Forestry, Faculty of
DSpace
UBCV
Guevara Rozo, Sydne
Doctor of Philosophy - PhD
University of British Columbia
Lignocellulosic feedstocks are pivotal to the transition to a biobased economy, however, the plant cell wall evolved to withstand stresses, microbial attack, and degradation, and therefore presents significant challenges for industrial biomass processing. Structural modifications of xylan, the major hemicellulose in angiosperms, have been shown to positively affect cell wall deconstruction by interfering with xylan-cellulose-lignin interactions. In contrast, the role of mannan, a major hemicellulose commonly found in gymnosperms (minor hemicellulose in angiosperm cell walls), which also interacts with cellulose and lignin, is still poorly understood. Genes involved in the mannan biosynthetic pathway have been described in Arabidopsis, where cellulose synthase-like family A (CSLA) proteins synthesize the mannan backbone. The functional validation of orthologous genes responsible for the synthesis of this polysaccharide in woody plants and the effects of changing its content on secondary cell wall stability remain largely unstudied. In this study, I identify genes from woody plants that are responsible for mannan biosynthesis and evaluate the effect of modifying the expression of these genes on plant growth and cell wall structure. Using a combination of bioinformatic and phylogenetic tools, I identified putative genes from spruce (Picea engelmannii × glauca × sitchensis; PG29) and poplar (Populus tremula × Populus alba; 717) that encode CSLAs and GDP-mannose pyrophosphorylase (GMP) proteins. I functionally characterized these genes by evaluating if they were able to restore mannan content in the Arabidopsis csla 2,3,9 and vtc1-1 mutants, respectively. Then, I evaluated their function in wood by mutating the poplar CSLA and GMP candidate genes via CRISPR Cas9 or ectopically expressing the spruce gene candidates in poplar. In this work, I demonstrate that mannan content can be manipulated in tree stems either by altering the expression of CSLA or GMP encoding genes, without significantly affecting plant growth. This research provides new avenues to investigate the contribution of mannan alongside other common hemicelluloses in maintaining plant cell wall functionality.
UNRAVELING MANNAN BIOSYNTHESIS IN POPLAR AND SPRUCE: FUNCTIONAL CHARACTERIZATION OF CELLULOSE SYNTHASE-LIKE FAMILY A (CSLA) AND GUANOSINE-DIPHOSPHATE-MANNOSE (GMP) GENES IN POPLAR by Sydne Guevara Rozo B.Sc., Universidad Nacional de Colombia, 2015 M.Sc., The University of Alberta, 2018 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Forestry) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) May 2024 © Sydne Guevara Rozo, 2024 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: Unraveling mannan biosynthesis in Poplar and Spruce: functional characterization of Cellulose Synthase-Like family A (CSLA) and Guanosine-diphosphate-Mannose (GMP) genes in Poplar submitted by Sydne Guevara Rozo in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Forestry Examining Committee: Dr. Shawn D. Mansfield, Professor, Wood Science, UBC Supervisor Dr. Lacey Samuels, Professor, Botany, UBC Supervisory Committee Member Dr. George Haughn, Professor, Botany, UBC Supervisory Committee Member Dr. Harry Brumer, Professor, Michael Smith Laboratories, UBC Supervisory Committee Member Dr. Joerg Bohlmann, Professor, Michael Smith Laboratories, UBC University Examiner Dr. Andrew Riseman, Professor, Plant Science, UBC University Examiner iii Abstract Lignocellulosic feedstocks are pivotal to the transition to a biobased economy, however, the plant cell wall evolved to withstand stresses, microbial attack, and degradation, and therefore presents significant challenges for industrial biomass processing. Structural modifications of xylan, the major hemicellulose in angiosperms, have been shown to positively affect cell wall deconstruction by interfering with xylan-cellulose-lignin interactions. In contrast, the role of mannan, a major hemicellulose commonly found in gymnosperms (minor hemicellulose in angiosperm cell walls), which also interacts with cellulose and lignin, is still poorly understood. Genes involved in the mannan biosynthetic pathway have been described in Arabidopsis, where cellulose synthase-like family A (CSLA) proteins synthesize the mannan backbone. The functional validation of orthologous genes responsible for the synthesis of this polysaccharide in woody plants and the effects of changing its content on secondary cell wall stability remain largely unstudied. In this study, I identify genes from woody plants that are responsible for mannan biosynthesis and evaluate the effect of modifying the expression of these genes on plant growth and cell wall structure. Using a combination of bioinformatic and phylogenetic tools, I identified putative genes from spruce (Picea engelmannii × glauca × sitchensis; PG29) and poplar (Populus tremula × Populus alba; 717) that encode CSLAs and GDP-mannose pyrophosphorylase (GMP) proteins. I functionally characterized these genes by evaluating if they were able to restore mannan content in the Arabidopsis csla 2,3,9 and vtc1-1 mutants, respectively. Then, I evaluated their function in wood by mutating the poplar CSLA and GMP candidate genes via CRISPR Cas9 or ectopically expressing the spruce gene candidates in poplar. In this work, I demonstrate that mannan content can be manipulated in tree stems either by altering the expression of CSLA or GMP encoding genes, without significantly affecting plant iv growth. This research provides new avenues to investigate the contribution of mannan alongside other common hemicelluloses in maintaining plant cell wall functionality. v Lay Summary Cell walls provide structural support and protection to plants; they are divided into primary and secondary cell walls, and are composed of different polysaccharides, including hemicelluloses. One of the major hemicelluloses in plants is mannan, which plays an essential role in plant development as well as in maintaining seed mucilage structure. Despite being one of the primary hemicelluloses found in trees, the mechanisms governing its biosynthesis in woody plants and the role it plays in secondary cell wall integrity remain largely unknown. By using genetic engineering tools, I identified mannan biosynthesis-related genes and explored how the modification of this polysaccharide affects plant growth and cell wall structure. We concluded that mannan is not essential for secondary cell wall formation and that deficiencies in this polysaccharide do not significantly affect plant growth or development. These findings form the foundation to our understanding of the contribution of mannan to cell wall architecture and provide valuable information for future efforts to breed or genetically engineer trees for improved industrial use. vi Preface Chapter 1 contains portions of a published review article that I co-authored: de Vries, L., Guevara-Rozo, S., Cho, M., Liu, L.-Y., Renneckar, S., & Mansfield, S. D. (2021). Tailoring renewable materials via plant biotechnology. Biotechnology for Biofuels, 14(1), 167. https://doi.org/10.1186/s13068-021-02010-z. Specifically, the sections describing the xyloglucan, mannan, and xylan function, and biological, and industrial relevance were written by me, and edited by Dr. Shawn Mansfield and the other co-authors. The research questions from Chapter 3 and Chapter 4 were formulated by me with assistance from Dr. Shawn Mansfield. I conducted all experimental procedures performed in these chapters, with the technical support and guidance of Dr. Faride Unda from the University of British Columbia, Dr. Chung-Jui Tsai from the University of Georgia, Dr. Ian Black from the Complex Carbohydrate Research Center at the University of Georgia, and Dr. Yoshihisa Yoshimi from the University of Cambridge. Most of the experiments involved in the carbohydrate gel electrophoresis (PACE) analysis in Chapter 3, were performed in the laboratory of Dr. Paul Dupree at the University of Cambridge. Multiple undergraduate students were involved in the genotyping of the Arabidopsis and poplar transgenic lines, including Ruby Hsu, Morgan Leon, Rebecca Saunders, and Radmnesh Khalili. vii Table of Contents Abstract ......................................................................................................................................... iii Lay Summary .................................................................................................................................v Preface ........................................................................................................................................... vi Table of Contents ........................................................................................................................ vii List of Tables .............................................................................................................................. xiii List of Figures ...............................................................................................................................xv List of Symbols .......................................................................................................................... xvii List of Abbreviations ............................................................................................................... xviii Acknowledgements .................................................................................................................. xxiii Dedication ...................................................................................................................................xxv Chapter 1: Introduction ................................................................................................................1 1.1 Plant cell walls ................................................................................................................ 1 1.2 Woody feedstocks ........................................................................................................... 3 1.3 Hemicelluloses ................................................................................................................ 6 1.3.1 Xyloglucan .................................................................................................................. 6 1.3.1.1 Xyloglucan structure and classification .............................................................. 6 1.3.1.2 Xyloglucan biosynthesis ..................................................................................... 7 1.3.1.3 Biological and industrial relevance of xyloglucan .............................................. 8 1.3.2 Xylan ........................................................................................................................... 9 1.3.2.1 Xylan structure and classification ....................................................................... 9 1.3.2.2 Xylan biosynthesis ............................................................................................ 10 1.3.2.3 Biological and industrial relevance of xylan .................................................... 11 viii 1.3.3 Mannan ..................................................................................................................... 13 1.3.3.1 Mannan structure and classification .................................................................. 13 1.3.3.2 Mannans diversity in plants and algae .............................................................. 14 1.3.3.3 Mannan biosynthesis ......................................................................................... 16 1.3.3.4 Biological relevance of mannans ...................................................................... 19 1.3.3.5 Industrial relevance of mannans ....................................................................... 22 1.4 Research questions ........................................................................................................ 23 Chapter 2: Materials and Methods ............................................................................................26 2.1 Bioinformatic and phylogenetic analyses ..................................................................... 26 2.1.1 Identification of putative CSLAs .............................................................................. 26 2.1.2 Identification of putative GMPs ................................................................................ 26 2.2 Gene expression constructs ........................................................................................... 27 2.2.1 Gene isolation from plant material ........................................................................... 27 2.2.2 CSLA plant expression constructs ............................................................................ 27 2.2.3 GMP plant expression constructs .............................................................................. 28 2.3 Gene expression constructs ........................................................................................... 29 2.3.1 gRNA design ............................................................................................................. 29 2.3.2 CRISPR Cas9 cloning ............................................................................................... 29 2.4 Arabidopsis mutants transformation ............................................................................. 31 2.4.1 Agrobacterium-mediated transformation .................................................................. 31 2.4.2 Genomic screening of Agrobacterium-transformed Arabidopsis plants .................. 31 2.5 Arabidopsis plant phenotyping ..................................................................................... 32 2.5.1 Carbohydrates content .............................................................................................. 32 ix 2.5.2 Alcohol insoluble residue (AIR) ............................................................................... 32 2.5.3 Hemicellulose extraction .......................................................................................... 33 2.5.4 Linkage Analysis ...................................................................................................... 34 2.5.5 Preparation of soluble hemicelluloses and PACE analysis ....................................... 36 2.6 Poplar transformation and genomic screening .............................................................. 36 2.6.1 Agrobacterium-mediated transformation .................................................................. 36 2.6.2 Genomic screening .................................................................................................... 37 2.6.3 Gene expression analysis of poplar GMPs and spruce CSLA .................................. 37 2.6.4 Production of CRISPR Cas9 poplar lines ................................................................. 38 2.6.5 Genomic screening of CRISPR poplar lines ............................................................. 39 2.6.6 Determination of mutant spectrums in CRISPR poplar lines ................................... 39 2.7 Poplar phenotyping ....................................................................................................... 39 2.7.1 Growth conditions and measurement........................................................................ 39 2.7.2 Carbohydrates and lignin content ............................................................................. 40 2.7.3 Cross-sectional staining ............................................................................................ 40 2.7.4 Mannan antibody detection ....................................................................................... 41 2.8 Statistical analysis ......................................................................................................... 41 Chapter 3: Characterization and expression of CSLAs ...........................................................43 3.1 Introduction ................................................................................................................... 43 3.2 Bioinformatics of putative CSLAs................................................................................ 45 3.3 Putative CSLAs from spruce and poplar increase mannose content in the Arabidopsis csla2,3,9 triple mutant............................................................................................................... 49 x 3.4 PgCSLA1 increases 4-Man and 4-Glu linkage content in the Arabidopsis csla2,3,9 triple mutant .............................................................................................................................. 50 3.5 PgCSLA1 produces a random patterned glucomannan in Arabidopsis csla2,3,9 triple mutant ....................................................................................................................................... 53 3.6 CRISPR poplar knockouts of putative CSLA ............................................................... 56 3.6.1 Generation of double and single knockout CSLA poplar lines ................................ 56 3.6.2 Mutation of CSLAs in hybrid poplar does not affect plant growth .......................... 60 3.6.3 Poplar CSLA knockouts exhibit changes in mannan cell wall content .................... 61 3.7 Expression of the putative CSLA from spruce in poplar .............................................. 64 3.7.1 PgCSLA1 expression in poplar does not lead to plant yield penalty ........................ 64 3.7.2 PgCSLA1 expression increases mannan content in poplar stems ............................ 65 3.8 Discussion ..................................................................................................................... 66 3.8.1 CSLA family members in poplar trees and their contribution to mannan biosynthesis ................................................................................................................................... 67 3.8.2 CSLA family in PG29 ............................................................................................... 68 3.8.3 Implications of eliminating mannan from poplar stem by mutating CSLAs ............ 69 3.8.4 Implications of expressing PgCSLA1 in Arabidopsis .............................................. 70 3.8.5 Implications of increasing mannan from poplar stems by expressing CSLAs ......... 72 3.9 Conclusion .................................................................................................................... 72 Chapter 4: Characterization and expression of GMPs ............................................................73 4.1 Introduction ................................................................................................................... 73 4.2 Phylogenetic analysis of putative GMPs ...................................................................... 74 xi 4.3 Putative GMPs from spruce and poplar increased mannose content in the Arabidopsis vtc1-1 mutant ............................................................................................................................ 78 4.4 CRISPR poplar knockouts of putative GMPs ............................................................... 79 4.4.1 Generation of GMP poplar knockout lines ............................................................... 79 4.4.2 Mutation of PtaGMP1 in hybrid poplar does not affect plant growth ...................... 82 4.4.3 Poplar PtaGMP1 knockout lines have reduced mannan content .............................. 83 4.5 Expression of putative GMP from spruce in poplar ..................................................... 85 4.5.1 Expression of PgGMP1 in poplar does not affect plant growth ............................... 85 4.5.2 PgGMP1 increases mannan content in poplar stems ................................................ 86 4.6 Discussion ..................................................................................................................... 87 4.6.1 Poplar AtVTC1 orthologs ......................................................................................... 87 4.6.2 White spruce P29 AtVTC1 orthologs ....................................................................... 87 4.6.3 Implication of mutating AtVTC1 orthologs in poplar............................................... 88 4.6.4 Implication of expressing PG29 AtVTC1 orthologs in poplar .................................. 89 4.7 Conclusion .................................................................................................................... 90 Chapter 5: Conclusions and future directions ..........................................................................92 5.1 Major findings in Chapter 3 and Chapter 4 .................................................................. 92 5.2 Mannan removal as a potential strategy to reduce biomass recalcitrance .................... 94 5.3 Mannan structural modifications as a potential strategy to improve biomass processing ....................................................................................................................................... 94 5.4 Mannan as a signal molecule and its implications in plant abiotic stress tolerance ..... 97 5.5 Mannan and other polymers in the cell wall ................................................................. 98 5.6 Mannan trafficking ........................................................................................................ 99 xii References ...................................................................................................................................101 Appendices ..................................................................................................................................129 Appendix A - Potri.008G026400 transcript sequence from poplar 717 ................................. 129 xiii List of Tables Table 2.1 Primers used for cloning of gene candidates ................................................................ 29 Table 2.2 Primers used for CRISPR Cas9 cloning ....................................................................... 30 Table 2.3 Primers used for qPCR of poplar GMPs. ...................................................................... 38 Table 3.1 Total of transmembrane domains (TMD) in CSLAs of Arabidopsis and putative CSLAs of poplar and spruce ......................................................................................................... 46 Table 3.2 Expression of CSLA poplar candidate genes in xylem of Galactinol Synthase (GolS) transgenic poplar and WT trees from Mansfield lab transcriptome database ............................... 47 Table 3.3 Structural polysaccharides composition of Arabidopsis WT plants, csla2,3,9 triple mutants, and csla2,3,9 mutants complemented with CESA7::PtaCSLA1 .................................... 50 Table 3.4 Structural polysaccharides composition of Arabidopsis WT plants, csla2,3,9 triple mutants, and csla2,3,9 mutants complemented with CESA7::PgCSLA1 ..................................... 50 Table 3.5 Overview of the targeted CSLA1 and CSLA2 locus in P. tremula and P. alba ........... 57 Table 3.6 Summary of mutations observed in single and double poplar knockout lines. ............ 60 Table 3.7 Mean growth measurement of single and double poplar CSLA knockout lines. ......... 60 Table 3.8 Structural cell wall carbohydrates in the xylem of single and double CSLA poplar knockouts ...................................................................................................................................... 62 Table 3.9 Lignin content in single and double poplar CSLA knockout lines ............................... 62 Table 3.10 Mean growth measurement poplar lines expressing PgCSLA1 .................................. 65 Table 3.11 Lignin content in poplar transformants expressing PgCSLA1 .................................... 66 Table 3.12 Structural cell wall carbohydrates of poplar lines expressing PgCSLA1. ................... 66 Table 4.1 Expression of GMP poplar candidate genes in xylem from the Galactinol Synthase (GolS) transgenic poplar and WT trees from the Mansfield lab transcriptome database ............. 76 xiv Table 4.2 Structural polysaccharides composition of Arabidopsis WT plants, vtc1-1 mutants, and vtc1-1 mutants complemented with CESA7::PtaGMP1 and CESA7::PgGMP1 .......................... 78 Table 4.3 Overview of the targeted CSLA1 and CSLA2 locus in P. tremula and P. alba ........... 80 Table 4.4 Summary of mutations observed in single PtaGMP1 poplar CRISPR Cas9 knockout lines ............................................................................................................................................... 81 Table 4.5 Mean growth measurement poplar lines expressing PgCSLA1 .................................... 82 Table 4.6 Lignin content in PtaMGP1 knockout poplar lines ...................................................... 83 Table 4.7 Structural cell wall sugars in the xylem of PtaGMP1 knockout poplar lines .............. 83 Table 4.8 Mean growth measurement of transgenic poplar lines expressing CESA7::PgGMP1…....................................................................................................................................................... 85 Table 4.9 Lignin content in lines expressing CESA7::PgGMP1 .................................................. 86 Table 4.10 Structural cell wall carbohydrates of poplar lines expressing CESA7::PgGMP1 ...... 86 xv List of Figures Figure 1.1 Structural model of plant secondary cell wall. .............................................................. 3 Figure 1.2 Mannan structures and nomenclature ........................................................................ 