL  Mtr04       DRKRMSKPLESLMIRAERHKLAMARESDLSEDG-KNVENQTISKIKFADIEKRPTRVPRPPPKPSGGGSVSTNSNPANGI  Egr01       DRSRVSRPLEALMIRAERHKLALEREKQISGDA-KADDPQTVSRMKLANVEKRPPRTPRPPPKPSGAAPSIANNAGAVTS  Spu02       DRSRTSRPLETLMLRAERHKLALEREMHVSNDG-KDVDSQKISKMKLAHIEKRPPRVPRPPPKPSAGVPVATNFN-----  Ptr05       DRSRSSRPLESLMIRAERHKLALEREKHISSDG-KDVDSQTVSKMKLAHTEKRAPRVPRPPPKSSAGAPVATNAN-----  Spu06       DRSRSSRPLESLMIRAERHKLALEREKRISSDG-KDVDSPTVSKMKLAHIEKRSPRVPRPPPKSSAAGPVATNAN-----  Smo03       ----------------DRHKLAVDSAPEA------------------------------------SSSSSAAA--ATASL  Ppa02       DSKFTRSPLEALIIRAERHKAAVEREQAIDAMPPLKRAESISKPLTPVEIAKREVRKANPPPKLNPPHPSQ----SPGGA  Ppa03       EDAFDSELLETVIVRSERHRAALEREKCIPEVGIVAKRNVEVAKMCPTEVEKRPLRIAKPPPKRSLLSSINVSTPVAGPL  Pac02       DSAHRHSPLESLMLRNERHKLAVAREKALVSANDLNPERPEVNRMKSSEIEKRAPRVARPPPKASAKGPSATNVLPAGGT  Pac03       DSAHRHSPLESLMLRNERHKLAVAREKALVSANDLNPERPEVNRMKSSEIEKRAPRVARPPPKASAKGPSATNVLPAGGT  Aal03       DSLHRHSPLESIMLRSDRHKLALAREKAIVASHDQNVERPEVNKMKSSEIEKRAPRVARPPPKASSKGPTPDNSLPAGVI  Pac04       DSLHRNSPLENLMLRSDRHKLALAREKAIVSSHDQNSERPEVNKMKFSEVEKRAPRVARPPPKASANAATSDIALPTGVV  Ppa01       D-GSQRTPLEALIIRQARHKAAIEREQAI--MPALKSGEVITKPITVAEVEKRELRKPRPPPKPSRPQPSVPAAPSGGGV  PEn06       DFGHAYGSLEALISRSDRHKLAMEREKAINHSKEHPSEIQEISKMKLAEVQKRPPRIAKPPPKPTVSSSPGFN--ATSGI  PiPa01      DFDHGHGSLEALISRSDRHKLAMEREKAINHSKDHPSEIQEISKMKLAEIQKRPPRVAKPPPKPTISSSPGSN--ATSGI  PJe07       DFDHGHGSLEALISRSDRHKLAMEREKAINHSKDQPSESQEISKMKLAEIQKRPTRVAKPPPKPTISSSPASN--VTSGI  PPo03       DFDHGHGSLEALISRSDRHKLAMEREKAINHSKDQPSEIQEISKMKLAEIQKRPTRVAKPPPKPTISSSPVSN--ATSGI  Smo01       DGGHRPSPLETILLRSDRHKLAVEREMAI--------KPEAVAKMTPAEVEKRELRVAKPPPKPSLAGPPT----PPRLA  Smo02       DGGHRISPLETILLRSDRHKLAVEREKAI--------KPEAVAKMTPAEVEKRELRVAKPPPKPSLAGPPT----PPRLA  Atr02       DSGHKHGA---LMIRADRHKLALEREKAIEQGNDGKVDPQVVSKIKLAQIEKRAPRVPRPPPRSSGASSIPTANPAPPPL  PiPa03      --------------------------------------------------------------------------------  PJe05       ----------------GRHNLSIQRENDIKNGNDRESKEPEISKMTFKGVQKRAPRVAKKPPKPSTGSLT-YSNAASHAI  PPo01       EAGHESSSLEFIMPISGRHNLSIQRENDIKNGNDRESKEPEISKMTFKGVQKRAPRVAKKPPKPSTGSLT-YSNAASHAI  Bra01       ----------------------KEAMDL-NNSACYSRQ----QF-RAPLVKSRSRRVPNPPPS-----------------  Osa02       ----------------------KEAMDL-NSTSCYSRQ----QF-RAPLVRSRAPRVPNPPPS-----------------  ZMa03       ----------------------KEAVDL-NSSLSHSRQ----QF-SVNQVRSRAPRVPNQPPN-----------------  SBi01       ----------------------KEAMDL-NSSLCHRRQ----QF-RATLVRSRAPRVPNPPPN-----------------  ZMa04       ----------------------KETTDL-SSSLCHRRQQ---QF-RVALVRSRAPRVPNPPPN-----------------  Mtr05       ----------------------CEKS-----SENQDFQD---SS-SPRKEKSSIVLPPTPPPK-----------------  Mtr06       ----------------------CEKS-----SENQDFQD---SS-SPRKEKSSIVLPPTPPPK-----------------  Ath01       -----------------------------------------------TK----GQEVRNPNPK-----------------  Egr05       ----------------------VEHN-----SPTQNLSP---RLAAAKKER--EERIPIPPLK-----------------  Ptr04       --------------------------------------------------------------------------------  Spu01       ----------------------VEHS-----SPRTSIQE------APRKEI--LAKVPNPPPK-----------------  Ptr01       ----------------------VEHS-----SPRPSIQE------AAIKEK--HAKVPNPPPR-----------------  Spu03       ----------------------VEHS-----SPRPSLQE------AARKEK--QAKVPNPPPR-----------------  SBi02       ---------------------------------------------SLAQLGAERPYIPPPPAPPPAAPFKPPPSPELSKL  ZMa02       ---------------------------------------------SLAQLDPPA------PPAPGPAPLKSPPSPELSKL  Bra04       ---------------------------------------------GSAQLGAEP--SAPPPMQRQTPAFMSSSSPELSKL  Osa01       ---------------------------------------------SLVQRG-PP--IPPPPPPVPPAAFKSAPSPELSKL  Bra03       ---------------------------------------------Q-KSVR---PPPPPPPPPPLPVSSKSSSS-ELIAK  Osa03       ---------------------------------------------GCSSVRPPPPPPPPPLPPPMPATFKSSSSSELSKL  SBi04       ---------------------------------------------GLSIVPPPAPPPPPPPPPPMPAPHKSS-SSELSKL  Ath03       N------LPEPITNQNGDIYRKDEIES--YSRSSNSEELTESS--SLSTVRSRVPRVPKPPPK--------L------PQ  Egr03       K------PSDAPASQSEEAVAAAEIERP-RHSRCNSEELAESVLS-SVATRSRAPRVPKPPPK--------L-------A  Mtr03       S------DHGIVMQK-DLKREFSETEKP-RHSRCNSEELADCHDSLNVNVRSRVPRVPNPPPK--------S---EQEIL  Ptr03       K------CTDIMSSS-KRLEENVETEKL-RHSRCNSEELTEST---L---SSRVPRVPKPPPK--------S------V-  Ptr06       K------FTDIVSSS-KRMEENVEIEKP-RHSRCNSEELTEST---LANLRSRVPRVPKPPPK--------S------V-  Spu05       K------CTEITSSS-KRMEENVEIEKP-RHSRCNSEELTETT---LASLRSRVPRVPKPPPK--------S------V-  Spu08       K------CTEITSSS-KRMEENAEIEKP-RHSRCNSEELTETT---LASLRSRVPRVPKPPPK--------S------V-   Appendix B.2 (continued) Amino acid alignment of homologous CPU sequences used for the maximum likelihood phylogenetic analysis. Black regions represent identical amino acids. Grey regions represent similar amino acids.   190                      330       340       350       360       370       380       390       400                    ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| Atr01       NNLFPPPPTQS--AERAPPPPPPPMP--AKLSRCGSMVRRVPEVVEFYHSLMRRDSKESESGPG-VSEIPAAANA-RNMI  PEn05       PPPPPPPPPPP--AGIAPPAPPVPPP--KLLMAGTRVMQRAPEVAELYHTLMKRQGKKDSSGGG-PVDTPDAANVRSNMI  PJe08       PPPPPPPPPPP--AGIAPPAPPAPPP--KQLIPGTRVMQRAPEVAELYHSLMKRQGKKDPSGGG-PVDAPDAANVRSNMI  PPo04       PPPPPPPPPPP--AGIAPPAPPAPPP--KQLMPGTRVMQRAPEVAELYHSLMKRQGKKDPSGGG-PVDAPDAANVRSNMI  Osa04       AA---PPKPPP--------PPPPPKF----STRNAGVMKRAPQVAELYHSLMRRDSKKDTSGSG-ICETANSANVRSSMI  SBi03       TV---PPRPPP--------PPPPPKF----SSKSTGVMKRAPQVAELYHSLMRRDTKKDTSSGG-ICEAANSANVRSSMI  ZMa01       TV---PPRPPP--------PPPPPKF----SSKSTGVMKRAPQVAELYHSLMRRDSKKDTSGGG-ICEAANSANVRSSMI  Egr02       --PPAPPPPPP--------PPPP-SK--LYGKRTSGAVKRAPQVVEFYHSLMKRDSRKDSLSGG-PCDASDVANVRSSMI  Ptr02       SLPPP--PPPP--------PPPPPKF--SVKNTTAGVVQRAPQVVEFYHSLMKRDSRKESSNGG-ICDASGVANVRSNMI  Spu07       SHPPPPPPPPP--------PPPPPKF--PVKSTTAGVVQRAPQVVEFYHSLMKRDSRKESSNEG-ICDASDVANVRSNMI  Ptr07       PL-PPPPPPPP--------PPPPPKF--SVRSTTAGVVQRAPQVVEFYHSLMKRDSRKESSNGG-ICEASDVANVRSNMI  Spu04       PLLLPPPPPPP--------PPPPPKF--SVRSTTAGVVQRAPQVVEFYHSLMKRDSRKESSNGG-ICEASNVGNVRSNMI  Mtr01       AQQPPPPPPPP----------PPMSF--ASR-GNTAMVKRAPQVVELYHSLMKRDSRRDSSSGG-LSDAPDVADVRSSMI  Atr03       TKAPPPPPPPP----------PPPKF--SMR-SSASVMQRAPEVVEFYHSLMKRDGRKDSAAGG-VCDAPGVTNVHSSMI  PEn01       ------PPPPP--------PQPPLHP--KFT-VRKDVMQKAPEVVKFYQSLMKRDAKKDSCGTG-LFDNQDVAIAHSDMI  PiPa06      SSPPP-PPPLP--------THHALHP--KFT-VRKDVVQKAPEVVKFYQSLMKRDAKKETCGTG-IFDNQDVAIAHSEMI  PJe03       SSPPAPPPPPP--------THHALHP--KFT-VRKDVVQKAPEVVKFYQSLMKRDAKKDTCGTG-IFDNQDVAIAHSDMI  PJe04       SSPPAPPPPPP--------THHALHP--KFT-VRKDVVQKAPEVVKFYQSLMKRDAKKDTCGTG-IFDNQDVAIAHSDMI  PPo02       SSPPAPPPPPP--------THHALHP--KFT-VRKDVVQKAPEVVKFYQSLMKRDAKKDACGTG-IFDNQDVAIAHSDMI  SBi05       -SGAQTPRAPGPPPGKGPPPPPPPPGALPRNLGGGDKVHRAPEIVEFYQSLMKREAKR-ET--SLGSMSSNVSDARSNMI  Bra02       -SGAPPPPRPPPPPGKGPPPPPPPPGSLSRSLAGGEKVHRAPEVVEFYQSLMKREAK-NTT--SLGSKTSSVSDNRSNMI  Osa05       -ALSPPPPRPPPPPGKGPPPPPPPPGSLPRNLAGGDKVHRAPEVVEFYQSLMKREAKKDTT--SLGSTTSSAFDVRSNMI  Osa06       -ALPPPPPRPPPPPGKGPPPPPPRPGSLPRNLAGGDKVHRAPEVVEFYQSLMKREAKKDTT--SLGSTTSSVSDVRSNMI  Mtr02       PS---------------VPSPPPPPGSLPRGAVGDDKVKRAPELVEFYQSLMKREAKKDAS--LLTSSTSNAADTRSNVI  Ath02       PGGGPPPPPPPPPPPPGPPPPPPPPGALGRGAGGGNKVHRAPELVEFYQSLMKRESKKEGAPSLISSGTGNSSAARNNMI  Mtr04       PSAPSIPPPPPGPPPPGPPPPPPPPRGLSKGAADDDKVHRAPQLVEFYQSLMKREAKKDTS-SLLVSSTGNTSDARNNMI  Egr01       AG-PPMPPPLPPPPPPGPPRPPPPPGSLPRGTGSGEKVHRAPELVEFYQTLMKREAKKDTS-SL-ISSTANVSDARSNMI  Spu02       PS-GRVPPPS----------PPPPPGSLPKGAGSGDKIRRAPELVEFYQALMKREAEKDTS-SL-ISSTSNVSDARSNMI  Ptr05       PS-GGVPPPPPPPPPPGPPRPPPPPGSLPRGAGSGDKVHRAPELVEFYQSLMKREAKKDTS-SL-ISSTSNVSHARSNMI  Spu06       PL-GGVPPPPPGAPPPGPPPPPPPPGSLPRGAGSGDKVHRAPELVEFYQSLMKREAKKDTS-SL-TSSTSNVSNARSNMI  Smo03       SARPP-PPPPPLPPPRVMSSSSP----LASSSDNKLRFQRAPQVIELYHAMTKRDVKKDAPSTATAAARVSVDEARSSII  Ppa02       PGGFVIPPPPPPPPPP------LKGSLSRTQGNHSDDVHRAPEVVEFYHSLMKRDSKSAVSNSG----GGTDPTARNNMI  Ppa03       GPPPPPPPPPPPPPPP-------TPGSL-IKGSGAEKMQRAPGVVEFYQSLMKRDAKQSLSSPG---GTVSNSEARNNII  Pac02       PGAPPPPPPPPPPPPP----------SMKSQGAGGDKVQRAPEVVEFYQSLMRREAKKDTSTGM---SDVNVSDARNSLI  Pac03       PGAPPPPPPPPPPPPP----------SMKSQGAGGDKVQRAPEVVEFYQSLMRREAKKDTSTGM---SDVNVSDARNSLI  Aal03       PGAPPPPPPPPPPPPP----------SIKSQGPAGDKVQRAPEVVEFYQSLMRREAKSNTAVGA---TDVNVSDARNNLI  Pac04       SGAPPPPPPPPPPPPP----------SMKSQSQGGDKVQRAPEVVEFYQSLMRREAKTNSAVGS---TDLNVSDARNSLI  Ppa01       PPPPPPPPPPRPP-----PPPPPMGGLS-KMGKKTDDVHRAPEVVEFYQSLMKRDAKSAVVNT----AGGNNPEARNNMI  PEn06       PMPPPRPPPRP-------PPPPPLGGMLKSQGPSGNKVHRAPELVEFYQSLMKREAKKEAATMASA--ASNVADVRNNMI  PiPa01      PIPPPRPPPLP-------PPPPPLGGVLKLQGPSGNKVHRAPELVEFYQSLMKREAKKEAATIASA--ASDVSDVRNNMI  PJe07       PMPPPRPPPLP-------PPPPPLGGMLKSQGPSGNKVHRAPGLVEFYQSLMKREAKKEAASMASA--ASDVSDVRNNMI  PPo03       PMPPPRPPPLP-------PPPPPLGGMLKSQGPSGNKVHRAPELVEFYQSLMKREAKKEAASMASA--ASDVSDVRNNMI  Smo01       GTAPPPPPPPPLPPGAAPPPPPPLPGMGKPQGQSGSKVQRAPEVVEFYQSLMKRDARKDAAVSS---SGNASSEARSNLI  Smo02       GTAPPPPPPPPLPPGAAPPPPPPLPGMGKPQGQSGSKVQRAPEVVEFYQSLMKRDARKDAAVSS---SGNASSEARSNLI  Atr02       PPGAPPPPPPPRPPPPSLP-----------KGSGGDKVHRAPELVEFYQSLMKREAKKDGSSVASS--TSNTADVRSNMI  PiPa03      --------------------------------------------------LVKREAQKEISNVAST-ASSNISDACSNMI  PJe05       PISPPPPPQPPTPPAPGIP--------IESKGRAGDKVQR---VIEFYQSLVKREVQKEISNVAST-ASTNISDACNNMI  PPo01       PISPPPPPQPPTPPAPGIP--------IESKGRAGDKVQRVPQVIEFYQSLVKREVQKEISNVAST-ASTNISDACNNMI  Bra01       -----PTCIQPTMKANPHPPPPPPLPSK--LLKSTKAVQRVPEVVELYRLLIRRESKND-----AGSMGIPVATNSRDMI  Osa02       -----PTYTQPIVNARPPP-PPPPLPSR--LLKSTKAVQRVPDVVELYRLLVRREGKND-----SGSMGIPAATNSREMI  ZMa03       -----PTSTQPKATVRPPP-PPPPLPSK--LQRSTKAIQRVPEVVELYRSLVRREGKNN-----SGSVGIPAATNSREMI  SBi01       -----PTCTQPKTTVRPPPPPPPPLPSK--LQRSTKAIQRVPEVVELYRSLVRREGKND-----SGSVGIPAATNSREMI  ZMa04       -----PTCTQPKTTVRPPPPPPPPLPSK--LQRSAKAIQRVPEVVELYRSLVRPEGKND-----SGSVGIPAATSSREMI  Mtr05       -----PASTLFSPSHKTAAPPPPPTPSK--SSIGLKTVRRVPEVIELYRSLTRKDANIE-----THHNGIPAVAFTRNMI  Mtr06       -----PASTLFSPSHKTAAPPPPPTPSK--SSIGLKTVRRVPEVIELYRSLTRKDANIE-----THHNGIPAVAFTRNMI  Ath01       -----PTIQG------TKPPPPPPLPSK--RTLGKRSVRRAPEVVEFYRALTKRESHMG-----INQNGVLSPAFNRNMI  Egr05       -----PNGNL--ASFKAPAPPPPPPPSR--SLSGIRAVRRVPEVVELYRALTRKDARME-----SNPTVVPTVTFTRNMI  Ptr04       -----------PSSPKAPAPPPPPPPPK--MSVGSKTVRRVPEVAEFYRLVTRRDVHME-----INSAAIPVVAFTPSMI  Spu01       -----PTSVA-PPSPK-SAPPPPPPPPK--MSVGSKTVRRVPEVVEFYRLLTRRDAHME-----INSAAIPVVAFTPSMI  Ptr01       -----PTYVA-PPSLK-SAPPPPPPPPN--MCAGSKAVRRVPEVVEFYRLLTRRDAHME-----TNSAAIPVVAFTPNMI  Spu03       -----PTYAA-PPSLK---PPPPPPPPN--MSSGSKAVRRVPEVVEFYRLLTRRDAHME-----ASSAAVPAVAFTPSMI  SBi02       PPPPPPLCHPPPPPPPPPP-PPPPPPAR----SGQCDVRRVPEVVEFYHSLMRRESKRD-GVG-A--TNGAGVATTRDMI  ZMa02       PPPPPPPCPP---PPPPPPPPPPPPPPA-GASSGQGDVRRVPEVVEFYHSLMRRESKRD-SGTAAANGGGGGAAATRDMI  Bra04       PPPPTLPRPPLPAS--AAPPPPPPPPPS------GSCVRRVPEVVEFYHSLMRRESKR--GGSGA--ANGGGAAATRDMI  Osa01       PPPPPPM----PA---AAPPPPPPPPPA------GPRVTRVPEVVEFYHSLMRRDSRSR-GSGGT--ANGGGVAATRDMI  Bra03       ----LPPPIPPPPSMPPPPPPPPPP------PLSGPCVRRVPEVVEFYHSLMRRDSKRD-GGGE---HGGSGAAAARDMI  Osa03       PPPPPPPPPPPMPRS-PPAPPPPPPPAASSAPASGPCVRRVPEVVEFYHSLMRRDSKRD-GGGGAEACPGGGAAAARDMI  SBi04       PPPPPPPPPPPPPTMPPPAPPPPPPPAAATAPAPAPCVRRVPEVVEFYHSLMRRDSRSR-GSGAGEAGSGGGAAAARDMI  Ath03       KSPPPPPPPPPQPPPPPPPPPPPPPPKS--LSIASAKVRRVPEVVEFYHSLMRRDSTNSDSTGGAAAEAILANSNARDMI  Egr03       PPPPPPPPPPQPPPPPPAPPPPPPPPAG--TRPVPAKVRRVPEVVEFYHSLMRRDSRRD----GAAADAPPATAKSRDMI  Mtr03       QPPAKTA----PPPRKAAPPPPPPPPKG--GKMPPAKVRKVPEVVEFYHSLMRRDSQTR----SGTAAEVPATANARDMI  Ptr03       ---SCPP------PPPVGPPPPPPPPKG--KRAGTEKVRRVPEVAEFYHSLMRRDS--R----SGVAEALPVTANARDMI  Ptr06       ---SGPP----PPPNPVAPPPPPPPPKG--RRVGAEKVRRVPEVVEFYHSLMRKNS--R----CGMAETLPASANARDMI  Spu05       ---SGPP----PPPNPVAPPPPPPPPKG--RRVGMAKVRRVPEVVEFYHSLMRKNS--R----CGMAETLPASSNARDMI  Spu08       ---SGPP----PPPNPVAPPPPPPPPKG--RRVGMAKVRRVPEVVEFYHSLMRKNS--R----CGMAETLPASSNARDMI   Appendix B.2 (continued) Amino acid alignment of homologous CPU sequences used for the maximum likelihood phylogenetic analysis. Black regions represent identical amino acids. Grey regions represent similar amino acids.    191                      410       420       430       440       450       460       470       480                    ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| Atr01       GEIENRSSHLLAIKSDVEKQGDFIRFLIREVQNASFSHIQDVLPFVKWLDDQLSCLVDERAVLKHFEWPEHKADALREAA  PEn05       GEIENRSSHLLAIKTDVETQGDFVRFLIKEVRSAVYLNIEDVVAFVKWLDDELSFLVDERAVLKHFDWPERKADALREAA  PJe08       GEIENRSSHLLAIKTDVETQGDFVRSLIKEVRAAVYINIEDVVAFVKWLDDELSYLVDERAVLKHFDWPERKADALREAA  PPo04       GEIENRSSHLLAIKTDVETQGDFVRSLIKEVRAAVYVNIEDVVAFVKWLDDELSYLVDERAVLKHFDWPERKADALREAA  Osa04       GEIENRSSHLQAIKADVETQGEFVKSLIKEVTNAAYKDIEDVVAFVKWLDDELGFLVDERAVLKHFDWPERKADTLREAA  SBi03       GEIENRSSHLQAIKADVETQGEFVKSLIKEVTSAAYKDIEDVVAFVKWLDDELGFLVDERAVLKHFDWPEKKADTLREAA  ZMa01       GEIENRSSHLQAIKADVETQGEFVKSLIKEVTSAAYKDIEDVVAFVKWLDDELGFLVDERAVLKHFDWPERKADTLREAA  Egr02       GEIENRSSHLLAIKADVETQGEFVNSLIREVNNAVYENIEDVVAFVKWLDDELCFLVDERAVLKHFDWPEKKADTFREAA  Ptr02       GEIENRSSHLIAIKADVETQGEFVNSLIREVNGAVYRDIEDVVAFVKWLDDELGFLVDERAVLKHFDWPEKKADTLREAA  Spu07       GEIENRSSHLIAIKADVETQGEFVNSLIREVNGAVYQDIEDVVAFVKWLDDELGFLVDERAVLKHFDWPEKKADTLREAA  Ptr07       GEIENRSSHLLAIKADIETQGEFVNSLIREVNNAVYQNIEDVVAFVKWLDDELGFLVDERAVLKHFDWPEKKADTLREAA  Spu04       GEIENRSSHLLAIKADVETQGEFVNSLIREVNSAAYQNIEDVVAFVKWLDDELGFLVDERAVLKHFDWPEKKADTLREAA  Mtr01       GEIENRSSHLLAIKADIETQGEFVNSLIREVNDAVYENIDDVVAFVKWLDDELGFLVDERAVLKHFDWPEKKADTLREAA  Atr03       GEIENRSSYLLAIKADVETQGEFVRSLIREVNNAVYRDIEDVVAFVKWLDDELCFLVDERAVLKHFEWPEGKADALREAA  PEn01       GEIKNRSAHLLAIKSDVETKGDFVRSLIREVQAAAYTDIDDVLAFVQWLDDELASLVDERAVLKHFDWPEKKADTMREAA  PiPa06      GEIKNRSAHLLAIKSDVETNGDFVRSLIREVQAAAFTDIDDVLAFVQWLDDELAFLVDERAVLKHFDWPEKKADIMREAA  PJe03       GEIKNRSAHLLAIKSDVETKGDFVRSLIREVQGAAYTDIDDVLAFVQWLDDELASLVDERAVLKHFDWPEKKADIMREAA  PJe04       GEIKNRSAHLLAIKSDVETKGDFVRSLIREVQGAAYTDIDDVLAFVQWLDDELASLVDERAVLKHFDWPEKKADIMREAA  PPo02       GEIKNRSAHLLAIKSDVETKGDFVRSLIREVQGAAYTDIDDVLAFVQWLDDELASLVDERAVLKHFDWPEKKADIMREAA  SBi05       GEIENRSTFLLAVKADVETQGEFVESLANEVRAASFVNIDDVVAFVNWLDEELSFLVDERAVLKHFDWPESKTDAIREAA  Bra02       GEIENRSTFLLAVKADVETQGDFVESLASEVRAARFVNIDDVVAFVHWLDEELAFLVDERAVLKHFDWPESKTDALREAA  Osa05       GEIENRSTFLLAVKADVETQGDFVESLANEVRAASFVNIDDVVAFVNWLDEELSFLVDERAVLKHFDWPESKTDALREAA  Osa06       GEIENRSTFLLAVKVDVETQGDFVESLANEVRAASFVNIDDVVAFVNWLDEELSFLVDERAVLKHFDWPESKTDALREAA  Mtr02       AEIENRSSFLLAVKADVETQGDFVMSLATEVRAASFSKIEDVVAFVNWLDEELSFLSDERAVLKHFDWPEGKSDALREAS  Ath02       GEIENRSTFLLAVKADVETQGDFVQSLATEVRASSFTDIEDLLAFVSWLDEELSFLVDERAVLKHFDWPEGKADALREAA  Mtr04       GEIENRSTFLLAVKADVETQGDFVTSLATEVRASSFSDIEDLVAFVNWLDEELSFLVDERAVLKHFDWPEGKADALREAA  Egr01       GEIENRSTFLLAVKADVESQGEFVQSLATEVRAASFAKIEDLVAFVNWLDEELSFLVDERAVLKHFDWPEGKADALREAA  Spu02       GGIENRSSFLLAVKADVETQGDFIQSLATEVRAASFSNIDDAVAFVNWLDEELSFLVDERAVLKHFDWPESKADALREAA  Ptr05       GEIENRSSFLLAVKADVETQGDFVQSLATEVRAASFSTIDDLVAFVNWLDEELSFLVDERAVLKHFDWPESKADALREAA  Spu06       GEIENRSSFLLAVKADVETQGDFVQSLATEVRAASFSNIDDLLAFVNWLDEELSFLVDERAVLKHFDWPESKADALREAA  Smo03       GEIENRSSHLLAIKADVENQRELVVSLAAEVRAADYTEMEDVLAFVTWLDGELALLVDERAVLKHFNWPEAKADALRESA  Ppa02       GEIENRSAHLLAIKADVETQGDFVMSLAVEVRAAEFTDIEDVVNFVRWLDDELSYLVDERAVLKHFDWPEGKADAMREAS  Ppa03       GEIENRSTHLLAIKADVETQGEFVESLAAEVRAASFSNIEEVVEFVVWLDEELSFLVDERAVLKYFDWPEGKVDALREAS  Pac02       GEIENRSAFVLAVKADVETQGDFVQSLAAEVRDAAYSDIEDVVAFVSWLDEELSFLVDERAVLKHFDWPESKADALREAS  Pac03       GEIENRSAFVLAVKADVETQGDFVQSLAAEVRDAAYSDIEDVVAFVSWLDEELSFLVDERAVLKHFDWPESKADALREAS  Aal03       GEIENRSAFLLAVKADVETQGEFVQSLAAEVRDAAYSDIEDVVAFVSWLDEELSFLVDERAVLKHFDWPENKADALREAS  Pac04       GEIENRSAFLLAVKADVETQGDFVQSLAAEVRDAAYTDIADVIAFVSWLDEELSFLVDERAVLKHFDWPENKADALREAS  Ppa01       GEIENRSTHLLAIKADVETQGEFVMSLAAEVRAAVYGDIKDVVEFVNWLDEELSFLVDERAVLKHFDWPEGKADAMREAA  