114 Figure 1.3 Mannan biosynthesis ................................................................................................... 18 Figure 3.1 Phylogenetic tree of the potential poplar and spruce CSLA orthologs. ...................... 46 Figure 3.2 Multiple sequence alignment of Arabidopsis CSLAs and the putative CSLAs from poplar and spruce .......................................................................................................................... 48 Figure 3.3 Hemicellulose extracts................................................................................................. 52 Figure 3.4 Linkage analysis of hemicellulose extracts using stems of wild-type plants. ............. 52 Figure 3.5 Linkage analysis of csla2,3,9 complemented lines using PgCSLA1 ........................... 53 Figure 3.6 Mannan backbone structure in csla2,3,9 PgCSLA1-complemented lines digested with CjMan26A..................................................................................................................................... 55 Figure 3.7 Mannan backbone structure in csla2,3,9 PgCSLA1-complemented lines digested with AnGH5 .......................................................................................................................................... 56 Figure 3.8 Gene diagram of putative poplar CSLA genes and target CRISPR sites .................... 57 Figure 3.9 Double-knockout via CRISPR Cas9 of putative poplar CSLA genes. Three poplar lines were obtained carrying mutations in both PtaCSLA1 and PtaCSLA2 .................................. 58 Figure 3.10 Single knockout via CRISPR Cas9 of putative poplar CSLA1 gene. One poplar line was obtained carrying a unique mutation in PtaCSLA1 ............................................................... 59 Figure 3.11 Immunofluorescence labelling of xylem tissue from wild-type poplar trees, single, and double CSLA knockout lines ................................................................................................. 63 Figure 3.12 Expression of PgCSLA1 in poplar lines carrying the CESA7::PgCSLA1 construct . 64 Figure 4.1 Phylogenetic tree of the potential Arabidopsis VTC1 orthologs ................................. 75 xvi Figure 4.2 Multiple sequence alignment of Arabidopsis VTC1 and the putative GMPs from poplar and spruce .......................................................................................................................... 77 Figure 4.3 Gene diagram of putative poplar GMP genes and target CRISPR sites...................... 79 Figure 4.4 Single-knockout of PtaGMP1 via CRISPR Cas9.. ..................................................... 81 Figure 4.5 Expression of PtaGMP1and PtaGMP2 in wild-type and knockout lines ................... 84 xvii List of Symbols cm Centimetres °C Degrees Celsius Da Dalton DSAC Degree of substitutions by acetyl groups g Grams h Hours m Metres µl Microlitres μM Micromolar mg Milligrams mL Millilitres min Minute M Molar nM Nanomolar mM Millimolar % Percentage pmol Picomoles pH Potential hydrogen g Relative centrifuge force rpm Rotations per minute v/v Volume to volume xviii List of Abbreviations AaXEG Aspergillus aculeatus Xyloglucanase ABRC Arabidopsis Biological Resource Center AcGGM Acetylated Galactoglucomannan AGAL α-galactosidases AIR Alcohol Insoluble Residue Ak Amorphophallus konjac AnGH5 Aspergillus nidulans GH5 ANOVA Analysis of Variance Ara Arabinose AsA Ascorbic Acid attB Attachment site on Bacteria AX Arabinoxylans AXY Altered Xyloglucan BA 6-Benzylaminopurine BGAL β-galactosidase βGGM β-GalactoGlucomannan BLAST Basic Local Alignment Search Tool Cc Coffea canephora Cd Cadmium CE Esterases CEL Cellulases xix CjMan26A Cellvibrio japonicus Man26A CRISPR Cas9 Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9 CSL Cellulose Synthase Like CTAB Cetyltrimethylammonium Bromide CURS2 Curcumin Synthase 2 DARX Deacetylase on Arabinosyl Sidechain of xylan DCM Dichloromethane DCS Diketide-CoA Synthase DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid ESK Eskimo EtOH Ethanol FPKM Fragments Per Kilobase of transcript per Million mapped reads FUT Fucosyltransferase FWD Forward Gal Galactose GAX Glucuronoarabinoxylans GC/MS Gas chromatography/Mass spectrometry GDP-Man Guanosine-diphosphate-Mannose GFP Green Fluorescent Protein GGMO Galactoglucomannan oligosaccharides xx GH Glycosyl Hydrolases Glc Glucose GlcA Glucuronic Acid GMP Mannose-1-phosphate guanyltransferase GMX Glucuronoxylan Methyltransferases gRNA Guide RNA GT Glycosyltransferase GUX Glucuronic Acid Substitution of Xylan GX Glucuronoxylans HEK Human Embryonic Kidney HPLC High-Performance Liquid Chromatography INRA National Research Institute for Agriculture, Food and Environment KJC Konjac proteins KO Knockout KOH Potassium hydroxide MAGT Mannan α-Galactosyl Transferase Man Mannose MAN Mannanase mCherry Monoclonal Cherry (Red) Protein MEGA-X Molecular Evolutionary Genetics Analysis software MeGlcA (Methyl)Glucuronic Acid xxi MOAT Mannan O-acetyltransferases MS Murashige and Skoog basal medium MSR Mannan Synthesis Related protein MUCI10 Mucilage-Related10 MUSCLE MUltiple Sequence Comparison by Log-Expectation NAA 1-Naphthaleneacetic Acid NaEDTA Ethylenediaminetetraacetic acid disodium salt dihydrate NaOH Sodium hydroxide NMR Nuclear Magnetic Resonance PACE Polysaccharide Analysis using Carbohydrate gel Electrophoresis PAM Protospacer Adjacent Motif PCR Polymerase Chain Reaction PCW Primary Cell Wall PG29 Picea engelmannii × glauca × sitchensis, genotype PG29 PMAAs Partially Methylated Alditol Acetates qPCR Quantitative real-time PCR REV Reverse Rha Rhamnose RNA Ribonucleic Acid RWA Reduced Wall Acetylation S2 Drosophila Schneider 2 cells SCW Secondary Cell Wall xxii TBL Trichome Birefringence-like TDZ Thidiazuron TFA Trifluoracetic Acid TMD Transmembrane Domains TMHMM Transmembrane Hidden Markov Model tool Tris HCL Tris Hydrochloride VND Vascular-Related NAC-Domain VTC Vitamin C Defective WT Wild Type XAT Xylan Arabinosyltransferases XEH Xyloglucan Endohydrolase XET/XTH Xyloglucan:Endo Transglycosylase/Hydrolase XLT Xylan Galactosyltransferase XOAT Xylan O-acetyltransferases XXT Xyloglucan xylosyltransferases XyG Xyloglucan Xyl Xylose xxiii Acknowledgements Firstly, I want to thank my supervisor, Dr. Shawn Mansfield, for his mentorship and for giving me the opportunity to work in his lab. Shawn, I truly appreciate the freedom you gave me during my research journey and the different opportunities you provided me to collaborate with other colleagues and receive training in many areas that allowed me to succeed in my project. Thanks for your support and guidance during all these years, I have grown immensely as a scientist and as a person as a result of all this. I wish to thank the members of my committee, Dr. Lacey Samuels, Dr. Harry Brumer, and Dr. George Haughn, for their insights and suggestions throughout my degree. I also want to thank the many amazing colleagues with whom I had the pleasure to work, who helped me to learn different techniques that I am proud to now know, specifically, thanks to Dr. Faride Unda, Dr. CJ Tsai, Dr. Ian Black, and Dr. Yoshihisa Yoshimi. Thank you to the members of the Mansfield lab, past and present, for providing me with a welcoming and supportive lab environment to do research, learn, teach, and be curious. I thank Melike Karaca, Xinyi Huang, Faride Unda, Lisanne de Vries, Madison Kaplen, Emilia Muirragui, and Elizabeth Mahon for your support and friendship. Melike, it has been a pleasure navigating these years with you. Thank you for your kindness, for always keeping me well-fed with delicious food, and for being a super fun and smart friend with whom to talk. In addition, I am immensely thankful for the love and support of my family. Thank you to my lovely husband, Nicolas Salcedo, who celebrated every single one of my milestones and provided me with his unconditional support throughout my degree. Thanks for our scientific discussions, your patience, positivity, and encouragement. You have undoubtedly made of my PhD experience a time of beautiful memories, even in the hardest times. Thank you, Mom, Nata, xxiv and Santi, for always reminding me of my capabilities and encouraging me to always go further than I think is possible, you are the best example I have of perseverance and kindness. Nata, thank you for moving to Vancouver, I cannot imagine the last years without you, thanks for always listening to my worries and providing me with your positive advice. Mami y Santi, gracias por estar tan cerca incluso desde lejos, gracias por siempre acordarme de cuál es mi norte. Your love and encouragement have been the cornerstone of my success, and I will always feel very fortunate to have you all by my side. xxv Dedication To Nico1 Chapter 1: Introduction In the pursuit of transitioning towards a biobased economy, understanding the synthesis and interactions of polymers within plant cell walls is crucial. This thesis aims to elucidate the genetic mechanisms underlying mannan biosynthesis and explore the impact of modulating mannan content on tree growth, thereby advancing our understanding of plant cell wall architecture and highlighting the potential that the genetic modification of its components may have in enhancing industrial biomass processing. 1.1 Plant cell walls Cell walls are complex structures that provide structural support and protection to plants; they are involved in critical processes such as water transport, cell-to-cell adhesion, and intercellular communication (Meents et al., 2018; Zhang et al., 2021; Zhong et al., 2019). Plant cell walls are commonly classified into two major types: primary walls (PCW) and secondary cell walls (SCW). PCW are formed during cell growth and are deposited to the outside of the plasma membrane. They provide support to plants during development and are primarily composed of cellulose (~25%), hemicelluloses (~30%), pectins (~40%), and glycosylated proteins (~2-5%) (Cosgrove, 2023; de Vries et al., 2021). SCWs account for most of the plant biomass; they are deposited after cell expansion and provide structural support and protection to plants (Meents et al., 2018; Zhong et al., 2019). The SCWs of xylem tissue in trees are primarily composed of cellulose (~40-60%), hemicelluloses (~30-45 %), and lignin (~15-30 %) (Fig. 1.1; de Vries et al., 2021; Meents et al., 2018). The composite of cellulose, hemicellulose, and lignin is known as lignocellulosic biomass and represents an important resource in the production of building materials, textiles, fuels, pulp, and 2 chemicals (Nanda et al., 2015). To produce these commodity compounds, lignocellulosic biomass needs to be subjected to chemical or physical degradation and/or pretreatments that ultimately break down the lignocellulosic matrix releasing the individual constituents that subsequently can be used to produce different products (Nanda et al., 2015). Despite the vast uses and society’s dependence on lignocellulosic biomass, the inherent complexity of plant cell walls makes them highly recalcitrant, which in turn imposes significant challenges to processing efficiency (Nanda et al., 2015). Factors such as lignin content and structure, cellulose crystallinity and degree of polymerization, wall accessibility, biomass porosity, and hemicellulose content are known to significantly contribute to biomass recalcitrance, therefore current studies in plant breeding and plant engineering are focused on understanding the complexity of the synthesis, regulation, and interaction of the components that constitute the cell walls (de Vries et al., 2021). 3 Figure 1.1 Structural model of plant secondary cell wall. The model shows several cellulose synthase complexes embedded in the plasma membrane depositing cellulose chains into the apoplast as microfibrillar bundles. Hemicelluloses, such as glucuronoxylan associate non-covalently to the surfaces of the cellulose microfibrils, while lignification occurs into the cellular voids remaining as the cells enter into programmed cell death. A schematic chemical makeup and linkage of several major and minor cell wall polymers in the secondary cell wall are also shown, including cellulose, hemicelluloses, and lignin. This figure was adapted from de Vries et., al, 2021, the Creative Commons license can be found at creativecommons.org/licenses/by/4.0/. 1.2 Woody feedstocks In recent years, there has been a growing interest in the use of plants as a renewable and sustainable alternative to replace petroleum-derived products. First-generation biofuels, derived from starch and sugar crops such as sugar cane, corn, and cassava, have been prominently used 4 to produce biogas and ethanol (Mat Aron et al., 2020). However, the use of these biofuels is controversial as they compete with food crops by limiting resources such as arable land to grow, and water supply. To resolve this problem, second-generation biofuels have emerged; these biofuels are created from forest residues and woody biomass that are rich in lignocellulose (Mat Aron et al., 2020). Poplar, a perennial fast-growing tree, is considered a promising woody feedstock due its inherent significant productivity and the substantial genetic resources available (An et al., 2021). Poplars are deciduous trees from the genus Populus that grow in a wide range along the northern hemisphere (Cronk, 2005). The genus Populus belongs to the Salicaceae family and comprises approximately 30 species that are distributed globally. Poplar trees are generally found in riparian habitats; however, they can also exist in a wide range of environments, temperatures, and precipitation conditions (Douglas, 2017). Poplars are dioecious trees and pollination is wind-dependent (Cronk, 2005). Members of the Populus genus are diploid and carry two sets of 19 chromosomes as a result of an ancient genome duplication (Tuskan et al., 2006). Hybridization in these trees occurs naturally among different species that exist in the same geographic area and has been widely exploited commercially for poplar breeding (Douglas, 2017). Poplars can be cultivated in short rotation cycles being harvested on a 3 to 5-year rotation or at longer rotations to maximize the recovery of large stem diameter trees when used for industrial proposes (An et al., 2021). Currently, they are considered economically important for the production of timber wood, plywood, and raw materials for the paper industry (Douglas, 2017). Due to their genetic diversity, fast growth, and economic importance, poplars are used as a plant model to study tree biology (Douglas, 2017). The genome sequence of P. trichocarpa was the first tree to be available for genetic studies (Tuskan et al., 2006), however, constraints regarding 5 its transformation efficiency have made hybrids the most used in experimental setups. One of the well-characterized hybrids in the genus Populus is the P. alba x P. tremula hybrid which is naturally found close to river floodplains in European regions. P. tremula x P. alba INRA 717-1B4 (hereafter Poplar 717), is a female clone derived from a breeding program at the INRA (National Research Institute for Agriculture, Food and Environment) in France (Mader et al., 2016). Poplar 717 is susceptible to Agrobacterium transformation and was the first tree to have its genome edited by CRISPR Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats CRISPR-associated protein 9) (Zhou et al., 2015). Similarly, the genome sequence of some gymnosperm trees, such as white spruce (Picea engelmannii × glauca × sitchensis, PG29), has been published in recent years (Birol et al., 2013; Warren et al., 2015). Conifers are dominant across large areas of the northern hemisphere and are among the longest-living plant species on the planet. White spruce is one of the most widely planted conifer species in Canada, and the PG29 genotype has been broadly used in tree-breeding programs in British Columbia (Warren et al., 2015). Although the availability of the genome sequence of white spruce and other tree species has allowed advances in the study of conifers, the efficient transformation system and the simplicity of life cycle have made angiosperms, such as poplar, the more commonly used platform for the study of tree biology (Rodríguez et al., 2022). The use of poplar hybrids in different studies of genetic engineering has enabled improvements in saccharification for biofuel production (de Vries et al., 2021). To date, most efforts to improve processing have been focused on modifying lignin content and lignin composition in trees (Bonawitz & Chapple, 2013; Huntley et al., 2003; Leplé et al., 2007; Van Acker et al., 2014; Wilkerson et al., 2014), however, in recent years, targeting hemicelluloses has appeared as a 6 good alternative to reduce cell wall recalcitrance (Busse‐Wicher et al., 2014; Lyczakowski et al., 2017; Pereira et al., 2017; Terrett & Dupree, 2019). 1.3 Hemicelluloses Hemicelluloses are defined as ꞵ-1,4-linked glycans with glycosyl substituents. Plant hemicellulose composition varies depending on species, tissue type, and developmental stage. In PCW, xyloglucan constitutes most of the hemicellulose content, while in SCW, xylan and mannan are more abundant (Pauly et al., 2013; Scheller & Ulvskov, 2010; Zhang et al., 2021). Among these hemicelluloses, xyloglucan and xylan have been extensively investigated, whereas our understanding of mannan remains limited. Next, I will provide a brief overview of some of the most relevant aspects of xyloglucan and xylan, and then delve into our current understanding of mannan structure, biosynthesis, and function in plants. 1.3.1 Xyloglucan 1.3.1.1 Xyloglucan structure and classification Xyloglucan (XyG) accounts for 20–30% (w/w) of the PCW in dicots, gymnosperms, and non-gramineous monocots, while in grasses, it constitutes only 2–10% (w/w) (Hayashi, 1989; Pauly & Keegstra, 2016). XyG consists of a β-1,4-linked glucan backbone partially decorated with xylose substituents at the O-6 position. The XyG backbone and xylose residues can be further substituted with galactosyl, fucosyl, galactofucosyl, arabinopyranosyl, or arabinofuranosyl groups (Hayashi, 1989; Pauly & Keegstra, 2016). XyG can also carry O-acetyl groups on the glucan backbone or on the galactosyl or arabinofuranosyl substituents. Based on their structure, most of the XyGs known to date can be classified into two types: fucogalactoxyloglucan and acetoxyloglucan. Fucogalactoxyloglucan is commonly present in most dicot species; it is 7 xylosylated at three of four backbone glucosyl residues and is often galactosylated and fucosylated. Acetoxyloglucan is common in grasses and is characterized as having xylosylations in two out of four or more glucosyl residues in the backbone (Hayashi, 1989; Pauly & Keegstra, 2016). 1.3.1.2 Xyloglucan biosynthesis Xyloglucan biosynthesis occurs in the Golgi apparatus, where the glucan backbone is elongated, decorated, and then delivered to the extracellular space where additional enzymes continue its modification (Hayashi, 1989; Pauly & Keegstra, 2016). In Arabidopsis, the xyloglucan backbone is elongated by Cellulose Synthase-Like C4 (CSLC4) (Cocuron et al., 2007), a XyG:synthase that uses UDP-Glucose as a substrate. Xylose residues are added to the glucose backbone by the action of XyG:xylosyltransferases (XXT1, 2, and 5), which use UDP-Xylose as a substrate. In Arabidopsis, xylose side chains can be decorated additionally with galactosyl residues via MURUS3 (MUR3) and Galactosyltransferase Position 2 (XLT2) (Hayashi, 1989; Pauly & Keegstra, 2016). Galactosyl decorations can be in turn substituted with fucosyl residues that are added by XyG:fucosyltransderases (FUT) using UDP-fucose) (Julian & Zabotina, 2022). The acetylation of XyG backbone is executed by the action of multiple acetyltransferases involving Reduced Wall Acetylation (RWA), Trichome Birefringence-like (TBL), and altered xyloglucan (AXY) proteins (Julian & Zabotina, 2022). Depending on plant species, xylosyl residues can be further decorated with galacturonic acid, arabinopyranose, and/or arabinofuranose (Julian & Zabotina, 2022). Once XyG biosynthesis is completed in the Golgi apparatus it is delivered to the apoplast via vesicle transport. There, XylG can be further modified by the action of multiple 8 enzymes such as XyG endotransglycosylases (XTHs), glycosyl hydrolases (GH), and expansins (Hayashi, 1989; Maris et al., 2011; Pauly & Keegstra, 2016). 1.3.1.3 Biological and industrial relevance of xyloglucan XyG plays an important role in cell growth and differentiation as it interacts with cellulose microfibrils in the PCW; it works as a tether allowing the loosening and tightening of cellulose microfibrils during cell maturation (Cosgrove, 2023; Hayashi & Kaida, 2011). In some seeds, XyG accounts for up to 42% of the total weight and serves as a storage polysaccharide that provides energy to the seedlings once they have germinated (Pauly & Keegstra, 2016). Industrially, XyG is currently used as a thickening, stabilizing, and gelling agent, and has more recently been considered a polysaccharide with potential in textile applications and the pharmaceutical industries, as well as for drug-delivery and packaging technologies (Mishra & Malhotra, 2009; Piqué et al., 2018). The loosening of XyG has been proposed as a strategy to modify and accelerate the saccharification of dominant cell wall carbohydrates in biomass, such as cellulose (Kaida et al., 2009; Zhou et al., 2007). Most of the efforts to limit XyG-cell wall polymer interactions have focused on the enzymes that act on XyG in the apoplast, such as XyG:Endo Transglycosylase/hydrolase (XET/XTHs) that exhibit XyG:endohydrolase (XEH) and XyG:endotransglycosylase (XET) activity which have been shown to alter the length of XyG chains (Eklöf & Brumer, 2010; Günl & Pauly, 2011; Maris et al., 2011). In poplar, transgenic trees expressing the fungal Aspergillus aculeatus Xyloglucanase 2 (AaXEG2) were shown to possess reduced XyG content but displayed increased stem height and cellulose content (Park et al., 2004; Yamamoto et al., 2011). Poplar trees overexpressing AaXEG2 had a 50% increase in 9 glucose yields and 60% cellulose conversion (Kaida et al., 2009). Similarly, modification of XET proteins in Arabidopsis increased saccharification yields and significantly altered secondary growth (Kushwah et al., 2020). In poplar, modification of XET proteins (PtxtXET16-34) caused changes in vessel elements making them significantly wider in transgenic trees than the corresponding wild-type controls (WT; Nishikubo et al., 2011). Similarly, modification of galactose side chains of xyloglucan has been shown to increase its gelation and gel strength properties. In Arabidopsis, AtBGAL10 was identified as a key β-galactosidase, and its absence significantly altered XyG composition and negatively affected plant growth (Sampedro et al., 2012). Genetic modification of xyloglucan is an area that continues to be explored in plant genetic engineering, as it appears to represent an effective way to reduce cell wall recalcitrance. 1.3.2 Xylan 1.3.2.1 Xylan structure and classification Xylan constitutes the most abundant hemicellulose in the SCW of dicots, accounting for approximately 20% (w/w) (Scheller & Ulvskov, 2010). Xylan is composed of repeating β-1,4 xylose residues as a backbone that can be decorated with acetate, (methyl)glucuronic acid ([Me]GlcA), and/or arabinose residues (Pauly et al., 2013; Scheller & Ulvskov, 2010; Zhang et al., 2021). Xylans can be grouped into glucuronoxylans (GX), glucuronoarabinoxylans (GAX), and arabinoxylans (AX) depending on the type and abundance of their decorations (Tryfona et al., 2023). Glucuronoxylan is one of the major components of the secondary wall in woody and herbaceous dicots, accounting for up to 30% of their dry mass weight. Glucuronoxylan backbone is decorated with GlcA and MeGlcA residues and can be partially acetylated in some xylose (Xyl) units (Pauly et al., 2013; Zhong et al., 2019b). Glucuronoarabinoxylan, on the other hand, is commonly found in grass species and gymnosperms. Its backbone is highly substituted by 10 GlcA/MeGlcA and arabinofuranose residues that can be further esterified with ferulic or p-coumaric acid groups (Pauly et al., 2013). Finally, AX is highly abundant in grass endosperm walls where it can account for up to 70 % of the biomass weight (Pauly et al., 2013). Arabinoxylan backbone is mainly decorated with acetyl and arabinose residues often with terminal ferulic acid groups (Pauly et al., 2013). In GX and GAX from secondary walls of dicots and gymnosperms, a reducing end composed of Xyl, rhamnose (Rha), and GlcA is present (β-Xyl-1-3-α-Rha-1-2-α-GalA-1-4-β-Xyl), and it is thought to play an important role in the synthesis of both polymers (Wierzbicki et al., 2019). 1.3.2.2 Xylan biosynthesis Xylan is synthesized in the Golgi apparatus by the action of several enzymes and its biosynthesis involves the formation of the sugar backbone and the assembly of the multiple decorations. In Arabidopsis, UDP-Xyl is used as a substrate for the synthesis of xylan backbone, this is synthesized in the cytosol and transported to the Golgi lumen by UDP-Xyl transporters (Zhao et al., 2018). Once in the Golgi, UDP-Xyl is used by Irregular Xylem 9, 14, and 10 xylosyltransferases to assembly the sugar backbone (Wu et al., 2009). Thereafter, multiple decorations can be added including glucuronic acid residues, arabinose residues, acetate, and methyl groups. In Arabidopsis, GlcA residues are added to the xylan backbone via Glucuronic Acid Substitution of Xylan1, 2, and 3 proteins (GUX1, GUX2, GUX3), while arabinose substitutions are added by arabinosyltransferases (XATs; Lee et al., 2012). Acetyl groups are incorporated into the sugar backbone by xylan O-acetyltransferases (XOAT) and include Eskimo 1 (ESK1), TBL33, and RWA proteins (Zhong et al., 2017). Finally, methyl groups are added to GlcA residues by Glucuronoxylan Methyltransferases (GMX; Lee et al., 2012). 