PEn06       GEIENRSAFLLSVKADVETQGDFVQALATEVRASAYKNIEDVVAFVNWLDEELSFLVDERAVLKHFDWPESKADALREAA  PiPa01      GEIENRSAFLLSVKADVETQGDFVQALATEVRACAYKNIEDVVAFVNWLDEELTFLVD----LKHFDWPESKADALREAA  PJe07       GEIENRSAFLLSVKADVETQGDFVQALATEVRACAYKKIEDVVAFVNWLDEELSFLVDERAVLKHFDWPESKADALREAA  PPo03       GEIENRSAFLLSVKADVETQGDFVQALATEVRACAYKKIEDVAAFVNWLDEELSFLVDERAVLKHFDWPESKADALREAA  Smo01       GEIENRSSHLLAIKADVETQGDFVNSLAAEVRAAVYSNIDDILAFVNWLDEELAFLVDERAVLKHFDWPEAKADALREAA  Smo02       GEIENRSSHLLAIKADVETQGDFVNSLAAEVRAAVYSNIDDILAFVNWLDEELAFLVDERAVLKHFDWPEAKADALREAA  Atr02       GEIENRSTFLLAVKADVETQGDFVQSLATEVRAATFNNIEDVLSFVNWLDEELSFLVDERAVLKHFDWPEGKADALREAA  PiPa03      GEFGNKSAYLLSVKADVETQSDFIQSLANEVRAAAYTNIKDVVDFVHWLDEELSFLVDERAVLKHFDWPEGKADALREAA  PJe05       GEFGNKSAYLLSVKADVETQGDFVQSLANEVRAAAYTNIKDVVDFVHWLDEELSFLVDERAVLKHFDWPEGKADTLREAA  PPo01       GEFGNKSAYLLSVKADVETQGDFVQSLANEVRAAAYTNIKDVVDFVHWLDEELSFLVDERAVLKHFDWPEGKADTLREAA  Bra01       GEIENRSAYVIAIKSDVENQGEFINFLAKEVQNAAYKEMADVEEFVKWLDGELSYLVDERAVLKHFNWPEKKADAMREAA  Osa02       GEIENKSAYVLAIKSDVENQSEFINFLAVEVKNAAYKEIADVEEFVKWLDGELSYLVDERAVLKHFNWPEKKADTMREAA  ZMa03       GEIENRSAYVLAIKSDVENQGNFVNFLASEVQNAAYKKIADVEEFVKWLDGELSYLVDERAVLKQFNWPEKKADALREAA  SBi01       GEIENRSAYVLAIKSDVENQGNFVNFLASEVQNAAYKEIADVEEFVKWLDGELSYLVDERAVLKHFNWPEKKADAMREAA  ZMa04       GEIENRSAYVLAIKSDVENQGNFVNFLASEVQNAAYREIADVEEFVKWLDGELSYLVDERAVLKHFNWPEKKADAMREAA  Mtr05       EEIENRSKHLSAIKSEVQSQKEFISFLIKQVESASYADISEVETFIKWLDGELSTLVDERSVLKHFQWPEQKVDALREAA  Mtr06       EEIENRSKHLSAIKSEVQSQKEFISFLIKQVESASYADISEVETFIKWLDGELSTLVDERSVLKHFQWPEQKVDALREAA  Ath01       GEIENRSKYLSDIKSDTDRHRDHIHILISKVEAATFTDISEVETFVKWIDEELSSLVDERAVLKHFKWPERKVDSLREAA  Egr05       GEIENRSSYLSAIKSDVERQGQFINFLIKEVESAAFKDMTDVEAFIKWLDEELSSLVDERAVLKHFHWPERKADAMREAA  Ptr04       GEIENRSTYLSAIKSDVEKQKEFINFLIKEVESAAFKEISDVKAFVKWLDDELSSLVDERAVLKHFQWPERKADALREAA  Spu01       GEIENRSSYLSAIKSDVEKQKEFINFLIKEVESAAFKEISDVKAFVKWLDDELSSLVDERAVLKHFQWPERKADALREAA  Ptr01       GEIENRSSYLSAIKSDVEKQKEFINFLIKEVESSAFKDISDVKAFVKWLDDELSSLVDERAVLKHFQWPERKADALREAA  Spu03       GEIENRSSYLSAIKSDVEKHKEFINFLIKEVESSAFNDISDVKAFVKWLDDELSSLVDERAVLKHFQWPERKADALREAA  SBi02       GEIENRSAHLLAIKSDVERQGDFIRFLIKEVEGAAFVGIEDVVSFVKWLDDELSRLVDERAVLKHFEWPEHKADALREAA  ZMa02       GEIENRSAHLLAIKSDVERQGDFIRFLIKEVEGAAFVDIEDVVSFVKWLDDELSRLVDERAVLKHFEWPENKADALREAA  Bra04       GEIENRSAHLLAIRSDVERQGDFIRFLIKEVEGAAFANIQDVVTFVKWLDNELSRLVDERAVLKHFEWPEQKADALREAA  Osa01       GEIENRSAHLLAIKSDVERQGDFIRFLIKEVEGAAFVDIEDVVTFVKWLDNELSRLVDERAVLKHFEWPENKEDALREAA  Bra03       GEIENRSSHLLAIRSDVERQGDFIRFLIKEVEGAAFADIDDVVTFVKWLDVELSRLVDERAVLKHFDWPEKKADALREAA  Osa03       GEIENRSAHLLAIKSDVERQGDFIRFLIKEVEGAAFVDIEDVVTFVKWLDVELSRLVDERAVLKHFEWPEQKADALREAA  SBi04       GEIENRSSHLLAIKSDVERQGDFIRFLIKEVQSAAFVDIEDVVTFVKWLDVELSRLVDERAVLKHFDWPEGKADALREAA  Ath03       GEIENRSVYLLAIKTDVETQGDFIRFLIKEVGNAAFSDIEDVVPFVKWLDDELSYLVDERAVLKHFEWPEQKADALREAA  Egr03       GEIENRSTHLLAIKTDVETQGDFIRSLIKEVEGAAFTSIEDVVPFVKWLDDELSYLVDERAVLKHFNWPEQKADALREAA  Mtr03       GEIENRSTHLLAIKTDVETQGDFIRYLIKEVEGAAFTDIEDVVPFVKWLDDELSYLVDERAVLKHFDWPEQKADAMREAA  Ptr03       GEIENRSSHLLAIKTDVEIQGDFIKFLIKEVEIAAFTDIEDVVPFVKWLDDELSYLVDERAVLKHFDWPEQKADALREAA  Ptr06       GEIENRSTHLLAIKTDVEIQGDFIRFLIKEVENAAFTGIEDVVPFVKWLDDELSYLVDERAVLKHFDWPEQKADALREAA  Spu05       GEIENRSTHLLAIKTDVEIQGEFIRFLIKEVENAAFTDIEDVVPFVKWLDDELSYLVDERAVLNHFDWPEQKADALREAA  Spu08       GEIENRSTHLLAIKTDVEIQGEFIRFLIKEVENAAFTDIEDVVPFVKWLDDELSYLVDERAVLKHFEWPEQKADALREAA   Appendix B.2 (continued) Amino acid alignment of homologous CPU sequences used for the maximum likelihood phylogenetic analysis. Black regions represent identical amino acids. Grey regions represent similar amino acids.   192                      490       500       510       520       530       540       550       560                    ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| Atr01       FGYCDLKKFEAEASSYRNDLRQPCAPALKKMQALLEKLEHCVYNLSRMREGAIKRYKGFNIPWEWMLDTGYVSQIKLASV  PEn05       CGYRDLKKLETEVSSYEADPHQTCDAAFKRMLSLLEKLERRVENFVQMRDMAMARYKEFQIPCDWMLDTGIIGKIKFASV  PJe08       CGYCDLKKLETEVSAYEAEPNQTCDAAFKRMLALLEKLERRVENFVQMRDMAMARYREFQIPCDWMLDTGIIGKIKFASV  PPo04       CGYCDLKKLETEVSAYEAEPNQTCDAAFKRMLALLEKLERRVENFVQMRDMAMARYREFQIPCDWMLDTGIIGKIKFASV  Osa04       FGYQDLKKLESEVSNYKDDPRLPCDIALKKMVTISEKTERSVYNLLRTRDATMRQCKEFNIPTDWMLDNNLIGKIKFSSV  SBi03       FGYQDLKKLESEVSNYKDDPRLPCEIALKKMVTLSEKTERGVYNLLRTREAMMRQCKEFNIPTDWMLDNNLISKIKFASV  ZMa01       FGYQDLKKLESEVSNYKDDPRLPCEIALKKMVALSEKTERGVYSLLRTRDAMMRQCKEFNIPTDWMLDNNLISKIKFASV  Egr02       FGYRDLKKLESEVCYYKDDLRIPCDVALKKMVNLSEKMEHTIYNLLRTRESLMRNCKEYQIPVDWMLDNGIITKIKIGSV  Ptr02       FGYSDLKKLESEVSYYKNDPRVPCDIALKKMVALSEKMERTVYNLLRTRESLMRNCKEFQIPSDWMLDNGIISKIKFGSV  Spu07       FGYSDLKKLESEVSYYKDDPRIPCDIALKKMVAVSEKMERTVYNLLRTRESLMRNSKEFQIPSDWMLDNGIISKIKFGSV  Ptr07       FGFSDLKKLESEVSYYKDDPRVPCDLALKKMVALSEKMEHTVYNLLRTRESLMRNCKESQIPSDWMLDNGIISKIKFGSV  Spu04       FGFSDLKKLESEVSYYKDDPRVPCDLALKKMVALSERMERTVYNLLRTRESLMRNCKEFQIPSDWLLDNGIISKIKLGSV  Mtr01       FGYQDLKKLESEVSSYKDDPRLPCDIALKKMVALSEKMERTVYTLLRTRDSLMRNCKEFQIPVEWMLDNGIIGKIKLGSV  Atr03       FGYRDLKKLESEVSCFEDDSRVPCDVALKKMLSLSEKMERGVYNLLRTRDVIMGHCKVFQIPTDWMLDSGIISKIKFSSV  PEn01       FGYQDSKRLESEVWSYEDDLHQPCDVALKKMTVLLEKLEQGVYNLLRKRETAITVYKEFDIPTDWMLDNGMISKIKVASV  PiPa06      FGYQDAKRLESEVWQYEDDLRQPCDVALKKMTVLLEKLEQSVYNLQRKRETAITVYKQFDIPTDWMLDSGMIRKIKVASV  PJe03       FGYQDAKKLESEVWQYEDDLRQPCDVALKKMTVLLEKLEQSVYNLLRKRETSITVYKQFHIPTDWMLDSGMISKIKVASV  PJe04       FGYQDAKKLESEVWQYEDDLRQPCDVALKKMTVLLEKLEQSVYNLLRKRETSITVYKQFHIPTDWMLDSGMISKIKVASV  PPo02       FGYQDAKKLESEVWQYEDDLRQPCDVALKKMTVLLEKLEQSVYNLLRKRETSITVYKQFHIPTDWMLDSGMISKIKVASV  SBi05       FEYHDLMKLQSKVSSFTDDPQLACEEALKKMYSLLEKVEQSVYALLRTRDMTVSRYKEYGIPFDWLSDSGVVGKIKLASV  Bra02       FEYQDLLKLENKATSFADDPKLPCEEALKKMYSLLEKVEQTVYALLRTRDMTTSRYKEYGIPVDWLSDSGKVGKIKLASV  Osa05       FEYQDLLKLEHKVSSFTDDPKLACEEALKKMYSLLEKVEQSVYALLRTRDMAISRYREYGLPVDWLSGSGVVGKIKLASV  Osa06       FEYQDLLKLEHKVSSFTDDPKLACEEALKKMYSLLETVEQSVYALLRTRDMAISRYREYGIPVDWLSDSGVVGKIKLASV  Mtr02       FEYQDLMKLEKQVSNFTDDPKLPCEDALQKMYSLLEKLEQSVYALLRTRDFAISRYKEFGVPVNWLLDSGVVGKIKLSSV  Ath02       FEYQDLMKLEKQVTSFVDDPNLSCEPALKKMYKLLEKVEQSVYALLRTRDMAISRYKEFGIPVDWLSDTGVVGKIKLSSV  Mtr04       FEYQDLMKLENRVSTFVDDPKLSCEAALKKMYSLLEKVEQSVYALLRTRDMAISRYREFGIPINWLQDAGVVGKIKLSSV  Egr01       FEYQDLIKLEKRVSSFSDDPKLPCEAALKKMYSLLEKVEQSVYALLRTRDMAMSRYRDFGIPVDWLLDSGVVGKIKLSSV  Spu02       FEYQDLMKLEKQVITYVDDPNLPCEAALKRMYKLLEKVENSVYALLRTRDMAVSRYKELRIPTNWLLDSGVVGKIKLSSV  Ptr05       FEYQDLMKLERQVTSFVDDPNLPCEAALKKMYKLLEKVENSVYALLRTRDMAVSRYREFGIPTNWLLDSGVVGKIKLSSV  Spu06       FEYQDLMKLERQVTSFVDDPNLPCEAALKKMYKLLEKVENSVYALLRTRDMAVSRYREFGIPTNWLLDSGVVGKIKLSSV  Smo03       FQYRDLRKLELELASFEDDYGMKRDPALNRMQTVMERTEHSIYCFLRTRDKAAIRYKESGIPTNWMLDGGLVGKMKESSV  Ppa02       FEFQDLTKLLAEVSHFEDRPEIPCDKALQKLLATLEKVEESVYGLLRTRDMAIARYREFGIPIQWMLDSGIVGKIKLASV  Ppa03       FEYRDLKKLQSEVSAFEDKPGLPCDAALLEILKCLEKMEKSVYELLRTRDTAIARYKDFSVPTQWMLDKGLVGKMKEVSV  Pac02       FEYQDLKRLEFEATSFEDDPRLPCEASLKKMLSLLEKVEQSVYALLRTRDMAIARYKEFNIPTQWMLDSGLVGKIKLATV  Pac03       FEYQDLKRLEFEATSFEDDPRLPCEASLKKMLSLLEKVEQSVYALLRTRDMAIARYKEFNIPTQWMLDSGLVGKIKLATV  Aal03       FEYQDLKKLEVEATSFQDDPRLPCELALKKMLSLLEKVESSVYALLRTRDMAIARYKEFGIPTQWMLDSGLVGKIKLATV  Pac04       FEYQDLKKLEVEATSFQDDPHLPCERALKKMLALLEKVESSVYALLRSRDMAVARYKEFGIPTQWMLDSGLVGKIKLATV  Ppa01       FEYQDLTKLLGEVSKFEDKSEMPCDKALKKMLTLLEKTEQSVYGLLRTRDMAMARYKEFNIPVQWMLDSGIVGKIKLASV  PEn06       FEYQDLKRLESVAASFVDNPNLSCDAALKKMYSLLEKVEQSVYALLRTRDMAIARYKEFNIPTDWLLDSGVVGKIKLASV  PiPa01      FEYQDLKKLESVVTSFVDNPKLSCDAALKKMYSLLEKVEQSVYALLRTRDMAIARYKEFNIPTDWLLDSGVVGKIKLASV  PJe07       FEYQDLKKLESVVASFADNPKLSCDAALKKMYSLLEKVEQSVYALLRTRDMAIARYKEFNIPTDWLLDSGVVGKIKLASV  PPo03       FEYQDLKKLESVVASFADNPKLSCDAALKKMYSLLEKVEQSVYALLRTRDMAIARYKEFNIPTDWLLDSGVVGKIKLASV  Smo01       FEYQDLQKLEADISSYKDDPRVPRDAALKRMFSLLEKVEQSVFALLRTRDMAIARYKEFNIPTYWMLDSGLIGKIKLASV  Smo02       FEYQDLQKLEADISSYKDDPRVPRDAALKRMFSLLEKVEQSVFALLRTRDMAIARYKEFNIPTYWMLDSGLIGKIKLASV  