11 1.3.2.3 Biological and industrial relevance of xylan Xylan is essential for plant growth and development as it provides structural support by interacting with cellulose and lignin (Busse‐Wicher et al., 2014; Pereira et al., 2017; Wierzbicki et al., 2019); xylan is highly important at the industrial level for the production of packing materials, hydrogels, and chemicals such as furfural, lactic acid, and ethanol (Naidu et al., 2018). Xylan binds directly to cellulose microfibrils via hydrogen bonds forming an interface between lignin and cellulose that is important to maintain secondary cell wall strength (Wierzbicki et al., 2019). However, the interaction of xylan with other polymers, presents a challenge for biomass processing as it reduces cellulose accessibility during enzymatic and chemical pretreatments. Hence, reductions in xylan content and/or alteration in its structure have been used as a strategy to improve biomass processing (Wierzbicki et al., 2019). Molecular dynamic simulations and nuclear magnetic resonance (NMR) studies have shown that xylan folds as a twofold screw when it binds to cellulose (Pereira et al., 2017; Simmons et al., 2016). Acetate and GlcA decoration patterns along the xylan backbone are important to maintain this conformation and permit docking of xylan with cellulose microfibrils, as well as to preserve microfibrils stability and orientation (Grantham et al., 2017). In Arabidopsis, mutation of the acetyltransferase ESK1, for example, prevented the interaction between xylan and cellulose, and caused dwarfism and collapsed xylem vessels in plants (Grantham et al., 2017). Similarly, in rice, the disruption of Deacetylase on Arabinosyl Sidechain of xylan1 (DARX1), an enzyme responsible for the deacetylation of the arabinosyl residues in GAX, increased the content of xylan threefold and reduced the mechanical strength of the stem as well as plant height (Zhang et al., 2019). In general, reductions in acetyl groups along the xylan backbone positively affects 12 biomass processing, although it is usually accompanied with a plant growth penalty (Smith et al., 2017). Attempts at reducing biomass recalcitrance by altering xylan structure have also focused on the modification of GlcA decorations. It is thought that in addition to interfering with xylan conformation, GluA substitutions also contribute to cell wall stability by binding single xylan chains through Ca2+-mediated crosslinks (Pereira et al., 2017). In Arabidopsis gux1 gux2 double mutants, GluA content was reduced without having a significant effect on plant growth (Mortimer et al., 2010). The reduced GluA substituents in these mutants increased glucose release by 30% and xylose released by 700% compared to wild-type plants (Mortimer et al., 2010). Likewise, modifications of ferulic acid decorations in arabinosyl substitutions of AX and GAX has shown a positive effect on biomass recalcitrance. Ferulic acid groups form cross-links between adjacent xylan chains and lignin (de Oliveira et al., 2015; Terrett & Dupree, 2019), and hence contribute to cell wall rigidity. In rice, the overexpression of OsAT10 (a putative p-coumaroyl coenzyme A transferase that alters the content of ferulic acid and p-coumaric acid in GAX) decreased ferulic acid levels and increased saccharification efficiency by 40%, without having negative effects on plant growth (Li et al., 2018). In poplar, multiple genes related to xylan biosynthesis have been identified and their modification has also shown to affect wood biomass recalcitrance (Lee et al., 2012; Ratke et al., 2015). For instance, down-regulation of PoGT43B and PoGT47C, two genes encoding xylosyltransferases, in poplar reduced wall thickness, xylan content, and increase the amount of glucose released after cellulase wood digestion (Lee et al., 2009, 2011). In contrast, the overexpression of the PtRWA-C in P. trichocarpa, increased acetylation, lignin content, and reduced saccharification efficiency (Zhang et al., 2023). 13 Like xylan, mannan also binds directly to cellulose microfibrils and interacts with lignin (Lyczakowski et al., 2017; Terrett et al., 2019; Yu et al., 2018). However, these interactions are still poorly understood. To date, it is not clear how mannans plays a role in stabilizing cell wall polymers in trees and how genetic modifications of the genes responsible for its synthesis may affect cell wall deconstruction. 1.3.3 Mannan 1.3.3.1 Mannan structure and classification Mannans present in plants are divided into four main distinct classes according to their backbone and side chain composition (Fig. 1.2a-d): linear mannan or homomannan, glucomannan, galactomannan, and galactoglucomannan (Ishida et al., 2023; Scheller & Ulvskov, 2010; Yu et al., 2022). Homomannan backbone contains primarily β-(1,4)-Mannose (Man) residues, while glucomannan backbones are constituted by a combination of β-(1,4)-Man and β-(1,4)-Glucose (Glc) residues. Galactomannan and galactoglucomannan backbones are made of β-(1,4)-Man, and β-(1,4)-Man and β-(1,4)-Glc residues, respectively, decorated with galactose (Gal) side chains at the carbon 6 (C-6) of Man units. Two types of galactoglucomannans have been identified. The first, an acetylated galactoglucomannan (AcGGM), has a random backbone made of β-(1,4)-Man and β-(1,4)-Glc residues with Man units decorated with O-acetyl groups predominantly at the C-2 and C-3 positions. The second, a β-GalactoGlucoMannan (β-GGM), has a repeating backbone made of [4)-Man-β-(1,4)-Glc-β-(1] residues (L. Yu et al., 2018, 2022). In this polysaccharide, Man units are decorated with di-galactosylated side chains or α-Gal substitutions at the C-6 position. A one-letter code nomenclature has been assigned to describe the arrangement of the sugars and side chains along the mannan backbone. The letters M and G represent unsubstituted Man and Glc residues, while the letters A and B denote Man units that 14 have been decorated with α-Gal or the Gal-β-(1,2)-Gal-α-(1,6) disaccharide, respectively (Fig. 1.2e;Yu et al., 2022). Figure 1.2 Mannan structures and nomenclature. a-d. Diversity of mannans found in plants. e. One-letter code nomenclature assigned to describe the arrangement of the sugars and side chains along the mannan backbone (Yu et al., 2022). The letters M and G represent unsubstituted Man and Glc residues, while the letters A and B denote Man units that have been decorated with α-Gal or the Gal-β-(1,2)-Gal-α-(1,6) disaccharide, respectively. The abundance of each type of mannan in plants is represented by plant/seed icons. AcGGM: acetylated galactoglucomannan; β-GGM: β-GalactoGlucoMannan. Green circles: mannose residues, blue circles: glucose residues, yellow circles: galactose residues, and red circles: acetyl groups. 1.3.3.2 Mannan diversity in plants and algae Mannans are highly conserved through plant evolution (Rodríguez-Gacio et al., 2012; Scheller and Ulvskov, 2010). Linear mannans associated with cellulose form the cell wall architecture of several species of red and green algae, such as Acetabularia acetabulum, Codium fragile, and Porphyra umbilicalis (Dunn et al., 2007; Zhong et al., 2019a). In the vegetative phase of algae species belonging to the Acetabularia and Codium genera, mannan in the form of microfibrils is 15 the dominant polysaccharide in the cell wall, which is the reason why algae species from these genera are also known as "mannan weeds” (Dunn et al., 2007; Zhong et al., 2019a). In the case of early land plants, such as mosses and ferns, mannans are also highly abundant. In the gametophytic stem and leaves of the umbrella moss Hypnodendron menziesii, Man is the most abundant monosaccharide detected (Chavan et al., 2021). Immunofluorescence microscopy with the monoclonal antibody LM21 of the moss stems showed that heteromannans are present in all the stem cell types, indicating that this polysaccharide is a major component of the cell walls (Chavan et al., 2021; Zhong et al., 2019a). Similarly, in ferns, Psilotum nudum was described to contain a glucomannan-rich primary cell wall with mannan also present in its SCW (Chernova et al., 2020). During the tracheophyte evolution, a decrease in the proportion of heteromannans in the primary cell wall seems to have occurred. In gymnosperms, mannans are a minor component of the PCW, while in the SCW remain as one of the main hemicelluloses, accounting for up to 18% of the wood cell walls (Rodríguez-Gacio et al., 2012; Scheller & Ulvskov, 2010; Timell, 1967). In spruce, AcGGM is the predominant form, and two different degrees of acetylations have been reported for this polysaccharide. The first is galactoglucomannans carrying a high degree of galactose substitutions, while the second one has a lower degree of galactosylation (mannose, glucose, galactose molar rations of 2.5-7:0.5-1.6:1 versus 17-40:3-10:1; Capek et al., 2000, 2002; Willför et al., 2003; Zhong et al., 2019a). Finally, in angiosperms, mannans are still present and their content varies highly depending on the plant tissue. In PCW they account for up to 3-5% (w/w) of the cell wall, while in SCW glucomannan constitutes 2-5% (w/w) (Rodríguez-Gacio et al., 2012; Scheller & Ulvskov, 2010; Timell, 1967). Homomannan and galactomannan are abundant in ivory nuts and guar seeds endosperms (Buckeridge et al., 2000; Stephen & Phillips, 2016; L. Yu et al., 2018). In monocots and eudiot plants, homomannan, glucomannan, 16 galactomannan, galactoglucomannan, and acetylated mannan are present, however, β-GGM seems to be restricted to only the PCW of dicot plants (Ishida et al., 2023; Yu et al., 2022). 1.3.3.3 Mannan biosynthesis Mannan biosynthesis has been well characterized in Arabidopsis. In general, this polysaccharide is synthesized in four major steps (Fig. 1.3): 1) formation of Guanosine-diphosphate-Mannose (GDP-Man) in the cytosol, 2) import of GDP-Man from the cytosol, 3) polymerization of a mannan backbone, and 4) addition of side chain substitutions (galactosyl residues or acetyl groups) to the backbone (Scheller & Ulvskov, 2010; Voiniciuc, 2022). In Arabidopsis, mannose-1-phosphate guanylyltransferase I/ Vitamin C Defective I (GMP1/VTC1) is responsible for the synthesis of GDP-Man in the cytosol and its enzymatic activity is stimulated by the KONJAC (KJC1, KJC2) proteins (Conklin et al., 1999, 2000; Sawake et al., 2015). Once GDP-Man is synthesized, it is (Jing et al., 2021) used by Cellulose Synthase-Like family A (CSLA) proteins to form the mannan backbone (Goubet et al., 2009; Voiniciuc, 2022). To date, it is not clear if GDP-Man is translocated from the cytosol by transporters located in the Golgi membrane or if it is used directly by the CSLA proteins. GDP-Man transporters have been identified in Arabidopsis, however, they seem to be mostly related to the glycosylation of sphingolipids (Baldwin et al., 2001; Jing et al., 2021; Yu et al., 2018). Due to the structural similarity of CSLAs with the Cellulose Synthase (CesA) enzymes in terms of the presence of the D,D,D, QxxRW motif and the number of transmembrane domains (Dhugga et al., 2004; Saxena & Brown, 1997), it is also possible that CSLAs synthesized the mannan backbone using GDP-man from the cytosol and directly secrete the mannan polymer into the Golgi lumen through a channel formed by its own transmembrane domains, similar to the mode 17 of synthesis displayed by CesA enzymes producing cellulose into the apoplastic space (Purushotham et al., 2016, 2020). CSLA proteins can employ either GDP-Man or a combination of GDP-Man and GDP-Glc to synthesize homomannan or glucomannan backbones, respectively (Goubet et al., 2009; Liepman et al., 2005). In the case of the CesA enzymes, substrate selectivity has been associated with the binding of UDP-Glc to a conserved pocket at the interface of the enzyme’s catalytic domain located at the cytosolic site and the transmembrane region (Verma et al., 2023). If the CSLAs are extruding the mannan backbone through a pore by using GDP-man and GDP-Glc from the cytosol, it is possible that a similar catalytic pocket may be needed to determine their substrate specificity. In Arabidopsis, nine genes have been identified as CSLAs (AtCSLA1, AtCSLA2, AtCSLA3, AtCSLA4, AtCSLA5, AtCSLA6, AtCSLA7, AtCSLA8, and AtCSLA9), however, only four have shown to be related with mannan production in planta: AtCSLA2, AtCSLA3, AtCSLA7, and AtCSLA9 (Goubet et al., 2009). Of these genes, only three seem to be important in the production of mannan in Arabidopsis stems (AtCSLA2, AtCSLA3, and AtCSLA9). CSLA9 appears to be responsible for the majority of glucomannan synthesis in this tissue, while CSLA2 and CSLA3 are required for the synthesis of a minor proportion (Goubet et al., 2009). The activity of CSLAs is influenced by mannan synthesis-related proteins (MSR), which can enhance their activity and manipulate the type of backbone CSLAs synthesized (Voiniciuc et al., 2019). To date, only two genes have been identified to play such a function in Arabidopsis: AtMRS1 and AtMRS2 (Voiniciuc et al., 2019; Y. Wang et al., 2013). Finally, acetyl and/or galactosyl residues are added to the mannan backbone via mannan O-acetyltransferases (MOAT1, MOAT2, MOAT3, and MOAT4) and Mannan α-Galactosyl Transferases (MAGT1/ MUCILAGE-RELATED10 18 (MUCI10), MAGT2), respectively (Nishigaki et al., 2021; Yu et al., 2018; Zhong et al., 2018, 2019a). Once mannan has been synthesized, it is transported to the extracellular matrix by exocytosis where it canremodeled by α-galactosidases (AGAL), carbohydrate esterases (CE), cellulases (CEL), and mannases (MAN). Figure 1.3 Mannan biosynthesis.The substrate for mannan synthesis, GDP-Mannose (GDP-Man), is synthesized in the cytosol by mannose-1-phosphate guanylyltransferase/ Vitamin C Defective I (GMP/VTC) protein, whose enzymatic activity can be stimulated by the KONJAC (KJC) proteins. GDP-Man can be used for the production of ascorbic acid in the cytosol, or it can be transported to the Golgi lumen to form mannan. In the Golgi, GDP-Man is used as a substrate by cellulose synthase-like family A (CSLAs) proteins to synthesize the mannan backbone. CSLAs also can integrate GDP-glucose (GDP-Glc) into the sugar backbone to form glucomannan. Activity of these proteins can be enhanced by the Mannan Synthesis Related proteins (MSR). Once the mannan backbone is synthesized it can be decorated by mannan α-galactosyl transferases (MAGT) with α-galactosyl side chains to form galactoglucomannan. Finally, acetyl units can be added to the mannan backbone via mannan O-acetyltransferases 19 (MOAT). Once mannan polymer is synthesized, it is delivered to the extracellular matrix by exocytosis where it can go under remodelling by a-galactosidases (AGAL), carbohydrate esterases (CE), cellulases (CEL), and mannases (MAN). CSC: Cellulose Synthase Complex. Green circles: mannose residues, blue circles: glucose residues, yellow circles: galactose residues, and red circles: acetyl groups. Light gray dotted arrows indicate processes where it is unknown how exactly they occur. Black dotted arrows indicate that several steps are involved in the process. 1.3.3.4 Biological relevance of mannans Mannans are present in multiple tissues of plants and seem to perform different functions according to their location and plant developmental stage. In seed embryos, for example, the expression CSLA7, a member of the CSLA family in Arabidopsis, is essential for normal growth. Arabidopsis csla7 mutants are embryo-lethal, they arrest their development at the globular stage and have defective cell patterning and proliferation as well as a reduced pollen tube (Goubet et al., 2003, 2009). The mannan produce by CSLA7 seems to be important in maintaining the correct extensibility and stability of the cell wall that reinforce the pollen tube, allowing its correct development (Goubet et al., 2003). In seed endosperms, mannans protect against dehydration, are involved in germination, and represent a source of carbon during early seedling development (Stephen et al., 2006; Yu et al., 2018). The low solubility of homomannans and galactomannans in the endosperm protect seeds from desiccation, however, their degradation is essential for germination. To germinate, mannans need to be degraded to allow the radicle to emerge. MANs are endohydrolases that catalyze the cleavage of the mannan linkage to produce galactoglucomannan oligosaccharides (GGMOs). In Arabidopsis, a total of eight genes have been identified to belong to the MAN gene family, but only four are highly expressed during germination (AtMAN2, AtMAN5, AtMAN6, and AtMAN7). In Arabidopsis, when AtMAN6 is mutated, germination is dramatically reduced, and only 50% of the seeds germinate. The cleavage of mannans by MANs is important for seed germination as it 20 weakens the micropylar endosperm and allows the radicle to emerge (Iglesias-Fernández et al., 2011). MANs also play an important role in seedling development by allowing the mobilization of mannans reserves. They are normally synthesized in the lateral endosperm instead of micropylar endosperm (Belotserkovsky et al., 2007; Nonogaki et al., 1998). Different lines of evidence also suggest the GGMOs created during mannan remodelling are important signal molecules required for plant development (Beňová-Kákošová et al., 2006; Zhang et al., 2023). In Zinnia elegans cultures, for example, the addition of GGMOs increased cell density and altered secondary cell wall patterning (Beňová-Kákošová et al., 2006). Similarly, when AtMAN6 is mutated in Arabidopsis, stems have thicker SCW than wild-type plants, while thinner SCWs were observed in lines overexpressing the same gene (Zhang et al., 2023). Likewise, in Poplar, the formation of thicker SCWs was observed when PtrMAN6 was mutated (Zhao et al., 2013). Based on these results, it is theorized that mannans act as signalling molecules that regulate the transition between PCW and SCW formation in plants. Mannans are essential to maintain seed mucilage architecture (Terrett et al., 2019; Terrett & Dupree, 2019; Voiniciuc et al., 2015; Yu et al., 2014). Even though mannans constitute only 5% (w/w) of total carbohydrates in the mucilage, drastic changes in mannan content or structure highly affect mucilage integrity (Voiniciuc et al., 2015; Yu et al., 2014). In Arabidopsis, AtCSLA2 is responsible for making the patterned β-GGM present in the seed mucilage ( Yu et al., 2014, 2018). When AtCSLA2 was mutated, Arabidopsis seeds possessed denser mucilage capsules and exhibited compromised cellulose organization. In these mutants, cellulose crystallinity was reduced, and the spatial distribution of pectin and cellulose was altered (Yu et al., 2014, 2018). 21 Mannan interacts with other polymers in the cell wall and its side chains may play an important role for these interactions. For example, mannans have been shown to bind directly to cellulose microfibrils (Terrett et al., 2019). Studies in spruce and Arabidopsis seeds have suggested that galactoglucomannan, upon binding to cellulose, adopts a two-fold screw-like conformation, similar to the one observed in xylan(Terrett et al., 2019; Yu et al., 2018). Galactosyl residues, similar to GlcAc and acetyl groups in xylan, seem to be important in maintaining the conformation that mannan adopts when interacting with cellulose. Consistent with this information, in Acetobacter, the supplementation of media with different types of mannans with varying Man:Gal ratios affected the ensuing cellulose molecular organization (Hackney et al., 1994). Adding low Man:Gal ratio mannans into the Acetobacter media induced the coalescence of cellulose microfibrils and reduced cellulose crystallinity by 55% (Hackney et al., 1994). The solubility of mannans increased when a higher number of Gal decorations was present along the sugar backbone (Hackney et al., 1994). Likewise, in Arabidopsis, when MUCI10 (an a-galactosyltransferase) was mutated, seed mucilage collapsed and cellulose crystallinity was reduced, similar to what occurred in the Arabidopsis csla2 mutant (Voiniciuc et al., 2015). In addition to interacting with cellulose, NMR studies have revealed that mannan is cross-linked to lignin via ether bonds (Nishimura et al., 2018), however, the specifics about how this interaction contributes to plant development remains unknown. When mannan was eliminated from lignifying tissues in Arabidopsis no effects in plant growth were observed (Goubet et al., 2009). Specifically, in the Arabidopsis csla 2,3,9 mutant, the absence of mannan did not affect plant morphology or xylem strength (Goubet et al., 2009). In general, mannan seems to play a role in 22 maintaining cell wall architecture by interacting with other polymers, however, it is still largely unknown how these interactions occur and what role they play in lignocellulose recalcitrance. 1.3.3.5 Industrial relevance of mannans Mannans are widely used in the food industry for the creation of different products, one of the most important is gums (from galactomannans) (Stephen & Phillips, 2016). Galactosyl decorations found along the mannan backbone play an important role in the properties of these gums, as they affect their viscosity and gel-forming ability with other polysaccharides such as agar and xanthan gums (Singh et al., 2018). Two of the main sources of galactomannan in plants are guar gum and locust bean gum. Guar gum comes from the seed endosperm of Cyamopsis tetragonolobus, a plant belonging to the Leguminosae family (Singh et al., 2018). This gum displays strong water absorption and solubility, can form highly viscous solutions, and is resistant to breakdown in low pH environments (Mudgil et al., 2014; Singh et al., 2018)). Therefore, guar gum is used as a stabilizer, food additive, emulsifier, and thickening agent in different products (Mudgil et al., 2014; Singh et al., 2018). For example, due to their water-binding properties, guar gums are used as ice cream stabilizers and additives in frozen products as they prevent the formation of ice crystals in the food (Mudgil et al., 2014; Singh et al., 2018). Similarly, locust bean gum is also obtained from the seed endosperm of Ceratonia siliqua. In contrast with guar gum, locust bean gum has a lower galactose content and hence limited water solubility (Mudgil et al., 2014; Singh et al., 2018). This property makes the locust bean gum an excellent thickening agent used to improve the stability and texture of, for example, non-fat and low-fat yogurt (Mudgil et al., 2014; Singh et al., 2018). When heat is applied, the solubility of locust gum increases, and in combination with other polysaccharides, it can also be used to produce edible films and coating products (Singh et al., 2018). 23 Mannans are used as dietary supplements to promote health. For instance, Konjac glucomannan, from the tubers of the Amorphophallus konjac plant, has been used to treat diabetes and obesity (Singh et al., 2018). As it contains non-digestible fiber, Konjac glucomannan flour is employed in the production of low-calorie foods, including tofu, noodles, and different dietary supplements (Singh et al., 2018). Likewise, spruce GGM has also been shown to be an excellent candidate as a prebiotic for gut microbiota (Zhao et al., 2020). Mannans are also of major industrial interest because of their potential to act as packaging materials and improve biomass conversion for bioenergy. Recent studies have shown that galactoglucomannan from woody plant species can be used to create latex with high transparency, hydrophobicity, and flexible properties, which make it an ideal material to replace petroleum-based packaging products (Yong et al., 2022). Similarly, mannans from tree biomass are considered an important source of fermentable sugars that can be converted into ethanol and butanol, as hexoses are more easily fermented by industrial yeast strains than pentoses (Saxena et al., 2023). 1.4 Research questions Although Arabidopsis has been widely used as a model system to study secondary xylem development, it seems that there are some regulatory processes unique to wood formation in trees that cannot be resolved in this system (De Meester et al., 2022; Lapierre et al., 2021; Taylor, 2002). For example, the expression of genes encoding the enzymes DIKETIDE-CoA SYNTHASE (DCS) and CURCUMIN SYNTHASE 2 (CURS2) in Arabidopsis, led to the synthesis and incorporation of curcumin in the lignified cell walls of the plants, without affecting their growth and increasing saccharification efficiency up to 24% (De Meester et al., 2022). When expressed in poplar, DCS and CURS2 also produced and incorporated curcumin in the 24 lignified cell walls of trees, however, transgenic trees exhibited altered growth phenotypes and did not show significant changes in saccharification efficiency (De Meester et al., 2022). Understanding the events that regulate the formation of the different components in tree cell walls is critical to making woody feedstocks, such as poplar, a cost-effective and competitive source to produce biofuels and bio-based materials. As previously mentioned, mannan is important for multiple biological processes, however, our knowledge about its synthesis and function in plants remains limited. To date, only a few genes involved in mannan biosynthesis in trees have been identified and none of them have been functionally characterized in planta. Therefore, this study aims to expand on our knowledge about mannan biosynthesis and function in trees. I aim to identify genes that are responsible for synthesizing mannan in poplar (Poplar 717) and spruce (PG29) and explore the consequences of changing the content of this polysaccharide in tree growth and cell wall architecture. Specifically, I intend to address the following questions: 1) What genes are responsible for synthesizing the mannan backbone in poplar and spruce? I will focus my attention on identifying genes that encode CSLAs. In poplar, five genes from the CSLA family have been predicted to exist: PtCSLA1, PtCSLA2, PtCSLA3, PtCSLA4, and PtCSLA5 (Liepman et al., 2005; Suzuki et al., 2006). The function of these genes has been tested only in vitro, in Drosophila Schneider 2 (S2) cells, where PtCSLA1 and PtCSLA3 were shown to possess β-mannan synthase activity while PtCSLA5 was not associated with such enzymatic activity (Liepman et al., 2005). I hypothesized that, similar to what occurs in Arabidopsis, multiple genes from this family are responsible for synthesizing mannan in poplar stems, each of them contributing differently to the production of the ensuing polysaccharide. In the case of spruce, I hypothesized that the 25 CSLA family consists of fewer gene members, due to the lack of genome duplication in these trees. 2) What genes are responsible for synthesizing GDP-Man in poplar and spruce? I will focus my efforts in functionally characterizing genes that are orthologues to VTC1, encoding proteins responsible for synthesizing GDP-Man. I hypothesized that both in poplar and spruce, similar to Arabidopsis, only a few genes are involved in this process. 3) What are the consequences of altering the mannan content on plant growth and cell wall architecture in trees? Using the previously identified genes, I will genetically engineer poplar to produce higher and lower concentrations of mannan in their stems. I hypothesize that since mannans have been shown to play an important role in maintaining mucilage architecture by interacting with other cell wall polymers as well as being signaling molecules, their alteration may result in changes in plant growth or cell wall composition. In Chapter 3, I address the first question by using a combination of bioinformatic and phylogenetic tools to identify putative CSLA genes in poplar and spruce, and assess their function in the Arabidopsis triple mutant csla 2,3,9. In addition, I also address the importance of mannan content to plant traits by expressing one spruce CSLA candidate in poplar and by mutating the poplar CSLA candidates using CRISPR Cas9. In Chapter 4, I address the second question of my thesis by identifying orthologs of VTC1 in spruce and poplar using bioinformatic and phylogenetic tools and evaluating their function in the Arabidopsis vtc1 mutant. Again, I attempt to address the impact of mannan content on plant growth and development using an alternative approach to increase and abolish mannan from trees, this time modulating the amount of substrate precursor available for mannan synthesis. 26 Chapter 2: Materials and Methods 2.1 Bioinformatic and phylogenetic analyses 2.1.1 Identification of putative CSLAs To identify putative functional CSLAs in poplar and spruce, sequences of CSLAs previously identified in Arabidopsis AtCSLA2 (AT5G22740), AtCSLA3 (AT1G23480), and AtCSLA9 (AT5G03760) were used to complete BLAST searches against the poplar (Populus trichocarpa v3) and spruce (PG29 v3) transcriptomes. BLAST searches were performed using the following parameters: Maximum target sequences: 500, Expect threshold: 0.05, Word size: 28, Maximum matches in a query range: 0, Gap costs: Linear. Sequences retrieved from these searches were evaluated by their similarity to the Arabidopsis CSLAs in terms of amino acid sequence, total transmembrane domains, and the occurrence of the highly conserved QxxRW motif. A multiple sequence alignment of the full-length proteins from genes having these characteristics was performed using MUSCLE in MEGA-X (Kumar et al., 2018). Then, a phylogenetic tree was constructed using IQ-Tree with an ultrafast bootstrap with 1000 replicates using the LG+G4 substitution model (Nguyen et al., 2015). Total transmembrane domains were predicted by TMHMM server v. 2.v (Krogh et al., 2001). The identification of the QxxRW motif was manually completed using the multiple sequence alignment created in MEGA-X. To narrow down the number of CSLA candidate genes from poplar, RNAseq expression data from poplar wild-type trees and mutant trees with low mannan content (hereafter GolS transcriptome database) (Unda et al., 2017) was used as a reference to select the most suitable candidates. 2.1.2 Identification of putative GMPs Sequences of the previously identified Arabidopsis GMP (AtVTC1) was used to complete BLAST searches against the poplar and spruce genomes to identify putative GMPs following the 27 parameters described above. Amino acid sequences of candidate genes were evaluated by the presence of the signature peptide PDOC00094 present in the C terminus of enzymes displaying transferase activity (Conklin et al., 1999; Lukowitz et al., 2001). Sequences carrying this peptide were aligned and a phylogenetic tree was constructed using the same parameters described above. For poplar, the same RNAseq GolS expression database was used to select the putative CSLA genes employed to select the final GMP candidates. 2.2 Gene expression constructs 2.2.1 Gene isolation from plant material Xylem tissue from spruce (Picea engelmannii × glauca × sitchensis, PG29), provided by the Bohlmann Lab at UBC, and poplar 717 (P. tremula x P. alba, INRA 717-1B4) was used to extract RNA using the CTAB-based protocol (Kolosova et al., 2004). This RNA was treated with DNase using Turbo DNA-free kit (Life Technologies, Carlsbad, CA, U.S.A) and subsequently exploited for the synthesis of cDNA with the iScript cDNA synthesis kit (Bio-Rad Laboratories Inc., Hercules, CA, U.S.A). 2.2.2 CSLA plant expression constructs The coding region of PgCSLA1 (GCHX01112248) was amplified using BestTaq polymerase (Applied Biological Materials, Richmond, BC, Canada) and the PgCSLA1_FWD_attB and PgCSLA1_REV_attB primers (Table 2.1) carrying the Gateway attB1 and attB2 cloning sites. PgCSLA1 was cloned into the pDONR221 (Invitrogen, Carlsbad, CA) Gateway entry vector by BP clonase recombination. PtaCSLA1 (Potri.008G026400) was synthesized by Azenta (New Jersey, USA) using Potri.008G026400 transcript sequence from poplar 717 as a reference (Appendix A). The accuracy of the sequence was validated by amplifying parts of the gene from poplar 717 cDNA and sequencing. The coding sequence of PtaCSLA1 was cloned from the 28 Azenta original vector, pUC-GW-Amp (Azenta), into the pDONR221 (Invitrogen, Carlsbad, CA) Gateway entry vector using the primers PtaCSLA1_FWD_attB and PtaCSLA1_REV_attB (Table 2.1). Coding sequences of all candidate genes were cloned into the pDONR221 plasmid, and subsequently transferred into the plant expression vector pk7WG2 driven by the Arabidopsis CESA7 promoter (Karimi et al., 2002; Smith et al., 2015) using LR recombination. Chemically competent Agrobacterium tumefaciens (strains GV3101 and EHA105) cells were transformed with each of the expression vectors carrying the putative genes following the freeze-thaw protocol (Wise et al., 2006). 2.2.3 GMP plant expression constructs The GMP candidate genes PgGMP1(BT102800) and PtaGMP1(Potri.010G198800) were amplified from PG29 and poplar 717 cDNA respectively from xylem tissue using BestTaq polymerase (Applied Biological Materials, Richmond, BC, Canada). PgGMP1 was amplified using the primers PgGMP1_FWD_attB and PgGMP1_REV_attB, while PtaGMP1 was amplified using the primers PtaGMP1_FWD_attB and PtaGMP1_REV_attB (Table 2.1). Coding sequences of all candidate genes were cloned into the pDONR221 plasmid, and subsequently transferred into the plant expression vector pk7WG2 driven by the Arabidopsis CESA7 promoter using LR recombination (Karimi et al., 2002; Smith et al., 2015). Chemically competent Agrobacterium (strains GV3101 and EHA105) cells were transformed with each of the expression vectors carrying the putative genes following the freeze-thaw protocol (Wise et al., 2006). 29 Table 2.1 Primers used for cloning of gene candidates. Plant/Gene Primer name Primer sequence Poplar 717/CSLA PtaCSLA1_FWD_attB 5′- GGGGACAAGTTTGTACAAAAAAGCAGGCTATGGATCGTTTATCATCTGCTAGT -3′ PtaCSLA1_REV_attB 5′- GGGGACCACTTTGTACAAGAAAGCTGGGTCTAAGAGCGGG -3′ PtaCSLA1_FWD 5′- GTCTGCTACCTACGCTTTCTTTG -3′ PtaCSLA1_REV 5′- CCCATTCATTCACTCTTCCTG -3′ Poplar 717/GMP PtaGMP1_FWD_attB 5′- GGGGACAAGTTTGTACAAAAAAGCAGGCTATGAAGGCACTAATCCTTGTTG -3′ PtaGMP1_REV_attB 5′- GGGGACCACTTTGTACAAGAAAGCTGGGTCTCACATAACTATCTCTGGCTTCAAA -3′ PtaGMP1_FWD 5′- ATGAAGGCACTAATCCTTGTTG -3′ PtaGMP1_REV 5′- TCACATAACTATCTCTGGCTTCAAA -3′ Spruce PG29/CSLA PgCSLA1_FWD_attB 5′- GGGGACAAGTTTGTACAAAAAAGCAGGCTATGGATCGGTTATCTTCTGCAAAT -3′ PgCSLA1_REV_attB 5′- GGGGACCACTTTGTACAAGAAAGCTGGGTCCTAGGATGTTGGTATAAATGTTCC -3′ PgCSLA1_FWD 5′- ATGGATCGGTTATCTTCTGCAAAT -3′ PgCSLA1_REV 5′- CTAGGATGTTGGTATAAATGTTCC -3′ Spruce PG29/GMP PgGMP1_FWD_attB 5′- GGGGACAAGTTTGTACAAAAAAGCAGGCTATGAAGGCCCTCATATTGGT -3′ PgGMP1_REV_attB 5′- GGGGACCACTTTGTACAAGAAAGCTGGGTCTCACATGACAATTTCTGGCTTG -3′ PgGMP1_FWD 5′- ATGAAGGCCCTCATATTGGT -3′ PgGMP1_REV 5′- TCACATGACAATTTCTGGCTTG -3′ 2.3 Gene expression constructs 2.3.1 gRNA design A database with all possible gRNAs targeting each of the genes of interest was created using the variant-sensitive Aspen CRISPR designer pipeline (Tsai & Xue, 2015). From these databases, the gRNA1 (5′-TGGATTATGATCCGGTCGGACGG-3′) targeting the second exon of the two putative poplars CSLA genes (Potri.008G026400, Potri.010G234100) was selected. This gRNA contained a total of nine predicted off-targets with at least three mismatches in the seed region. For the putative GMPs (Potri.010G198800 and Potri.008G060100) the gRNA2 (5′-TTAGGGACACTGAGAGTCAA-3′) was employed. This last gRNA targeted the first exon of both genes and had zero mismatches with Potri.010G198800, and only one mismatch in position fifteen with Potri.008G060100. A total of twenty-one possible off-targets were predicted for gRNA2 all carrying at least three mismatches and being present in the seed region. 2.3.2 CRISPR Cas9 cloning Knockout constructs were generated by Gibson assembly following the protocol published by Jacobs & Martin, 2016. Briefly, the Medicago truncatula U6 promoter and the scaffold fragment 30 were amplified from the pUC gRNA Shuttle plasmid (Addgene plasmid #47024) using the primers SwaI.MtU6F and MtU6R and SpeI.hpR and hpF, respectively (Table 2.2). Then, these two fragments were cloned alongside each of the selected gRNAs into the p201N::Cas9 plasmid (Addgene plasmid # 59175) using the NEBuilder HiFi DNA Assembly Cloning Kit (New England Biolabs). A total of two different constructs were constructed: p201NCas9::gRNA1 and p201NCas9::gRNA2. Following transformation of TOP10 E. coli (Invitrogen) competent cells with 3µL of the Gibson reaction, PCR-positive colonies were confirmed by Sanger sequencing (Genewiz) of two different fragments, the first one using the primers: SpeI.hpR and gRNA1.SF.R-CSLAs/gRNA2.SF.R-GMPs and the second one using the primers: 2X35Shyb and gRNA1.U6.F-CSLAs/gRNA2.U6.F-GMPs (Table 2.2). Subsequently, confirmed vectors were transferred into A. tumefaciens strain EAH105 for poplar transformation. Table 2.2 Primers used for CRISPR Cas9 cloning. Primer name Primer sequence SwaI.MtU6F 5′- GATATTAATCTCTTCGATGAAATTTATGCCTATCTTATATGATCAATGAGG -3′ MtU6R 5′- AAGCCTACTGGTTCGCTTGAAG -3′ SpeI.hpR 5′- GTCATGAATTGTAATACGACTCAAAAAAAGCACCGACTCGGTG -3′ hpF 5′- GTTTTAGAGCTAGAAATAGCAAGTT -3′ gRNA1.SF.R-CSLAs 5′- AACTTGCTATTTCTAGCTCTAAAACGTCGGGATGGTGTTACCGGC -3′ gRNA2.SF.R-GMPs 5′- AACTTGCTATTTCTAGCTCTAAAACACCTGAGGCAAGGACAAGCAC -3′ gRNA1.U6.F-CSLAs 5′-TCAAGCGAACCAGTAGGCTTGCCGGTAACACCATCCCGAC -3′ gRNA2.U6.F-GMPs 5′- TCAAGCGAACCAGTAGGCTTGTGCTTGTCCTTGCCTCAGGT -3′ 2X35Shyb 5′- CTCCACCATGTTCACATCAATC -3′ StUbiP‐R 5′- ACATGCACCTAATTTCACTAGATG -3′ Cas9_p59175_FWD 5′- CTCAAGAGGCGCCGATATAC -3′ Cas9_p59175_FWD 5′- AGCCGATTGATGTCCAGTTC -3′ CRISPR_PtaCSLA1_FWD 5′- TCCCTACTGGTCGCGATG -3′ CRISPR_PtaCSLA1_REV 5′-TAGCTGTGCTTCATGCCTTC -3′ CRISPR_PtaCSLA2_FWD 5′- TCCTATGGTTCTGGTGCAAG -3′ CRISPR_PtaCSLA2_REV 5′- GCTTGGCAACTCAGCTTATC -3′ CRISPR_PtaGMP1_FWD 5′- CTCTGACTGTTGTGATGAACAGG -3′ CRISPR_PtaGMP1_REV 5′- TCCTTGCCTCAGGTTGGTAG -3′ CRISPR_PtaGMP2_FWD 5′- GCCTGGATGAATGTGTCAAG -3′ CRISPR_PtaGMP2_REV 5′- GACAGAGCATGGAATTGTTACG -3′ 31 2.4 Arabidopsis mutants transformation 2.4.1 Agrobacterium-mediated transformation The Arabidopsis mutant csla 2,3,9 was provided by Paul Dupree Lab from the University of Cambridge. The vtc1-1 mutant was obtained from the Arabidopsis Biological Resource Center (ABRC), stock number CS8326 (Conklin et al., 1996). Transformation of Arabidopsis mutants was completed following the floral dip method (Clough & Bent, 1998) using the GV3101 strains carrying the expression vectors. Seeds were plated on ½ concentration Murashige and Skoog basal medium (MS media) (Murashige & Skoog, 1962) plates containing 50 μg/mL kanamycin, stratified at 4°C in the dark for two days, and subsequently germinated under constant light. Germinated seeds were transferred to soil and grown for six weeks under long-day conditions (16 h light / 8 h dark) (Zhang et al., 2006). 2.4.2 Genomic screening of Agrobacterium-transformed Arabidopsis plants Genomic DNA was extracted from 7-week-old leaf tissue from Arabidopsis complemented mutants. Leaf tissue was ground and 400 μL of DNA extraction buffer containing 25 mM NaEDTA, 250 mM NaCl, 200 mM Tris HCl, and 0.5% Sodium Dodecyl Sulfate. Samples were centrifugated at 13,000 rpm for 3 min and the supernatant was placed in a new 1.5 mL tube. DNA precipitation was achieved by adding 300µL of Isopropanol and 10 min incubation followed by centrifugation at 13,000 rpm for 5 min. The supernatant was discarded, and the pellet was washed by adding 500 μL of 70% EtOH. After centrifugation at 13,000 rpm for 2 min, the pellet was dried and then resuspended in autoclaved distilled water. Genomic screening was performed using the following gene-specific primers on the extracted DNA (Table 2.1): PtaCSLA1 (PtaCSLA1_FWD and PtaCSLA1_REV), PgCSLA1 (PgCSLA1_FWD and 32 PgCSLA1_REV), PtaGMP1 (PtaGMP1_FWD and PtaGMP1_REV), and PgGMP1 (PgGMP1_FWD and PgGMP1_REV). 2.5 Arabidopsis plant phenotyping 2.5.1 Carbohydrate content A modified Klason procedure was used to determine the carbohydrate content in Arabidopsis stems (Glass et al., 2015). The bottom 13 cm of 7-week-old Arabidopsis stems were collected and dried at 50 °C for 48h. Stems were pooled for each line and wild-type plants, ground in a 2000 Geno/Grinder® (SPEX® SamplePrep, USA), and extracted overnight with acetone using a Soxhlet apparatus. Approximately 10 mg of extracted ground stem tissue was treated with 100 μL of 72% sulphuric acid for 5 min, then vortexed and briefly centrifuged on a bench-top centrifuge. Samples were incubated at 30 °C for 1 h at 500 rpm, and then 2 mL of nanopure water was added. Next, samples were vortexed and incubated at 130 °C for 75 min followed by centrifugation at max speed for 5 min. Finally, the supernatant was collected and diluted (1:10) in nanopure water. Carbohydrate content was determined by high-performance anion exchange chromatography (HPLC) of the filtered aliquots. A dilution series of the external standards (arabinose, rhamnose, galactose, glucose, xylose, and mannose) was used to create standard curves and determine sugar concentrations in the samples. The samples and standards were run on a Dionex Dx-600 anion-exchange HPLC (Dionex, Sunnyvale, CA) on a CarboPac PA1 column at a flow rate of 0.8 mL/ min. Meso-Erythritol was used as an internal standard. 2.5.2 Alcohol insoluble residue (AIR) Stems of five different transformed Arabidopsis plants were pooled together and ground to create a biological replicate for chemical analysis. Three samples (each with the stem tissue of five plants) from each of the three lines transformed with the PgCSLA1 gene were used for the AIR 33 procedure. With each sample, the process was as follows: 250 mg of the ground stem tissue was EtOH extracted at 70°C for 30 min. Samples were centrifugated at 2000 g for 10 min and the supernatant was discarded. Then, the pellet was resuspended in a chloroform: methanol (3:2 v/v) solution and mixed at room temperature overnight. Samples were centrifuged at 2000 g for 10 min and the supernatant was discarded. The pellet was resuspended again in a chloroform: methanol (3:2 v/v) solution and mixed at room temperature for 1h. The samples were then centrifuged at 2000 g for 10 min and the supernatant was discarded. Subsequently, the pellet was washed in 100% EtOH and centrifuged again. Then, the pellet was treated with 65% EtOH, centrifuged, and resuspended in 80% EtOH, and centrifuged again. Finally, the pellet was washed with 100% EtOH, centrifuged, and dried at 45°C in an oven. 2.5.3 Hemicellulose extraction To get a mannan-rich extract from the plant cell walls, Arabidopsis AIR extracts were subjected to a series of sequential extractions with different reagents following the protocol described by Pattathil et al., 2012. Briefly, 200 mg of the AIR extract was resuspended in 50mM of ammonium oxalate (pH 5.0) and incubated at room temperature for 24h with constant shaking. After incubation, the samples were centrifuged, and the supernatant was stored at 4°C (Ammonium Oxalate Extract). Then, the residual pellet was washed with 20 mL of deionized water, re-pelleted by centrifugation, and resuspended in 50 mM of sodium carbonate containing 0.5% sodium borohydride. After incubation for 24 h with constant shaking, the samples were centrifuged and the supernatant was stored at 4°C (Carbonate Extract), and the remaining pellet was washed with deionized water and re-pellet by centrifugation. The pellet was then subjected to 1M KOH solution containing 1.0% sodium borohydride treatment and incubated for 24 h with constant shaking. After incubation, the supernatant was decanted and stored at 4°C (1M KOH 34 Extract). The remaining pellet was resuspended directly in 4M KOH containing 1.0% sodium borohydride and incubated for 24 h. After incubation, the supernatant was decanted and stored at 4°C (4M KOH Extract). All extracts were dialyzed using 3,500 Da molecular weight tubing (S632724, Spectrum Laboratories Inc., CA, USA) and then lyophilized for 2 days. Once dried, these extracts were used for the linkage analysis. 2.5.4 Linkage Analysis Linkage analysis was performed following the protocol described by Black et al., 2021. Approximately, 400 µg of lyophilized 1M KOH and 4 M KOH extracts were placed into a borosilicate glass screw-cap test tube with a magnetic stir bar and resuspended in 400 µL of dry DMSO for 2 days. Then, 400 µL of NaOH/DMSO base solution was added to the sample resuspended in DMSO and stirred for 20 min followed by the addition of 100µL of iodomethane and stirred for 40 min. The addition of the base and the iodomethane was repeated once to ensure complete methylation of the sample. Next, 2 µL of water was added to the sample and the iodomethane was evaporated by sparging with nitrogen for 1 min. Then, 2 mL of DCM (dichloromethane) was added, the sample was vortexed for 20 s, and then the aqueous and organic layers were separated by low-speed centrifugation. The aqueous layer was discarded, and 2 mL of fresh water was added. After a total of five washes, the bottom DCM layer was transferred into a new tube and dried under a stream of nitrogen. For hydrolysis, 200 µL of 2M TFA (trifluoracetic acid) was added to the sample followed by 20 µL of inositol (1mg/mL) and incubated at 120°C for 2 h. Samples were then cooled and dried under a stream of nitrogen followed by the addition of 100 µL of isopropanol. The sample was evaporated again under nitrogen, and an additional step of isopropanol and evaporation of the sample was completed. For reduction, 300 µL of 10 mg/mL solution of sodium borodeuteride in 1M of ammonium 35 hydroxide was added to the sample and incubated at room temperature overnight. Then, the samples were neutralized by adding 5 drops of glacial acetic acid and 5 drops of methanol and finally dried under a stream of nitrogen. Next, 100µL of 9:1 methanol: acetic acid solution was added to the sample and dried down again. This step was repeated three more times until a crusty white residue was observed at the bottom of the tube. For acetylation, 250 µL of acetic anhydride was added to the sample and vortex. Next, 230 µL of concentrated TFA were added and the sample was incubated at 50°C for 20 min. Once cool, 1 mL of isopropanol was added to the sample and dried with nitrogen. This last step was repeated to ensure the removal of the TFA. Once dried, 2 mL of water followed by 2 mL of DCM were added to the sample, vortexed, and centrifuged for 1 min. The aqueous layer was removed, and 2 mL of water was added again. After a total of four washes, the organic bottom DCM layer was removed and transferred into a new tube. Finally, the sample was dried under a stream of nitrogen, diluted in 150 µL of DCM, and transferred to a GC tube. The GC/MS analysis of the partially methylated alditol acetates (PMAAs) was performed using an Agilent 7890A GC interfaced to a TQ8030 MSD. The composition analysis employed a Supelco SP-2330 fused silica capillary column (30 m × 0.25 mm ID) and was performed under the following conditions: initial temperature of 60 °C for 1 min, and then the temperature was increased at 27.5°C/min until 170 °C, and then slowed to 4°C/min until it reached 235 °C and held for 2 min. Finally, the GC was ramped at a rate of 3°C/min until the GC oven reached a final temperature of 240 °C and held for 12 min. A commercially available glucomannan extract was used to determine the elution time of the linkages of interest and their identity was confirmed by examining at their ion profile. The spectral database for PMAAs from the Complex Carbohydrate Research Center at the University of Georgia was used to identify the ion profile for each linkage. 36 2.5.5 Preparation of soluble hemicelluloses and PACE analysis Preparation of soluble hemicelluloses and a carbohydrate gel electrophoresis (PACE) analysis was carried out following the protocol described by Goubet et al., 2002. Briefly, twenty milligrams of AIR were treated with 1 mL of 4M KOH at room temperature for 1 h and then centrifuged at 4000 rpm for 5 min. The supernatant was loaded onto a PD-10 desalting column (GE Healthcare, IL, USA) and eluted with 50 mM ammonium acetate (pH 6.0). The eluted hemicelluloses were digested with 2µL of Cellvibrio japonicus Man26A (CjMan26A) mannanase (University of Newcastle) or Aspergillus nidulans GH5 (AnGH5) mannanase (Novozymes) in 50 mM ammonium acetate (pH = 6.0) at 37°C overnight. For sequential digestions, 2µL of β-glucosidase (β-Glc), β-mannosidase (β -Man), and α-galactosidase (α-Gal) enzymes were added after the digestion with either CjMan26A or AnGH5. Enzymes were deactivated after digestion with a heat treatment at 100°C for 20 min. Samples were then derivatized with 8-aminonaphthalene-1,3,6-tresulfonic acid overnight and dried at 60°C in a speedvac. Samples were then resuspended in 100 µL of 3M urea and 2µL were loaded onto a 20% acrylamide resolving gel. The electrophoresis was carried out at 200 V for 30 min and then 1000 V for 120 min. 2.6 Poplar transformation and genomic screening 2.6.1 Agrobacterium-mediated transformation Poplar leaf discs were transformed following the protocol by Wilkerson et al., 2014. Briefly, discs were cut from five-week-old poplar 717 leaves and co-cultivated for 1 hour at 28 °C with each of the A. tumefaciens strains (EAH105; OD600nm= 0.1-0.2) transformed with the expression vectors. Leaf discs were placed abaxial-side-up on WPM media (0.1 μM NAA, 0.1 μM BA, and 0.1 μM TDZ) and incubated in the dark for two days. Then, leaf discs were transferred to WPM 37 media (0.1 μM NAA, 0.1 μM BA, and 0.1 μM TDZ) containing 250 μg mL-1 cefotaxime and 500 μg mL-1 carbenicillin to eliminate A. tumefaciens and maintained in darkness for two days. Afterward, leaf discs were placed on WPM (0.1 μM NAA, 0.1 μM BA, and 0.1 μM TDZ) selection media containing 50 μg mL-1 of kanamycin for five weeks. Next, leaf discs were transferred to WPM shooting media (0.01 μM BA, 250 μg mL-1 cefotaxime, 500 μg mL-1 carbenicillin, and 50 μg mL-1 kanamycin) for four weeks and subsequently moved to WPM rooting media (0.01 μM NAA, 250 μg mL-1 cefotaxime, 500 μg mL-1 carbenicillin and 50 μg mL-1 kanamycin). After five weeks of growth, plants were genomically screened using the gene-specific primers (Table 2.1) and positive transformants were propagated in antibiotic-free WPM media (0.01 μM NAA). Apical stem pieces from propagated transformants (eight biological replicates per line) were grown for four weeks before transferring the plantlets to the soil. This protocol was concomitantly followed using wild-type poplar 717 leaf discs as a control, with the exception that all media were antibiotics-free. 2.6.2 Genomic screening Genomic DNA was extracted from leaves of transformed poplar in tissue culture following the same procedure mentioned previously. The genomic DNA target genes were detected by employing gene-specific primers (Table 2.1). 