Atr02       FEYQDLMKLERQVSLFVDDLGLHYEKALKKMYSLLEKVEQSVYALLRTRDMATSRYREFGIPVDWLSDSGVVGKIKLASV  PiPa03      FEYQDLQRLESEIASFVDDPRLSRDTALHRMYSLLESVEKSMYALLRTRDMVVARYKEFNIPIYWLLDSGLVGKIKFACV  PJe05       FEYQDLQRLESEIASFVDDPILSRDTALHRMYSLLESVEKSMYALLRTRDIVVARYKEFNIPIYWLLDSGLVGKIKFACV  PPo01       FEYQDLQRLESEIASFVDDPILSRDTALHRMYSLLESVEKSMYALLRTRDIVVARYKEFNIPIYWLLDSGLVGKIKFACV  Bra01       FTYRDLKNLESEASSFHDDRRLATPMAFKRMQALQDKIEQGIHNTEKIRDSASGRYKDLMIPWDWMLDSGIIKQLKSASL  Osa02       FTYRDLKNLESEASSFHDDRRVATPMALKRMQALQDKIEQGIHNTERARDSASGRYKDLKIPWEWMLDSGIISQLKMASL  ZMa03       FNYRDLKNIESEASSFHDDRRVATPMALKRMQALQDKIEQGIHNTERVRDSASGRYKDLKIPWEWMLDSGVISQLKMASL  SBi01       FNYRDLKNLESEASSFHDDRRVATPMALKRMQALQDKIEQGIHNTERVRDSASGRYKDLKIPWEWMLDSGVISQLKKASL  ZMa04       FNYRDLKNLESEASSFHDDRRVATPMALKRMQALQDKIEQGIHNTERVRDSAGGRYKDLKIPWEWMLDSGVISQLKMASL  Mtr05       CNYRELKNLESEVSSYEDNPKEPISMALKRIQALQDRLEGSVSSKERIRESSSKKYRNFHIPWEWMMDTGLVGQIKLCSL  Mtr06       CNYRELKNLESEVSSYEDNPKEPISMALKRIQALQDRLEGSVSSKERIRESSSKKYRNFHIPWEWMMDTGLVGQIKLCSL  Ath01       CNYKRPKNLGNEILSFKDNPKDSLTQALQRIQSLQDRLEESVNNTEKMRDSTGKRYKDFQIPWEWMLDTGLIGQLKYSSL  Egr05       FSYRDLRNLESEVSSFQDNPKEPLIPALRRMQALQDRLEQSINNVERMRESTSKKYRDLQIPWQWMLDTGLIGQMKLSSL  Ptr04       FNYRDLINLESEVSSFQDNKKEPLIRALGRMQALQDRLERSVNNTERTRESMIKRYRDLQIPWEWLLNTGLIGQMKLSSL  Spu01       FNYRDLTNFESEVSSFQDNTKEPLIQSLGRMQALQDRLERSVNITERTRESMIKRYRDLQIPWEWLLNTGLIGQMKLSSL  Ptr01       FNYRDLMNLESEVSSFQDNPKDLLTLALGRMQALQDRLERSIDNMERTRESMIKRYRDFQIPWEWLLNTGLIGEMKLSSL  Spu03       FNYRDLINLESEVSSFQDNPKDPLILALGRMQALQDRLERSIDNMERTRESMIKRYRDFQIPWEWLLDTGLIGQMKLSSF  SBi02       FGYCDLRKLEAEAASFRDDARQPCAAALKKMQALFEKLEHGVYNLARVRDAATSRYTRFQIPWEWMKDTGIVSQIKLQSV  ZMa02       FGYCDLKKLEREAASFRDDARQPCAAALKKMQALFEKLEHGVYNLARVRDAATGRYTRFQIPWEWMKDTGIVSQIKLQSV  Bra04       FGYCDLKKLEVEASSFRDDARQPCAAELKKMQALFEKLEHGVYNLARGRDGATSRYSRFQIPWDWMQDTGIVSQIKLQSV  Osa01       FGYCDLKKLEVEASSFRDDARQPCSTALKKMQALFEKLEHGVYNLARFRDGATGRYSRFQIPCEWMQDTGIVSQIKLQSV  Bra03       FGYRDLKKVETEAAAFCDDPRQPCSSALKKMQALFEKLEHGVYSLARVRDGAMSRYRGYQIPFEWMQDTGIISQIKIQSV  Osa03       FGYRDLKKIEEEASSFCDDPRQPCSSALKKMQALFEKLEHGVYSLARVRDGAMNRYRGYHIPWEWMQDTGIVSQIKLQSV  SBi04       FGYRDLKKIESEASSFCDDPRQPCSSALKKMQALFEKLEHGVYSLVRVRDGAMSRYRGYQIPWEWMQDTGIVSQIKLQSV  Ath03       FCYFDLKKLISEASRFREDPRQSSSSALKKMQALFEKLEHGVYSLSRMRESAATKFKSFQIPVDWMLETGITSQIKLASV  Egr03       FGYCDLKKLESEASSFRDDPRQMCGPALKKMQALLEKLEHGVYNLSRMRESAGKRYKGFRIPMDWMLDSGIVNQIKLASV  Mtr03       FGYCDLKKLESEASSFRDDPRQLCGPALKKMQTLFEKLEHGVYNISRMRESATKRFKVFQIPVDWLLDSGYATKIKLASV  Ptr03       FGYYDLKKLESEASLFRDNPRQPCGPALKKMQALLEKLEHGVYNLSRMRESATMRYKGFQIPTDWMLETGIVSQIQLASV  Ptr06       FGYCDLKKVESEALLFRDDPRQPCGPALKKMQALLEKLERGVYNLSKMRESATMRYKGFQIPTDWMLETGIVSQMKLASV  Spu05       FGYCDLKKVESEALLSRDDPRQPCGPALKKMQALLEKLERGVYNLSKMRESATMRYKGFQIPTDWMLETGIVSQIKLASV  Spu08       FGYCDLKKVESEALLSRDDPRQPCGPALKKMQALLEKLERGVYNLSKMRESATMRYKGFQIPTDWMLETGIVSQIKLASV   Appendix B.2 (continued) Amino acid alignment of homologous CPU sequences used for the maximum likelihood phylogenetic analysis. Black regions represent identical amino acids. Grey regions represent similar amino acids.   193                      570       580       590       600       610       620       630                   ....|....|....|....|....|....|....|....|....|....|....|....|....|....|. Atr01       KLAKKYIKRVASELGAAM----SPEEKEELIVQGVRFAFRVHQFAGGFDVDTMRAFQELREKAGSCHAECD  PEn05       KLARMYMKRVAVEVESMG-DFEKEVTREFLMFQGVRFAFRVHQFAGGFDSESMSVFKHFRN----------  PJe08       KLARIYMKRVAVEVESMG-DSEREVTREFLMFQGVRFAFRVHQFAGGFDSESMSVFKHFKNLAET------  PPo04       KLARIYMKRVAVEVESMG-DSEREVTREFLMFQGVRFAFRVHQFAGGFDSESMSVFKHFKNLAET------  Osa04       KLAKMYMKRVAMELQYMG-PLNKDPALEYMLLQAVRFAFRMHQFAGGFDPETMDAFEELRNLVHVRNSTQ-  SBi03       KLAKMYMKRVAMELQYMG-PLNKDPALEYMLLQAVRFAFRMHQFAGGFDPETMDAFEELRNLVHVRNSTQ-  ZMa01       KLAKMYMKRVAMELQYMG-PLNKDPALEYMLLQAVRFAFRMHQFAGGFDPETMDAFEELRNLVHVRNSTQ-  Egr02       KLAQKYMKRVAMELQLKA-TADKDPAMDYMLLQGVRFAFRIHQFAGGFDSETMQAFEELRNLANLLNKK--  Ptr02       KLAKKYMKRVATEIQSKAAALEKDPALDYMLLQGVRFAFRIHQFAGGFDAETMHAFEELRNLAHLLNKK--  Spu07       KLAKKYMKRVATEIQSKAAALEKDPALDYMLLQGVRFAFRIHQFAGGFDAETMHAFEELRNLAHLLNKK--  Ptr07       KLAKKYMKRVATEIQSKAAALEKDPALDYMLLQGVRFAFRIHQFAGGFDAETMHAFEELRNLAHLLNKK--  Spu04       KLAKKCMKRVATIIQSKAAALEKDPALDYMLLQGVRFAFRIHQFAGGFDAETMHAFEELRNLAHLLNKK--  Mtr01       KLAKKYMKRVAIEVQTKS-AFDKDPAMDYMVLQGVRFAFRIHQFAGGFDAETMHAFEELRNLASLLNKT--  Atr03       KLAKKYMKRVAMELQAKG-ASDKDPSLEYMLLHGVRFAFRIHQFAGGFDAETMQAFEELRNLAHIRNNK--  PEn01       KLAKKYMNRISIELESMG-SPEKEPAQEFLLLQGVRFAFRAHQFAGGFDEETMHAFEELRQLA--------  PiPa06      KLAKKYMNRISVELESMG-SPEKEPALEFLLLQGVRFAFRAHQFAGGFDEETMHAFEELRKLA--------  PJe03       KLAKKYMNRISIELESMG-NPEKEPALEFLLLQGVRFAYRAHQFAGGFDEETMHAFEELRKLA--------  PJe04       KLAKKYMNRISIELESMG-NPEKEPALEFLLLQGVRFAYRAHQFAGGFDEETMHAFEELRKLA--------  PPo02       KLAKKYMNRISIELESMG-NPEKEPALEFLLLQGVRFAYRAHQFAGGFDEETMHAFEELRKLA--------  SBi05       QLANKYMKRVASELDALEG-TEKEPNREFLLLQGVRFAFRVHQFAGGFDAESMKAFEELRSKMTTQSSAP-  Bra02       QLAKKYMERVASELDALEG-TEKEPNREFLLLQGVRFAFRVHQFAGGFDADSMKVFEELRSKMTTQTSGPP  Osa05       QLAKKYMKRVATELDALQG-TEKEPNREFLLLQGVRFAFRVHQFAGGFDEESMKAFEELRSKMST-QTSAP  Osa06       QLAKKYMNRVATELDALQG-TEKEPNREFLLLQGVRFAFRVHQFAGGFDEESMKAFEELRSKMCTTQTSAP  Mtr02       QLANKYMKRIASEIDTLSG-PENEPTREFLILQGVRFSFRVHQFAGGFDTESMKAFEELRNNIHVQAGEYN  Ath02       QLAKKYMKRVAYELDSVSG-SDKDPNREFLLLQGVRFAFRVHQFAGGFDAESMKAFEELRSRAKTESGDNN  Mtr04       QLARKYMKRVASELDALSG-PEKEPAREFLILQGVRFAFRVHQFAGGFDAESMKAFEDLRSRIQTPQAPQV  Egr01       QLARNYMKRVADELDGLGG-SDKEPNREFLLLQGVRFAFRVHQFAGGFDAESMKAFEELRNRVRSQTEEET  Spu02       QLARKYMKRVASELDAMSG-PEKEPNREFLVLQGVRFAFRVHQFTGGFDAESMKAFEELRSRVSSQMGEEN  Ptr05       QLARKYMKRVASELDTMSG-PEKEPNREFLVLQGVRFAFRVHQFAGGFDAESMKAFEELRSRVRSQMGEEN  Spu06       QLARKYMKRVASELDTVSG-PEKEPNREFLVLQGVRFAFRVHQFAGGFDAESMKAFEELRSRVRSQMGEEN  Smo03       KLAEKFMKRVVLELDGAG---SDELVEEFLLLQGVRFAFRVHQFAGGFDDKTMQAFEELRSRARRTT--PS  Ppa02       TLARLYVKRVASQLNQTL--PIKETVREFLLLQGVRFAFRVHQFAGGFDPESMHAFMALRASSDGPIVSPP  Ppa03       RLAQLYMKRVSGELDKLAGGSDKEPLREFLLVQGVRFAFRVHQFAGGLNSESMAAFEALRQRASQQESPES  Pac02       QLARKYMKRVAAELDASTASASQDPQREFLLLQGVRFAFRVHQFAGGFDAESMRTFEELRDRV--------  Pac03       QLARKYMKRVAAELDASTASASQDPQREFLLLQGVRFAFRVHQFAGGFDAESMRTFEELRDRV--------  Aal03       QLARKYMKRVAAELDASTASTSQDPQREFLLLQGVRFAFRVHQFAGGFDAESMRTFEELRDRI--------  Pac04       QLARKYMKRVAAELDASTASPTQDPQREFLLLQGVRFAFRVHQFAGGFDAESMRTFEELRDRIRA------  Ppa01       KLARLYMKRVSTELEQVGS--LNEPVREFLLLQGVRFAFRVHQFAGGFDPESMQAFESLRACANRPSNPPD  PEn06       QLARKYMKRVTSELDAALNDPDKEPIKEFLLLQGVRFAFRVHQFAGGFDAESMNAFEDLRNRVHGQAEETD  PiPa01      QLARKYMKRVTSELDAALDAPDKEPIREFLLLQGVRFAFRVHQFAGGFDAESMNAFEDLRNRVHGQAEETD  PJe07       QLARKYMKRVTSELDATLNVPDKEPIREFLLLQGVRFAFRVHQFAGGFDAESMNAFENLRNRVHGQAEETD  PPo03       QLARKYMKRVTSELDATLNVPDKEPIREFLLLQGVRFAFRVHQFAGGFDAESMNAFENLRNRVHGQAEETD  Smo01       KLAQQYMNRVIKELDSV---QDKEPLREFLLLQGVRFAFRVHQFAGGFDPESMRTFEELRNRAQSEQLKRS  Smo02       KLAQQYMNRVIKELDSV---QDKEPLREFLLLQGVRFAFRVHQFAGGFDPESMRTFEELRNRAQIAWSMIS  Atr02       QLARKYMKRVASELDALSG-PEKEPTKEFLLLQGVRFAFRVHQFAGGFDAESMRAFEELRGRVNAQAAEGN  PiPa03      QLARKYMERVASELDGFANISEKEPVREFLLLQGVRFAFRVHQFAGGFDEESMRAFESLRRRAQEQTREAN  PJe05       QLARKY-----------------------------------------------------------------  PPo01       QLARKYMERVASELDGIANISEKEPVREFLLLQGVRFAFRVHQFAGGFDEESMRAFESLRRRAQEQTGEAN  Bra01       KLAKEYMNRIMNALKSDPVND----E--ELLLQGVRFAFRIHQLAGGFDEDCRKAFQELKTYASKSE----  Osa02       KLAREFMNRVVNALKSDPTND----E--ELLLQGVRFAFRIHQLAGGFDEGCRKAFQELKMYASKSD----  ZMa03       KLAKEYMNRIVKTLKSDPANE----E--ELLLQGVRFAFRIHQLAGGFDEGCRKAFQELNTNASKSE----  SBi01       KLAKEYMNRIVNTLKSDPAND----E--ELLLQGVRFAFRIHQLAGGFDEGCRKAFQELKTYASKSE----  ZMa04       KLAKEYMNRIASTLKSDPAND----E--ELLLQGVRFAFRVHQLAGGFDEGCRKAFQELKTYASKSE----  Mtr05       RLAKEFMKRITKEIKSHELHEDN--N--NLLLQGVKFAFRVHQVQ--------------------------  Mtr06       RLAKEFMKRITKEIKSHELHEDN--N--NLLLQGVKFAFRVHQFAGGFDPDTTQTFLELKKVGCAVPSNSN  Ath01       