2.6.3 Gene expression analysis of poplar GMPs and spruce CSLA Total RNA was isolated from poplar xylem scrapings using the PureLinkTM RNA Mini Kit (Invitrogen). RNA was treated with DNase using the Turbo DNA-free kit (Ambion, Life Technologies), and then, it was used to synthesize cDNA with the iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA). Relative expression levels of PtaGMP1 (Potri.010G198800; primers qPCR_PtaGMP1_FWD and qPCR_PtaGMP1_REV), PtaGMP2 38 (Potri.008G060100; primers qPCR_PtaGMP2_FWD and qPCR_PtaGMP2_REV), and PgCSLA1 (GCHX01112248; primers qPCR_PgCSLA1_FWD and qPCR_PgCSLA1_REV) were quantified using BlasTaq 2X qPCR MasterMix (Applied Biological Materials, Richmond, BC) (Table 2.2). PCR reactions consisted of 5 μl BlasTaq 2X master mix, 0.3 µL of reverse and forward primers (10 μM), 1 μL cDNA, and deionized water to a volume of 10 µL. Three technical replicates were used per sample. qPCR parameters were as follows: 5 min at 95°C, 39 cycles of 95°C for 5 s, and 60°C for 30 s, then 1 cycle of 94°C for 10 s, followed by a melt curve cycle of 56°C to 95°C at 0.5°C increments for 5 s. Average expression of the reference gene PtrEF1β (Potri.009G018600; primers PtEF1β-qPCR-FWD and PtEF1β-qPCR-REV) was used to calculate the relative expression of the target genes (Table 2.3). Finally, fold-change was calculated relative to the lowest-expression line by subtracting the normalized expression from the highest reported target gene. Table 1.3. Primers used for qPCR of poplar GMPs and spruce CSLA. Plant/Gene Primer name Primer sequence Spruce- CSLA1 qPCR_PgCSLA1_FWD 5'- CGCGGCTATGTGAAGGAATG -3' qPCR_PgCSLA1_REV 5'-GAGCCACTTCAGGATTGTGC -3' Poplar 717- GMPs qPCR_PtaGMP1_FWD 5'- GAGGGTTGTTTGATTGGACCTG -3' qPCR_PtaGMP1_REV 5'- AACTCCACGCATCACAGAGC -3' qPCR_PtaGMP2_FWD 5'- TCCTCATCAAAGCTGGCCAC -3' qPCR_PtaGMP2_REV 5'- TCGACAATGCATCCTGGTCC -3' Poplar 717- reference gene Elongation Factor 1β (EF1β) PtEF1β-qPCR-FWD 5'- GGCATTAAGTTTTGTCGGTCTG -3' PtEF1β-qPCR-REV 5'- TTGACGAGCATGACAGAGGC -3' 2.6.4 Production of CRISPR Cas9 poplar lines Poplar leaf discs were transformed with the p201NCas9::gRNA1 and p201NCas9::gRNA2 constructs following the protocol described previously. A total of 1500 leaf discs were transformed with each construct. 39 2.6.5 Genomic screening of CRISPR poplar lines Genomic DNA was extracted from leaves of tissue culture poplar trees as described previously. Genetic screening of the plants was completed by amplifying a fragment of the CAS9 gene using the primers Cas9_p59175_FWD and Cas9_p59175_REV. Only positive lines were subsequently analyzed by Sanger sequencing to confirm the mutations. 2.6.6 Determination of mutant spectrums in CRISPR poplar lines Using Genomic DNA isolated from lines that proved positive after the genomic screening of Cas9, a PCR was performed using the primers CRISPR_PtaCSLA1_FWD and CRISPR_PtaCSLA1_REV to amplify the PtaCSLA1 sequence, CRISPR_PtaCSLA2_FWD and CRISPR_PtaCSLA2_REV to amplify the PtaCSLA2, CRISPR_PtaGMP1_FWD and CRISPR_PtaGMP1_REV for PtaGMP1, and CRISPR_PtaGMP2_FWD and CRISPR_PtaGMP2_REV to amplify PtaGMP2. PCR products were sequenced by Sanger using the same primers employed for amplification. The confirmation of a biallelic or monoallelic mutation of each gene was completed by analyzing the chromatograms and comparing them with the known gene sequences of each possible allele. 2.7 Poplar phenotyping 2.7.1 Growth conditions and measurement The apical meristems of six-week-old transformed plants and corresponding controls were transferred to two-gallon pots containing perennial soil mix (50% peat, 25% fine bark, and 25% pumice; pH 6.0). Trees were grown on flooding tables under 16 h of light at the UBC greenhouse for five months. At the time of harvest, stem height, stem diameter, and weight of fresh biomass were recorded. Height was measured from root collar to tree apex, while diameter was measured 10 cm above root collar with digital calipers. The weight of fresh biomass was recorded using 40 the whole tree cut 10 cm above the root collar. Developing xylem was collected by scraping the debarked stems and subsequently flash-frozen in liquid nitrogen. Stems were air-dried for two weeks and then a 10-cm piece from each stem was ground using a Wiley mill to pass a 40-mesh sieve. 2.7.2 Carbohydrate and lignin content Cell wall composition was quantified using the secondary acid hydrolysis procedure (Coleman et al., 2008). Briefly, 40-mesh wood powder was Soxhlet extracted with acetone for 24h. Extractive-free tissue (200 mg) was dried at 50°C overnight and treated with 3 mL of 72% sulphuric acid for 2 h with constant manual stirring with a glass rod every 10 min. Next, 112 mL of water were added, and samples were autoclaved at 121°C for 60 min. After autoclaving, the samples were filtered through pre-weighed coarseness crucibles, and the remaining residues were rinsed with 150 mL of water, dried overnight at 105°C, and weighed to determine acid-insoluble lignin. The filtered aliquot was used to determine the acid-soluble lignin by measuring its UV absorbance at 205 nm. Carbohydrate content was determined by HPLC of the filtered aliquots. A dilution series of the external standards (arabinose, rhamnose, galactose, glucose, xylose, and mannose) was used to create standard curves and determine sugar concentrations in samples. The samples and standards were run in a Dionex Dx-600 anion-exchange HPLC column (Dionex, Sunnyvale, CA) at a flow rate of 0.8 mL/ min with post-column detection. Fucose was used as an internal standard. 2.7.3 Cross-sectional staining Wood samples from five months old transgenic poplar trees were soaked overnight in dH2O. Samples were then cut into 40 μm cross sections with a Spencer AO860 hand sliding microtome (Spencer Lens Co., Buffalo, NY. USA) and stored in falcon tubes with dH2O. Samples were 41 treated with 0.1% Pontamine Fast Scarlet 4B for 10 min and then washed twice with TBST (10 mM Tris-buffer, 0.25 M NaCl, pH 7, with 0.1% Tween). Then, the sections were mounted onto glass slides and visualized on an Olympus FV1000 MPE laser scanning microscope (Olympus Corp., Tokyo, Japan) employing Olympus Fluoview software (FV10-ASW2). 2.7.4 Mannan antibody detection The prepared cross-section samples described above were also subjected to antibody labelling. Briefly, immunolabeling was performed as follows: wood cross-sections were treated with 1 M KOH solution for 1h, washed out with TBST, and then blocked with 5% BSA in TBST for 1h and 30 min at room temperature. Then, sections were incubated with diluted (1:20) Rat LM21 anti-(1,4)-β-mannan primary antibody (Kerafast, US) and Mouse CCRC-M138 anti-xylan primary antibody (Carbosource) in the blocking solution at 4°C overnight, and subsequently washed twice with TBST for 5 min. Next, samples were treated with diluted (1:400) Goat anti-Rat IgG Alexa FluorTM 568 and diluted (1:1000) Goat anti-Mouse IgG Alexa FluorTM 488 secondary antibodies (Invitrogen) in the blocking solution and incubated for 1 h at room temperature. Samples were washed three times with TBST and mounted on glass slides for visualization. Poplar stem cross sections were visualized on an Olympus FV1000 MPE laser scanning microscope (Olympus Corp., Tokyo, Japan) using the Olympus Fluoview software (FV10-ASW2). 2.8 Statistical analysis Square-root or natural-log transformations were used on data to meet model assumptions of normality and homoscedasticity. When met, the means of more than two groups were compared using a one-way analysis of variance (ANOVA). If the ANOVA showed a significant difference, a Tukey HSD post-hoc analysis with a Bonferroni correction was conducted to identify the 42 significant pairs. Data where the assumptions were not met, was evaluated with a non-parametric test, using a Kruskal-Wallis test followed by a Dunn’s post-hoc analysis using also a Bonferroni correction. The statistical significance of the p-values was assessed with an experiment-wise error rate of 0.05. All analyses were conducted in the R Software Environment (version 4.3.1). 43 Chapter 3: Characterization and expression of CSLAs 3.1 Introduction Mannans are highly conserved through plant evolution; they are the predominant cell wall polymer in several green algae, are highly abundant in the cell walls of early land plants such as mosses and lycophytes, and are also present in the cell walls of gymnosperms and angiosperms (Rodríguez-Gacio et al., 2012; Scheller & Ulvskov, 2010). Mannans play an essential role in plant embryonic development, as well as in maintaining the seed mucilage architecture (Goubet et al., 2009; Voiniciuc et al., 2015). Recently, they have been also associated with the regulation of the transition between primary cell wall and secondary cell wall deposition by acting as signal molecules (Zhang et al., 2023). Mannans, as it occurs with xylan, also interact with other cell wall polymers in plant tissues to confer form and function (Yu et al., 2022). Recent studies have shown that mannans can bind directly to cellulose microfibrils and are cross-linked to lignin via ether bonds (Terrett et al., 2019; Terrett & Dupree, 2019). Although mannans have been widely studied in seed mucilage and primary cell walls (Voiniciuc et al., 2015; Yu et al., 2014, 2018, 2022), their role in secondary cell wall stability and the genes responsible for their synthesis are still largely unknown in woody plants. Having a better understanding of the events that regulate the formation of the different components of cell walls is essential to transition into a sustainable biobased economy where lignocellulosic feedstocks play a pivotal role. The mannan backbone is synthesized in the Golgi lumen by mannan synthases from the glycosyltransferase (GT) superfamily 2 called cellulose synthase-like (CSL) A proteins. These enzymes can catalyze the synthesis of mannan or glucomannan polymers by using guanosine diphosphate (GDP)-mannose and GDP-glucose as substrates (Scheller & Ulvskov, 2010; Voiniciuc, 2022). A total of nine CSLAs have been identified in Arabidopsis (AtCSLA1 to 44 AtCSLA9), from which AtCSLA7 was shown to be essential for embryo development, while AtCSLA2, AtCSLA3, and AtCSLA9 were responsible for producing mannan in seed mucilage and plant stems (Goubet et al., 2003, 2009). In the perennial and fast-growing tree, poplar, five genes belonging to the CSLA family have been predicted to exist (PtCSLA1 to PtCSLA5) (Liepman et al., 2005; Suzuki et al., 2006), yet none of them have been functionally characterized in planta. This study aimed to advance our understanding of mannan biosynthesis and its biological role in cell walls by identifying CSLA candidate genes from different tree species and manipulating their expression in poplar stems. We hypothesized that mannan present in the xylem of trees may play an important role in maintaining the cell wall integrity either by interacting with other cell wall polymers or by serving as a signal molecule that regulates plant development. Using a combination of bioinformatic and phylogenetic tools, we identified two putative genes from spruce (Picea engelmannii × glauca × sitchensis; PG29) and poplar (Populus tremula × Populus alba; 717) that encode CSLA proteins. These genes were functionally assessed in the Arabidopsis triple mutant csla 2,3,9 depleted of mannan. We further evaluated the effects on plant growth and cell wall composition by eliminating mannan from xylem tissue by mutating poplar CSLA candidates via CRISPR Cas9. Finally, we assessed the effect of increasing mannan in the secondary cell walls of trees by expressing the spruce CSLA candidate in poplar stems. Our results suggest that mannan is not essential for SCW formation, as we did not observe major changes in growth or cell wall composition in the transgenic trees devoid of mannan. This research opens new avenues to investigate the contribution of mannan along with other hemicelluloses to cell wall architecture. 45 3.2 Bioinformatics of putative CSLAs In poplar, a total of five CSLA genes were identified: Potri.008G026400, Potri.010G234100, Potri.006G116900, Potri.009G149700, and Potri.004G1890 (Fig. 3.1). In the case of spruce, we identified only one gene as a likely candidate: GCHX01112248 (Fig. 3.1). These putative CSLAs had similar characteristics to the known Arabidopsis CSLAs in terms of their amino acid sequence, the number of transmembrane domains (Table 3.1), and the occurrence of the QxxRW motif (Fig. 3.2). To narrow the CSLA candidates for mannan synthesis in poplar xylem, we used as a reference, the RNA-seq expression database (hereafter GolS transcriptome database) generated by Unda et al., 2017. This expression database was generated from poplar trees expressing the Arabidopsis GolS3 galactinol synthase that manifested in higher cellulose and a significantly reduced lignin and mannose content compared to wild-type trees, making them ideal for identifying genes that are likely involved in mannan biosynthesis. From the poplar candidates, Potri.008G026400 (hereafter PtaCSLA1) and Potri.010G234100 (hereafter PtaCSLA2) were differentially expressed in the poplar xylem when examining the GolS transcriptome database; the highest expressed gene in wild-type trees, as well as the most significantly downregulated gene in the transgenic trees was PtaCSLA1 (Table 3.2). Hence, PtaCSLA1 and GCHX01112248 (hereafter PgCSLA1) were selected to be functionally evaluated in the Arabidopsis triple mutant csla2,3,9. 46 Figure 3.1 Phylogenetic tree of the potential poplar and spruce CSLA orthologs. CSLAs from Arabidopsis are marked in cyan colour (AT5G22740: CSLA2; AT1G23480: CSLA3; AT5G03760: CSLA9). Poplar candidates are in dark blue and the Spruce PG29 candidate selected for the functional analysis in Arabidopsis is in green. Numbers in the labels of the nodes correspond to the UFBoot support. The scale at the bottom shows phylogenetic distance of the associations. The tree was constructed using the LG+G4 substitution model. Table 3.1. Total of transmembrane domains (TMD) in CSLAs of Arabidopsis and the putative CSLAs of poplar and spruce. Transmembrane domains were predicted using the TMHMM server v. 2.v. Plant Species Accession number/ Gene Total of TMD predicted Arabidopsis thaliana AT5G22740/CSLA2 4 Arabidopsis thaliana AT1G23480/CSLA3 5 Arabidopsis thaliana AT5G03760/CSLA9 5 Populus trichocarpa Potri.008G026400/CSLA1 6 Populus trichocarpa Potri.010G234100/CSLA2 5 Picea engelmannii × glauca × sitchensis GCHX01112248/CSLA1 5 47 Table 3.2. Expression of CSLA poplar candidate genes in xylem of the Galactinol Synthase (GolS) transgenic poplar and WT trees from the Mansfield lab transcriptome database. Expression levels are represented as fragments per kilobase of transcript per million mapped reads (FPKM). Gene Accession number Wild Type Mutant Log2Fold Potri.008G026400 399.063 53.801 -2.890 Potri.010G234100 68.984 14.001 -2.300 Potri.009G149700 17.474 11.214 -0.639 Potri.006G116900 13.062 5.696 -1.197 Potri.004G189000 9.102 5.068 -0.844 48 Figure 3.2 Multiple sequence alignment of Arabidopsis CSLAs and the putative CSLAs from poplar and spruce. In red is the QxxRW motif characteristic from known CSLAs. 49 3.3 Putative CSLAs from spruce and poplar increase mannose content in the Arabidopsis csla2,3,9 triple mutant The Arabidopsis csla2,3,9 triple mutants were transformed with the CESA7::PtaCSLA1 and CESA7::PgCSLA1 constructs via Agrobacterium-mediated transformation. Two lines were obtained carrying the CESA7::PtaCSLA1 construct, while four positive lines were obtained carrying the CESA7::PgCSLA1 construct. Each line was confirmed by genomic screening using leaf tissue and gene-specific primers (Table 2.1). Complemented lines grew similar to the wild type and csla2,3,9 mutant Arabidopsis plants, no changes in the growth rate, overall plant size, or flowering time were observed. To determine if the complemented lines recovered the phenotype of the csla2,3,9 mutants depleted of mannan, a polysaccharide compositional analysis (Klason) was performed using the bottom part of 7-week-old stems of positive transgenic lines, wild type, and the triple mutant plants. It was apparent that the triple mutants were complemented by both constructs, CESA7::PtaCSLA1 or CESA7::PgCSLA1, as the mannose content increased relative to the triple mutant plants. In the two lines carrying the CESA7::PtaCSLA1 construct, mannose content reached 19.39 µg/mg of extracted tissue (Table 3.3) relative to 19.58 µg/mg found in the corresponding wild-type control plants, while in the lines complemented with the CESA7::PgCSLA1 construct, the recovered mannose content was as much as 23.52 µg/mg relative to the 21.24 µg/mg found in the corresponding wild-type control plants (Table 3.4). In both experiments, no mannose was detected in the csla2,3,9 triple mutant, as previously described (Goubet et al., 2009). No significant changes in other compositional sugars between the complemented lines and the wild-type plants were observed. Together these results suggest that both PtaCSLA1 and PgCSLA1 encode functional mannan synthases capable of producing mannan in the secondary cell walls of Arabidopsis. 50 Table 3.3. Structural polysaccharide composition of Arabidopsis WT plants, csla2,3,9 triple mutants, and csla2,3,9 mutants complemented with CESA7::PtaCSLA1. Values shown in bold are significantly different from the csla2,3,9 triple mutant (Kruskal-Wallis and post-hoc analysis, p-value <0.05, n=3-4 for each line). The standard error is represented in brackets. Values are represented in µg/mg of extracted tissue. WT: wild-type Col-0 Arabidopsis, Ara: Arabinose, Rha: Rhamnose, Gal: Galactose, Glc: Glucose, Xyl: Xylose, Man: Mannose. Line Ara Rha Gal Glc Xyl Man WT 14.65 (5.30) 8.05 (0.60) 15.30 (1.48) 353.13 (14.49) 121.62 (6.28) 22.45 (0.57) csla2,3,9 9.91 (0.93) 9.58 (0.90) 15.29 (1.13) 343.37 (5.95) 151.05 (7.17) - Line 1 11.33 (0.37) 10.66 (0.39) 16.72 (0.31) 328.65 (19.99) 137.41 (2.47) 22.39 (0.91) Line 2 10.46 (0.86) 10.46 (0.59) 15.13 (0.80) 337.80 (9.57) 138.08 (2.82) 14.30 (0.80) Table 3.4. Structural polysaccharide composition of Arabidopsis WT plants, csla2,3,9 triple mutants, and csla2,3,9 mutants complemented with CESA7::PgCSLA1. Values shown in bold are significantly different from the csla2,3,9 triple mutant (Kruskal-Wallis and post-hoc analysis, p-value <0.05, n=3-4 for each line). The standard error is represented in brackets. Values are represented in µg/mg of extracted tissue. Ara: Arabinose, Rha: Rhamnose, Gal: Galactose, Glc: Glucose, Xyl: Xylose, Man: Mannose. 3.4 PgCSLA1 increases 4-Man and 4-Glu linkage content in the Arabidopsis csla2,3,9 triple mutant To examine the linkages of the mannan produced by the lines complemented with PgCSLA1, a methylation analysis was performed. To obtain a rich mannan extract suitable for the linkage analysis, mannan was isolated from the cell walls of 7-week-old soil-grown Arabidopsis stems Line Ara Rha Gal Glc Xyl Man WT 10.04 (1.13) 9.16 (0.47) 15.45 (1.58) 368.33 (16.93) 123.65 (5.57) 23.84 (0.75) csla2,3,9 11.37 (0.50) 10.79 (0.51) 16.29 (0.17) 364.84 (6.63) 150.17 (4.15) - Line 1 11.91 (0.99) 10.03 (0.49) 16.65 (0.33) 300.38 (31.48) 132.65 (3.94) 18.61 (2.87) Line 2 9.89 (0.72) 9.74 (1.03) 17.02 (0.48) 307.45 (8.28) 127.85 (4.34) 25.79 (1.24) Line 3 12.24 (0.46) 10.49 (0.66) 16.66 (0.33) 317.28 (7.24) 121.87 (3.41) 23.36 (1.08) Line 4 13.05 (1.56) 9.12 (0.73) 16.46 (1.56) 308.82 (32.26) 142.40 (11.58) 22.27 (2.75) 51 following the protocol described by Pattathil et al., 2012. We used wild-type tissue to first determine which of the extraction fractions containing hemicelluloses, the 1M KOH or the 4M KOH extract, was the most enriched with mannan (Fig. 3.3). We determined this by examining the amounts of 4-Man and 4-Glc linkages present in these extracted fractions, which are diagnostic for glucomannan (Fig. 3.4). A commercially available glucomannan extract was used to determine the elution time of the linkages of interest and their identity was confirmed by examining their parent ion profile. By employing this commercial glucomannan it was determined that 4-Man had an elution time of 19.54 min, while 4-Glc eluded at 20.52 min. For both linkages, we used the 118 and 233 ions as the diagnostic ions. Inositol was used as an internal standard and eluded at 28.02 min. When performing the linkage analysis using the wild-type sample from both extracts, it was apparent that the peaks of the expected linkages were only visible in the 4M KOH extract (Fig. 3.4). Therefore, moving forward, only the 4M KOH extracts were subsequently used to perform the linkage analysis employing stem tissue from the complemented lines, the csla2,3,9 mutants, and the wild type plants. It was observed that the 4-Man peak was present in wild-type and PgCSLA1-complemented mutant plants, but it was absent in the triple mutants, as predicted (Fig. 3.5). The 4-Glc linkage was higher in the complemented lines, but it was very similar in the wild-type and mutant plants. Other peaks with linkages corresponding to xylan were also present in all the samples. In general, the presence of the 4-Man peak in the complemented lines supports the previous results observed during the Klason procedure. Specifically, glucomannan is being produced in the csla2,3,9 triple mutants as a consequence of the expression of PgCSLA1 in Arabidopsis. 52 Figure 3.3 Hemicellulose extracts. Using wild-type Arabidopsis stems, two extracts, 1M KOH and 4 M KOH, were collected to determine which was enriched in mannan. 1M KOH 4M KOH 7x1000.250.50.7511.251.51.752+EI TIC Scan GLUCOMANNAN_LK100.D * 19.545* 28.026* 20.522 8x1000.250.50.7511.251.51.752+EI TIC Scan WT_1M_LK100.D * 17.975* 21.154 * 28.027* 14.054 8x1000.250.50.7511.251.51.752+EI TIC Scan WT_4M_LK100.D * 17.979* 19.541 * 20.518 * 28.028* 14.058 * 24.294Counts vs. Acquisition Time (min)14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 314-Man 4-Glc Glucomannan 1M KOH - WT 4M KOH - WT Figure 3.4 Linkage analysis of hemicellulose extracts using stems of wild-type plants. Using a commercially available glucomannan extract, the elution time of the diagnostic peaks, 4-Man and 4-Glc, was determined (19.54 min and 20.52 min, respectively; top chromatogram). From the two extracts collected using wild-type tissue, the diagnostic peaks were only observed in the 4M KOH extract, indicating that this fraction was enriched in mannan. 53 Figure 3.5 Linkage analysis of csla2,3,9 complemented lines using PgCSLA1. A linkage analysis was performed using the 4M KOH fraction from wild-type Arabidopsis plants, csla2,3,9 mutants, and csla2,3,9 complemented mutants with PgCSLA1. The 4-Man diagnostic peak for mannan was only observed in the wild-type and complemented lines, confirming the production mannan in the complemented lines as a consequence of the expression of PgCSLA1. 3.5 PgCSLA1 produces a random patterned glucomannan in Arabidopsis csla2,3,9 triple mutant In Arabidopsis, CSLA2 and CSLA9 produce glucomannan with different characteristics. On one hand, CSLA2 synthesizes a patterned glucomannan that can be decorated with galactosyl residues, while CSLA9 produces a random patterned glucomannan backbone where galactose decorations are normally absent ( Yu et al., 2022). To date, it is not clear yet whether the capability of synthesizing a particular backbone structure is intrinsically specific to the CSLA 7x100123+EI TIC Scan GLUCOMANNAN_LK100.D * 19.545* 28.0267x100123+EI TIC Scan WT_4M_LK100.D * 17.979* 19.541* 28.028* 21.154* 14.058* 24.2947x100123+EI TIC Scan CSLA234_4M_LK100.D * 17.978* 14.058 * 28.029* 24.296* 20.5227x100123+EI TIC Scan 2248_4M_LK100.D * 17.975* 20.522* 28.026* 19.545 * 21.154 * 24.297* 14.058Counts vs. Acquisition Time (min)13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31csla 2,3,9 +EI TIC Scan 2248_4M_LK100.D * 17.975* 20.522* 28.026* 19.545 * 21.154 * 24.297* 14.058 Glucomannan WT – Col 0 4-Man 4-Glc csla 2,3,9 + PgCSLA1 54 enzymes or influenced by external factors, like the MSR proteins (Voiniciuc et al., 2019). We performed a PACE analysis to characterize the type of mannan that PgCSLA1 was producing in the complemented csla2,3,9 Arabidopsis lines. Two enzymes were employed for this analysis, CjMan26A and AnGH5 β-mannanases. The former cleaves β-Man-(1,4)-β-Glc bonds, but it is not able to cleave β-Glc-(1,4)-β-Man; this enzyme cannot tolerate Gal or acetate substitutions at the -1 position. In contrast, AnGH5 cleaves β-Man-(1,4)-β-Glc and does not digest β-Glc-(1,4)-β-Man bonds, however, it tolerates single Gal substitutions at the -1 position. In addition, sequential digestions were completed using β-glucosidase (β-Glc), β-mannosidase (β -Man), and α-galactosidase (α-Gal). The main oligosaccharides released after treating wild type and complemented lines samples with CjMan26A corresponded to M, MM, GMM, and MGMM (Fig. 3.5a); which is consistent with the patterns previously observed in the Arabidopsis csla2 single mutant, where mannan production depends on CSLA9 activity and the oligosaccharides liberated have a high degree of polymerization. In the csla2,3,9 mutant, no detectable oligosaccharides were apparent. When treating the CjMan26A digested sample with β-Glc and β-Man, all oligosaccharides were converted into mono- and disaccharides (Fig. 3.5b). Next, we treated the complemented lines with AnGH5 and subsequently with β-Glc, β-Man, and α-Gal. It was again observed that, similar to the CjMan26A digestion, all oligosaccharides were converted to either G, M, or MM (Fig. 3.6a). Only in the WT tissue, a faint band corresponding to GBGM was observed, indicating the presence of a trace of primary cell wall tissue in this sample (Fig. 3.6b). In general, our results are similar to those observed in the csla2 Arabidopsis mutant, where CSLA9-dependent oligosaccharides are formed, suggesting that PgCSLA1, when expressed in the secondary cell walls of Arabidopsis, synthesizes a non-patterned glucomannan absent of galactosyl side chains. 55 Figure 3.6 Mannan backbone structure in csla2,3,9 PgCSLA1-complemented lines digested with CjMan26A. a) Arabidopsis stem material from three different complemented lines (L1, L2, and L3) with PgCSLA1 was analyzed by PACE. Hemicelluloses were extracted from wild-type plants, the csla2,3,9 triple mutants, and complemented lines using alkali before being hydrolyzed with CjMan26A. The products were derivatized with 8-amino-naphthalene-1,3,6-trisulfonic acid and separated by PACE gel electrophoresis. The three complemented lines yielded oligosaccharides with a high degree of polymerization, showing a similar pattern to that of Arabidopsis csla2 mutants. b) Characterization of CjMan26A digested products by PACE using α-galactosidase (α-Gal), β-glucosidase (β-Glc), and β-mannosidase (β-Man) enzymes. Most of the oligosaccharides are reduced to mono- and disaccharides in the complemented line (only Line 1 is shown), suggesting that the mannan synthesized by PgCSLA1 lacks a discernible pattern and remains undecorated with Gal residues. M, Man; G, Glc; DP, Degree of polymerization; Mannan oligosaccharide standards M to M6. a) b) 56 Figure 3.7 Mannan backbone structure in csla2,3,9 PgCSLA1-complemented lines digested with AnGH5. a) Arabidopsis stem material from three different complemented lines (L1, L2, and L3) with PgCSLA1 was analyzed by PACE. Similar to CjMan26, enzymatic digestion produced oligosaccharides with a high degree of polymerization. b) Characterization of AnGH5 products by PACE using α-galactosidase (α-Gal), β-glucosidase (β-Glc), and β-mannosidase (β-Man) enzymes. In the complemented line it was observed that most of the oligosaccharides are reduced to mono- and disaccharides (just Line 1 is shown). In the wild-type sample only was a trace of GBGA observed, indicating the presence of residual primary cell wall in the sample, where β-GGM is present. These results suggest that the mannan synthesized by PgCSLA1 is similar to that of the mannan produced by CSLA9 in Arabidopsis: a non-patterned glucomannan. 3.6 CRISPR poplar knockouts of putative CSLA 3.6.1 Generation of double and single knockout CSLA poplar lines To confirm the activity of the putative poplar CSLA genes functionally evaluated in Arabidopsis csla2,3,9 mutants and to further understand the role of mannan in cell wall integrity, hybrid poplar 717 trees were transformed with the CRISPR Cas9 construct carrying the gRNA1 (3′-TGGATTATGATCCGGTCGGA-5′). This gRNA targeted a conserved region in exon two of the a) b) 57 CSLA poplar candidates and was designed such that the target region was SNP-free (Fig. 3.7; Table 3.5). After tree regeneration, PCR was employed to confirm the presence of the CAS9 gene in the tissue culture trees, and lines that were positive were sent for Sanger sequencing. During the plant regeneration process, we observed that multiple trees were able to grow in the selection media, however, most of them were not positive when Cas9 was amplified, nor did they carry a mutation in the target sequence when sequenced. Figure 3.8 Gene diagram of putative poplar CSLA genes and target CRISPR sites. Two genes were identified in poplar as putative CSLAs: Potri.008G026400 (PtaCSLA1) and Potri.010G234100 (PtaCSLA2). These genes share 93% amino acid sequence identity and carry the QxxRW motif. gRNA1 (3′-TGGATTATGATCCGGTCGGA-5′) was used to target the second exon of both genes to create a mutation via CRISPR Cas9 technology. Table 3.5. Overview of the targeted CSLA1 and CSLA2 locus in P. tremula and P. alba. Underlined is the sequence targeted by gRNA1, where the biallelic mutations were desired. Highlighted in gray is the PAM. The dark blue region emphasizes some of the differences found between the alleles of each gene. gRNA1gRNA1QxxRW motifPotri.008G026400 - PtaCSLA1Potri.010G234100 - PtaCSLA2QxxRW motifWild Type Gene Sequence P. alba PtaCSLA1 TTGGATTATGATCCGGTCGGACGGCCAAGAAAGCCCACA P. tremula TTGGATTATGATCCGGTCGGACGGCCAAGAAAGCCCGCA P. alba PtaCSLA2 CTGGATTATGATCCGGTCGGACGGCCATGAAAGGCCACA P. tremula TTGGATTATGATCCGGTCGGACGGCCATGAAAGGCCACA 58 According to the results obtained following Sanger sequencing, three lines with indels in Potri.008G026400 and Potri.010G234100 were obtained (hereafter PtaCSLA1/2 KO), as well as one line with a unique mutation in Potri.008G026400 (hereafter PtaCSLA1 KO). These four lines contained biallelic frameshift mutations in the target genes, and the indels were either a single nucleotide insertion or deletion relatively close to the PAM region (Fig. 3.8 and 3.9). Lines were positive after the Cas9 PCR had a 100% success rate of mutation for at least one of the targeted genes. A summary of the mutations in the lines obtained is found in Table 3.6. All successful CRISPR-modified poplar lines were clonally propagated and transferred to soil and grown in the greenhouse. Figure 3.9 Double-knockout via CRISPR Cas9 of putative poplar CSLA genes. Three poplar lines were obtained carrying mutations in both PtaCSLA1 and PtaCSLA2. In all cases, indels were either single nucleotide insertions or single deletions, causing a frameshift mutation. Here, the results of the double mutation in Line 2 are shown. In this line, PtaCSLA1 had an adenine (A) insertion, while PtaCSLA2 had either a thymine (T) or an adenine 59 (A) insertion (orange bars highlight these changes). Corroboration of biallelic mutations was completed by confirming that both alleles were amplified and visible in the chromatograms. The blue bar highlights a position where the different alleles for PtaCSLA1 were expected to differ. Finally, the grey bars emphasize the PAM position of the sequences of the different genes. Figure 3.10 Single knockout via CRISPR Cas9 of putative poplar CSLA1 gene. One poplar line was obtained carrying a unique mutation in PtaCSLA1. In this line, the indel corresponded to a single nucleotide deletion where a guanine (G) was removed. In the left chromatogram, this nucleotide change is highlighted by the orange bar, while in the chromatograms on the right, it is apparent that no changes occurred in the PtaCSLA2 gene sequence targeted by gRNA1. Grey bars emphasize the PAM position of the sequences of the different genes. 60 Table 3.6. Summary of mutations observed in single and double poplar CRISPR Cas9 knockout lines. For each poplar line, the target sequences of the CRISPR Cas9 constructs are shown. Both alleles of PtaCSLA1 and PtaCSLA2 are shown for each line (the upper sequence is the P. alba allele, and the lower sequence is the P. tremula allele). Underlined is the gRNA1 and the PAM is highlighted in gray. For each line, the indel patterns are shown and are highlighted in dark green. All lines were biallelically edited in the CSLA1 and CSLA2 locus, or only in the CSLA1 locus for the single mutant. Single/Double knockout Line Gene Changes in the target sequence (N20NGG) Indel Single knockout Line 1 PtaCSLA1 TTGGATTATGATCCGGTC - GACGGCCAAGAAAGCCCACA TTGGATTATGATCCGGTC - GACGGCCAAGAAAGCCCGCA -1 -1 PtaCSLA2 CTGGATTATGATCCGGTCGGACGGCCATGAAAGGCCACA TTGGATTATGATCCGGTCGGACGGCCATGAAAGGCCACA 0 0 Double knockout Line 1 PtaCSLA1 TTGGATTATGATCCGGTCTGGACGGCCAAGAAAGCCCACA TTGGATTATGATCCGGTCTGGACGGCCAAGAAAGCCCGCA +1 +1 PtaCSLA2 CTGGATTATGATCCGGTCGGGACGGCCATGAAAGGCCACA TTGGATTATGATCCGGTCGGGACGGCCATGAAAGGCCACA +1 +1 Line 2 PtaCSLA1 TTGGATTATGATCCGGTCAGGACGGCCAAGAAAGCCCACA TTGGATTATGATCCGGTCAGGACGGCCAAGAAAGCCCGCA +1 +1 PtaCSLA2 CTGGATTATGATCCGGTCTGGACGGCCATGAAAGGCCACA TTGGATTATGATCCGGTCAGGACGGCCATGAAAGGCCACA +1 +1 Line 3 PtaCSLA1 TTGGATTATGATCCGGTCGGGACGGCCAAGAAAGCCCACA TTGGATTATGATCCGGTCTGGACGGCCAAGAAAGCCCGCA +1 +1 PtaCSLA2 CTGGATTATGATCCGGTCTGGACGGCCATGAAAGGCCACA TTGGATTATGATCCGGTCCGGACGGCCATGAAAGGCCACA +1 +1 3.6.2 Mutation of CSLAs in hybrid poplar does not affect plant growth Both wild-type and knockout lines were grown and harvested after 5 months of growth in the greenhouse. In general, no substantial alterations in height, diameter, and fresh biomass were detected in the transformed lines compared to the wild-type trees (Table 3.7). Table 3.7. Mean growth measurement of single and double poplar CSLA knockout lines. Growth measurements of the single and double knockout lines are shown. Single knockout targeted only the CSLA1 gene, while in the double knockout both PtaCSLA1 and PtaCSLA2 were mutated. One-way ANOVA with a Tukey post-hoc test was used to evaluate statistical differences. No significant changes were observed when comparing the 61 knockout lines to the corresponding the wild-type trees (p <0.05, n=5-6 per line). The standard error is shown in brackets. KO: knockout; WT: wild type 717 poplar. Line Height (cm) Stem diameter (cm) Fresh biomass (g) WT 166.2 (3.53) 10.1 (3.53) 202.9 (11.92) PtaCSLA1 KO line 1 171.3 (4.65) 10.6 (0.40) 216.1 (14.58) PtaCSLA1/2 KO line 1 168.8 (6.05) 10.8 (0.16) 226.0 (14.89) PtaCSLA1/2 KO line 2 158.5 (2.32) 9.9 (0.55) 184.9 (13.30) PtaCSLA1/2 KO line 3 163.5 (4.52) 9.5 (0.46) 205.5 (15.83) 3.6.3 Poplar CSLA knockouts exhibit changes in mannan cell wall content The xylem cell wall composition of the transformed lines was ascertained by performing a Klason using dried, extract-free wood powder. Analysis of the individual carbohydrates by HPLC revealed that in the single and double knockouts, mannose was absent compared to the wild-type trees (Table 3.8). No significant differences were observed in the content of other structural sugars when the mutated lines were compared to the wild-type control (Table 3.8). In addition, immunofluorescence labelling of mannan polysaccharides was performed on stem sections of the knockouts and wild-type control trees using the LM21 antibody. As shown in Fig. 3.10, mannan labelling intensity was significantly reduced in both single and double knockouts compared to the wild-type control, but xylan and cellulose remained similar. Mannose content from the HPLC results and mannan intensity from the immunolabelling was indistinguishable between single and double knockout lines. In terms of lignin content, we did not observe statistically significant differences (Table 3.9), however, we do see that in the single and double knockouts that the total lignin increased slightly (from 20.602% up to 21.979% total cell wall content). Together, these results confirm that PtaCSLA1 is related to mannan biosynthesis in poplar and suggest that this gene is the most significant contributor to the synthesis of mannan in the secondary cell wall of poplar. 62 Table 3.8. Structural cell wall carbohydrates in the xylem of single and double CSLA poplar knockouts. Values are shown in µg/mg of extract-free xylem tissue across five biological replicates per line. A Kruskal-Wallis and post-hoc analysis was used to evaluate differences in mannan content in the wild-type trees and the knockout lines (p-value <0.05, n=5-6 per line). One-way ANOVA with a Tukey post-hoc test was used to evaluate statistical differences in the other sugars (p <0.05, n=5-6 per line). Significant differences are shown in bold. The standard error is represented in brackets. KO: knockout; WT: wild-type poplar 717. Line Ara Rha Gal Glc Xyl Man WT 2.08 (0.23) 2.89 (0.18) 6.54 (0.36) 446.90 (3.07) 176.93 (2.76) 20.75 (1.02) PtaCSLA1 KO line 1 1.83 (0.21) 3.15 (0.24) 6.68 (0.27) 451.27 (3.75) 182.36 (5.80) - PtaCSLA1/2 KO line 1 3.15 (0.28) 3.87 (0.21) 8.55 (0.24) 438.75 (4.53) 185.22 (1.62) - PtaCSLA1/2 KO line 2 2.77 (0.21) 3.75 (0.12) 7.79 (0.53) 435.66 (3.25) 186.41 (1.64) - PtaCSLA1/2 KO line 3 2.05 (0.35) 3.09 (0.38) 6.60 (0.64) 448.20 (6.56) 180.70 (5.96) - Table 3.9. Lignin content in single and double poplar CSLA knockout lines. Soluble, insoluble, and total lignin were determined by Klason in the single and double CSLA knockouts generated via CRISPR/Cas9. One-way ANOVA with a Tukey post-hoc test was used to evaluate statistical differences. No significant changes were observed when comparing the knockout lines versus the wild-type trees (p <0.05, n=5-6 per line). The standard error is represented in brackets. KO: knockout; WT: wild type 717 poplar. Line Insoluble lignin (%) Soluble lignin (%) Total Lignin (%) WT 17.97 (0.25) 2.63 (0.20) 20.60 (0.09) PtaCSLA1 KO line 1 18.40 (0.49) 2.99 (0.16) 21.40 (0.39) PtaCSLA1/2 KO line 1 19.53 (0.42) 2.28 (0.13) 21.82 (0.33) PtaCSLA1/2 KO line 2 19.45 (0.35) 2.15 (0.08) 21.61 (0.30) PtaCSLA1/2 KO line 3 19.16 (0.68) 2.82 (0.21) 21.98 (0.71) 63 Figure 3.11 Immunofluorescence labelling of xylem tissue from wild-type poplar trees, single, and double CSLA knockout lines. Transverse stem sections of poplar 717 wild-type trees and single and double knockout lines of CSLA genes were labelled with LM21 antibody to evaluate mannan presence. Mannan was noticeably absent in both the single and double knockout lines, and it was not possible to distinguish any differences in the immunolabelling among the lines. No discernible alterations in xylan or cellulose were detected when comparing wild-type and knockout lines. Xylan was labelled with the CCRC-M138 antibody, while cellulose was stained with Pontamine. Bar 100 µm. KO, knockout. 64 3.7 Expression of the putative CSLA from spruce in poplar 3.7.1 PgCSLA1 expression in poplar does not lead to plant yield penalty Following on the results obtained after the Arabidopsis csla2,3,9 mutant complementation with the CESA7::PgCSLA1 construct, we transformed poplar 717 using Agrobacterium as previously described. A total of five positive transgenic lines were obtained. RT-qPCR was used to measure the transgene expression in developing xylem tissue and revealed that Line 3 had the highest expression (Fig. 3.12). Positive lines were transferred to the greenhouse to grow in soil for 5 months. After this time, trees were harvested, and growth measurements were taken. Transgenic lines did not display significant differences in stem diameter, height, and biomass when compared to the wild-type trees (Table 3.10). Figure 3.12 Expression of PgCSLA1 in poplar lines carrying the CESA7::PgCSLA1 construct. Relative expression of the PgCSLA1 gene was measured by RT-qPCR. No expression was detected in the WT control. Three to four biological replicates were analyzed for each line using technical triplicates. 65 Table 3.10. Mean growth measurement of transgenic poplar lines expressing PgCSLA1. Growth measurements of five independent poplar lines expressing PgCSLA1 are shown. One-way ANOVA with a Tukey post-hoc test was used to evaluate statistical differences. No significant changes were observed when comparing the overexpression lines versus the wild-type trees (p <0.05, n=4-5 per line). The standard error is shown in brackets. WT: wild-type poplar 717. Line Height (cm) Stem diameter (mm) Fresh biomass (g) WT 183.2 (5.51) 9.7 (0.34) 209.1 (14.41) PgCSLA1 – Line 1 200.1 (5.52) 9.8 (0.20) 200.1 (25.97) PgCSLA1 – Line 2 197.2 (197.25) 10.1 (0.31) 249.0 (7.06) PgCSLA1 – Line 3 194.2 (11.64) 10.7 (0.31) 258.8 (28.58) PgCSLA1 – Line 4 180.0 (9.20) 10.0 (0.31) 215.4 (16.82) PgCSLA1 – Line 5 173.2 (3.10) 9.1 (0.34) 189.6 (4.72) 3.7.2 PgCSLA1 expression increases mannan content in poplar stems To investigate the impact of PgCSLA1 expression in poplar cell wall composition, a Klason analysis was performed using the extract-free xylem tissue of the wild-type and the five transgenic poplar lines generated. No significant differences were noted in acid-insoluble, acid-soluble, or total lignin content when comparing the transgenic lines to the wild-type controls (Table 3.11). In terms of structural sugars, four lines showed higher amounts of mannose compared to the wild-type trees, however, only Line 3 (p-value <0.001) and Line 9 (p-value =0.0012) were significantly different. These two lines showed an increase of up to 11.6% in mannan content when compared to the wild-type trees; in these lines, mannose content was found to be 35.23 µg/mg of extracted tissue compared to 31.58 µg/mg present in the wild-type trees (Table 3.12). In Line 3 the content of xylose was significantly lower (p-value= 0.042), while in Line 9 glucose was significantly higher (p-value= 0.017). Besides Line 6 which had a lower amount of rhamnose (p-value= 0.003) and galactose (p-value= 0.016), no significant differences were observed in the content of other structural polysaccharides (Table 3.12). 66 Together, these results confirmed that PgCSLA1 is related to mannan biosynthesis and demonstrate that its expression of PgCSLA1 in poplar secondary cell walls can indeed increase mannan content without negatively impacting plant yield. Table 3.11. Lignin content in poplar transformants expressing PgCSLA1. Acid-soluble, acid-insoluble, and total lignin were determined by Klason in independent lines expressing the CESA7::PgCSLA1 construct. One-way ANOVA with a Tukey post-hoc test was used to evaluate statistical differences. No significant changes were observed when comparing the knockout lines versus the wild-type trees (p <0.05, n=4-5 per line). The standard error is shown in brackets. WT: wild type 717 poplar. Line Insoluble lignin (%) Soluble lignin (%) Total Lignin (%) WT 17.84 (0.17) 3.20 (0.05) 21.05 (0.19) PgCSLA1 - Line 1 18.90 (0.56) 3.06 (0.04) 21.96 (0.54) PgCSLA1 - Line 2 18.17 (0.32) 3.11(0.11) 21.28 (0.22) PgCSLA1 - Line 3 17.66 (0.45) 3.15 (0.11) 20.81 (0.41) PgCSLA1 - Line 4 17.73 (0.63) 3.25 (0.03) 20.98 (0.62) PgCSLA1 - Line 5 17.83 (0.22) 2.92 (0.08) 20.75 (0.23) Table 3.12. Structural cell wall carbohydrates of poplar lines expressing PgCSLA1. Values are shown in µg/mg of extract-free xylem tissue across four biological replicates per line. One-way ANOVA with a Tukey post-hoc test was used to evaluate statistical differences. Values shown in bold are significantly different from wild-type trees (p-value <0.05, n=4-5 per line). The standard error is shown in brackets. WT: wild type 717 poplar. Line Ara Rha Gal Glc Xyl Man WT 4.71 (0.16) 5.72 (0.08) 11.27 (0.32) 451.37 (2.61) 190.68 (0.99) 31.58 (0.57) PgCSLA1 - Line 1 3.72 (0.12) 4.79 (0.15) 8.87 (0.42) 459.59 (3.04) 190.00 (0.72) 31.11 (0.27) PgCSLA1 - Line 2 3.81 (0.23) 5.05 (0.11) 9.08 (0.50) 458.97 (1.31) 187.43 (1.35) 32.04 (0.29) PgCSLA1 - Line 3 4.18 (0.22) 4.83 (0.20) 10.60 (0.25) 461.81 (2.06) 186.09 (0.55) 35.23 (0.76) PgCSLA1 - Line 4 4.64 (0.30) 5.59 (0.13) 11.03 (0.99) 456.04 (1.47) 190.25 (1.40) 33.72 (0.54) PgCSLA1 - Line 5 4.11 (0.12) 5.51 (0.22) 10.06 (0.11) 463.84 (3.45) 191.06 (0.90) 35.11 (0.50) 3.8 Discussion In this study, we identified CSLA genes that are responsible for mannan biosynthesis in woody species by validating genes identified through bioinformatics with functional characterization in 67 planta. We demonstrate that PtaCSLA1 from poplar and PgCSLA1 from Picea engelmannii × glauca × sitchensis can complement the Arabidopsis csla2,3,9 triple mutant and can alter the mannan content in poplar xylem without affecting plant growth. I will discuss the implications of these findings and their alignment with our original hypotheses. 3.8.1 CSLA family members in poplar trees and their contribution to mannan biosynthesis In poplar, bioinformatic analysis implicated five genes from the CSLA family in mannan biosynthesis (PtCSLA1, PtCSLA2, PtCSLA3, PtCSLA4, and PtCSLA5), two of them encoding proteins with demonstrated β-mannan synthase activity in vitro (PtCSLA1 and PtCSLA3; Liepman et al., 2007; Suzuki et al., 2006). In the current study, our phylogenetic results were consistent with previous reports (Suzuki et al., 2006). We also found that the CSLA family in poplar is composed of five genes that are grouped into three different subgroups, where PtCSLA1 and PtCSLA2 compose one of the subgroups and have a high expression in the xylem. In our study PtaCSLA1 and PtaCSLA2 corresponded to the PtCSLA1 and PtCSLA2 reported by Suzuki et al., 2006. In contrast with the expression data reported in this study, we only found PtaCSLA1 and PtaCSLA2 as the highest expressed putative CSLAs in the poplar xylem according with the GolS transcriptome database, but not PtaCSLA5. Also, we observed that PtaCSLA1 and PtCSLA2 were grouped with AtCSLA3 in our phylogenetic tree instead of being grouped with AtCSLA9 as reported by Suzuki et al., 2006. Research conducted in Arabidopsis has shown that even though several genes can be classified into a CSLA family, not necessarily all of them contribute equally to mannan production in plants. For example, in the model organism Arabidopsis, AtCSLA9 is the main contributor to mannan synthesis in stems, while AtCSLA2 and AtCSLA3 only produce a minor portion of the 68 glucomannan present in this tissue. Stems of Arabidopsis csla9 mutants have approximately 20% of the wild-type mannose content by weight, while csla2 and csla3 double mutants have 86% (Goubet et al., 2009). Even though some of the poplar CSLAs have been characterized in vitro (Suzuki et al., 2006), our understanding of their contribution to mannan synthesis in planta remained limited. In the current study we demonstrated that, when expressed in planta, PtaCSLA1 can recover the mannan content of Arabidopsis mutants with low mannan phenotypes. In addition, we showed that when PtaCSLA1 and PtaCSLA2 are mutated in poplar, mannan is absent in the xylem tissue. We also demonstrated that the mutation of PtaCSLA1 alone is sufficient to eliminate mannan from the tree xylem making the single and double knockout lines generated via CRISPR Cas9 indistinguishable. In general, our results aligned with our original hypothesis, the CSLA family in poplar is composed of several genes that contribute differentially to the mannan production in tree stems. Here, we confirmed the function of PtCSLA1 as a β-mannan synthase in poplar and provided evidence about its unique role as the main gene responsible for mannan production in trees. Our results suggest that the contribution of other CSLAs to the synthesis of this polysaccharide in tree xylem is likely null or negligible. Future studies should evaluate if the expression of genes that encode proteins with demonstrated β-mannan synthase activity, such as PtCSLA3, may be important in other stages of plant development or other tissues. 3.8.2 CSLA family in PG29 In the case of Picea engelmannii × glauca × sitchensis, we identified and functionally characterized at least one of the putative genes composing the CSLA family in this gymnosperm: PgCSLA1. Although we did not evaluate their functionality in the present study, GCHX01322020 (PgCSLA2) and GCHX01181033 (PgCSLA3), found in our phylogenetical 69 analysis, may also be part of this family. However, their functional characterization needs to be confirmed in future studies. Our results agree with reports of CSLAs found in other pine species such as loblolly pine (Pinus taeda), where two genes were identified and characterized in vitro as CSLAs (Liepman et al., 2007). In general, we observed that CSLAs from spruce display a high degree of conserved sequence homology with other plant species and always have the QxxRW motif present. The CSLA family, however, may differentiate between poplar and spruce by the fact that few genes form the gene family, which is likely a result of the absence of genome duplications in these tree species. 3.8.3 Implications of eliminating mannan from poplar stem by mutating CSLAs Studies in Arabidopsis seeds have shown that alterations in mannan content highly impact mucilage integrity, and NMR studies support that mannan interacts with cellulose and lignin suggesting a role for mannan in maintaining cell wall architecture (Terrett et al., 2019; Yu et al., 2018). In the current study, we explored how the absence of mannan affects poplar tree growth and cell wall composition. In general, our results do not support that mannan plays an essential role in poplar tree growth or xylem development. We did not observe significant changes in tree height, stem diameter, or total biomass in the lines where mannan was absent from poplar stems. These results are consistent with previous reports in Arabidopsis where the absence of mannan did not affect plant morphology or stem strength (Goubet et al., 2009). Even though our results show that mannan by itself does not seem to play a critical role in maintaining xylem architecture, its function is not likely to be confined solely to seed mucilage and embryogenesis. Recent studies in Arabidopsis have shown that when mannan is absent along with xyloglucan in plants, mutants display exacerbated phenotypes (Yu et al., 2022). Arabidopsis csla2 xxt1 xxt2 mutants manifest in smaller rosette diameter, significantly shorter stems and siliques than the 70 xxt1 xxt2 or the csla2 mutants, showing a functional connection between β-GGM and XyG (Yu et al., 2022). The tree lines created in the current study are an excellent platform to study the contribution of mannan alongside different hemicelluloses to assess plant cell wall functionality. Similarly, different lines of evidence have associated mannan as an important signal molecule implicated in plant development. In Arabidopsis, the mutation of AtMan6 was shown to modulate the transition between PCW and SCW formation. Arabidopsis man6 mutants looked morphologically like wild-type plants, but showed higher amounts of lignin, xylose, and mannose (Zhang et al., 2023). In the current study, even though we did not target enzymes in the plasma membrane associated with mannan remodelling, we expected that the CRISPR Cas9 poplar lines created may have an effect on the oligosaccharides involved in signalling. Even though we did not find statistical differences in the cell wall composition between the wild-type and CSLA knockout lines (outside of mannan content), we did observe small increases in lignin as well as xylose, such as those observed in the Arabidopsis man6 mutant. In general, our results indicate that the absence of mannan does not drastically affect SCW formation, suggesting that it may play only a minor or complemented role when working as a signalling molecule. In general, this research opens new avenues to investigate the impact of other carbohydrate-synthesizing enzymes in concert with mannan to affect plant cell wall architecture and strength. 