RLAQEYMKRIAKELESNGGKE----G--NLMLQGVRFAYTIHQFAGGFDGETLSIFHELKKITTGETRG--  Egr05       KLAKEYMKRIAREMRSSESQE----D--NLMLQGVRFAFRVHQFSGGFDDETIGAFELLKMAGSGYQKQQN  Ptr04       RLAKDYLKRITKELQLNESGE----E--NLLLQGARFAYRVHQFAGGFDAETTHAFQELKKIGMGSLKQ--  Spu01       RLAKDYLKRITKELQLNESGE----E--NLLLQGARFAYRIHQFAGGFDAETIHAFQELKKIGMGSVKQ--  Ptr01       RLAKVYLKRITKELQLNESGE----D--NLLLQGARFAYRVHQFAGGFDAETIRAFQELKKIGMGSLKQ--  Spu03       RLAKVYLKRTTKELQLNEPGE----D--NLLLQGARFAYRVHQFAGGFDAETIRAFQELKKIGMDSLKE--  SBi02       KLAMKYLKRVSSELEVIKG-PEE--E--ELMLQGVRFAFRVHQFAGGFDVDTMRAFQELKEKASMCRIQRH  ZMa02       KLAMKHLKRVSSELEVIKG-PEE--EEQELMLQGVRFAFRVHQFAGGFDVDTMRAFQELKEKASMCRVQRQ  Bra04       KLARKYLERVSSELEAIKG-PAE--E--ELMLQGVRFAFRVHQFANGFDADTMRAFQELKEKASMCRFQRQ  Osa01       KLAMKYLKRVSSELEAIKG-PDE--E--ELMLQGVRFAFRVHQFAGGFDVDTMRAFQELKEKASMCRIQRQ  Bra03       KLARKYLRRVSSELEAIQG-PDE--E--ELMLQGVRFAFRVHQFAGGFDGDTMRAFQEIKEKASAFQSQRD  Osa03       KLAMKYLRRVSSELEAIKG-PDE--E--ELMLQGVRFAFRVHQFAGGFDGDTMRAFQELKEKASTFQSQRE  SBi04       KLAMKYLRRVSSELEAIQG-PDE--E--ELVLQGVRFAFRVHQFAGGFDGDTMRAFQELKEKASTFQQ-RG  Ath03       KLAMKYMKRVSAELEAIEGGPEE--E--ELIVQGVRFAFRVHQFAGGFDAETMKAFEELRDKARSCHVQCQ  Egr03       KLAMKYMKRVSAELEMGAGGPEE--E--ELIVQGVRFAFRVHQFAGGFDVETMKAFQELRDKANSCHVQCQ  Mtr03       KLAMKYMKRVSAELETVGG-PEE--E--ELIVQGVRFAFRVHQFASGFDADTMRAFQELRDKARSCHVQCH  Ptr03       KLAMKFLKRVSSELETVGG-PEE--E--ELIVQGVRYAFRVHQFAGGFDAETMRAFRELRDKARSCHVQCQ  Ptr06       KLAMKYMKRVSAELETGGGGPEE--E--ELIVQGVRYAFRVHQFAGGFDVETMRAFQELREKAGSCHVQCQ  Spu05       KLAMKYMKRVSAELETAGGPPEE--E--ELIVQGVRYAFRVHQFAGGFDGETMRAFQELRDKAGSC----Q  Spu08       KLAMKYMKRVSAELETAGGPPEE--E--ELIVQGVRYAFRVHQFAGGFDGETMRAFQELRDKAGSC----Q   Appendix B.2 (continued) Amino acid alignment of homologous CPU sequences used for the maximum likelihood phylogenetic analysis. Black regions represent identical amino acids. Grey regions represent similar amino acids.194  Appendix B.3 Entire list of genes co-expressed with PtCPU in developing P. trichocarpa wood transcriptomes at a PCC of at least 0.60  Poplar ID (v3.0) PCC MapMan Annotation Potri.001G279000 1.00  FUNCTIONS IN: molecular_function unknown  Potri.001G448400 0.76  EMB2301 (EMBRYO DEFECTIVE 2301); transcription activator/ transcription factor  Potri.009G060600 0.74  NF-YA1 (NUCLEAR FACTOR Y, SUBUNIT A1); transcription factor  Potri.003G151700 0.73  ATGH9A1 (ARABIDOPSIS THALIANA GLYCOSYL HYDROLASE 9A1); cellulase/ hydrolase, hydrolyzing O-glycosyl compounds  Potri.001G333600 0.73  protein binding  Potri.008G145900 0.73  protein kinase family protein  Potri.006G092600 0.73  ROPGEF7; Rho guanyl-nucleotide exchange factor  Potri.001G354900 0.72  PGP9 (P-GLYCOPROTEIN 9); ATPase, coupled to transmembrane movement of substances  Potri.010G003100 0.72  hydroxyproline-rich glycoprotein family protein  Potri.001G401300 0.71  LAC17 (laccase 17); laccase  Potri.006G211200 0.71  rac GTPase activating protein, putative  Potri.001G416300 0.71  ATK3 (ARABIDOPSIS THALIANA KINESIN 3); ATPase/ microtubule binding / microtubule motor  Potri.019G078400 0.71  leucine-rich repeat transmembrane protein kinase, putative  Potri.008G144900 0.71  ATP binding / protein binding / protein kinase/ protein serine/threonine kinase  Potri.005G230900 0.71  transferase family protein  Potri.003G070200 0.70  unknown protein  Potri.006G204700 0.70  aspartyl protease family protein  Potri.010G184000 0.70  unknown protein  Potri.011G066100 0.70  WAVE2  Potri.003G141900 0.70  FUNCTIONS IN: molecular function unknown  Potri.016G051900 0.70  ATNEK5 (NIMA-RELATED KINASE5); ATP binding / kinase/ protein kinase/ protein serine/threonine kinase/ protein tyrosine kinase  Potri.013G107700 0.70  C2 domain-containing protein  Potri.010G184100 0.70  DEAD/DEAH box helicase, putative (RH18)  Potri.017G000300 0.69  MTHFR2 (METHYLENETETRAHYDROFOLATE REDUCTASE 2); methylenetetrahydrofolate reductase (NADPH)  Potri.T137300 0.69  unknown protein Potri.010G244900 0.69  FLA17 (FASCICLIN-LIKE ARABINOGALACTAN PROTEIN 17 PRECURSOR)  Potri.002G036200 0.69  kinase  Potri.005G256000 0.69  XCP2 (xylem cysteine peptidase 2); cysteine-type peptidase/ peptidase  Potri.008G170800 0.69  arpc2b (actin-related protein C2B); structural molecule  Potri.014G146200 0.69  unknown protein  Potri.001G333200 0.69  FUNCTIONS IN: molecular_function unknown  Potri.006G018000 0.69  ATMAP70-5 (microtubule-associated proteins 70-5); microtubule binding  Potri.008G038900 0.69  zinc finger (GATA type) family protein  Potri.010G112800 0.69  PIN3 (PIN-FORMED 3); auxin:hydrogen symporter/ transporter  Potri.007G098500 0.69  EXPRESSED IN: 22 plant structures  Potri.017G079600 0.69  IQD31 (IQ-domain 31); calmodulin binding  Potri.018G107800 0.69  unknown protein  Potri.016G134000 0.69  armadillo/beta-catenin repeat family protein / U-box domain-containing protein  Potri.016G085900 0.68  myosin heavy chain-related  Potri.014G087200 0.68  CRCK2; ATP binding / kinase/ protein kinase/ protein serine/threonine kinase  Potri.015G060900 0.68  unknown protein  Potri.002G257900 0.68  CESA4 (CELLULOSE SYNTHASE A4); cellulose synthase/ transferase, transferring glycosyl groups  Potri.001G463000 0.68  polygalacturonase, putative / pectinase, putative  Potri.010G252400 0.68  merozoite surface protein-related  Potri.014G029900 0.68  transferase, transferring glycosyl groups  Potri.001G228100 0.68  protein kinase family protein  Potri.003G124800 0.68  leucine-rich repeat transmembrane protein kinase, putative   195  Appendix B.3 (continued) Entire list of genes co-expressed with PtCPU in developing P. trichocarpa wood transcriptomes at a PCC of at least 0.60  Poplar ID (v3.0) PCC MapMan Annotation Potri.008G012400 0.68  FLA17 (FASCICLIN-LIKE ARABINOGALACTAN PROTEIN 17 PRECURSOR)  Potri.004G211900 0.68  LOCATED IN: membrane  Potri.007G046100 0.68  transport protein-related  Potri.004G059600 0.68  IRX1 (IRREGULAR XYLEM 1); cellulose synthase/ transferase, transferring glycosyl groups  Potri.004G049300 0.68  ANAC073 (ARABIDOPSIS NAC DOMAIN CONTAINING PROTEIN 73); transcription activator/ transcription factor  Potri.001G174900 0.68  hydrolase, alpha/beta fold family protein  Potri.011G159000 0.67  polygalacturonase  Potri.006G131000 0.67  IRX9 (IRREGULAR XYLEM 9); transferase, transferring glycosyl groups / xylosyltransferase  Potri.003G155200 0.67  vacuolar sorting receptor, putative  Potri.011G120300 0.67  LAC17 (laccase 17); laccase  Potri.011G067400 0.67  MRH1 (morphogenesis of root hair 1); ATP binding / protein binding / protein kinase/ protein serine/threonine kinase/ protein tyrosine kinase  Potri.017G075600 0.67  glycosyltransferase family 14 protein / core-2/I-branching enzyme family protein  Potri.004G057100 0.67  WAVE2  Potri.002G113100 0.67  RXF12; endo-1,4-beta-xylanase/ hydrolase, hydrolyzing O-glycosyl compounds  Potri.008G198100 0.67  endonuclease/exonuclease/phosphatase family protein  Potri.007G032700 0.67  BLH2 (BEL1-LIKE HOMEODOMAIN 2); DNA binding / transcription factor  Potri.001G416800 0.67  GAUT12 (GALACTURONOSYLTRANSFERASE 12) Potri.014G161700 0.67  auxin-responsive protein-related  Potri.006G109900 0.67  (1-4)-beta-mannan endohydrolase, putative  Potri.014G175200 0.67  FUNCTIONS IN: molecular_function unknown  Potri.002G023000 0.67  unknown protein  Potri.017G128100 0.67  armadillo/beta-catenin repeat family protein  Potri.018G024800 0.67  PAP26 (PURPLE ACID PHOSPHATASE 26); acid phosphatase/ protein serine/threonine phosphatase  Potri.013G083400 0.67  KT2 (POTASSIUM TRANSPORTER 2); potassium ion transmembrane transporter  Potri.015G060100 0.67  IRX6  Potri.001G018200 0.67  catalytic  Potri.008G038200 0.66  pal1 (Phe ammonia lyase 1); phenylalanine ammonia-lyase  Potri.006G229300 0.66  AtGLDP1 (Arabidopsis thaliana glycine decarboxylase P-protein 1); catalytic/ glycine dehydrogenase (decarboxylating)/ pyridoxal phosphate binding  Potri.010G221600 0.66  EMB1144 (embryo defective 1144); chorismate synthase  Potri.006G115200 0.66  AtGRF4 (GROWTH-REGULATING FACTOR 4); transcription activator  Potri.019G130700 0.66  C4H (CINNAMATE-4-HYDROXYLASE); trans-cinnamate 4-monooxygenase  Potri.012G126500 0.66  ANAC007 (ARABIDOPSIS NAC 007); transcription factor  Potri.010G058700 0.66  unknown protein Potri.001G000800 0.66  ATP binding / microtubule motor  Potri.008G149300 0.66  RHF1A (RING-H2 GROUP F1A); protein binding / ubiquitin-protein ligase/ zinc ion binding  Potri.013G050900 0.66  unknown protein  Potri.011G153300 0.66  EMB2301 (EMBRYO DEFECTIVE 2301); transcription activator/ transcription factor  Potri.007G107200 0.66  PGSIP1 (PLANT GLYCOGENIN-LIKE STARCH INITIATION PROTEIN 1); transferase, transferring glycosyl groups  Potri.014G024700 0.66  FRA1 (FRAGILE FIBER 1); microtubule motor  Potri.005G065200 0.66  dehydration-responsive protein-related  Potri.016G066500 0.66  FLA17 (FASCICLIN-LIKE ARABINOGALACTAN PROTEIN 17 PRECURSOR)  Potri.003G223800 0.66  ATP binding / microtubule motor  Potri.005G061600 0.66  PGSIP1 (PLANT GLYCOGENIN-LIKE STARCH INITIATION PROTEIN 1); transferase, transferring glycosyl groups  Potri.001G036900 0.66  4CL2 (4-COUMARATE:COA LIGASE 2); 4-coumarate-CoA ligase  Potri.003G113000 0.66  ANAC007 (ARABIDOPSIS NAC 007); transcription factor  Potri.008G182700 0.66  DIS1 (DISTORTED TRICHOMES 1); ATP binding / actin binding / protein binding / structural constituent of cytoskeleton  Potri.011G156100 0.66  MUR4 (MURUS 4); UDP-arabinose 4-epimerase/ catalytic  Potri.011G153700 0.66  MOR1 (MICROTUBULE ORGANIZATION 1); microtubule binding   196  Appendix B.3 (continued) Entire list of genes co-expressed with PtCPU in developing P. trichocarpa wood transcriptomes at a PCC of at least 0.60  Poplar ID (v3.0) PCC MapMan Annotation Potri.010G223300 0.66  zinc finger (GATA type) family protein  Potri.002G222600 0.66  auxin-responsive protein-related  Potri.010G081800 0.65  unknown protein  Potri.005G194200 0.65  CESA9 (CELLULOSE SYNTHASE A9); cellulose synthase/ transferase, transferring glycosyl groups  Potri.008G177500 0.65  unknown protein  Potri.006G033300 0.65  CYP98A3 (cytochrome P450, family 98, subfamily A, polypeptide 3); monooxygenase/ p-coumarate 3-hydroxylase  Potri.006G181900 0.65  IRX3 (IRREGULAR XYLEM 3); cellulose synthase  Potri.008G018600 0.65  unknown protein  Potri.011G068300 0.65  unknown protein  Potri.007G032500 0.65  zinc ion binding  Potri.006G120400 0.65  unknown protein Potri.004G161100 0.65  kinesin-related protein (MKRP2)  Potri.016G136500 0.65  HSL1 (HSI2-LIKE 1); transcription factor  Potri.015G045700 0.65  unknown protein  Potri.017G016000 0.65  DNAJ heat shock family protein  Potri.010G119400 0.65  LPP3 (LIPID PHOSPHATE PHOSPHATASE 3); phosphatidate phosphatase  Potri.010G160500 0.65  calcineurin-like phosphoesterase family protein  Potri.002G101800 0.65  zinc finger (C3HC4-type RING finger) family protein  Potri.005G236200 0.65  iqd32 (IQ-domain 32); calmodulin binding  Potri.010G095900 0.65  protein kinase family protein  Potri.002G160000 0.65  unknown protein  Potri.004G159300 0.65  BLH7 (bell1-like homeodomain 7); DNA binding / transcription factor  Potri.016G079500 0.65  unknown protein  Potri.006G167200 0.65  ATR2 (ARABIDOPSIS P450 REDUCTASE 2); NADPH-hemoprotein reductase  Potri.003G164800 0.65  unknown protein  Potri.016G138600 0.65  (1-4)-beta-mannan endohydrolase, putative  Potri.008G201600 0.64  RFR1 (REF4-related 1)  Potri.005G070400 0.64  EXPRESSED IN: 22 plant structures  Potri.007G046900 0.64  leucine-rich repeat transmembrane protein kinase, putative  Potri.009G006500 0.64  FRA8 (FRAGILE FIBER 8); glucuronosyltransferase/ transferase  Potri.001G189500 0.64  PEN3 (PENETRATION 3); ATPase, coupled to transmembrane movement of substances / cadmium ion transmembrane transporter  Potri.001G121300 0.64  WRKY11; calmodulin binding / transcription factor  Potri.002G257800 0.64  PHR1 (PHOSPHATE STARVATION RESPONSE 1); transcription factor  Potri.007G031500 0.64  GNS1/SUR4 membrane family protein  Potri.018G103900 0.64  IRX3 (IRREGULAR XYLEM 3); cellulose synthase  Potri.008G034800 0.64  scpl49 (serine carboxypeptidase-like 49); serine-type carboxypeptidase  Potri.001G068100 0.64  GUT2; catalytic/ glucuronoxylan glucuronosyltransferase  Potri.006G108300 0.64  HSL1 (HSI2-LIKE 1); transcription factor  Potri.015G106000 0.64  AML1 (ARABIDOPSIS MEI2-LIKE PROTEIN 1); RNA binding / protein binding  Potri.014G035800 0.64  PDE135 (pigment defective embryo 135); transmembrane transporter  Potri.010G224100 0.64  pal1 (Phe ammonia lyase 1); phenylalanine ammonia-lyase  Potri.015G017500 0.64  GLT1; glutamate synthase (NADH)  Potri.014G110400 0.64  GLP10 (GERMIN-LIKE PROTEIN 10); manganese ion binding / nutrient reservoir  Potri.010G092300 0.64  RHF1A (RING-H2 GROUP F1A); protein binding / ubiquitin-protein ligase/ zinc ion binding  Potri.005G044900 0.64  VAP27-2 (VAMP/SYNAPTOBREVIN-ASSOCIATED PROTEIN 27-2); structural molecule  Potri.017G130300 0.64  MYB43 (myb domain protein 43); DNA binding / transcription factor  Potri.007G011400 0.64  transmembrane transporter  Potri.014G153700 0.64  galactosyltransferase family protein  197  Appendix B.3 (continued) Entire list of genes co-expressed with PtCPU in developing P. trichocarpa wood transcriptomes at a PCC of at least 0.60  Poplar ID (v3.0) PCC MapMan Annotation Potri.001G112200 0.64  KNAT7 (KNOTTED-LIKE HOMEOBOX OF ARABIDOPSIS THALIANA 7); DNA binding / transcription activator/ transcription factor  Potri.009G068600 0.64  hydroxymethylglutaryl-CoA lyase, putative / 3-hydroxy-3-methylglutarate-CoA lyase, putative / HMG-CoA lyase, putative  Potri.016G006700 0.64 unknown protein Potri.013G003700 0.64  protein kinase family protein  Potri.011G146300 0.64  subtilase family protein  Potri.013G127200 0.64  IQD13 (IQ-domain 13); calmodulin binding  Potri.011G158700 0.64  FAD-binding domain-containing protein  Potri.010G252500 0.63  merozoite surface protein-related  Potri.002G109200 0.63  SHM2 (SERINE HYDROXYMETHYLTRANSFERASE 2); catalytic/ glycine hydroxymethyltransferase/ pyridoxal phosphate binding  Potri.001G352300 0.63  O-acetyltransferase family protein  Potri.014G093700 0.63  ATPOB1; protein binding  Potri.008G094700 0.63  kinesin light chain-related  Potri.001G127100 0.63  zinc finger (C3HC4-type RING finger) family protein  Potri.002G261700 0.63  unknown protein  Potri.009G009500 0.63  unknown protein  Potri.013G157900 0.63  C4H (CINNAMATE-4-HYDROXYLASE); trans-cinnamate 4-monooxygenase  Potri.016G029100 0.63  hydroxyproline-rich glycoprotein family protein  Potri.002G216000 0.63  GDSL-motif lipase/hydrolase family protein  Potri.014G044100 0.63  invertase/pectin methylesterase inhibitor family protein  Potri.008G156600 0.63  SULTR3;1 (SULFATE TRANSPORTER 3;1); secondary active sulfate transmembrane transporter/ sulfate transmembrane transporter/ transporter  Potri.014G066600 0.63  unknown protein  Potri.003G188500 0.63  4CL2 (4-COUMARATE:COA LIGASE 2); 4-coumarate-CoA ligase  Potri.011G094800 0.63  zinc finger (C3HC4-type RING finger) family protein  Potri.017G012600 0.63  zinc finger (C3HC4-type RING finger) family protein  Potri.004G235000 0.63  unknown protein  Potri.008G026400 0.63  ATCSLA09; mannan synthase/ transferase, transferring glycosyl groups  Potri.006G035500 0.63  transcription elongation factor-related  Potri.001G057800 0.63  calmodulin binding / transcription regulator  Potri.001G005500 0.63  SUB1; calcium ion binding  Potri.016G071000 0.63  ATEXO70A2 (exocyst subunit EXO70 family protein A2); protein binding  Potri.001G358200 0.63  ATBAG3 (ARABIDOPSIS THALIANA BCL-2-ASSOCIATED ATHANOGENE 3); protein binding  Potri.005G021100 0.63  ATP binding / microtubule motor  Potri.014G100400 0.63  kinesin light chain-related  Potri.001G320800 0.63  FLA12  Potri.014G185300 0.63  unknown protein  Potri.014G074700 0.63  PLDBETA1 (PHOSPHOLIPASE D BETA 1); phospholipase D  Potri.012G113700 0.63  unknown protein  Potri.003G116200 0.63  pentatricopeptide (PPR) repeat-containing protein  Potri.005G162800 0.63  2-dehydro-3-deoxyphosphoheptonate aldolase, putative / 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, putative / DAHP