3.8.4 Implications of expressing PgCSLA1 in Arabidopsis Mannan in angiosperms and gymnosperms is different in terms of content and composition. In angiosperms, glucomannan is found in very low abundance, whereas in gymnosperms, galactoglucomannan is abundant (Scheller & Ulvskov, 2010). In the present study, we identified PgCSLA1 as one of the most plausible candidates responsible for the synthesis of 71 galactoglucomannan in spruce. We showed that when expressed in planta, this gene was able to recover the Arabidopsis csla2,3,9 mutant phenotype, increasing mannan content to WT levels or even higher. Despite the widespread distribution of mannan in plants, it is not completely understood what the genetic factors are that determine the presence and structure of a specific type of mannan. Even though in spruce galactoglucomannan is prevalent, in the current study we only detected a non-patterned glucomannan in the Arabidopsis lines complemented with PgCSLA1. Two of the factors that have been shown to contribute to the galactosylation of the mannan backbone are the pattern of the sugars that constitute the backbone and the activity of an appropriate MAGT (Ishida et al., 2023). In Arabidopsis mucilage, for example, only patterned mannan is galactosylated (Nishigaki et al., 2021; Voiniciuc et al., 2015; Yu et al., 2018). The ability of CSLAs to produce a particular type of backbone seems to be modulated by different elements. In yeast, it has been shown that one of these elements is the presence of the MSR proteins (Voiniciuc et al., 2019). In Komagataella pastoris, Amorphophallus konjac (Ak) CSLA3 protein produces glucomannan, while AtCSLA2 produces pure mannan, and Coffea canephora (Cc) MANS1 remains inactive. When AtMSR1 was expressed alongside these proteins in yeast, the glucomannan content and structure changed. For instance, AtCSLA2 expressed alongside AtMRS1 produced glucomannan instead of pure mannan. Similarly, yeast cells expressing AkCSLA3 and AtMRS1 had up to 70 % more glucomannan compared to cells expressing AkCSLA3 alone. The activity of CcMANS1 was, however, not affected by AtMSR1, but was drastically enhanced when the native MSR protein (CcMSR1) was used (Voiniciuc et al., 2019). In the current study, the presence or absence of a particular MSR protein in the Arabidopsis csla2,3,9 mutant does not seem to affect the ability of PgCSLA1 to synthesize mannan but could 72 have influenced the type of backbone synthesized and hence its galactosylation. Finally, the lack of a particular MAGT able to decorate the backbone produced by PgCSLA1 cannot be discarded. 3.8.5 Implications of increasing mannan from poplar stems by expressing CSLAs One of the strategies used in the current study to increase mannan content in tree stems was the expression of the spruce gene PgCSLA1 in poplar stems. We demonstrated that expressing PgCSLA1 in poplar xylem is sufficient to increase mannan content and that this change does not affect plant yield or cell wall composition. Future studies should evaluate how this change may have affected the interaction of the other cell wall polysaccharides and hence the integrity of the cell wall matrix. 3.9 Conclusion In this study we identified two CSLA enzymes, one from poplar, PtaCSLA1, and one from spruce, PgCSLA1. We demonstrated that these two enzymes can recover mannan content in the Arabidopsis csla 2,3,9 mutant that is depleted in mannan. We showed that when mutated, PtaCSLA1 can cause a drastic reduction in the mannan content of xylem tissue without affecting tree growth. Our results support the idea that PtaCSLA1 is the main gene responsible for mannan biosynthesis in poplar xylem. Similarly, we showed that when expressed in poplar, PgCSLA1 can increase the content of mannan without affecting plant growth. This study provides new insights into the role of mannan in poplar trees, suggesting that is not essential for SCW formation and that it may play a minor or complemented role when acting as a signalling molecule. 73 Chapter 4: Characterization and expression of GMPs 4.1 Introduction Mannan is the primary hemicellulose found in the secondary cell wall of gymnosperms and is also present in different tissues of angiosperms (Scheller & Ulvskov, 2010). Mannans are essential for plant embryo development, play an important role in maintaining seed mucilage architecture, and can regulate plant development by acting as signaling molecule (Voiniciuc et al., 2015; Yu et al., 2022; Zhang et al., 2023). The mannan backbone is synthesized by cellulose synthase-like (CSL) A proteins that use GDP-mannose (GDP-Man) and GDP-glucose (GDP-Glc) as substrates (Scheller & Ulvskov, 2010). GDP-Man is synthesized by GDP-mannose pyrophosphorylase (GMP) in the cytosol. Along with being essential for mannan production, GDP-mannose is required for the N-glycosylation of proteins and plays a crucial role in the synthesis of L-ascorbic acid (AsA), an important antioxidant involved in plant growth and development (Conklin et al., 1999, 2000; Kka et al., 2018; Lukowitz et al., 2001; Scheller & Ulvskov, 2010). To date, it is not clear what genes are responsible for the production of GDP-Man in trees, and the effect that its mis-regulation has on cell wall composition. The present study seeks to understand how GDP-Man is synthesized in trees and how it contributes to mannan biosynthesis in the secondary cell wall. In Arabidopsis, Vitamin C Defective 1 (VTC1; AT2G39770) has been identified as the main enzyme responsible for catalyzing the reaction from Man1-phosphate (Man1-P) to GDP-Man (Conklin et al., 2000). Arabidopsis vtc1-1 mutants exhibited significant reductions in mannose content (35% of wild type) and AsA production (29% of wild type), as well as increased sensitivity to ozone due to their deficiency in AsA (Conklin et al., 1999, 2000). The activity of VTC1 is stimulated by KONJAC1 (KJC1) and KJC2 proteins (Sawake et al., 2015). We 74 hypothesized that similar to Arabidopsis, only a few genes are responsible for encoding enzymes related to GDP-Mane biosynthesis in trees and propose that by altering their expression substantial changes in mannan cell wall composition can be accomplished. In the present study, we identified one putative GMP from each of poplar (Populus tremula × Populus alba; 717) and spruce (Picea engelmannii × glauca × sitchensis; PG29) that are orthologs of Arabidopsis VTC1. We functionally evaluated their in-planta activity in the Arabidopsis vtc1-1 mutant by assessing their ability to recover the mannose content in the stems of these mutant plants. We also evaluated the effect on plant growth and mannan production when poplar GMP candidate genes were mutated via CRISPR Cas9. Finally, we expressed the spruce GMP candidate in poplar stems as a strategy to increase mannan content in plants and evaluate the function of this polysaccharide in cell wall architecture. Our results suggest that by manipulating the amount of GDP-Manin the cytosol is possible to alter mannan production in the cell walls and that such modification does not negatively impact plant growth. This work provides valuable information about the genes involved in GDP-Man biosynthesis in trees and offers an alternative pathway to investigate the contribution of this molecule in different biological processes, such as the production of AsA in trees. 4.2 Phylogenetic analysis of putative GMPs Two genes from spruce (BT102800 and BT107981) and four from poplar (Potri.010G198800, Potri.008G060100, Potri.006G090300, and Potri.008G006700) were identified as putative GMPs according to our phylogenetic analysis (Fig. 4.1). All putative candidate genes had the PDOC00094 signature peptide (PROSITE, PDOC00094, transferase hexapeptide-repeat) present in the C terminus, similar to already characterized enzymes that display the transferase activity (Fig. 4.2). Similar to the putative CSLAs from poplar (Chapter 3), the GMP candidate genes 75 were differentially expressed in poplar xylem when examining the GolS transcriptome database. Of the four candidate genes, Potri.010G198800 (hereafter PtaGMP1), was the highest expressed in wild-type trees and the most significantly downregulated in the poplar transgenics (Table 4.1; Unda et al., 2017). This gene shared high sequence similarity with Potri.008G060100 (92% similarity; hereafter PtaGMP2). Hence, we selected Potri.010G198800 and BT102800 (hereafter PgGMP1) to be functionally assessed in the Arabidopsis vtc1-1 mutant. Figure 4.1 Phylogenetic tree of the potential Arabidopsis VTC1 orthologs. Arabidopsis VTC1 is marked in cyan colour (AT2G39770). Poplar candidates are in dark blue and PG29 candidates are in green. Numbers in the labels of the nodes correspond to the UFBoot support. The scale at the bottom shows the phylogenetic distance of the associations. The tree was constructed using the LG+G4 substitution model. 76 Table 4.1. Expression of GMP poplar candidate genes in xylem from the Galactinol Synthase (GolS) transgenic poplar and WT trees from the Mansfield lab transcriptome database. Expression levels are represented as fragments per kilobase of transcript per million mapped reads (FPKM). Gene Wild Type Mutant Log2Fold Potri.010G198800 - PtaGMP1 292.707 86.880 -1.7 Potri.008G060100 - PtaGMP2 119.375 56.825 -1.07 Potri.006G090300 - PtaGMP3 26.839 24.246 -0.146 Potri.008G006700 - PtaGMP4 0.115 0.0734 -0.649 77 Figure 4.2 Multiple sequence alignment of Arabidopsis VTC1 and the putative GMPs from poplar and spruce. In blue is the hexapeptide-repeat PDOC00094 characteristic of enzymes with transferase activity. 78 4.3 Putative GMPs from spruce and poplar increased mannose content in the Arabidopsis vtc1-1 mutant To ascertain the function of the putative poplar and spruce GMPs, we complemented the Arabidopsis mutant vtc1-1 with the candidate genes. Mutant lines were transformed with the CESA7::PtaGMP1 and CESA7::PgGMP1 constructs via the Agrobacterium floral dip method. A total of three lines were obtained carrying the CESA7::PtaGMP1 construct, while only one was obtained carrying the CESA7::PgGMP1 construct. In the lines carrying the CESA7::PtaGMP1 construct, mannose was higher in stem tissues compared to the vtc1-1 mutant (Table 4.2); complete recovery of mannose content was only achieved in line 3, while lines 1 and 2 had a partial recovery. In the complemented line carrying the PgGMP1 gene, mannose content was completely recovered, and it was even higher than in the wild-type plants (p-value=0.025). No significant differences were observed in the content of other structural sugars when comparing the complemented and wild-type Arabidopsis plants. In general, these results suggest that PtaGMP1 and PgGMP1 are related to mannan biosynthesis and putatively encoding GDP-Man pyrophosphorylases. Table 4.2. Structural polysaccharides composition of Arabidopsis stem from WT plants, vtc1-1 mutants, and vtc1-1 mutants complemented with CESA7::PtaGMP1 and CESA7::PgGMP1. Values shown in bold are significantly different from the vtc1-1 mutant (Kruskal-Wallis and Dunn’s post-hoc analysis, p-value <0.05, n=3 for each line). The standard error is represented in brackets. Values represent µg/mg of extracted tissue. WT: wild-type Col-0 Arabidopsis, Ara: Arabinose, Rha: Rhamnose, Gal: Galactose, Glc: Glucose, Xyl: Xylose, Man: Mannose. Line Ara Rha Gal Glc Xyl Man WT 8.03 (0.65) 8.91 (0.70) 14.63 (1.48) 357.01 (13.47) 119.64 (6.10) 23.04 (1.58) vtc1-1 11.93 (0.32) 9.87 (0.15) 15.57 (0.41) 336.06 (4.76) 130.50 (1.22) 10.66 (0.27) PtaGMP1 Line 1 10.69 (0.36) 10.43 (0.14) 15.51 (0.23) 330.50 (6.42) 118.58 (3.96) 17.23 (1.34) PtaGMP1 Line 2 10.65 (0.04) 8.88 (0.46) 16.43 (0.57) 344.07 (15.83) 121.06 (3.49) 17.15 (0.74) PtaGMP1 Line 3 18.77 (6.34) 10.22 (0.62) 14.92 (0.61) 353.78 (12.21) 136.64 (4.71) 22.32 (1.37) PgGMP1 Line 1 10.68 (0.17) 10.48 (0.06) 15.28 (0.15) 368.64 (3.85) 138.07 (1.31) 23.74 (1.07) 79 4.4 CRISPR poplar knockouts of putative GMPs 4.4.1 Generation of GMP poplar knockout lines To confirm the function of PtaGMP1 and PtaGMP2 in mannan biosynthesis, poplar knockouts were created using CRISPR Cas9 targeting both genes. The first exon of both genes was targeted by using the gRNA2 (3′- TTAGGGACACTGAGAGTCAA-5′), which targeted a sequence free of SNPs (Fig. 4.3; Table 4.3). This gRNA matched perfectly to the target sequence of PtaGMP1 and had only one mismatch at position 15 within the target region of PtaGMP2. Following poplar transformation, trees were grown in the tissue culture room. PCR was employed to confirm the presence of the Cas9 gene in the tissue culture trees and positive lines were sent for Sanger sequencing. Similar to our observations with CSLAs (Chapter 3), during the selection in rooting media of the transformed plants, multiple trees developed superficial roots, however, they were not positive for Cas9 or exhibited a mutation in the target sequence when sequenced. Figure 4.3 Gene diagram of putative poplar GMP genes and target CRISPR sites. Two genes were identified in poplar as putative GMPs: Potri.010G198800 (PtaGMP1) and Potri.008G060100 (PtaGMP2). gRNA2 (3′-TTAGGGACACTGAGAGTCAA-5′) was used to target the second exon of both genes and create a mutation via CRISPR Cas9 technology. Potri.010G198800 - PtaMGP1Potri.008G060100 - PtaMGP2gRNA2gRNA280 Table 4.3. Overview of the targeted GMP1 and GMP2 locus in P. tremula and P. alba. Underlined is the sequence targeted by gRNA2 where the biallelic mutations were desired. Highlighted in gray is the PAM. The mismatch of gRNA2 with PtaGMP2 is shown in dark green. A total of three lines with indels in PtaGMP1 were obtained after confirmation by Sanger sequencing (hereafter PtaGMP1 KO lines). These three lines contained biallelic frameshift mutations in PtaGMP1 and all indels corresponded to insertions in the target sequence (Fig. 4.4). Lines that resulted positive after the Cas9 PCR had a 100% success rate of mutation for at least one of the targeted genes. No lines with indels in PtaGMP2 were obtained. A summary of the mutations in the PtaGMP1 KO lines is shown in Table 4.4. All successful CRISPR-modified poplar lines were clonally propagated and transferred to soil to be grown in the greenhouse for a period of 5 months. Wild Type Gene Target sequence P. alba PtaGMP1 CAAGTGGTTTAGGGACACTGAGAGTCAACGGCCTCAACC P. tremula CAAGTGGTTTAGGGACACTGAGAGTCAACGGCCTCAACC P. alba PtaGMP2 CTAGTGGTTTAGGAACACTGAGAGTCAACGGCCTCAACC P. tremula CTAGTGGTTTAGGAACACTGAGAGTCAACGGCCTCAACC 81 Figure 4.4 Single-knockout of PtaGMP1 via CRISPR Cas9. Three poplar lines were obtained carrying mutations in PtaGMP1. In all cases, indels were single nucleotide insertions causing a frameshift mutation. Here, the results of wild-type (WT) trees versus Line 2 are shown. In this line, PtaGMP1 had a thymine (T) insertion (chromatograms on the left) while PtaGMP2 did not display any mutations (chromatograms on the right). The corroboration of biallelic mutations was done by confirming that both alleles were amplified and visible in the chromatograms. The orange bar highlights the position where the mutation occurred. Gray bars emphasize the PAM position of the sequences of the different genes. The purple bar emphasizes the mismatch of the gRNA2 with PtaGMP2. Table 4.4. Summary of mutations observed in single PtaGMP1 poplar CRISPR Cas9 knockout lines. For each poplar line, the target sequences of the CRISPR Cas9 construct are shown. Both alleles of PtaGMP1 and PtaGMP2 are shown for each line (the upper sequence is the P. alba allele, and the lower sequence is the P. tremula allele). Underlined is the gRNA2, while the PAM is highlighted in gray. For each line, the indel patterns are highlighted in 82 dark green. All lines were biallelically edited in the GMP1 locus, while no mutations were observed in the GMP2 locus. Single knockout line Gene Changes in the target sequence (N20NGG) Indel Line 1 PtaGMP1 CAAGTGGTTTAGGGACACTGAGAGTTCAACGGCCTCAACC CAAGTGGTTTAGGGACACTGAGAGTTCAACGGCCTCAACC +1 PtaGMP2 CTAGTGGTTTAGGAACACTGAGAGTCAACGGCCTCAACC CTAGTGGTTTAGGAACACTGAGAGTCAACGGCCTCAACC 0 Line 2 PtaGMP1 CAAGTGGTTTAGGGACACTGAGAGTTCAACGGCCTCAACC CAAGTGGTTTAGGGACACTGAGAGTTCAACGGCCTCAACC +1 PtaGMP2 CTAGTGGTTTAGGAACACTGAGAGTCAACGGCCTCAACC CTAGTGGTTTAGGAACACTGAGAGTCAACGGCCTCAACC 0 Line 3 PtaGMP1 CAAGTGGTTTAGGGACACTGAGAGTTCAACGGCCTCAACC CAAGTGGTTTAGGGACACTGAGAGTTCAACGGCCTCAACC +1 PtaGMP2 CTAGTGGTTTAGGAACACTGAGAGTCAACGGCCTCAACC CTAGTGGTTTAGGAACACTGAGAGTCAACGGCCTCAACC 0 4.4.2 Mutation of PtaGMP1 in hybrid poplar does not affect plant growth Positively confirmed PtaGMP1 knockouts and wild-type control trees were harvested after 5 months in the greenhouse, where growth measurements were taken. No significant differences in height, diameter, and biomass were observed between the knockout lines and wild-type trees (Table 4.5). Table 4.5. Mean growth measurement poplar lines the PtaGMP1 knockout. Growth measurements of three independent PtaGMP1 knockout poplar lines are shown (n=5). A Kruskal-Wallis with Dunn’s post-hoc analysis test was used to evaluate statistical differences. No significant differences were found when comparing the transformed lines versus the wild-type trees (p <0.05, n=5-6 per line). The standard error is shown in brackets. KO: knockout; WT: wild type 717 poplar. Line Height (cm) Stem diameter (mm) Fresh biomass (g) WT 168.5 (3.34) 10.2 (0.436) 203.6 (14.6) PtaGMP1 KO line 1 172.3 (4.00) 10.2 (0.320) 204.7 (12.5) PtaGMP1 KO line 2 175.6 (5.24) 9.7 (0.166) 206.1 (5.98) PtaGMP1 KO line 3 169.5 (4.25) 10.0 (0.301) 221.0 (14.4) 83 4.4.3 Poplar PtaGMP1 knockout lines have reduced mannan content The xylem cell wall composition of PtaGMP1 knockout lines was determined by performing a Klason analysis using extract-free xylem flour. Total Klason lignin content, as well as soluble and insoluble lignin did not show significant differences when comparing the knockout lines to the wild-type controls (Table 4.6). HPLC analyses of the structural sugars revealed a significant decrease in mannose content between transgenic lines and the control trees (Table 4.7). The most significant reduction in mannose content was observed in line 2, where mannose was decreased by around 19.75% when compared to wild-type trees. Although all transgenic lines showed lower levels of mannan compared to wild-type trees, only line 2 and line 3 were statistically significantly different (p-value=0.009 and p-value=0.031, respectively). No significant changes in other sugars were observed between the knockout lines and the wild-type control trees. In conclusion, these results confirm that PtaGMP1 is related to mannan biosynthesis, presumably encoding a GMP enzyme in the cytosol. Table 4.6. Lignin content in PtaGMP1 knockout poplar lines. Insoluble, soluble, and total lignin were determined by performing a Klason analysis using xylem from the three PtaGMP1 knockout poplar lines and wild-type trees. A Kruskal-Wallis with Dunn’s post-hoc analysis test was used to evaluate statistical differences. No significant changes were observed when comparing the knockout lines versus the wild-type trees (p-value <0.05, n=4-5 per line). The standard error is represented in brackets. KO: knockout; WT: wild type 717 poplar. Poplar Line Insoluble lignin (%) Soluble lignin (%) Total Lignin (%) WT 18.60 (0.61) 3.31(0.79) 21.60 (0.62) PtaGMP1 KO line 1 17.30 (0.26) 3.64 (0.15) 21.01 (0.34) PtaGMP1 KO line 2 18.70 (0.16) 3.45 (0.05) 22.20 (0.11) PtaGMP1 KO line 3 18.20 (0.73) 3.46 (0.14) 21.60 (0.72) Table 4.7. Structural cell wall sugars in the xylem of PtaGMP1 knockout poplar lines. Values are expressed as µg/mg of extract-free xylem tissue across four or five biological replicates per line. A Kruskal-Wallis with Dunn’s 84 post-hoc analysis test was used to evaluate statistical differences. Significant differences are shown in bold (p-value <0.05, n=4-5 per line). The standard error is represented in brackets. KO: knockout; WT: wild-type poplar 717. Line Ara Rha Gal Glc Xyl Man WT 3.41 (0.24) 4.13 (0.28) 8.17 (0.39) 433.71 (1.77) 183.27 (2.03) 23.80 (0.65) PtaGMP1 KO line 1 3.15 (0.10) 3.60 (0.06) 7.98 (0.18) 442.33 (4.45) 186.78 (1.01) 20.79 (0.64) PtaGMP1 KO line 2 3.10 (0.13) 3.47 (0.06) 7.83 (0.16) 442.37 (4.31) 186.89 (0.79) 19.10 (0.39) PtaGMP1 KO line 3 3.22 (0.06) 4.27 (0.14) 7.90 (0.16) 438.03 (2.78) 184.42 (2.29) 19.37 (0.31) We evaluate the expression of PtaGMP1 and PtaGMP2 in the PtaGMP1 KO lines by performing a RT-qPCR (Fig. 4.5). In these lines the primers used for the RT-qPCR targeted a region located earlier than the stop codon caused by the frameshift mutation. In general, we observed that the expression of the two genes was lower when compared to the wild-type lines. Only in line 3, PtaGMP2 showed a higher expression. In general, these results suggest that PtaGMP2 does not seem to compensate for mutations only in PtaGMP1. Figure 4.5 Expression of PtaGMP1 and PtaGMP2 in wild-type and knockout lines. Relative expression of the PtaGMP1and PtaGMP2 genes was measured by RT-qPCR. In general, the expression of both genes was reduced in the knockout lines compared to the wild-type trees. Only in line 3, PtaGMP2 showed a higher expression. Three to four biological replicates were analyzed for each line using technical triplicates. 85 4.5 Expression of putative GMP from spruce in poplar 4.5.1 Expression of PgGMP1 in poplar does not affect plant growth We hypothesized that one of the limiting factors for producing mannan in the cell wall could be related to the lack of sufficient substrate available for the CSLA enzymes to synthesize the mannan backbone. Hence, we elected to transform poplar 717 with the CESA7::PgGMP1 construct using Agrobacterium. Two positive transgenic lines were obtained and once they were regenerated in tissue culture, they were transferred to the greenhouse to grow in soil for 5 months. Upon the completion of this growth phase, the plants were harvested, and their growth parameters and cell wall composition were assessed. In comparison with the control trees, no major developmental defects were observed in the transgenic lines carrying the CESA7::PgGMP1 construct. Specifically, we did not observe changes in stem height, diameter, and biomass, suggesting that expressing this gene in trees does not affect plant growth (Table 4.8). Table 4.8. Mean growth measurement of transgenic poplar lines expressing CESA7::PgGMP1. Growth measurements of two independent poplar lines expressing CESA7::PgGMP1 are shown. A Kruskal-Wallis with Dunn’s post-hoc analysis was used to evaluate differences in growth in the wild-type trees and the lines expressing PgGMP1. No significant changes were observed when comparing the lines carrying the PgGMP1 versus the wild-type trees (p <0.05, n=4-5 per line). The standard error is shown in brackets. WT: wild-type poplar 717. Poplar Line Height (cm) Stem diameter (mm) Fresh biomass (g) WT 183.2 (5.50) 9.7 (0.34) 209.1 (12.41) PgGMP1 Line 1 179.6 (4.27) 9.2 (0.37) 200.6 (12.88) PgGMP1 Line 2 186.7 (5.75) 9.6 (0.15) 205.7 (15.92) 86 4.5.2 PgGMP1 increases mannan content in poplar stems The xylem cell wall composition was again determined by a Klason analysis. Compared to the WT trees, there were no significant differences in total, insoluble, or acid-soluble lignin content in the transgenic lines (Table 4.9). The composition of structural polysaccharides was only different in line 2 in terms of mannose content (p-value=0.035; Table 4.10). In this line, the mannose content exhibited a 14% increase in comparison to the wild type, rising from 25.03 µg/mg of extracted tissue in wild-type trees to 28.51 µg/mg in the transgenic line. No changes were observed in other polysaccharides when comparing the transgenic lines to the wild-type trees (Table 4.10). Overall, these results suggest that PgGMP1 encodes a GMP enzyme and demonstrates that its expression within the secondary cell wall of poplar may be effective in augmenting mannan content in trees. Table 4.9. Lignin content in lines expressing CESA7::PgGMP1. Acid-soluble, acid-insoluble, and total lignin were determined by Klason analysis in independent lines expressing the CESA7::PgGMP1 construct. A Kruskal-Wallis with Dunn’s post-hoc analysis was used to evaluate statistical differences. No significant changes were observed when comparing the lines carrying the CESA7::PgGMP1 construct versus the wild-type trees (p <0.05, n=4-5 per line). The standard error is shown in brackets. WT: wild type 717 poplar. Poplar Line Insoluble lignin (%) Soluble lignin (%) Total Lignin (%) WT 18.09 (0.43) 3.10 (0.02) 21.19 (0.42) PgGMP1 - Line1 18.54 (0.37) 3.20 (0.11) 21.75 (0.45) PgGMP1 - Line2 18.46 (0.40) 2.98 (0.07) 21.44 (0.34) Table 4.10. Structural cell wall carbohydrates of poplar lines expressing CESA7::PgGMP1. Poplar 717 was transformed with the CESA7::PgGMP1 construct. Two lines were recovered, and sugars extracted from xylem tissue are shown in µg/mg of extract-free wood tissue. A Kruskal-Wallis with Dunn’s post-hoc analysis was used to evaluate statistical differences. Significant changes were observed in the mannan amount in line 2 versus the wild 87 type (p <0.05, n=4-5 per line). No significant changes were observed when comparing other sugars (p <0.05, n=4-5 per line). The standard error is shown in brackets. WT: wild type 717 poplar. Line Ara Rha Gal Glc Xyl Man WT 3.84 (0.17) 4.32 (0.16) 8.95 (0.19) 383.11 (1.77) 156.66 (1.19) 25.03 (0.45) PgGMP1 - Line1 3.46 (0.27) 4.09 (0.08) 8.26 (0.22) 391.62 (3.91) 156.25 (1.93) 25.42 (0.64) PgGMP1 - Line2 3.45 (0.19) 4.10 (0.11) 8.21 (0.20) 392.29 (4.38) 154.58 (0.98) 28.51 (0.51) 4.6 Discussion 4.6.1 Poplar AtVTC1 orthologs In the current study, we aimed to identify genes related to the synthesis of GDP-Man in poplar. We showed that at least four genes are putative orthologs of VTC1 in poplar: PtaGMP1 to PtaGMP4. We functionally characterized PtaGMP1 as one of the contributors to GDP-Man biosynthesis in this tree. We demonstrate that this gene, when expressed in Arabidopsis vtc1-1 mutant, can recover the mutant mannan content in cell walls. Similarly, when mutated in poplar, PtaGMP1 caused a reduction in the mannan content in xylem tissue. Our results suggest that PtaGMP1 is responsible, at least in part, for producing GDP-Man in the cytosol. Future studies should evaluate the transferase catalytic activity of the other potential orthologs identified, and determine their contribution to GDP-Man biosynthesis in trees. 4.6.2 White spruce P29 AtVTC1 orthologs In the case of PG29, we identified and functionally characterized PgGMP1 as an ortholog of AtVTC1. Similar to PtaGMP1, we showed that PgGMP1 can recover Arabidopsis vtc1-1 phenotype, at least the mannan production, and demonstrate that its expression in poplar also contributes to the synthesis of this polysaccharide in the secondary cell wall. Our bioinformatic results also suggest that at least two other genes may be implicated in the synthesis of GDP-Man 88 in PG29: BT10798 (PgGMP2) and GCHX01496356 (PgGMP3). Future studies should evaluate the functionality of these genes and their contribution to GDP-Man synthesis in spruce. 4.6.3 Implication of mutating AtVTC1 orthologs in poplar In the current study, we explored how modifications in the amount of GDP-Man produced in the cytosol can affect the synthesis of mannan in the tree cell walls. In Arabidopsis seed coat mucilage it has been shown that the structure of galactoglucomannan is dictated by the activity of the glycosyltransferases and not the availability of the nucleotide sugar pool, while the nucleotide precursors can impact the overall amount of the polysaccharides (Nishigaki et al., 2021). Here, we were able to demonstrate that a mutation in PtaGMP1 in poplar trees manifested in a reduction of approximately 20 % in mannan content in the secondary cell walls. Due to the fact that a complete reduction in mannan was not achieved by mutating this gene, it is conceivable that the other candidate orthologs may be contributing to the synthesis of GDP-Man or compensating. Specifically, due to its high expression in the xylem, we suspect PtaGMP2 could be a significant contributor to the synthesis of GDP-Man. Although we targeted both PtaGMP1 and PtaGMP2 using the gRNA2, multiple factors may have contributed to the absence of double mutants in our study. One of the limitations of CRISPR Cas9 technology is the generation of off-target cleavage sites; these can be reduced by employing a single gRNA when multiple genes are targeted (Leplé et al., 2007). The use of this methodology, however, limits the number gRNAs that can be employed and match perfectly with the targeted genes. Different studies have shown that the location of single mismatches in the gRNA sequence can affect the rate of mutation differently (Pattanayak et al., 2013; Zheng et al., 2017). In the gRNAs the interrupted 12 nt sequence at the 3′ end of the spacer segment is known as the seed region (Zheng et al., 2017). In general, it is 89 considered that single mismatches away from the PAM and seed regions are tolerated and do not compromise cleavage efficacies significantly (Zheng et al., 2017). In the current study, however, we were not able to recover knockout lines of PtaGMP2. One possible explanation for this can be related to the mismatch at the +15 position in the gRNA2. Despite not being in a key recognition area, this mismatch might be sufficient to avoid the cleavage of PtaGMP2 (Pattanayak et al., 2013). In addition, since GDP-Man affects ascorbic acid production and protein N-glycosylation, it is possible that plants with a double mutation in both GMP genes were not able to survive during the plant regeneration process. Finally, the low efficiency in the poplar transformation process might also explain the lack of PtaGMP1 and PtaGMP2 double knockouts generated. Ascorbic acid is a known antioxidant important for cell wall division, photosynthesis, and response to biotic and abiotic stresses (Bilska et al., 2019). We suspected that the reduction of GDP-Man in the transgenic PtaGMP1 knockout lines could also have decreased the AsAc production and hence affected their growth. However, we did not observe significant changes in plant biomass, stem diameter, height, or cell wall composition in these lines. We hypothesized that the reduction in GDP-Man levels in these lines was not sufficient to affect growth processes that depend on this antioxidant, whose production could also depend on other factors. One of the limitations of our study, however, is that we did not directly measure GDP-Man content, and hence future studies should confirm that PtaGMP1 is directly associated with the production of this component in the cytosol. 4.6.4 Implication of expressing P29 AtVTC1 orthologs in poplar An associated objective of this study was to assess whether expressing PG29 AtVTC1 orthologs in poplar could enhance mannan content in trees. We assumed that GDP-Man may be a limiting 90 factor facilitating significant increases in the content of this polysaccharide in the cell wall. Our results showed that by expressing PgGMP1 in poplar stems, mannose content can be increased up to 14% compared to wild-type trees, and that this change does not affect plant growth or the composition of other sugars in the cell wall. This increase in mannan content is very interesting considering that other factors in the mannan biosynthetic pathway that can limit this process were not altered. For example, in Arabidopsis, previous studies have shown that KJC1 and KJC2 are important proteins that stimulate the activity of VTC1 (Sawake et al., 2015). The kjc1 mutation reduced the activity of VTC1 to 10% and caused a 60% reduction in the content of AsAc (Sawake et al., 2015). Similarly, and although it is not clear yet how this process occurs, the transport of GDP-Man into the Golgi lumen has been suggested to be an additional limitation in the production of mannan. Nevertheless, it seems that none of these factors drastically limited the increase of mannan in the PgGMP1 transgenic lines. Future studies should evaluate how the modification of KONJAC and other enzymes involved in mannan biosynthesis can boost the production of mannan in trees. 4.7 Conclusion In the current study, we elucidated genes that are likely related to the synthesis of GDP-Man in poplar and spruce. We identified and functionally characterized at least one putative GMP from poplar, PtaGMP1, and one from spruce, PgGMP1. We showed that the mutation of PtaGMP1 in poplar significantly reduces mannan content in the cell wall, while the expression of PgGMP1 increases mannan content in tree stems. The changes caused by the modification of these two genes in trees do not appear to affect plant growth or cell wall composition significantly. We conclude that by manipulating the expression of PtaGMP1 and PgGMP1 changes can be made in mannan composition, however not substantive enough to impact growth or development. Future 91 studies are needed to confirm our findings and expand on the direct relationship of these two genes with the production of GDP-Man in the cytosol. 92 Chapter 5: Conclusions and future directions 5.1 Major findings in Chapter 3 and Chapter 4 One of the biggest limitations to understanding the functional role(s) of mannan in trees is the fact that the genes involved in its synthesis have largely not been functionally tested. In the present study, I focused my attention on identifying genes that putatively encode CSLAs and GMPs in poplar and spruce, and on examining the consequences of altering their expression on growth and cell wall composition in the xylem of plants. In Chapter 3, my phylogenetic analyses suggested that the CSLA family in poplar is composed of five candidate genes (PtCSLA1, PtCSLA2, PtCSLA3, PtCSLA4, and PtCSLA5), while spruce is composed of three (PgCSLA1 to PgCSLA3). The variability in the number of genes forming this family in these tree species is likely related to the presence/absence of a recent genome duplications and consequently, it agrees with previous reports in Arabidopsis and loblolly pine, where multiple genes were cataloged as CSLAs in the former while only a few were suggested for the latter (Liepman et al., 2007). In Chapter 4, I focused my attention on identifying putative GMP candidates. In this chapter, I showed that a total of four putative GMP gene candidates exist in poplar (PtaGMP1 to PtaGMP4), while three were classified as AtVTC1 orthologs in spruce (PgGMP1 to PgGMP3). As a first attempt to functionally characterize some of the putative CSLAs and GMP identified, in Chapter 3 and Chapter 4, I complemented the Arabidopsis csla 2,3,9 and vtc1-1 mutants, respectively. In Chapter 3, I showed that PtCSLA1 and PgCSLA1 successfully recovered mannan content in the stems of the Arabidopsis csla 2,3,9 mutant, while in Chapter 4, I showed that PtaGMP1 and PgGMP1 recovered the phenotype of the vtc1-1 mutants. In this work, I used two different approaches to manipulate the mannan content in tree xylem. Our results showed that, to some extent, mannan content can be significantly changed by altering either 93 of these gene families, however, more drastic changes were observed when manipulating genes encoding CSLAs. Specifically, in Chapter 3, I showed that a mutation to PtaCSLA1 alone was sufficient to abolish mannan in poplar xylem. This result not only suggests that PtaCSLA1 is the main enzyme responsible for synthesizing this polysaccharide in poplar xylem, but also allowed us to understand some of the consequences of eliminating mannan from the cell walls on poplar plant growth. Our results indicate that an absence of mannan does not drastically affect SCW formation, suggesting that it may play only a minor role in poplar trees, and it also does not appear to possess a major signalling role, as no observable phenotypes were apparent. Specifically, we showed that eliminating mannan from xylem did not affect tree height, stem diameter, or biomass as well as the whole composition of the secondary cell wall. In agreement with these findings, in Chapter 3, we showed that expressing PgCSLA1 in poplar can increase the content of mannan in the xylem, again, without affecting plant growth. Continuing with the overarching strategy to modify mannan, in Chapter 4, I demonstrated that a significant reduction in the content of this polysaccharide manifests when PtaGMP1 is mutated. However, in contrast with the PtaCSLA1 mutants, mannan was not completely abolished from the xylem. As discussed in the thesis, one of the reasons why we suspected mannan was not eliminated is because PtaGMP2 is likely still contributing to the production of GDP-Man in these plants. In this chapter, we showed that a small reduction did not affect plant growth or cell wall composition. Finally, in an attempt to increase mannan content in trees, we expressed PgGMP1 in poplar. We observed that by overexpressing this gene with a xylem specific promoter, approximately a 14% in mannan was attainable with no major effects on plant yield or cell wall structure. Despite our results suggesting that PgGMP1 may be responsible for producing GDP-Man in trees, more studies are needed to directly associate this gene with the sole production of this substrate. 94 Overall, this research addresses some questions regarding the role of mannan in trees, showing that it does not appear to be essential for the development of the SCW and that its elimination or increase, at least in the ranges achieved in the current study, do not appear to affect plant growth. Our research forms the foundation for future research to further improve our understanding of the functional role of mannan in plants. 5.2 Mannan removal as a potential strategy to reduce biomass recalcitrance Facilitating enzyme access to the hydrophobic face of cellulose microfibrils and microfibril bundles is essential for the enzymatic hydrolysis of cellulose into monomeric glucose for fermentation to value-added fuels (Liu et al., 2022). One of the strategies to improve the efficiency of biomass saccharification is by genetically modifying the factors that contribute to the cell wall complexity. Highly cross-linked lignin-carbohydrate complexes have been shown to play an important role in determining the microporous structure of the cell wall that often prevents the access of cellulose-degrading enzymes (Li et al., 2019; Liu et al., 2022; Lyczakowski et al., 2017). Since mannan is one of the polysaccharides that is intricately associated with both lignin and cellulose, we hypothesized that it may potentially influence biomass processing (Terrett et al., 2019; Terrett & Dupree, 2019). In the present study, we generated poplar lines that presented variable levels of mannan content, including trees where mannan was absent or at least undetectable. Future studies should evaluate how the lack of mannan in the xylem of poplar trees may contribute to a reduction in cross-linked lignin-carbohydrate complexes and how this may or may not affect biomass recalcitrance via saccharification assays. 5.3 Mannan structural modifications as a potential strategy to improve biomass processing In xyloglucan and xylan, side chains have been shown to play an essential role in maintaining the interactions that these polymers have with cellulose, hence influencing cell wall recalcitrance and 95 plant growth. In xylan specifically, the mutation of the acetyltransferase ESK1 prevented the interaction between xylan and cellulose and caused dwarfism and collapsed xylem vessels in Arabidopsis plants (Grantham et al., 2017). In poplar, the overexpression of PtRWA-C, a xylan O-acetyltransferase, increased lignin content and altered the S/G ratio leading to a decrease in the saccharification efficiency (Zhang et al., 2023). Similarly, the mutation in MUR3, a xyloglucan galactosyltransferase, significantly perturbed Arabidopsis plant growth (Xiang et al., 2023). Mutants of MUR3 showed reduced cellulose content, different cellulose microfibril pattering when compared to wild-type plants, and impaired hypocotyl elongation and stem development (Xiang et al., 2023). In the case of mannan, the presence of galactosylations and acetylations also interferes with the associative behaviour of this polysaccharide with other cell wall polymers. For example, galactose substitutions along the mannan backbone contributed to the aqueous solubility of this polymer, as well as influenced cellulose crystallinity (Berglund et al., 2020; Voiniciuc et al., 2015). In support of this in planta observation, mannans from locust bean gum and kojac glucomannan chemically modified to have different degrees of acetylation were also shown to alter mannan solubility when different solvents were used during extraction (Berglund et al., 2020). One of the future directions of mannan research should focus on modifying the structure of this polysaccharide by altering the extent of its side chain decorations. To date, while it is known that galactose side chains contribute to the aqueous solubility of mannan (Berglund et al., 2020; Voiniciuc et al., 2015), the enzymes responsible for this process remain unidentified. By employing a methodology akin to that employed in the present study (i.e. phylogenetics, Arabidopsis mutant complementation, and CRISPR in poplar trees), it would be possible to identify the genes implicated in this process. 96 Regarding the genes responsible for encoding the enzymes involved in decorating the mannan backbone with acetyl groups in trees, there is more information available. In Populus trichocarpa (Ptr), four genes have been identified as MOATs belonging to the DUF231 family (PtrMOAT1, GenBank accession number: MK876281; PtrMOAT2, GenBank accession number: MK876282; PtrMOAT3, GenBank accession number: MK876283; and PtrMOAT4 GenBank accession number: MK876284; Zhong et al., 2019). Recombinant proteins of these DUF231 genes were generated in human embryonic kidney (HEK) 293 cells and exhibited O-acetyltransferase activity toward mannohexaose, however, to date, none of these genes have been characterized in planta (Zhong et al., 2019a). Future studies should corroborate the in vitro function of these enzymes by complementing the Arabidopsis moat1,2,3,4 mutant using the CESA7 promoter or a similar xylem-specific promoter to express these candidate genes. Along the same lines, another intriguing aspect to explore would be evaluating the impact of altering the content of acetyl groups on the mannan backbone in biomass processing. The degree of substitutions by acetyl groups (DSAC) in mannans varies depending on the plant species (Zhong et al., 2019). In Arabidopsis, the DSAC is 0.25, while in poplar it is 0.30, and in spruce it ranges from 0.27-0.32 (Zhong et al., 2019). Although no significant changes in plant growth were observed when the expression of MOAT genes were suppressed in Arabidopsis, Berglund et al., 2020 highlighted that some important properties of mannans can be altered by changing the content of acetyl groups. In this study, different types of chemically modified glucomannans were created, one classified as raw glucomannan (unacetylated), another one with a low degree of acetylations (DSAC=0.21), one with a middle degree (DSAC=0.58), and finally one with a high degree of acetyl groups (DSAC=2.85). They showed that the dispersibility and solubility of glucomannan with high levels of acetyl groups (DSAC=2.85), was 95% better when polar 97 solvents such as dimethyl sulfoxide were used. This type of solvent is normally used during biomass processing for the production of hydroxymethylfurfural, one of the components used for the generation of biofuels (Shuai & Luterbacher, 2016). By using the CESA7 promoter to express the putative MOATs of poplar, we could increase the extent of mannan acetylation and potentially increase the efficiency in the extraction of components of industrial interest derived from woody feedstocks. Similarly, one could enhance the degree of acetylations in the lines that presented higher amounts of mannan that were created in this study. 5.4 Mannan as a signal molecule and its implications in plant abiotic stress tolerance Galactoglucomannan oligosaccharides are produced by the cleavage of the mannan linkages by mannanase (MAN) enzymes in the plasma membrane, normally presenting a degree of polymerization between 4-8 backbone sugar units (Zhao et al., 2013). Multiple studies support that GGMOs are important signaling molecules that regulate plant growth and development, as well as plant-adaptive responses to abiotic and biotic stress (Chen et al., 2015; Kučerová et al., 2014; Zhang et al., 2023). GGMOs seem to be important in alleviating cadmium (Cd) toxicity in plant roots, which is a toxic soil pollutant present in multiple phosphate fertilizers used in agriculture (Kučerová et al., 2014). The application of GGMOs to Arabidopsis and maize plants was shown to reduce the concentration of Cd in leaves, increase plant growth, and the concentration of photosynthetic pigments (Kučerová et al., 2014; Vivodová et al., 2023). In Arabidopsis, the overexpression of MAN3 led to an enhanced Cd tolerance, while mutation of this gene caused a reduction in the tolerance to Cd in these plants (Chen et al., 2015). Furthermore, the addition of GGMOs has been even shown to stimulate root growth under non-stress conditions in Arabidopsis plants (Kučerová et al., 2014). In addition to their biological function, GGMOs are also of interest 98 for their prebiotic properties, as currently, they are used as a feed additive to prevent pathogen colonization in the digestive tract of humans and animals (Singh, Ghosh, et al., 2018). Alterations in the production of GGMOs in trees have been manipulated by mutating and overexpressing MANs present in the plasma membrane. In P. trichocarpa specifically, eight MANs have been predicted (PtrMAN1 to PtrMAN8; Yuan et al., 2006). These genes were differentially expressed in multiple tissues including differentiating xylem and phloem, as well as leaves and shoot tips (Zhao et al., 2013). In transgenic lines where PtrMAN6 (Potri.016G138600) was overexpressed, lignin deposition was significantly delayed and a reduction in crystalline cellulose was also observed, suggesting that the modification of this enzyme in trees is associated with the suppression of SCW thickening during xylem development (Zhao et al., 2013). Although in the present study, we did not observe significant changes in cell wall composition when mannan was abolished or reduced in the xylem, we did not evaluate the consequences of increasing mannan amount and its subsequent cleavage. Future studies should delve into the consequences of simultaneously overexpressing CSLAs and MANs. Increasing the production of GGMOs in trees can be achieved, for example, by creating mutants where PgGMP1, PgCSLA1, and PtrMAN6 are concurrently expressed. In this way, plants should produce higher concentrations of these oligosaccharides. Increasing the number of GGMOs in trees may expand our knowledge about their role in plant Cd tolerance and cell wall integrity, as well as serve to create products of industrial interest. 5.5 Mannan and other polymers in the cell wall It is very intriguing for us the reason why mannan has been conserved along plant evolution in the secondary cell wall of all multiple groups of plants, including angiosperms. In our study, we were not able to detect major changes in growth or cell wall composition in the lines where mannan was 99 eliminated from stem tissues, which raises new questions about the functional role of this polysaccharide in this tissue. In recent studies examining primary cell wall development, the role of mannan was only evident when XyG was also removed from plants, suggesting an association between these two hemicelluloses (Yu et al., 2022). Our study provides an excellent platform to study the contribution of mannan along with other polymers to the cell wall architecture. Future studies can examine the role that mannan plays in secondary cell wall synthesis by modifying genes associated with the synthesis of other polymers, such as xylan (Lee, Zhong, et al., 2012b; Ratke et al., 2018), and this should start with the transgenic knockout poplar lines generated in the present study. 5.6 Mannan trafficking The Golgi apparatus and the trans-Golgi network are required for the biosynthesis of various matrix polysaccharides and their delivery to the cell wall (Avci, 2022). They constitute an important and sensitive system that ensures the timing and regular delivery of these components to the plasma membrane needed for normal plant development (Avci, 2022). In the case of xylan, it has been proposed that its biosynthesis and packaging occur in different domains of the Golgi, whose structure changes once the deposition of the secondary cell wall has initiated (Meents et al., 2019). Arabidopsis irx9 mutants, for instance, showed dramatic changes in Golgi structure with an increase in cisternal fenestration and tubulation (Meents et al., 2019). To date, the mechanisms involved in the transport of other polysaccharides that constitute the cell wall remain unknown. In the case of mannan trafficking, it is still not clear if there is a differential participation of the Golgi stacks in its synthesis and their association with the SCW once mannan starts to be deposited. Likewise, no information is available regarding the type of vesicles involved in its transport to the 100 secondary cell wall. Improving our knowledge of these mechanisms will allow us to better understand the dynamic organization of the cell wall. The Arabidopsis model system that uses the inducible transcription factor VASCULAR-RELATED NAC-DOMAIN 7 (VND7) has been used to study the localization and movement of different enzymes and therefore, constitutes an excellent tool to study mannan trafficking (Meents et al., 2019; Watanabe et al., 2015; Yamaguchi et al., 2010). VND7 is a master regulator of protoxylem development, which when activated causes the differentiation of cells into protoxylem tracheary elements with helical secondary cell walls (Yamaguchi et al., 2010). In Arabidopsis, one of the genes that can be targeted to study mannan trafficking is CSLA9. 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New Phytologist, 208(2), 298–301. https://doi.org/10.1111/nph.13470 129 Appendices Appendix A - Potri.008G026400 transcript sequence from poplar 717 This DNA sequence was used as a reference for gene synthesis by Azenta 5′ATGGATCGTTTATCATCTGCTAGTAACATTTTTCCTGCCGGTCGGGATGGTGTTACCGGCCAACTTGGATTGATTTGGCAAGTAATAATGGAACCATTGATCGTGCCGGTCTTGAAGTTATTGGTGGTAGTGTGTTTGGGCATGTCAATAATGCTTTTCATTGAACGAGTGTACATGGGGGTTGTTATAGTTTTTGTGAAGTTGTTCGGGAAAAAACCAAATAAGAGCTACAAATGGGAGCCTATAAAGGATGATATTGAGCTTGGCAACTCAGCTTATCCTATGGTTCTGGTGCAAGTTCCAATGTACAATGAAAAGGAGGTTTATCAGTTGTCTATTGGAGCTGCCTGCGGGCTTTCTTGGCCGTCCGACCGGATCATAATCCAAGTTCTTGATGACTCAACAGACCCTGCCATTAAGAGCATGGTGGAAGTAGAATGTCAGAGATGGGCAAGCAAAGGCATCAATATAAAGTATGAGATCAGGGACAGCAGGAATGGATACAAGGCAGGAGCTCTCAAGGAAGGCATGAAGCACAGCTATGTTAAAGAATGTGATTACGTGGCCATATTCGACGCTGATTTTCAGCCAGAACCTGACTTTCTTTGGCGCACCATACCATTTCTAGCTCACAATCCTGAAATTGCTCTTGTTCAAGCTCGCTGGAAATTCGTCAATTCTGATGAGTGCTTAATGACAAGGATGCAAGAGATGTCATTGGATTACCATTTCACTGTGGAACAAGAAGTGGGGTCTGCTACCTACGCTTTCTTTGGATTCAATGGTACTGCTGGTGTGTGGAGAATTTCAGCAGTCAATGAAGCTGGAGGGTGGAAGGCTCGAACAACGGTGGAGGACATGGATTTGGCTGTCAGGGCTAGCCTCCAAGGCTGGAAGTTTGTCTATGTTGGTGACCTCAAGGTGAAAAATGAATTGCCAAGCACTTTCAAAGCATATCGATATCAGCAACATAGATGGTCTTGTGGGCCAGCAAATCTCTTCAGGAAAATGGCTATAGAAATAGTAAAAAACAAGAAAGTGTCAGTGTGGAAGAAATGTTATGTGATCTACAGTTTCTTCTTCGTCCGCAAGATTGTGGCACATCTTGTCACGTTTTTATTCTACTGTGTTGTTTTGCCTGCAACTGTTCTGTTTCCTGAAGTAACTGTTCCTAAATGGGGAGCTGTCTACATTCCTTCAGTTGTAACTCTCCTCAATGCTGTTGGAACCCCTAGATCACTCCATCTGATGGTTTTCTGGATCCTATTTGAGAATGTTATGTCTCTGCACCGGACAAAGGCGACATTCATAGGTCTTCTGGAGGCAGGAAGAGTGAATGAATGGGTTGTCACTGAAAAATTAGGAGATGCTCTTAAGGCCAAATTACCTGCTAAAGCTACCAAGAAACCGCGGATTAGGATCGGAGAAAGATTACATTTGCTAGAACTCTGTGCTGGAGCCTATCTCTTCTTCTGTGGATGCTATGATTTTGCCTTTGGAAAGAACAGATATTACATATTCCTCTTCCTCCAATCCATTGCCTTCTTCATTGCAGGATTTGGTTACATTGGCACCTTTGTCCCCCGCTCTTAG –′3
Thesis/Dissertation
eng
Forestry
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Mansfield, Shawn D., 1969-
Unraveling mannan biosynthesis in poplar and spruce : functional characterization of cellulose synthase-Like family A (CSLA) and guanosine-diphosphate-mannose (GMP) genes in poplar
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