synthetase, putative  Potri.001G295800 0.63  unknown protein  Potri.010G105900 0.63  ADL1C (ARABIDOPSIS DYNAMIN-LIKE PROTEIN 1C); GTP binding / GTPase  Potri.004G217700 0.63  FUNCTIONS IN: molecular function unknown  Potri.006G200300 0.62  FLA17 (FASCICLIN-LIKE ARABINOGALACTAN PROTEIN 17 PRECURSOR)  Potri.014G017500 0.62  unknown protein  Potri.015G135500 0.62  ATBAG1 (ARABIDOPSIS THALIANA BCL-2-ASSOCIATED ATHANOGENE 1); protein binding  Potri.011G136700 0.62  TOR1 (TORTIFOLIA 1); microtubule binding  Potri.003G131700 0.62  glycoside hydrolase family 28 protein / polygalacturonase (pectinase) family protein  Potri.001G320400 0.62  SHM4 (serine hydroxymethyltransferase 4); catalytic/ glycine hydroxymethyltransferase/ pyridoxal phosphate binding   198  Appendix B.3 (continued) Entire list of genes co-expressed with PtCPU in developing P. trichocarpa wood transcriptomes at a PCC of at least 0.60  Poplar ID (v3.0) PCC MapMan Annotation Potri.016G112000 0.62  IRX12 (IRREGULAR XYLEM 12); laccase  Potri.004G079900 0.62  unknown protein  Potri.002G059000 0.62  protein kinase family protein  Potri.017G057900 0.62  BGAL9 (Beta galactosidase 9); beta-galactosidase/ catalytic/ cation binding / sugar binding  Potri.014G114700 0.62  SAM-2 (S-ADENOSYLMETHIONINE SYNTHETASE 2); methionine adenosyltransferase  Potri.006G152700 0.62  anac075 (Arabidopsis NAC domain containing protein 75); transcription factor  Potri.007G135300 0.62  ANAC073 (ARABIDOPSIS NAC DOMAIN CONTAINING PROTEIN 73); transcription activator/ transcription factor  Potri.006G168400 0.62  unknown protein  Potri.002G005700 0.62  XCP2 (xylem cysteine peptidase 2); cysteine-type peptidase/ peptidase  Potri.005G001100 0.62  unknown protein Potri.002G099200 0.62  2-dehydro-3-deoxyphosphoheptonate aldolase, putative / 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, putative / DAHP synthetase, putative  Potri.001G118800 0.62  MYB42 (myb domain protein 42); transcription factor  Potri.011G132700 0.62  AtATG18f  Potri.006G194900 0.62  ATP binding / microtubule motor  Potri.013G156100 0.62  unknown protein Potri.007G085900 0.62  protein binding / zinc ion binding  Potri.018G039600 0.62  FAD-binding domain-containing protein  Potri.019G076900 0.62  unknown protein  Potri.004G013400 0.62  ADT1 (arogenate dehydratase 1); arogenate dehydratase/ prephenate dehydratase  Potri.014G096100 0.62  GT72B1; UDP-glucosyltransferase/ UDP-glycosyltransferase/ transferase, transferring glycosyl groups  Potri.013G061800 0.62  ATMS1; 5-methyltetrahydropteroyltriglutamate-homocysteine S-methyltransferase/ methionine synthase  Potri.010G038200 0.62  GLP7 (GERMIN-LIKE PROTEIN 7); manganese ion binding / nutrient reservoir  Potri.010G193100 0.62  IRX12 (IRREGULAR XYLEM 12); laccase  Potri.016G055700 0.62  unknown protein  Potri.016G020100 0.62  unknown protein  Potri.014G066200 0.62  zinc finger (C2H2 type) family protein  Potri.017G082900 0.62  AtHB34 (ARABIDOPSIS THALIANA HOMEOBOX PROTEIN 34); DNA binding / transcription factor  Potri.016G133900 0.62  ATSR1 (ARABIDOPSIS THALIANA SERINE/THREONINE PROTEIN KINASE 1); ATP binding / kinase/ protein kinase/ protein serine/threonine kinase  Potri.014G040300 0.62  PARVUS (PARVUS); polygalacturonate 4-alpha-galacturonosyltransferase/ transferase, transferring glycosyl groups / transferase, transferring hexosyl groups  Potri.005G216900 0.62  unknown protein  Potri.005G110900 0.62  3-dehydroquinate synthase, putative  Potri.006G056300 0.62  ATNEK5 (NIMA-RELATED KINASE5); ATP binding / kinase/ protein kinase/ protein serine/threonine kinase/ protein tyrosine kinase  Potri.016G141700 0.62  auxin efflux carrier family protein  Potri.014G095800 0.62  unknown protein  Potri.002G011800 0.61  BSK1 (BR-SIGNALING KINASE 1); ATP binding / binding / kinase/ protein kinase/ protein tyrosine kinase  Potri.014G074500 0.61  XSP1 (xylem serine peptidase 1); identical protein binding / serine-type endopeptidase  Potri.002G073400 0.61  SCL1 (SCARECROW-LIKE 1); transcription factor  Potri.018G107600 0.61  potassium channel tetramerisation domain-containing protein  Potri.001G099800 0.61  AtMYB103 (myb domain protein 103); DNA binding / transcription activator/ transcription factor  Potri.006G051000 0.61  unknown protein  Potri.002G110300 0.61  lyase  Potri.007G047500 0.61  IRX14 (irregular xylem 14); transferase, transferring glycosyl groups / xylosyltransferase  Potri.017G053900 0.61  unknown protein Potri.005G129500 0.61  BLH2 (BEL1-LIKE HOMEODOMAIN 2); DNA binding / transcription factor  Potri.009G160200 0.61  DCP1 (decapping 1); m7G(5)pppN diphosphatase/ protein homodimerization  Potri.010G067100 0.61  arpc2b (actin-related protein C2B); structural molecule  Potri.001G381500 0.61  ALIS5 (ALA-Interacting Subunit 5)  Potri.010G118300 0.61  serine/threonine protein kinase, putative   199  Appendix B.3 (continued) Entire list of genes co-expressed with PtCPU in developing P. trichocarpa wood transcriptomes at a PCC of at least 0.60  Poplar ID (v3.0) PCC MapMan Annotation Potri.018G068700 0.61  anac075 (Arabidopsis NAC domain containing protein 75); transcription factor  Potri.009G073600 0.61  FUNCTIONS IN: molecular_function unknown  Potri.012G006400 0.61  ATOMT1 (O-METHYLTRANSFERASE 1); caffeate O-methyltransferase/ myricetin 3-O-methyltransferase/ quercetin 3-O-methyltransferase  Potri.004G197500 0.61  ATP binding / protein binding / protein kinase/ protein serine/threonine kinase/ protein tyrosine kinase  Potri.001G231000 0.61  zinc finger (C3HC4-type RING finger) family protein  Potri.016G091100 0.61  pal1 (Phe ammonia lyase 1); phenylalanine ammonia-lyase  Potri.014G032300 0.61 unknown protein Potri.008G069900 0.61  ESK1 (ESKIMO 1)  Potri.014G066100 0.61  zinc finger (C2H2 type) family protein  Potri.001G177000 0.61  aminotransferase class I and II family protein  Potri.017G130600 0.61  LRR1; ATP binding / kinase/ protein serine/threonine kinase  Potri.002G091700 0.61  LOCATED IN: vacuole  Potri.005G117500 0.61  FAH1 (FERULIC ACID 5-HYDROXYLASE 1); ferulate 5-hydroxylase/ monooxygenase  Potri.003G009400 0.61 unknown protein Potri.012G016200 0.61  iqd21 (IQ-domain 21); calmodulin binding  Potri.003G059200 0.61  esterase/lipase/thioesterase family protein  Potri.001G265100 0.61  unknown protein  Potri.010G105700 0.61  FKF1 (FLAVIN-BINDING, KELCH REPEAT, F BOX 1); signal transducer/ two-component sensor/ ubiquitin-protein ligase  Potri.008G124000 0.61  unknown protein  Potri.006G263000 0.61  glycosyltransferase family 14 protein / core-2/I-branching enzyme family protein  Potri.002G228900 0.61  galactosyltransferase family protein  Potri.010G090800 0.61  leucine-rich repeat family protein  Potri.010G141600 0.61  chitinase  Potri.003G085000 0.61  FUNCTIONS IN: molecular function unknown  Potri.012G111500 0.61  cupin family protein  Potri.011G116300 0.61  protein kinase, putative  Potri.009G011100 0.61  LOCATED IN: membrane  Potri.016G113600 0.61  AUX1 (AUXIN RESISTANT 1); amino acid transmembrane transporter/ auxin binding / auxin influx transmembrane transporter/ transporter  Potri.005G053500 0.61  UNE12 (unfertilized embryo sac 12); DNA binding / transcription factor  Potri.010G038900 0.61  binding  Potri.001G062700 0.61  unknown protein  Potri.004G086100 0.61  LRR1; ATP binding / kinase/ protein serine/threonine kinase  Potri.006G087100 0.61  LAC17 (laccase 17); laccase  Potri.004G007600 0.61  aspartyl protease family protein  Potri.013G066200 0.61  UNE7 (unfertilized embryo sac 7); acetylglucosaminyltransferase/ transferase, transferring glycosyl groups  Potri.008G012000 0.61  unknown protein Potri.005G045500 0.61  protein kinase family protein  Potri.008G070400 0.60  ATHPP2C5; catalytic/ protein serine/threonine phosphatase  Potri.014G158200 0.60  unknown protein  Potri.006G228800 0.60  unknown protein  Potri.005G089200 0.60  ATM1 (ARABIDOPSIS THALIANA MYOSIN 1); motor  Potri.018G026900 0.60  epsin N-terminal homology (ENTH) domain-containing protein  Potri.002G132900 0.60  PARVUS (PARVUS); polygalacturonate 4-alpha-galacturonosyltransferase/ transferase, transferring glycosyl groups / transferase, transferring hexosyl groups  Potri.019G002100 0.60  aspartyl protease family protein  Potri.002G024400 0.60  unknown protein  Potri.010G187600 0.60  ESK1 (ESKIMO 1)  Potri.013G156200 0.60  LBD15 (LOB DOMAIN-CONTAINING PROTEIN 15)  Potri.003G183900 0.60  HCT (HYDROXYCINNAMOYL-COA SHIKIMATE/QUINATE HYDROXYCINNAMOYL TRANSFERASE   200  Appendix B.3 (continued) Entire list of genes co-expressed with PtCPU in developing P. trichocarpa wood transcriptomes at a PCC of at least 0.60  Poplar ID (v3.0) PCC MapMan Annotation Potri.002G153700 0.60  armadillo/beta-catenin repeat family protein  Potri.017G081600 0.60  glutaredoxin family protein  Potri.018G142800 0.60  epsin N-terminal homology (ENTH) domain-containing protein / clathrin assembly protein-related  Potri.002G146300 0.60  transketolase, putative  Potri.012G124400 0.60  GRL (GNARLED); transcription activator  Potri.009G075400 0.60  protein kinase family protein  Potri.002G025200 0.60  iqd32 (IQ-domain 32); calmodulin binding  Potri.001G075600 0.60  vacuolar sorting receptor, putative  Potri.001G258400 0.60  zinc finger (CCCH-type) family protein  Potri.001G332200 0.60  plastocyanin-like domain-containing protein  Potri.018G059100 0.60  unknown protein  Potri.011G058400 0.60  ANAC073 (ARABIDOPSIS NAC DOMAIN CONTAINING PROTEIN 73); transcription activator/ transcription factor  Potri.008G197000 0.60  SPL7 (SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 7); DNA binding / transcription factor  Potri.005G141300 0.60  unknown protein     201     Appendix B.4 Wood anatomy for wild-type (WT) and transgenic poplar 35S:PtCPU over-expression lines. Stem cross-sections (50 µm) of WT (left) and a representative transgenic poplar 35S:PtCPU over-expression line (line 14, right) stained with 0.25% toluidine blue. No phenotypic differences in wood anatomy were evident. Scale bar is 10 µm.  202    1 cm WT 35S:YFP-PtCPU  Appendix B.5 Over-expression of 35S:YFP-PtCPU in Arabidopsis (Col-0) produced rounded leaves and shorter siliques compared to wild-type (WT) (top panel). Cellular phenotypes in the vascular bundle were not observed from cross-section analysis of basal stems from six-week-old T2 plants stained with toluidine blue-O (bottom panel). Scale bar represents 50 µm. IFF: interfascicular fibres; Ph: phloem; X: xylem.  Ph X IFF Ph X IFF 203      Appendix B.6 Phenotypes of ten-day-old seedlings of wild-type, cpu, cmu1cmu2, and cpucmu1cmu2. Col-0 wild-type (A), cpu (B), cmu1cmu2 double mutant (C), cmu1cmu2 double mutant from Liu et al. (2016), designated here as cmu1-1cmu2 (D), and cpucmu1cmu2 triple mutant (E) were grown on half-strength Murashige and Skoog with plates upright. Root skewing is apparent in the cmu1cmu2 double mutant and the cpucmu1cmu2 triple mutant. Scale bar is 2 cm. A B D E C 204                         170       180       190       200       210       220       230       240                     ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| PtNPF8.1     TTGCAGACAAGCGAACTACTGGAGGATGGAAAGCTGCTCCTTTTATTATAGTGAATGAAGTTGCTGAGAGGCTAGCATTT  PtaNPF8.1    CTGCAGACAAGCGAACTACTGGAGGATGGAAAGCTGCTCCTTTTATTATAGTGAATGAAGTTGCTGAGAGGCTAGCATTC  PtNPF8.1.2   TTGCAGACAAGAGAACTACTGGAGGATGGAAAGCTGCTCCTTTTATTATAGTGAATGAAGTTGCTGAGAGGTTAGCATTT  PtaNPF8.1.2  TTGCAGACAAGAGAACTACTGGAGGATGGAAAGCTGCTCCTTTTATTATAGTGAATGAAGTTGCTGAGAGGTTAGCATTT                        250       260       270       280       290       300       310       320                     ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| PtNPF8.1     TTTGGTATAGCTGTGAACATGGTAGCTTACTTGGTCTTTGAAATGCATCAGTCACTTCCAAATGCTGCAACTCATGTGAC  PtaNPF8.1    TTTGCTATAGCTGTGAACATGGTGGCTTACTTGGTCTTTGAAATGCATCAGTCACTTCCAAATGCTGCAGCTCATGTGAC  PtNPF8.1.2   TATGCTATAGCTGTGAACATGGTAGCTTACTTGGTCTTTCAAATGCATCAATCACTTCCAGATGCTGCAACTCATGTGAC  PtaNPF8.1.2  TATGCTATAGCTGTGAACATGGTAGCTTACTTGGTCTTTCAAATGCACCAATCACTTCCAGATGCTGCAACTCATGTGAC                        330       340       350       360       370       380       390       400                     ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| PtNPF8.1     TGACTGGATTGGAGCTGCCTATGTCCTCACTCTCTTTGGAGCGTTTTGTGCTGATGCTTACCTGGGCCGATTCAGGACCA  PtaNPF8.1    TGACTGGATTGGAGCTGCCTATGTCCTCACTCTCTTTGGAGCGTTTTGTGCTGATGCTTACCTCGGCCGATTCAGGACCA  PtNPF8.1.2   TGACTGGATTGGAGCTGCTTTTGTCCTTACACTTTTTGGAGCGTTTTGTGCCGATGCTTACCTGGGCCGATTCAAGACCA  PtaNPF8.1.2  TGACTGGATT--------------------------------------------------------------CAAGACCA                        410       420       430       440       450       460       470       480                     ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| PtNPF8.1     TCATTGTTTTCTCTTGCATCTATACCGTAGGAATGGTATTATTGACACTCTCAGCTTCTATAGACAGCTTACGTCCACCA  PtaNPF8.1    TCATTGTTTTCTCTTGCATCTATACAGTAGGAATGGTATTATTGATACTCTCAGCTTCTATAGACAGCTTACGTCCACCA  PtNPF8.1.2   TCATTATTTTCTCTTGCATCTATGCAGTGGGAATGGTACTATTGACGCTCTCAGCTTCTATAGACAGCTTACGTCCACCA  PtaNPF8.1.2  TCGTTATTTTCTCTTGCATCTATACAGTGGGAATGGTATTATTGACGCTCTCAGCTTCTATAGACAGCTCACGTCCACCA                                    ....|. PtNPF8.1     AAATGC  PtaNPF8.1    AAATGC  PtNPF8.1.2   CAATGC  PtaNPF8.1.2  CGATGC       PtNPF6.1 PtaNPF6.1 PtNPF6.2 PtaNPF6.2      PtNPF6.1 - 0.976 0.934 0.743 PtaNPF6.1 0.976 - 0.924 0.736 PtNPF6.2 0.934 0.924 - 0.776 PtaNPF6.2 0.743 0.736 0.776 -        B A PtNPF6.1 PtaNPF6.1 PtNPF6.2 PtaNPF6.2 PtNPF6.1PtaNPF6.1PtNPF6PtaNPF6.2 PtNPF6.1PtaNPF6.1PtNPF6PtaNPF6.2 PtNPF6.1PtaNPF6.1PtNPF6PtaNPF6.2 PtNPF6.1PtaNPF6.1PtNPF6PtaNPF6.2 Appendix C. Additional materials for Chapter 4          Appendix C.1 Sequence information for PtNPF6.1 and PtNPF6.2. (A) Partial nucleotide sequence alignment for PtNPF6.1 and PtNPF6.2, and PtaNPF6.1 and PtaNPF6.2 from 717 (P. tremula x P. alba; Pta). Pta sequences were obtained from Aspen DB (aspendb.uga.edu). The region used to construct the hairpin RNA cassette is underlined. (B) Sequence identity matrix for NPF6.1 and NPF6.2 sequences in (A).  205    Appendix C.2 Unidentified anthocyanin peaks detected from transgenic poplar RNAi PtNPF6.1  lines exposed to natural outdoor high-light/UV-B radiation  Line Peak Area (mAU*min) g-1 DW Peak 2 Peak 3    WT 30.10 (5.02) 9.27 (1.92) A4 37.33 (5.70) 9.28 (1.55) B2 30.09 (5.15) 7.06 (1.53) B4 42.15 (10.78) 13.34 (5.63) D5 36.73 (7.83) 8.36 (1.56)    Mean and standard error of the mean (in parentheses) for three to four clonal replicates.  No significant differences from WT based on a Student’s t-test (P < 0.05).  DW: dry weight.                206   Appendix C.3 Phenotypes of wild-type (WT) and RNAi PtNPF6.1 lines fertilized with high (HN, 10 mM) or low (LN, 0.10 mM) ammonium nitrate for eight weeks. Chlorosis is evident in leaves of trees under the 0.10 mM treatment. Images on the right are of a representative line to show the differences in leaf chlorophyll accumulation.                         HN (10 mM NH4NO3) WT   A4  B2 LN (0.1 mM NH4NO3) WT   A4  B2 207   Appendix C.4 Wild-type (WT) and RNAi PtNPF6.1 lines fertilized with 10 mM or 0.10 mM ammonium nitrate for eight weeks did not exhibit biomass differences   WT RNAi line A4 RNAi line B2  10 mM NH4NO3 height (cm) 72.50 (1.85) 67.50 (0.84) 67.75 (2.17) stem diameter (mm) 6.74 (0.23) 6.71 (0.04) 6.24 (0.06) shoot mass 2.75 (0.29) 2.22 (0.03) 2.12 (0.09) total root mass (g DW) 4.69 (0.65) 3.87 (0.10) 3.46 (0.26) root:shoot ratio 1.68 (0.10) 1.74 (0.04) 1.63 (0.12)  0.10 mM NH4NO3 height (cm) 64.38 (2.56) 62.13 (0.88) 58.88 (1.28) stem diameter (mm) 6.09 (0.07) 6.03 (0.05) 5.37 (0.08) shoot mass 3.03 (0.38) 2.61 (0.15) 2.41 (0.13) total root mass (g DW) 5.47 (0.16) 4.37 (0.57) 3.89 (0.41) root:shoot ratio 1.87 (0.19) 1.66 (0.16) 1.60 (0.12)  10 mM ammonium nitrate: high nitrogen treatment; 0.10 mM ammonium nitrate: low nitrogen treatment. Mean and standard error of the mean (in parentheses) of four clonal replicates.  No significant differences from WT based on a Student’s t-test (P < 0.05).  DW: dry weight.              

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